The action of physiological and synthetic steroids on the calcium channel CatSper in human sperm

The sperm-specific channel CatSper (cation channel of sperm) controls the intracellular Ca2+ concentration ([Ca2+]i) and plays an essential role in sperm function. It is mainly activated by the steroid progesterone (P4) but is also promiscuously activated by a wide range of synthetic and physiological compounds. These compounds include diverse steroids whose action on the channel is so far still controversial. To investigate the effect of these compounds on CatSper and sperm function, we developed a high-throughput screening (HTS) assay to measure changes in [Ca2+]i in human sperm and screened 1,280 approved and off-patent drugs including 90 steroids from the Prestwick chemical library. More than half of the steroids tested (53%) induced an increase in [Ca2+]i and reduced the P4-induced Ca2+ influx in human sperm in a dose-dependent manner. Ten of the most potent steroids (activating and P4-inhibiting) were selected for a detailed analysis of their action on CatSper and their ability to act on sperm acrosome reaction (AR) and penetration in viscous media. We found that these steroids show an inhibitory effect on P4 but not on prostaglandin E1-induced CatSper activation, suggesting that they compete for the same binding site as P4. Pregnenolone, dydrogesterone, epiandrosterone, nandrolone, and dehydroepiandrosterone acetate (DHEA) were found to activate CatSper at physiologically relevant concentrations within the nanomolar range. Like P4, most tested steroids did not significantly affect the AR while stanozolol and estropipate slightly increased sperm penetration into viscous medium. Furthermore, using a hybrid approach integrating pharmacophore analysis and statistical modelling, we were able to screen in silico for steroids that can activate the channel and define the physicochemical and structural properties required for a steroid to exhibit agonist activity against CatSper. Overall, our results indicate that not only physiological but also synthetic steroids can modulate the activity of CatSper with varying potency and if bound to CatSper prior to P4, could impair the timely CatSper activation necessary for proper fertilization to occur

High Abundance Proteins Depletion vs Low Abundance Proteins Enrichment: Comparison of Methods to Reduce the Plasma Proteome Complexity

BACKGROUND: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. CONCLUSIONS/SIGNIFICANCE: Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol

The French Didactic Tradition in Mathematics

This chapter presents the French didactic tradition. It first describes theemergence and development of this tradition according to four key features (role ofmathematics and mathematicians, role of theories, role of design of teaching andlearning environments, and role of empirical research), and illustrates it through two case studies respectively devoted to research carried out within this traditionon algebra and on line symmetry-reflection. It then questions the influence of thistradition through the contributions of four researchers from Germany, Italy, Mexicoand Tunisia, before ending with a short epilogue

Gaia Data Release 3: G_RVS photometry from the RVS spectra

Gaia Data Release 3 (DR3) contains the first release of magnitudes estimated from the integration of Radial Velocity Spectrometer (RVS) spectra for a sample of about 32.2 million stars brighter than G_RVS~14 mag (or G~15 mag). In this paper, we describe the data used and the approach adopted to derive and validate the G_RVS magnitudes published in DR3. We also provide estimates of the G_RVS passband and associated G_RVS zero-point. We derived G_RVS photometry from the integration of RVS spectra over the wavelength range from 846 to 870 nm. We processed these spectra following a procedure similar to that used for DR2, but incorporating several improvements that allow a better estimation of G_RVS. These improvements pertain to the stray-light background estimation, the line spread function calibration, and the detection of spectra contaminated by nearby relatively bright sources. We calibrated the G_RVS zero-point every 30 hours based on the reference magnitudes of constant stars from the Hipparcos catalogue, and used them to transform the integrated flux of the cleaned and calibrated spectra into epoch magnitudes. The G_RVS magnitude of a star published in DR3 is the median of the epoch magnitudes for that star. We estimated the G_RVS passband by comparing the RVS spectra of 108 bright stars with their flux-calibrated spectra from external spectrophotometric libraries. The G_RVS magnitude provides information that is complementary to that obtained from the G, G_BP, and G_RP magnitudes, which is useful for constraining stellar metallicity and interstellar extinction. The median precision of G_RVS measurements ranges from about 0.006 mag for the brighter stars (i.e. with 3.5 < G_RVS < 6.5 mag) to 0.125 mag at the faint end. The derived G_RVS passband shows that the effective transmittance of the RVS is approximately 1.23 times better than the pre-launch estimate.Comment: 16 pages, 18 figures. Accepted for publication in A&

