Unconventional isoquinoline-based SERMs elicit fulvestrant-like transcriptional programs in ER+ breast cancer cells.

Estrogen receptor alpha (ERα) is a ligand-dependent master transcriptional regulator and key driver of breast cancer pathology. Small molecule hormones and competitive antagonists favor unique ERα conformational ensembles that elicit ligand-specific transcriptional programs in breast cancer and other hormone-responsive tissues. By affecting disparate ligand binding domain structural features, unconventional ligand scaffolds can redirect ERα genomic binding patterns to engage novel therapeutic transcriptional programs. To improve our understanding of these ERα structure-transcriptional relationships, we develop a series of chemically unconventional antagonists based on the antiestrogens elacestrant and lasofoxifene. High-resolution x-ray co-crystal structures show that these molecules affect both classical and unique structural motifs within the ERα ligand binding pocket. They show moderately reduced antagonistic potencies on ERα genomic activities but are effective anti-proliferative agents in luminal breast cancer cells. Interestingly, they favor a 4-hydroxytamoxifen-like accumulation of ERα in breast cancer cells but lack uterotrophic activities in an endometrial cell line. Importantly, RNA sequencing shows that the lead molecules engage transcriptional pathways similar to the selective estrogen receptor degrader fulvestrant. This advance shows that fulvestrant-like genomic activities can be achieved without affecting ERα accumulation in breast cancer cells.

First Precision Measurement of the Parity Violating Asymmetry in Cold Neutron Capture on

We report the first precision measurement of the parity-violating asymmetry in the direction of proton momentum with respect to the neutron spin, in the reaction ^{3}He(n,p)^{3}H, using the capture of polarized cold neutrons in an unpolarized active ^{3}He target. The asymmetry is a result of the weak interaction between nucleons, which remains one of the least well-understood aspects of electroweak theory. The measurement provides an important benchmark for modern effective field theory and potential model calculations. Measurements like this are necessary to determine the spin-isospin structure of the hadronic weak interaction. Our asymmetry result is A_{PV}=[1.55±0.97(stat)±0.24(sys)]×10^{-8}, which has the smallest uncertainty of any hadronic parity-violating asymmetry measurement so far.

Imaging Fluorescence of He_{2}

We show unequivocal evidence for formation of He_{2}^{*} excimers in liquid He II created by ionizing radiation produced through neutron capture. Laser beams induce fluorescence of the excimers. The fluorescence is recorded at a rate of 55.6 Hz by a camera. The location of the fluorescence is determined with an uncertainty of 5 µm. The technique provides an opportunity to record the flow of He_{2}^{*} excimers in a medium with very small viscosity and enables measurement of turbulence around macroscopic liter size objects or vortex matter in three dimensions.

Specific stereochemistry of OP-1074 disrupts estrogen receptor alpha helix 12 and confers pure antiestrogenic activity.

Complex tissue-specific and cell-specific signaling by the estrogen receptor (ER) frequently leads to the development of resistance to endocrine therapy for breast cancer. Pure ER antagonists, which completely lack tissue-specific agonist activity, hold promise for preventing and treating endocrine resistance, however an absence of structural information hinders the development of novel candidates. Here we synthesize a small panel of benzopyrans with variable side chains to identify pure antiestrogens in a uterotrophic assay. We identify OP-1074 as a pure antiestrogen and a selective ER degrader (PA-SERD) that is efficacious in shrinking tumors in a tamoxifen-resistant xenograft model. Biochemical and crystal structure analyses reveal a structure activity relationship implicating the importance of a stereospecific methyl on the pyrrolidine side chain of OP-1074, particularly on helix 12.

First Observation of P-odd γ Asymmetry in Polarized Neutron Capture on Hydrogen.

We report the first observation of the parity-violating gamma-ray asymmetry A_{γ}^{np} in neutron-proton capture using polarized cold neutrons incident on a liquid parahydrogen target at the Spallation Neutron Source at Oak Ridge National Laboratory. A_{γ}^{np} isolates the ΔI=1, ^{3}S_{1}→^{3}P_{1} component of the weak nucleon-nucleon interaction, which is dominated by pion exchange and can be directly related to a single coupling constant in either the DDH meson exchange model or pionless effective field theory. We measured A_{γ}^{np}=[-3.0±1.4(stat)±0.2(syst)]×10^{-8}, which implies a DDH weak πNN coupling of h_{π}^{1}=[2.6±1.2(stat)±0.2(syst)]×10^{-7} and a pionless EFT constant of C^{^{3}S_{1}→^{3}P_{1}}/C_{0}=[-7.4±3.5(stat)±0.5(syst)]×10^{-11} MeV^{-1}. We describe the experiment, data analysis, systematic uncertainties, and implications of the result.

