Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta

Background - During natural fertilization, sperm fusion with the oocyte induces long lasting intracellular calcium oscillations which in turn are responsible for oocyte activation. PLCZ1 has been identified as the factor that the sperm delivers into the egg to induce such a response. We tested the hypothesis that PLCZ1 cRNA injection can be used to activate bovine oocytes. Results - Mouse and bovine PLCZ1 cRNAs were injected into matured bovine oocytes at different concentrations. Within the concentrations tested, mouse PLCZ1 injection activated bovine oocytes at a maximum rate when the pipette concentration of cRNA ranged from 0.25 to 1 μg/μL, while bovine PLCZ1 was optimal at 0.1 μg/μL. At their most effective concentrations, PLCZ1 induced parthenogenetic development at rates similar to those observed using other activation stimuli such as Ionomycin/CHX and Ionomycin/DMAP. Injection of mouse and bovine PLCZ1 cRNA induced dose-dependent sperm-like calcium oscillations whose frequency increased over time. Injection of bovine and mouse PLCZ1 cRNA also induced IP3R-1 degradation, although bovine PLCZ1 cRNA evoked greater receptor degradation than its mouse counterpart. Conclusion - Injection of PLCZ1 cRNA efficiently activated bovine oocytes by inducing a sperm-like calcium oscillatory pattern. Importantly, the high rate of aneuploidy encountered in parthenogenetic embryos activated by certain chemical means was not observed in PLCZ1 activated embryos

Inositol 1,4,5-trisphosphate receptor 1, a widespread Ca2+ channel, is a novel substrate of polo-like kinase 1 in eggs

AbstractTo initiate embryo development, the sperm induces in the egg release of intracellular calcium ([Ca2+]i). During oocyte maturation, the inositol 1,4,5-trisphosphate receptor (IP3R1), the channel implicated, undergoes modifications that enhance its function. We found that IP3R1 becomes phosphorylated during maturation at an MPM-2 epitope and that this persists until the fertilization-associated [Ca2+]i responses cease. We also reported that maturation without ERK activity diminishes IP3R1 MPM-2 reactivity and [Ca2+]i responses. Here, we show that IP3R1 is a novel target for Polo-like kinase1 (Plk1), a conserved M-phase kinase, which phosphorylates it at an MPM-2 epitope. Plk1 and IP3R1 interact in an M-phase preferential manner, and they exhibit close co-localization in the spindle/spindle poles area. This co-localization is reduced in the absence of ERK activity, as the ERK pathway regulates spindle organization and IP3R1 cortical re-distribution. We propose that IP3R1 phosphorylation by Plk1, and possibly by other M-phase kinases, underlies the delivery of spatially and temporally regulated [Ca2+]i signals during meiosis/mitosis and cytokinesis

DATA OPTIMIZATION FOR 3D MODELING AND ANALYSIS OF A FORTRESS ARCHITECTURE

Thanks to the recent worldwide spread of drones and to the development of structure from motion photogrammetric software, UAV photogrammetry is becoming a convenient and reliable way for the 3D documentation of built heritage. Hence, nowadays, UAV photogrammetric surveying is a common and quite standard tool for producing 3D models of relatively large areas. However, when such areas are large, then a significant part of the generated point cloud is often of minor interest. Given the necessity of efficiently dealing with storing, processing and analyzing the produced point cloud, some optimization step should be considered in order to reduce the amount of redundancy, in particular in the parts of the model that are of minor interest. Despite this can be done by means of a manual selection of such parts, an automatic selection is clearly much more viable way to speed up the final model generation. Motivated by the recent development of many semantic classification techniques, the aim of this work is investigating the use of point cloud optimization based on semantic recognition of different components in the photogrammetric 3D model. The Girifalco Fortress (Cortona, Italy) is used as case study for such investigation. The rationale of the proposed methodology is clearly that of preserving high point density in the model in the areas that describe the fortress, whereas point cloud density is dramatically reduced in vegetated and soil areas. Thanks to the implemented automatic procedure, in the considered case study, the size of the point cloud has been reduced by a factor five, approximately. It is worth to notice that such result has been obtained preserving the original point density on the fortress surfaces, hence ensuring the same capabilities of geometric analysis of the original photogrammetric model