A Modern Mode of Activation for Nucleic Acid Enzymes

Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain) such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes), a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process

Gaia Data Release 3

CONTEXT: Gaia Data Release 3 (Gaia DR3) contains the second release of the combined radial velocities. It is based on the spectra collected during the first 34 months of the nominal mission. The longer time baseline and the improvements of the pipeline made it possible to push the processing limit from GRVS = 12 in Gaia DR2 to GRVS = 14 mag. AIMS: We describe the new functionalities implemented for Gaia DR3, the quality filters applied during processing and post-processing, and the properties and performance of the published velocities. METHODS: For Gaia DR3, several functionalities were upgraded or added to the spectroscopic pipeline. The calibrations were improved in order to better model the temporal evolution of the straylight and of the instrumental point spread function (PSF). The overlapped spectra, which were mostly discarded in Gaia DR2, are now handled by a dedicated module. The hot star template mismatch, which prevented publication of hot stars in Gaia DR2, is largely mitigated now, down to GRVS = 12 mag. The combined radial velocity of stars brighter than or equal to GRVS = 12 mag is calculated in the same way as in Gaia DR2, that is, as the median of the epoch radial velocity time series. The combined radial velocity of the fainter stars is measured from the average of the cross-correlation functions. RESULTS: Gaia DR3 contains the combined radial velocities of 33 812 183 stars. With respect to Gaia DR2, the temperature interval has been expanded from Teff ∈ [3600, 6750] K to Teff ∈ [3100, 14 500] K for the bright stars (GRVS ≤ 12 mag) and [3100, 6750] K for the fainter stars. The radial velocities sample a significant part of the Milky Way: they reach a few kiloparsecs beyond the Galactic centre in the disc and up to about 10−15 kpc vertically into the inner halo. The median formal precision of the velocities is 1.3 km s−1 at GRVS = 12 and 6.4 km s−1 at GRVS = 14 mag. The velocity zeropoint exhibits a small systematic trend with magnitude that starts around GRVS = 11 mag and reaches about 400 m s−1 at GRVS = 14 mag. A correction formula is provided that can be applied to the published data. The Gaia DR3 velocity scale agrees satisfactorily with APOGEE, GALAH, GES, and RAVE; the systematic differences mostly remain below a few hundred m s−1. The properties of the radial velocities are also illustrated with specific objects: open clusters, globular clusters, and the Large Magellanic Cloud. For example, the precision of the data allows mapping the line-of-sight rotational velocities of the globular cluster 47 Tuc and of the Large Magellanic Cloud

Regulation of healthcare ethics committees in Europe

In this article, the question is discussed if and how Healthcare Ethics Committees (HECs) should be regulated. The paper consists of two parts. First, authors from eight EC member countries describe the status quo in their respective countries, and give reasons as to the form of regulation they consider most adequate. In the second part, the country reports are analysed. It is suggested that regulation of HECs should be central and weak. Central regulation is argued to be apt to improve HECs’ accountability, relevance and comparability. To facilitate biomedical citizenship and ethical reflection, regulation should at the same time be weak rather than strict. Independence of HECs to deliberate about ethical questions, and to give solicited and unsolicited advice, should be supported and only interfered with by way of exception. One exception is when circumstances become temporary adversarial to ethical deliberation in healthcare institutions. In view of European unification, steps should be taken to develop consistent policies for both Eastern and Western European countries

<i>Gaia</i> Data Release 1. Summary of the astrometric, photometric, and survey properties

