Peptide profiling: Correlating estrogen receptor conformation with biological response

Abstract only availableChemicals found in the environment have been found to behave like the body's natural estrogen, estradiol. These exogenous estrogen-mimicking compounds have been termed xenoestrogens. Both estradiol and xenoestrogens can bind two estrogen receptors (ERs), ER alpha and ER beta, to elicit biological responses. The receptors are ligand inducible transcription factors that exhibit unique biological actions. While estradiol binds both receptors equally, some xenoestrogens have been shown to bind ER beta preferentially. When the ER is bound, the ligand induces a unique ER shape and in turn causes an array of tissue-specific biological responses. For example, the ligand tamoxifen, a commonly used breast cancer pharmaceutical, exhibits an ER antagonist response in the breast and an ER agonist response in the bone. This dual ligand quality characterizes what is now known as a selective estrogen receptor modulator (SERM). Peptide profiling, a novel ER ligand screening assay, is a method that can potentially identify SERMS by correlating in vitro ER conformation with in vivo biological response. Each ligand is screened using a two-hybrid fusion protein reporter gene assay. Upon ligand binding, the ER assumes a conformation; with this induced shape, some ER-interacting peptides will be able to bind while others will not. After screening a ligand against a library of fifteen different peptides, a unique peptide profile will figuratively illustrate the induced ER conformation. Eight xenoestrogens were screened in this experiment: estradiol, a natural physiological estrogen; resveratrol and genistein, two phytoestrogens; MPP, bisphenol A, and 4-hydroxytamoxifen, all synthetic estrogens; α-endosulfan and methoxychlor, both insecticides used on crops. Each ligand was found to have a unique peptide profile and, implicitly, a distinct ER conformation. The next step will be to determine each ligand's tissue specific activity and identify the unique peptide fingerprint that predicts its in vivo biological response. By correlating a ligand's tissue specific estrogenic activity with its unique ER conformation, peptide profiling will not only further elucidate tissue-specific ER activity differences but could also be used as a high-throughput screening tool for other potential environmental xenoestrogens and identify novel therapeutic SERMs.Life Sciences Undergraduate Research Opportunity Progra

Effect of reflood prediction uncertainties on LOFT cladding oxidation

The FLOOD4 and RELAP4/MOD6 computer codes, which are used to perform LOFT reflood analysis, have been compared to FLECHT-SET and Semiscale gravity feed tests to provide an evaluation of core reflood prediction techniques and an identification of phenomena important to LOFT reflood behavior. These comparisons provide a basis for estimating uncertainty in cladding temperature history during the LOFT loss-of-coolant experiments (LOCEs). The bounds on the cladding temperature response are then utilized to estimate a range of expected cladding oxidation and embrittlement which is essential for identifying special equipment needed during replacement, storage, and post-test examination of LOFT fuel modules. FLOOD4 couples the system hydraulic response with core heat transfer and steam generation. Four heat transfer correlations simulate the boiling curve and liquid entrainment, fallback and vaporization in the steam generators are modeled. FLOOD4 requires user input multipliers to specify the dispersed flow heat transfer, liquid entrainment and correlations to describe liquid fallback from the upper plenum region. The fraction of liquid vaporized in the steam generators must also be user input

LOFT fuel rod thermal/mechanical behavior predictions

Best-estimate predictions of LOFT fuel rod behavior during the first LOFT nuclear test series have been made with both RELAP/FLOOD4/FRAP-T and RELAP/SCORE/MOXY computer code combinations. The purpose of the predictions are to test the best-estimate code accuracy by comparison of predictions to subsequent test data and to develop plans for post-test fuel replacements and examinations. The FRAP-T3 computer code was utilized which couples the thermal, mechanical, internal gas, and material property behavior of a nuclear rod during various postulated accident sequences. The cladding deformation models account for elastic-plastic behavior. FRAP-T3 requires coolant conditions to be specified as input boundary conditions. The coolant conditions for the blowdown and refill phases of a loss-of-coolant experiment (LOCE) were calculated with RELAP4/MOD6 and for the reflood portion with FLOOD4

A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability

The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34(+) cord blood using non-viral, non-integrating methods.We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34(+) cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64-89%) of cardiac troponin I(+) cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine

Present state and future perspectives of using pluripotent stem cells in toxicology research

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed

Individual, social, and environmental factors affecting salivary and fecal cortisol levels in captive pied tamarins (Saguinus bicolor)

This is the peer reviewed version of the following article: Price, E., Coleman, R., Ahsmann, J., Glendewar, G., Hunt, J., Smith, T. & Wormell, D. (2019). Individual, social, and environmental factors affecting salivary and fecal cortisol levels in captive pied tamarins (Saguinus bicolor). American Journal of Primatology, 81(8), which has been published in final form at https://doi.org/10.1002/ajp.23033. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-ArchivingPied tamarins (Saguinus bicolor) are endangered New World primates, and in captivity appear to be very susceptible to stress. We measured cortisol in 214 saliva samples from 36 tamarins and in 227 fecal samples from 27 tamarins, and investigated the effects of age, sex, pregnancy, rearing history, social status, weight, group composition, and enclosure type using generalized linear mixed models. There was no effect of age on either fecal or salivary cortisol levels. Female pied tamarins in late pregnancy had higher fecal cortisol levels than those in early pregnancy, or nonpregnant females, but there was no effect of pregnancy on salivary cortisol. Females had higher salivary cortisol levels than males, but there was no effect of rearing history. However, for fecal cortisol, there was an interaction between sex and rearing history. Hand‐reared tamarins overall had higher fecal cortisol levels, but while male parent‐reared tamarins had higher levels than females who were parent‐ reared, the reverse was true for hand‐reared individuals. There was a trend towards lower fecal cortisol levels in subordinate individuals, but no effect of status on salivary cortisol. Fecal but not salivary cortisol levels declined with increasing weight. We found little effect of group composition on cortisol levels in either saliva or feces, suggesting that as long as tamarins are housed socially, the nature of the group is of less importance. However, animals in off‐show enclosures had higher salivary and fecal cortisol levels than individuals housed on‐show. We suggest that large on‐show enclosures with permanent access to off‐exhibit areas may compensate for the effects of visitor disturbance, and a larger number of tamarins of the same species housed close together may explain the higher cortisol levels found in tamarins living in off‐show accommodation, but further research is needed

Provisional Application of the Energy Charter Treaty: The Yukos Arbitration and the Future Place of Provisional Application in International Law

Section I discusses the basis, use, and advantages of the provisional application of treaties in international law. Section II will provide a general overview of the ECT, with a specific emphasis on the treaty provisions likely to arise in the Yukos Arbitration; and Section III presents a brief factual background to the Yukos Arbitration. Section IV analyzes the Yukos Arbitration by examining how the characterization of the obligations imposed by provisional application under the ECT should be resolved in light of the purpose behind provisional application of treaties. Section V will discuss how the outcome of the Yukos Arbitration has the potential to impact foreign investment under the ECT. The Comment concludes with a general discussion of the future place of provisional application in international treaty law