Characterisation of Osteopontin in an In Vitro Model of Embryo Implantation

At the onset of pregnancy, embryo implantation is initiated by interactions between the endometrial epithelium and the outer trophectoderm cells of the blastocyst. Osteopontin (OPN) is expressed in the endometrium and is implicated in attachment and signalling roles at the embryo–epithelium interface. We have characterised OPN in the human endometrial epithelial Ishikawa cell line using three different monoclonal antibodies, revealing at least nine distinct molecular weight forms and a novel secretory pathway localisation in the apical domain induced by cell organisation into a confluent epithelial layer. Mouse blastocysts co-cultured with Ishikawa cell layers served to model embryo apposition, attachment and initial invasion at implantation. Exogenous OPN attenuated initial, weak embryo attachment to Ishikawa cells but did not affect the attainment of stable attachment. Notably, exogenous OPN inhibited embryonic invasion of the underlying cell layer, and this corresponded with altered expression of transcription factors associated with differentiation from trophectoderm (Gata2) to invasive trophoblast giant cells (Hand1). These data demonstrate the complexity of endometrial OPN forms and suggest that OPN regulates embryonic invasion at implantation by signalling to the trophectoder

Cluster size distributions in particle systems with asymmetric dynamics

We present exact and asymptotic results for clusters in the one-dimensional totally asymmetric exclusion process (TASEP) with two different dynamics. The expected length of the largest cluster is shown to diverge logarithmically with increasing system size for ordinary TASEP dynamics and as a logarithm divided by a double logarithm for generalized dynamics, where the hopping probability of a particle depends on the size of the cluster it belongs to. The connection with the asymptotic theory of extreme order statistics is discussed in detail. We also consider a related model of interface growth, where the deposited particles are allowed to relax to the local gravitational minimum.Comment: 12 pages, 3 figures, RevTe

Osmotic stress induces JNK-dependent embryo invasion in a model of implantation

In vitro culture during assisted reproduction technologies (ARTs) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2 h in medium with osmolarity raised by 400 mosmol induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety

Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model?Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium.In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models.This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells.Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P < 0.05, P < 0.01 and P < 0.001.Hatched E4.5 mouse blastocysts exhibited weak attachment to confluent Ishikawa cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5.5 also resulted in intermediate and stable attachment from E5.5 + 4 h; however, these embryos did not go on to breach the Ishikawa cell layer, even when co-culture was extended to E7.5 (P < 0.01). Blastocysts cultured from E4.5 in permeable transwell inserts above Ishikawa cells before transfer to direct co-culture at E5.5 went on to attach but failed to breach the Ishikawa cell layer by E6.5 (P < 0.01). Gene expression analysis at E5.5 demonstrated that direct co-culture with Ishikawa cells from E4.5 resulted in downregulation of trophectoderm transcription factors Cdx2 (P < 0.05) and Gata3 (P < 0.05) and upregulation of the TGC transcription factor Hand1 (P < 0.05). Co-culture with non-endometrial human fibroblasts did not alter the expression of these genes.None.The in vitro model used here combines human carcinoma-derived endometrial cells with mouse embryos, in which the cellular interactions observed may not fully recapitulate those in vivo. The data gleaned from such models can be regarded as hypothesis-generating, and research is now needed to develop more sophisticated models of human implantation combining multiple primary endometrial cell types with surrogate and real human embryos.This study implicates blastocyst apposition to endometrial epithelial cells as a critical step in trophoblast differentiation required for implantation. Understanding this maternal regulation of the embryonic developmental programme may lead to novel treatments for infertility.This work was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for SCB from the Anatomical Society. No conflict of interest is declared

The state of workplace union reps organisation in Britain today

This article provides a brief evaluation of the state of workplace union reps’ organization in Britain as we approach the second decade of the 2000s. It documents the severe weakening of workplace union organization over the last 25 years, which is reflected in the declining number of reps, reduced bargaining power and the problem of bureaucratization. But it also provides evidence of the continuing resilience, and even combativity in certain areas of employment, of workplace union reps organization, and considers the future potential for a revival of fortunes

Precise Stellar Radial Velocities of an M Dwarf with a Michelson Interferometer and a Medium-resolution Near-infrared Spectrograph

