Measurement of the Permanent Electric Dipole Moment of the Neutron.

We present the result of an experiment to measure the electric dipole moment (EDM) of the neutron at the Paul Scherrer Institute using Ramsey's method of separated oscillating magnetic fields with ultracold neutrons. Our measurement stands in the long history of EDM experiments probing physics violating time-reversal invariance. The salient features of this experiment were the use of a ^{199}Hg comagnetometer and an array of optically pumped cesium vapor magnetometers to cancel and correct for magnetic-field changes. The statistical analysis was performed on blinded datasets by two separate groups, while the estimation of systematic effects profited from an unprecedented knowledge of the magnetic field. The measured value of the neutron EDM is d_{n}=(0.0±1.1_{stat}±0.2_{sys})×10^{-26} e.cm.

Prolactin receptors in Rip-cre cells, but not in AgRP neurones, are involved in energy homeostasis.

Among its many functions, prolactin has been implicated in energy homeostasis, particularly during pregnancy and lactation. The arcuate nucleus is a key site in the regulation of energy balance. The present study aimed to examine whether arcuate nucleus neuronal populations involved in energy homeostasis are prolactin responsive and whether they can mediate the effects of prolactin on energy homeostasis. To determine whether Agrp neurones or Rip-Cre neurones are prolactin responsive, transgenic mice expressing the reporter td-tomato in Agrp neurones (td-tomato/Agrp-Cre) or Rip-Cre neurones (td-tomato/Rip-Cre) were treated with prolactin and perfused 45 minutes later. Brains were processed for double-labelled immunohistochemistry for pSTAT5, a marker of prolactin-induced intracellular signalling, and td-tomato. In addition, Agrp-Cre mice and Rip-Cre mice were crossed with mice in which the prolactin receptor gene (Prlr) was flanked with LoxP sites (Prlrlox/lox mice). The Prlrlox/lox construct was designed such that Cre-mediated recombination resulted in deletion of the Prlr and expression of green fluorescent protein (GFP) in its place. In td-tomato/Rip-Cre mice, prolactin-induced pSTAT5 was co-localised with td-tomato, indicating that there is a subpopulation of Rip-Cre neurones in the arcuate nucleus that respond to prolactin. Furthermore, mice with a specific deletion of Prlr in Rip-Cre neurones had lower body weights, increased oxygen consumption, increased running wheel activity and numerous cells in the arcuate nucleus had positive GFP staining indicating deletion of Prlr from Rip-Cre neurones. By contrast, no co-localisation of td-tomato and pSTAT5 was observed in td-tomato/Agrp-Cre mice after prolactin treatment. Moreover, Prlrlox/lox /Agrp-Cre mice had no positive GFP staining in the arcuate nucleus and did not differ in body weight compared to littermate controls. Overall, these results indicate that Rip-Cre neurones in the arcuate nucleus are responsive to prolactin and may play a role in the orexigenic effects of prolactin, whereas prolactin does not directly affect Agrp neurones.

Osteoprotective Effects of Estrogen in the Maxillary Bone Depend on ERα.

Estrogen deficiency results in disruption of maxillary alveolar bone microarchitecture. Most of the actions of estrogen in long bones occur via estrogen receptor α (ERα). However, the function of ERα in the maxillary bone has not been defined. We aimed to investigate the role and underlying mechanisms of ERα in the physiological and mechanically induced alveolar bone remodeling in female and male mice. Wild-type (WT) and ERα(-/-) (ERKOα) mice were subjected to mechanically stimulated bone remodeling by inducing orthodontic tooth movement (OTM). The maxillary bone was analyzed using histomorphometric analysis, micro-computed tomography, quantitative polymerase chain reaction, and energy-dispersive spectroscopy. Bone marrow cells (BMCs) from WT and ERKOα mice were tested for their capacity to differentiate into osteoblasts and osteoclasts. Both male and female ERKOα mice exhibited marked reduction of alveolar bone mass and increased OTM. This response was associated with an increased number of osteoclasts and reduced number of apoptotic cells and osteoblasts in the periodontium and alveolar bone. Consistently, ERKOα mice exhibited lower levels of calcium in bone and increased expression of IL-33 (interleukin-33), TNF-α (tumor necrosis factor α), and IL-1ß (interleukin-1ß) and decreased expression of dentin matrix acidic phosphoprotein and alkaline phosphatase in periodontal tissues. Moreover, the differentiation of osteoclasts and osteoblasts in vitro was significantly higher in BMCs obtained from ERKOα. ERα is required to maintain the microarchitecture of maxillary alveolar bone. This process is linked to bone cell differentiation and apoptosis, as well as local production of inflammatory molecules such as IL-33, TNF-α, and IL-1ß.