Precision determination of absolute neutron flux.

A technique for establishing the total neutron rate of a highly-collimated monochromatic cold neutron beam was demonstrated using an alpha-gamma counter. The method involves only the counting of measured rates and is independent of neutron cross sections, decay chain branching ratios, and neutron beam energy. For the measurement, a target of 10B-enriched boron carbide totally absorbed the neutrons in a monochromatic beam, and the rate of absorbed neutrons was determined by counting 478 keV gamma rays from neutron capture on 10B with calibrated high-purity germanium detectors. A second measurement based on Bragg diffraction from a perfect silicon crystal was performed to determine the mean de Broglie wavelength of the beam to a precision of 0.024%. With these measurements, the detection efficiency of a neutron monitor based on neutron absorption on 6Li was determined to an overall uncertainty of 0.058%. We discuss the principle of the alpha-gamma method and present details of how the measurement was performed including the systematic effects. We also describe how this method may be used for applications in neutron dosimetry and metrology, fundamental neutron physics, and neutron cross section measurements.

Improved determination of the neutron lifetime.

The most precise determination of the neutron lifetime using the beam method was completed in 2005 and reported a result of τ(n)=(886.3±1.2[stat]±3.2[syst]) s. The dominant uncertainties were attributed to the absolute determination of the fluence of the neutron beam (2.7 s). The fluence was measured with a neutron monitor that counted the neutron-induced charged particles from absorption in a thin, well-characterized 6Li deposit. The detection efficiency of the monitor was calculated from the areal density of the deposit, the detector solid angle, and the evaluated nuclear data file, ENDF/B-VI 6Li(n,t)4He thermal neutron cross section. In the current work, we measure the detection efficiency of the same monitor used in the neutron lifetime measurement with a second, totally absorbing neutron detector. This direct approach does not rely on the 6Li(n,t)4He cross section or any other nuclear data. The detection efficiency is consistent with the value used in 2005 but is measured with a precision of 0.057%, which represents a fivefold improvement in the uncertainty. We verify the temporal stability of the neutron monitor through ancillary measurements, allowing us to apply the measured neutron monitor efficiency to the lifetime result from the 2005 experiment. The updated lifetime is τ(n)=(887.7±1.2[stat]±1.9[syst]) s.

RAC3 is a pro-migratory co-activator of ERα.

Estrogen receptor alpha (ERα) is a ligand-dependent nuclear receptor that is important in breast cancer genesis, behavior and response to hormone-based therapies. A T7 phage display screen against full-length human ERα, coupled with genome-wide exon arrays, was used to identify RAC3 as a putative ERα co-regulator. RAC3 is a Rho family small GTPase that is associated with cytoskeletal rearrangement. We demonstrate a novel role for nuclear RAC3 as an ERα transcriptional activator, with prognostic implications for metastatic disease. Through in vitro and cell-based studies, RAC3 was shown to exist in a GTP-bound state and act as a ligand specific ERα co-activator of E2-induced transcription. Overexpression of RAC3 induced pro-growth and pro-migratory genes that resulted in increased migration of ERα-positive breast cancer cells. Chemical inhibition and genetic knockdown of RAC3 antagonized E2-induced cell proliferation, cell migration and ERα mediated gene expression, indicating that RAC3 is necessary for full ERα transcriptional activity. In agreement with the molecular and cellular data, RAC3 overexpression in ERα-positive breast cancers correlated with a significant decrease in recurrence free survival and a significant increase in the odds ratio of metastasis. In conclusion, RAC3 is a novel ERα co-activator that promotes cell migration and has prognostic value for ERα-positive breast cancer metastasis. RAC3 may also be a useful therapeutic target for ERα-positive breast cancers.

Characterization of the ovarian and reproductive abnormalities in prepubertal and adult estrogen non-responsive estrogen receptor alpha knock-in (ENERKI) mice.

Estrogen non-responsive estrogen receptor alpha (ERalpha) knock-in (ENERKI) mice have a mutation (glycine 525 to leucine, G525L) in the ligand-binding domain of ERalpha. The mutant ERalpha protein has a significantly lower affinity and response to endogenous estrogens, while not altering growth factor activated ligand-independent pathways. ENERKI females demonstrated signs of early follicle development as determined by a significant increase in antral follicle formation by 20 days of age. Adult ENERKI females were infertile, had hemorrhagic ovarian follicular cysts, and failed to develop corpora lutea in response to a superovulation regimen. These results illustrate the importance of ERalpha ligand-induced signaling for ovarian development and for estrogen feedback on the hypothalamus and pituitary. Although ERalpha ligand-induced signaling by endogenous estrogens is lost in ENERKI females, the ERalpha selective agonist propyl pyrazole triol (PPT), a synthetic nonsteroidal compound, is still able to activate G525L ERalphain vivo to increase uterine weight. To test whether PPT could restore ligand-dependent receptor activation, ENERKI females were treated with PPT and evaluated for spontaneous ovulation, ovarian hemorrhagic cysts, and LH serum levels. Daily PPT treatments beginning on day 4 of life prevented formation of ovarian hemorrhagic cysts in adult ENERKI animals. In accordance with this result, preputial gland weight and LH levels were also lowered in these animals, indicating PPT treatments most likely led to restoration of ERalpha negative feedback of the hypothalamic-pituitary axis.