Context. At about 1000 days after the launch of Gaia we present the first Gaia data release, Gaia DR1, consisting of astrometry and photometry for over 1 billion sources brighter than magnitude 20.7. Aims. A summary of Gaia DR1 is presented along with illustrations of the scientific quality of the data, followed by a discussion of the limitations due to the preliminary nature of this release. Methods. The raw data collected by Gaia during the first 14 months of the mission have been processed by the Gaia Data Processing and Analysis Consortium (DPAC) and turned into an astrometric and photometric catalogue. Results. Gaia DR1 consists of three components: a primary astrometric data set which contains the positions, parallaxes, and mean proper motions for about 2 million of the brightest stars in common with the HIPPARCOS and Tycho-2 catalogues – a realisation of the Tycho-Gaia Astrometric Solution (TGAS) – and a secondary astrometric data set containing the positions for an additional 1.1 billion sources. The second component is the photometric data set, consisting of mean G-band magnitudes for all sources. The G-band light curves and the characteristics of ∼3000 Cepheid and RR-Lyrae stars, observed at high cadence around the south ecliptic pole, form the third component. For the primary astrometric data set the typical uncertainty is about 0.3 mas for the positions and parallaxes, and about 1 mas yr−1 for the proper motions. A systematic component of ∼0.3 mas should be added to the parallax uncertainties. For the subset of ∼94 000 HIPPARCOS stars in the primary data set, the proper motions are much more precise at about 0.06 mas yr−1. For the secondary astrometric data set, the typical uncertainty of the positions is ∼10 mas. The median uncertainties on the mean G-band magnitudes range from the mmag level to ∼0.03 mag over the magnitude range 5 to 20.7. Conclusions. Gaia DR1 is an important milestone ahead of the next Gaia data release, which will feature five-parameter astrometry for all sources. Extensive validation shows that Gaia DR1 represents a major advance in the mapping of the heavens and the availability of basic stellar data that underpin observational astrophysics. Nevertheless, the very preliminary nature of this first Gaia data release does lead to a number of important limitations to the data quality which should be carefully considered before drawing conclusions from the data

Structure-function analysis in nuclear RNase P RNA

Eukaryotic ribonuclease P (RNase P) enzymes require both RNA and protein subunits for activity in vivo and in vitro . We have undertaken an analysis of the complex RNA subunit of the nuclear holoenzyme in an effort to understand its structure and its similarities to and differences from the bacterial ribozymes. Phylogenetic analysis, structure-sensitive RNA footprinting, and directed mutagenesis reveal conserved secondary and tertiary structures with both strong similarities to the bacterial consensus and distinctive features. The effects of mutations in the most highly conserved positions are being used to dissect the functions of individual subdomains.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43252/1/11033_2004_Article_BF00988722.pd

Hereditary renal adysplasia, pulmonary hypoplasia and Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome: a case report

<p>Abstract</p> <p>Background</p> <p>Hereditary renal adysplasia is an autosomal dominant trait with incomplete penetrance and variable expression that is usually associated with malformative combinations (including Müllerian anomalies) affecting different mesodermal organs such as the heart, lung, and urogenital system.</p> <p>Case report</p> <p>A case showing pulmonary hypoplasia, hip dysplasia, hereditary renal adysplasia, and Mayer-Rokitansky-Kuster-Hauser syndrome in adulthood is reported here. The i.v. pyelography showed right renal agenesis with a normal left kidney and ureter. Ultrasound and Magnetic Resonance Imaging also showed right renal agenesis with multicystic embryonary remnants in the right hemipelvis probably corresponding to a dysgenetic kidney. An uretrocystoscopy showed absence of ectopic ureter and of the right hemitrigone. She was scheduled for a diagnostic laparoscopy and creation of a neovagina according to the McIndoe technique with a prosthesis and skin graft. Laparoscopy confirmed the absence of the uterus. On both sides, an elongated, solid, rudimentary uterine horn could be observed. Both ovaries were also elongated, located high in both abdominal flanks and somewhat dysgenetics. A conventional cytogenetic study revealed a normal female karyotype 46, XX at a level of 550 GTG bands. A CGH analysis was performed using a 244K oligoarray CGH detecting 11 copy number variants described as normal variants in the databases. The 17q12 and 22q11.21 microdeletions described in other MRKH patients were not present in this case. Four years after operation her evolution is normal, without symptoms and the neovagina is adequately functional. The geneticists have studied her family history and the pedigree of the family is shown.</p> <p>Conclusions</p> <p>We suggest that primary damage to the mesoderm (paraaxil, intermediate, and lateral) caused by mutations in a yet unidentified gene is responsible for: 1) skeletal dysplasia, 2) inappropriate interactions between the bronchial mesoderm and endodermal lung bud as well as between the blastema metanephric and ureteric bud, and eventually 3) Müllerian anomalies (peritoneal mesothelium) at the same level. These anomalies would be transmitted as an autosomal dominant trait with incomplete penetrance and variable expressivity.</p