Observation of Gravitationally Induced Vertical Striation of Polarized Ultracold Neutrons by Spin-Echo Spectroscopy.

We describe a spin-echo method for ultracold neutrons (UCNs) confined in a precession chamber and exposed to a |B0|=1 µT magnetic field. We have demonstrated that the analysis of UCN spin-echo resonance signals in combination with knowledge of the ambient magnetic field provides an excellent method by which to reconstruct the energy spectrum of a confined ensemble of neutrons. The method takes advantage of the relative dephasing of spins arising from a gravitationally induced striation of stored UCNs of different energies, and also permits an improved determination of the vertical magnetic-field gradient with an exceptional accuracy of 1.1 pT/cm. This novel combination of a well-known nuclear resonance method and gravitationally induced vertical striation is unique in the realm of nuclear and particle physics and should prove to be invaluable for the assessment of systematic effects in precision experiments such as searches for an electric dipole moment of the neutron or the measurement of the neutron lifetime.

Highly stable atomic vector magnetometer based on free spin precession.

We present a magnetometer based on optically pumped Cs atoms that measures the magnitude and direction of a 1 µT magnetic field. Multiple circularly polarized laser beams were used to probe the free spin precession of the Cs atoms. The design was optimized for long-time stability and achieves a scalar resolution better than 300 fT for integration times ranging from 80 ms to 1000 s. The best scalar resolution of less than 80 fT was reached with integration times of 1.6 to 6 s. We were able to measure the magnetic field direction with a resolution better than 10 µrad for integration times from 10 s up to 2000 s.

A Bell-Bloom experiment with polarization-modulated light of arbitrary duty cycle.

We report on a study of polarization-modulation experiments on the 4 → 3 hyperfine component of the D1 transition in Cs vapor contained in a paraffin-coated cell. The laser beam's polarization was switched between left- and right-circular polarization at a rate of 200 Hz. Variations of the transmitted light power were recorded while varying the amplitude of a transverse magnetic field. The power shows electromagnetically induced transparency (EIT) resonances when the atomic Larmor frequency matches a harmonic of the modulation frequency. We made a quantitative study of the resonance amplitudes with square-wave modulations of various duty cycles, and find an excellent agreement with recent algebraic model predictions.

Is the proton radius a player in the redefinition of the International System of Units?