Beamline Performance Simulations for the Fundamental Neutron Physics Beamline at the Spallation Neutron Source.

Monte Carlo simulations are being performed to design and characterize the neutron optics components for the two fundamental neutron physics beamlines at the Spallation Neutron Source. Optimization of the cold beamline includes characterization of the guides and benders, the neutron transmission through the 0.89 nm monochromator, and the expected performance of the four time-of-flight choppers. The locations and opening angles of the choppers have been studied using a simple spreadsheet-based analysis that was developed for other SNS chopper instruments. The spreadsheet parameters are then optimized using Monte Carlo techniques to obtain the results presented in this paper. Optimization of the 0.89 nm beamline includes characterizing the double crystal monochromator and the downstream guides. The simulations continue to be refined as components are ordered and their exact size and performance specifications are determined.

Measurement of Parity Violation in np Capture: the NPDGamma Experiment.

The NPDGamma experiment will measure the parity-violating directional gamma ray asymmetry A γ in the reaction [Formula: see text]. Ultimately, this will constitute the first measurement in the neutron-proton system that is sensitive enough to challenge modern theories of nuclear parity violation, providing a theoretically clean determination of the weak pion-nucleon coupling. A new beam-line at the Los Alamos Neutron Science Center (LANSCE) delivers pulsed cold neutrons to the apparatus, where they are polarized by transmission through a large volume polarized (3)He spin filter and captured in a liquid para-hydrogen target. The 2.2 MeV gamma rays from the capture reaction are detected in an array of CsI(Tl) scintillators read out by vacuum photodiodes operated in current mode. We will complete commissioning of the apparatus and carry out a first measurement at LANSCE in 2004-05, which would provide a statistics-limited result for A γ accurate to a standard uncertainty of ±5 × 10(-8) level or better, improving on existing measurements in the neutron-proton system by a factor of 4. Plans to move the experiment to a reactor facility, where the greater flux would enable us to make a measurement with a standard uncertainty of ±1 × 10(-8), are actively being pursued for the longer term.

Commissioning of the NPDGamma Detector Array: Counting Statistics in Current Mode Operation and Parity Violation in the Capture of Cold Neutrons on B 4 C and (27) Al.

The NPDGamma γ-ray detector has been built to measure, with high accuracy, the size of the small parity-violating asymmetry in the angular distribution of gamma rays from the capture of polarized cold neutrons by protons. The high cold neutron flux at the Los Alamos Neutron Scattering Center (LANSCE) spallation neutron source and control of systematic errors require the use of current mode detection with vacuum photodiodes and low-noise solid-state preamplifiers. We show that the detector array operates at counting statistics and that the asymmetries due to B4C and (27)Al are zero to with- in 2 × 10(-6) and 7 × 10(-7), respectively. Boron and aluminum are used throughout the experiment. The results presented here are preliminary.

New Pulsed Cold Neutron Beam Line for Fundamental Nuclear Physics at LANSCE.

The NPDGamma collaboration has completed the construction of a pulsed cold neutron beam line on flight path12 at the Los Alamos Neutron Science Center (LANSCE). We describe the new beam line and characteristics of the beam. We report results of the moderator brightness and the guide performance measurements. FP12 has the highest pulsed cold neutron intensity for nuclear physics in the world.

Measurement of the Neutron Lifetime by Counting Trapped Protons.

We measured the neutron decay lifetime by counting in-beam neutron decay recoil protons trapped in a quasi-Penning trap. The absolute neutron beam fluence was measured by capture in a thin (6)LiF foil detector with known efficiency. The combination of these measurements gives the neutron lifetime: τ n = (886.8 ± 1.2 ± 3.2) s, where the first (second) uncertainty is statistical (systematic) in nature. This is the most precise neutron lifetime determination to date using an in-beam method.

Measurement of Neutron Decay Parameters-The abBA Experiment.