Precise near-infrared radial velocimetry enables efficient detection and transit verification of low-mass extrasolar planets orbiting M dwarf hosts, which are faint for visible-wavelength radial velocity surveys. The TripleSpec Exoplanet Discovery Instrument, or TEDI, is the combination of a variable-delay Michelson interferometer and a medium-resolution (R=2700) near-infrared spectrograph on the Palomar 200" Hale Telescope. We used TEDI to monitor GJ 699, a nearby mid-M dwarf, over 11 nights spread across 3 months. Analysis of 106 independent observations reveals a root-mean-square precision of less than 37 m/s for 5 minutes of integration time. This performance is within a factor of 2 of our expected photon-limited precision. We further decompose the residuals into a 33 m/s white noise component, and a 15 m/s systematic noise component, which we identify as likely due to contamination by telluric absorption lines. With further development this technique holds promise for broad implementation on medium-resolution near-infrared spectrographs to search for low-mass exoplanets orbiting M dwarfs, and to verify low-mass transit candidates.Comment: 55 pages and 13 figures in aastex format. Accepted by PAS

A Magnetic Transition Probed by the Ce Ion in Square-Lattice Antiferromagnet CeMnAsO

We examined the magnetic properties of the square-lattice antiferromagnets CeMnAsO and LaMnAsO and their solid solutions La1-xCexMnAsO by resistivity, magnetic susceptibility, and heat capacity measurements below room temperature. A first-order phase transition is observed at 34.1 K, below which the ground-state doublet of the Ce ion splits by 3.53 meV. It is likely that Mn moments already ordered above room temperature are reoriented at the transition, as reported for related compounds, such as NdMnAsO and PrMnSbO. This transition generates a large internal magnetic field at the Ce site in spite of the fact that simple Heisenberg interactions should be cancelled out at the Ce site owing to geometrical frustration. The transition takes place at nearly the same temperature with the substitution of La for Ce up to 90%. The Ce moment does not undergo long-range order by itself, but is parasitically induced at the transition, serving as a good probe for detecting the magnetism of Mn spins in a square lattice.Comment: 11 pages, 5 figures, to be published in J. Phys. Soc. Jp

Solution of the Kwiecinski evolution equations for unintegrated parton distributions using the Mellin transform

The Kwiecinski equations for the QCD evolution of the unintegrated parton distributions in the transverse-coordinate space (b) are analyzed with the help of the Mellin-transform method. The equations are solved numerically in the general case, as well as in a small-b expansion which converges fast for b Lambda_QCD sufficiently small. We also discuss the asymptotic limit of large bQ and show that the distributions generated by the evolution decrease with b according to a power law. Numerical results are presented for the pion distributions with a simple valence-like initial condition at the low scale, following from chiral large-N_c quark models. We use two models: the Spectral Quark Model and the Nambu--Jona-Lasinio model. Formal aspects of the equations, such as the analytic form of the b-dependent anomalous dimensions, their analytic structure, as well as the limits of unintegrated parton densities at x -> 0, x -> 1, and at large b, are discussed in detail. The effect of spreading of the transverse momentum with the increasing scale is confirmed, with growing asymptotically as Q^2 alpha(Q^2). Approximate formulas for for each parton species is given, which may be used in practical applications.Comment: 18 pages, 6 figures, RevTe

Glucose influences endometrial receptivity to embryo implantation through O-GlcNAcylation-mediated regulation of the cytoskeleton

Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycaemia on these processes remains poorly understood. Here we show that physiological levels of glucose (5mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in co-cultures at 17mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarised epithelium. 2D and 3D morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of RhoA-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signalling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes

Analysis of Photovoltaic System Energy Performance Evaluation Method

Documentation of the energy yield of a large photovoltaic (PV) system over a substantial period can be useful to measure a performance guarantee, as an assessment of the health of the system, for verification of a performance model to then be applied to a new system, or for a variety of other purposes. Although the measurement of this performance metric might appear to be straight forward, there are a number of subtleties associated with variations in weather and imperfect data collection that complicate the determination and data analysis. A performance assessment is most valuable when it is completed with a very low uncertainty and when the subtleties are systematically addressed, yet currently no standard exists to guide this process. This report summarizes a draft methodology for an Energy Performance Evaluation Method, the philosophy behind the draft method, and the lessons that were learned by implementing the method