It is now recognized that the International System of Units (SI units) will be redefined in terms of fundamental constants, even if the date when this will occur is still under debate. Actually, the best estimate of fundamental constant values is given by a least-squares adjustment, carried out under the auspices of the Committee on Data for Science and Technology (CODATA) Task Group on Fundamental Constants. This adjustment provides a significant measure of the correctness and overall consistency of the basic theories and experimental methods of physics using the values of the constants obtained from widely differing experiments. The physical theories that underlie this adjustment are assumed to be valid, such as quantum electrodynamics (QED). Testing QED, one of the most precise theories is the aim of many accurate experiments. The calculations and the corresponding experiments can be carried out either on a boundless system, such as the electron magnetic moment anomaly, or on a bound system, such as atomic hydrogen. The value of fundamental constants can be deduced from the comparison of theory and experiment. For example, using QED calculations, the value of the fine structure constant given by the CODATA is mainly inferred from the measurement of the electron magnetic moment anomaly carried out by Gabrielse's group. (Hanneke et al. 2008 Phys. Rev. Lett. 100, 120801) The value of the Rydberg constant is known from two-photon spectroscopy of hydrogen combined with accurate theoretical quantities. The Rydberg constant, determined by the comparison of theory and experiment using atomic hydrogen, is known with a relative uncertainty of 6.6×10(-12). It is one of the most accurate fundamental constants to date. A careful analysis shows that knowledge of the electrical size of the proton is nowadays a limitation in this comparison. The aim of muonic hydrogen spectroscopy was to obtain an accurate value of the proton charge radius. However, the value deduced from this experiment contradicts other less accurate determinations. This problem is known as the proton radius puzzle. This new determination of the proton radius may affect the value of the Rydberg constant . This constant is related to many fundamental constants; in particular, links the two possible ways proposed for the redefinition of the kilogram, the Avogadro constant N(A) and the Planck constant h. However, the current relative uncertainty on the experimental determinations of N(A) or h is three orders of magnitude larger than the 'possible' shift of the Rydberg constant, which may be shown by the new value of the size of the proton radius determined from muonic hydrogen. The proton radius puzzle will not interfere in the redefinition of the kilogram. After a short introduction to the properties of the proton, we will describe the muonic hydrogen experiment. There is intense theoretical activity as a result of our observation. A brief summary of possible theoretical explanations at the date of writing of the paper will be given. The contribution of the proton radius puzzle to the redefinition of SI-based units will then be examined.

Nicotinic stimulation of catecholamine synthesis and tyrosine hydroxylase phosphorylation in cervine adrenal medullary chromaffin cells.

The synthesis and secretion of catecholamines by the adrenal medulla is of major importance in the stress response. Tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, has been extensively studied in adrenal medullary chromaffin cells from a number of species. Cervine chromaffin cells are of interest because the deer is known to be a relatively stress-prone reactive species. We report the first characterisation of tyrosine hydroxylase regulation in cervine chromaffin cells. Nicotinic receptor activation resulted in a time- and concentration-dependent increase in catecholamine synthesis, which was significantly reduced by the extracellular signal-regulated kinase (ERK)1/2 signalling pathway inhibitor PD98059 and the calcium/calmodulin protein kinase II inhibitor KN-93, but not by H89 or bisindolylmaleimide I, inhibitors of protein kinase A and C, respectively. Nicotinic stimulation also increased the phosphorylation of ERK1/2 and tyrosine hydroxylase. This latter response occurred on serine residues 19, 31 and 40 of the enzyme. The nicotinic-induced phosphorylation of ERK1/2 and serine 31 of tyrosine hydroxylase was suppressed by PD98059 but not bisindolylmaleimide I. These data indicate that nicotinic stimulation of tyrosine hydroxylase involves the phosphorylation of serine 31 via an ERK1/2-dependent, protein kinase C-independent pathway. Protein kinase C activation by phorbol 12-myristate 13-acetate also caused an ERK1/2-dependent increase in the serine 31 phosphorylation of tyrosine hydroxylase but, in contrast to the nicotinic response, was not accompanied by an increase in enzyme activity. Thus, ERK1/2-mediated serine 31 phosphorylation of tyrosine hydroxylase appears necessary but not sufficient for nicotinic activation of catecholamine synthesis in cervine chromaffin cells. These data present potentially important similarities and differences between the regulation of catecholamine synthesis in cervine and the more widely studied bovine adrenal medulla.

Complementary methods to investigate the development of clogging within a horizontal sub-surface flow tertiary treatment wetland.