We are developing an experiment to measure the correlations a, A, and B, and the Fierz interference term b in neutron decay, with a precision of approximately 10(-4). The experiment uses an electromagnetic spectrometer in combination with two large-area segmented silicon detectors to detect the proton and electron from the decay in coincidence, with 4π acceptance for both particles. For the neutron-polarization-dependent observables A and B, precision neutron polarimetry is achieved through the combination of a pulsed neutron beam, under construction at the SNS, and a polarized (3)He neutron polarizer. Measuring a and A in the same apparatus provides a redundant determination of λ = gA/gV . Uncertainty in λ dominates the uncertainty of CKM unitarity tests.

Neutron-detected tomography of impurity-seeded superfluid helium.

We describe a neutron radiography technique that can be used to map the distribution of 3He impurities in liquid 4He, providing direct and quantitative access to underlying transport processes. Images reflecting finite normal- and superfluid-component 4He velocity fields are presented.

A structural explanation for ERalpha/ERbeta SERM discrimination.

Many NRs have multiple subtypes that possess distinct expression patterns and that regulate distinct target genes. Antagonists generated through the addition of bulky side chains to agonist scaffolds are limited to being antagonistic on one or more subtypes of a particular NR. The passive antagonism mechanism, as revealed in our studies through direct comparison of the two THC-ER LBD complexes, suggests a new approach to achieving NR antagonism. Compounds could be designed to selectively stabilize the inactive conformations of certain NR subtypes and the active conformations of others. Such ligands are likely to exert novel biological and therapeutic effects.

Measurement of the neutron lifetime using a proton trap.

We report a new measurement of the neutron decay lifetime by the absolute counting of in-beam neutrons and their decay protons. Protons were confined in a quasi-Penning trap and counted with a silicon detector. The neutron beam fluence was measured by capture in a thin 6LiF foil detector with known absolute efficiency. The combination of these simultaneous measurements gives the neutron lifetime: tau(n)=(886.8+/-1.2[stat]+/-3.2[syst]) s. The systematic uncertainty is dominated by uncertainties in the mass of the 6LiF deposit and the 6Li(n,t) cross section. This is the most precise measurement of the neutron lifetime to date using an in-beam method.

Estrogen receptor pathways to AP-1.

Estrogen receptor (ER) binds to estrogen response elements in target genes and recruits a coactivator complex of CBP-pl60 that mediates stimulation of transcription. ER also activates transcription at AP-1 sites that bind the Jun/Fos transcription factors, but not ER. We review the evidence regarding mechanisms whereby ER increases the activity of Jun/Fos and propose two pathways of ER action depending on the ER (alpha or beta) and on the ligand. We propose that estrogen-ERalpha complexes use their activation functions (AF-1 and AF-2) to bind to the p 160 component of the coactivator complex recruited by Jun/Fos and trigger the coactivator to a higher state of activity. We propose that selective estrogen receptor modulator (SERM) complexes with ERbeta and with truncated ERalpha derivatives use their DNA binding domain to titrate histone deacetylase (HDAC)-repressor complexes away from the Jun/Fos coactivator complex, thereby allowing unfettered activity of the coactivators. Finally, we consider the possible physiological significance of ER action at AP-1 sites.

Estrogen inhibits vascular smooth muscle cell-dependent adventitial fibroblast migration in vitro.

BACKGROUND: Mounting experimental evidence suggests that estrogen treatment protects against neointima formation in response to vascular injury in vivo. Previous studies have suggested that this process includes the activation and migration of adventitial fibroblasts. The present in vitro study was designed to establish a mechanism whereby estrogen attenuates migration of adventitial fibroblasts. METHDS AND RESULTS: Primary cultures of vascular smooth muscle cells (VSMCs) and adventitial fibroblasts were derived from female Sprague-Dawley rats. Reverse transcriptase-polymerase chain reaction and Western blotting were used to determine that expression of the estrogen receptor (ER) was restricted to early-passage VSMCs. Migration of transduced (retrovirally mediated) fibroblasts was determined by counting the number of blue lacZ-expressing cells attached to Boyden-type chambers preconditioned under defined experimental conditions. Compared with growth medium alone, chambers treated with medium conditioned by VSMCs demonstrated a 2-fold increase in fibroblast migration, suggesting that VSMCs release soluble factor(s) competent to bind the Transwell membrane and promote fibroblast migration. In contrast, treatment of VSMCs with 17beta-estradiol (10(-9) to 10(-7) mol/L) before preconditioning of the chamber induced a dose-dependent inhibition of fibroblast migration. Cotreatment of VSMCs with 17beta-estradiol and the ER antagonist ICI-182780 (10(-7) mol/L) blocked the inhibitory effect of estrogen on fibroblast migration. CONCLUSIONS: These observations suggest a novel mechanism of hormonal vasoprotection by which estrogen directly modulates VSMC expression of factor(s) controlling migration of adventitial fibroblasts via an ER-dependent mechanism.