Disciplina radnje, evokacija s fusnotama uloga vlaka, u školovanju

Tion. b) Pipette advanced into the oocyte; cytoplasm is aspirated to break the plasma membrane. c) Aspirated cytoplasm and Texas Red dextran are injected into the oocyte. d) Schematic representation of the microscope reticulum used as guide to control the injected volume. The oocyte is represented in yellow and the pipette in blue. The red lines indicate the volume introduced into the oocyte which, calculated measuring the pipette internal diameters at both ends, is 5.9 pL. e) An oil drop of the same size as the injected volume is shown next to an oocyte. f) Oocytes injected using Texas Red dextran. g) From left to right, oocyte injected 2X and 1X the normal volume of Texas Red dextran. h) Fluorescent intensity profile of the line shown in f. i) Fluorescent intensity profile of the line shown in g. j) Developmental rates of injected and uninjected bovine oocytes after activation using ionomycin/DMAP.<p><b>Copyright information:</b></p><p>Taken from "Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta"</p><p>http://www.biomedcentral.com/1471-213X/8/16</p><p>BMC Developmental Biology 2008;8():16-16.</p><p>Published online 19 Feb 2008</p><p>PMCID:PMC2266721.</p><p></p

Modification of the nanostructure of lignocellulose cell walls via a non-enzymatic lignocellulose deconstruction system in brown rot wood-decay fungi

Abstract Wood decayed by brown rot fungi and wood treated with the chelator-mediated Fenton (CMF) reaction, either alone or together with a cellulose enzyme cocktail, was analyzed by small angle neutron scattering (SANS), sum frequency generation (SFG) spectroscopy, Fourier transform infrared (FTIR) analysis, X-ray diffraction (XRD), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Results showed that the CMF mechanism mimicked brown rot fungal attack for both holocellulose and lignin components of the wood. Crystalline cellulose and lignin were both depolymerized by the CMF reaction. Porosity of the softwood cell wall did not increase during CMF treatment, enzymes secreted by the fungi did not penetrate the decayed wood. The enzymes in the cellulose cocktail also did not appear to alter the effects of the CMF-treated wood relative to enhancing cell wall deconstruction. This suggests a rethinking of current brown rot decay models and supports a model where monomeric sugars and oligosaccharides diffuse from the softwood cell walls during non-enzymatic action. In this regard, the CMF mechanism should not be thought of as a “pretreatment” used to permit enzymatic penetration into softwood cell walls, but instead it enhances polysaccharide components diffusing to fungal enzymes located in wood cell lumen environments during decay. SANS and other data are consistent with a model for repolymerization and aggregation of at least some portion of the lignin within the cell wall, and this is supported by AFM and TEM data. The data suggest that new approaches for conversion of wood substrates to platform chemicals in biorefineries could be achieved using the CMF mechanism with >75% solubilization of lignocellulose, but that a more selective suite of enzymes and other downstream treatments may be required to work when using CMF deconstruction technology. Strategies to enhance polysaccharide release from lignocellulose substrates for enhanced enzymatic action and fermentation of the released fraction would also aid in the efficient recovery of the more uniform modified lignin fraction that the CMF reaction generates to enhance biorefinery profitability

Inositol 1,4,5-tirphosphate Receptor 1 Degradation in Mouse Eggs and Impact on [Ca2+]i Oscillations

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca(2+)](i)), which is almost entirely mediated by inositol 1,4,5-trisphosphate receptor 1 (IP(3)R1). In mammalian eggs, fertilization-induced [Ca(2+)](i) responses exhibit a periodic pattern that are called [Ca(2+)](i) oscillations. These [Ca(2+)](i) oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP(3)R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP(3)R1 degradation and examined the impact of the IP(3)R1 levels on the pattern of [Ca(2+)](i) oscillations. Using microinjection of IP(3) and of its analogs and conditions that prevent the development of [Ca(2+)](i) oscillations, we show that IP(3)R1 degradation requires uniform and persistently elevated levels of IP(3). We also established that progressive degradation of the IP(3)R1 results in [Ca(2+)](i) oscillations with diminished periodicity while a near complete depletion of IP(3)R1s precludes the initiation of [Ca(2+)](i) oscillations. These results provide insights into the mechanism involved in the generation of [Ca(2+)](i) oscillations in mouse eggs