A combination of experimental methods was applied at a clogged, horizontal subsurface flow (HSSF) municipal wastewater tertiary treatment wetland (TW) in the UK, to quantify the extent of surface and subsurface clogging which had resulted in undesirable surface flow. The three dimensional hydraulic conductivity profile was determined, using a purpose made device which recreates the constant head permeameter test in-situ. The hydrodynamic pathways were investigated by performing dye tracing tests with Rhodamine WT and a novel multi-channel, data-logging, flow through Fluorimeter which allows synchronous measurements to be taken from a matrix of sampling points. Hydraulic conductivity varied in all planes, with the lowest measurement of 0.1md(-1) corresponding to the surface layer at the inlet, and the maximum measurement of 1550md(-1) located at a 0.4m depth at the outlet. According to dye tracing results, the region where the overland flow ceased received five times the average flow, which then vertically short-circuited below the rhizosphere. The tracer break-through curve obtained from the outlet showed that this preferential flow-path accounted for approximately 80% of the flow overall and arrived 8h before a distinctly separate secondary flow-path. The overall volumetric efficiency of the clogged system was 71% and the hydrology was simulated using a dual-path, dead-zone storage model. It is concluded that uneven inlet distribution, continuous surface loading and high rhizosphere resistance is responsible for the clog formation observed in this system. The average inlet hydraulic conductivity was 2md(-1), suggesting that current European design guidelines, which predict that the system will reach an equilibrium hydraulic conductivity of 86md(-1), do not adequately describe the hydrology of mature systems.

Test of Lorentz invariance with spin precession of ultracold neutrons.

A clock comparison experiment, analyzing the ratio of spin precession frequencies of stored ultracold neutrons and 199Hg atoms, is reported. No daily variation of this ratio could be found, from which is set an upper limit on the Lorentz invariance violating cosmic anisotropy field b perpendicular < 2 x 10(-20) eV (95% C.L.). This is the first limit for the free neutron. This result is also interpreted as a direct limit on the gravitational dipole moment of the neutron |gn| < 0.3 eV/c2 m from a spin-dependent interaction with the Sun. Analyzing the gravitational interaction with the Earth, based on previous data, yields a more stringent limit |gn| < 3 x 10(-4) eV/c2 m.

Is it safe to discharge patients from accident and emergency using a rapid point of care Triple Cardiac Marker test to rule out acute coronary syndrome in low to intermediate risk patients presenting with chest pain?

PURPOSE: To determine whether patients presenting with chest pain who are at low to intermediate risk for ACS can safely be discharged from Accident and Emergency using Triple Cardiac Marker [TCM] [CK-MB, myoglobin, troponin I] without increasing risk and cost effective use of coronary care facilities. METHODS: Retrospective review of consecutive patients presented to A&E between Dec 2003 and July 2004 was performed and these patients were prospectively followed for six months for future coronary events and hospital admissions. A total of 325 patients presented to A&E with chest pain that were at low to intermediate risk for ACS. Paired TCM and ECGs were performed 2 h apart and the results were used to determine whether hospital admission was required. Follow up data was collected from hospital records, hospital database, GPS and patient interviews. RESULTS: 325 consecutive patients [225 men, 100 women; aged 18-97 years, median-68 years] were included in the study. Paired TCM was negative in 100 patients [30%] and they were discharged from A&E. The re-admission rate for this group of patients was 1% with ACS and no deaths from cardiac cause at six months. 36 [11%] had single TCM negative and were sent home with 3% re-admission rate with ACS and no death at six months. Subgroup analysis shows sensitivity and specificity of 85.7% and 96.5% respectively for TCM to diagnose ACS in this setting. CONCLUSION: Almost one third of patients who presented with chest pain and low to intermediate probability of ACS were safely discharged from A&E following paired negative TCM. Six month re-admission rate with ACS in this group of patients was only 1% with no death. Therefore paired TCM can be used to safely discharge this group of patients. This marker has the potential to significantly reduce hospital admissions.

Direct experimental limit on neutron-mirror-neutron oscillations.

In case a mirror world with a copy of our ordinary particle spectrum would exist, the neutron n and its degenerate partner, the mirror neutron n', could potentially mix and undergo nn' oscillations. The interaction of an ordinary magnetic field with the ordinary neutron would lift the degeneracy between the mirror partners, diminish the n' amplitude in the n wave function and, thus, suppress its observability. We report an experimental comparison of ultracold neutron storage in a trap with and without superimposed magnetic field. No influence of the magnetic field is found and, assuming negligible mirror magnetic fields, a limit on the oscillation time taunn' > 103 s (95% C.L.) is derived.

Redox-mediated substrate recognition by Sdp1 defines a new group of tyrosine phosphatases.

Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation. Here we describe how the stress-inducible MAPK phosphatase, Sdp1, presents an unusual solution to this apparent paradox by acquiring enhanced catalytic activity under oxidative conditions. Structural and biochemical evidence reveals that Sdp1 employs an intramolecular disulphide bridge and an invariant histidine side chain to selectively recognize a tyrosine-phosphorylated MAPK substrate. Optimal activity critically requires the disulphide bridge, and thus, to the best of our knowledge, Sdp1 is the first example of a cysteine-dependent phosphatase that couples oxidative stress with substrate recognition. We show that Sdp1, and its paralogue Msg5, have similar properties and belong to a new group of phosphatases unique to yeast and fungal taxa.

Hydrazine and amphetamine binding to amine oxidases: old drugs with new prospects.

Tranylcypromine (TCP), an amphetamine, is a reversible inhibitor of copper-containing amine oxidases. We have solved the structure of the complex of TCP with the amine oxidase from E. coli (ECAO) and shown that only the (+)-enantiomer of TCP binds. Kinetic studies on 2-phenylethylamine and TCP binding to wild-type ECAO and mutational variants fully support the model in which binding of the protonated amine is the first step in the catalytic cycle. Hydrazines are irreversible inhibitors of copper-containing amine oxidases. Binding of hydrazines leads to an adduct ("Adduct 1") with a chromophore at 430 nm which converts at higher pH to another adduct ("Adduct 2") with a chromophore at 520 nm. We have determined the structures of Adduct 1 and 2 for 2-hydrazinopyridine reacted with ECAO. It has been found that Adduct 1 corresponds to the hydrazone and azo tautomers whilst Adduct 2 corresponds to the azo tautomer coordinated to the active site copper. The implications of these results in developing more specific drugs are discussed.

Electrochemical screening of self-assembling beta-sheet peptides using supported phospholipid monolayers.

In the context of the medical applications of beta-sheet self-assembling peptides, it is important to be able to predict their activity at the biological membrane level. A study of the interaction of four systematically varied 11-residue (P11-1, P11-2, P11-6 and P11-7) and one 13-residue (P13-1) designed beta-sheet self-assembling peptides with DOPC monolayers on a mercury electrode is reported in this paper. Experiments were carried out in 0.1 mol dm(-3) KCl electrolyte with added phosphate buffer (0.001 mol dm(-3)) at pH approximately 7.6. The capacity-potential curves of the coated electrode in the presence and absence of the different peptides were measured using out-of-phase ac voltammetry. The frequency dependence of the complex impedance of the coated electrode surfaces in the presence and absence of the peptides was estimated between 65,000 and 0.1 Hz at -0.4V versus Ag/AgCl 3.5 mol(-3) dm(-3) KCl. The monolayer permeabilising properties of the peptides were studied by following the reduction of Tl(I) to Tl(Hg) at the coated electrode. Of the five peptides studied, P11-2, P11-7 and P13-1 interact most strongly with the DOPC layer. P11-1 which has a polar primary structure shows no obvious interaction with the phospholipid but surprisingly, it permeabilises the phospholipid layer to Tl(+).

Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution.

Galactose oxidase (GO; E.C. 1.1.3.9) is a copper- containing enzyme that oxidizes a range of primary alcohols to aldehydes. This broad substrate specificity is reflected in a high K(M) for substrates. Directed evolution has previously been used to select variants of GO that exhibit enhanced expression and kinetic properties. In assays using unpurified enzyme samples, the variant C383S displayed a 5-fold lower K(M) than wild-type GO. In the present study, we have constructed, expressed, purified and characterized a number of single, double and triple mutants at residues Cys383, Tyr436 and Val494, identified in one of the directed evolution studies, to examine their relative contributions to improved catalytic activity of GO. We report kinetic studies on the various mutant enzymes. In addition, we have determined the three-dimensional structure of the C383S variant. As with many mutations identified in directed evolution experiments, the availability of structural information does not provide a definitive answer to the reason for the improved K(M) in the C383S variant protein.

Cofactor processing in galactose oxidase.

Galactose oxidase (GO; EC 1.1.3.9) is a monomeric 68 kDa enzyme that contains a single copper and an amino acid-derived cofactor. The mechanism of this radical enzyme has been widely studied by structural, spectroscopic, kinetic and mutational approaches and there is a reasonable understanding of the catalytic mechanism and activation by oxidation to generate the radical cofactor that resides on Tyr-272, one of the copper ligands. Biogenesis of this cofactor involves the post-translational, autocatalytic formation of a thioether cross-link between the active-site residues Cys-228 and Tyr-272. This process is closely linked to a peptide bond cleavage event that releases the N-terminal 17-amino-acid pro-peptide. We have shown using pro-enzyme purified in copper-free conditions that mature oxidized GO can be formed by an autocatalytic process upon addition of copper and oxygen. Structural comparison of pro-GO (GO with the prosequence present) with mature GO reveals overall structural similarity, but with some regions showing significant local differences in main chain position and some active-site-residue side chains differing significantly from their mature enzyme positions. These structural effects of the pro-peptide suggest that it may act as an intramolecular chaperone to provide an open active-site structure conducive to copper binding and chemistry associated with cofactor formation. Various models can be proposed to account for the formation of the thioether bond and oxidation to the radical state; however, the mechanism of prosequence cleavage remains unclear.

Molecular findings in symptomatic and pre-symptomatic Alexander disease patients.

BACKGROUND AND OBJECTIVE: Alexander disease is a slowly progressive CNS disorder that most commonly occurs in children. Until recently, the diagnosis could only be established by the histologic finding of Rosenthal fibers in brain specimens. Mutations in the glial fibrillary acidic protein (GFAP) gene have now been shown in a number of biopsy- or autopsy-proven patients with Alexander disease. A prospective study on patients suspected to have Alexander disease was conducted to determine the extent to which clinical and MRI criteria could accurately diagnose affected individuals, using GFAP gene sequencing as the confirmatory assay. METHODS: Patients who showed MRI white matter abnormalities consistent with Alexander disease, unremarkable family history, normal karyotype, and normal metabolic screening were included in this study. Genomic DNA from patients was screened for mutations in the entire coding region, including the exon-intron boundaries, of the GFAP gene. RESULTS: Twelve of 13 patients (approximately 90%) were found to have mutations in GFAP. Seven of those 12 patients presented in infancy with seizures and megalencephaly. Five were juvenile-onset patients with more variable symptoms. Two patients in the latter group were asymptomatic or minimally affected at the time of their initial MRI scan. The mutations were distributed throughout the gene, and all involved sporadic single amino acid heterozygous changes that changed the charge of the mutant protein. Four of the nine changes were novel mutations. CONCLUSIONS: In symptomatic and asymptomatic patients with a predominantly frontal leukoencephalopathy by MRI, GFAP gene mutation analysis should be included in the initial diagnostic evaluation process for Alexander disease.

Crystal structure of the precursor of galactose oxidase: an unusual self-processing enzyme.

Galactose oxidase (EC ) is a monomeric enzyme that contains a single copper ion and catalyses the stereospecific oxidation of primary alcohols to their corresponding aldehydes. The protein contains an unusual covalent thioether bond between a tyrosine, which acts as a radical center during the two-electron reaction, and a cysteine. The enzyme is produced in a precursor form lacking the thioether bond and also possessing an additional 17-aa pro-sequence at the N terminus. Previous work has shown that the aerobic addition of Cu(2+) to the precursor is sufficient to generate fully processed mature enzyme. The structure of the precursor protein has been determined to 1.4 A, revealing the location of the pro-sequence and identifying structural differences between the precursor and the mature protein. Structural alignment of the precursor and mature forms of galactose oxidase shows that five regions of main chain and some key residues of the active site differ significantly between the two forms. The precursor structure provides a starting point for modeling the chemistry of thioether bond formation and pro-sequence cleavage.