Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo

In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2-/- embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7-21% form blastocysts compared to 76.2-83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages

Purification, partial characterization of rat kidney hyaluronic acid binding protein and its localization on the cell surface

Hyaluronic acid binding protein (HABP) has been purified to homogeneity from normal adult rat kidney by hyaluronate Sepharose affinity chromatography, and its apparent molecular mass was found to be 68 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of HABP under reducing as well as nonreducing conditions revealed a single protein band of 34 kDa, thus indicating that kidney HABP is a homodimer and lacks interchain disulfide bond. Its glycoprotein nature was demonstrated by Con-A binding analysis. The pI value of kidney HABP was 6, indicating its acidic nature. Polyclonal antibodies were raised against it, and the monospecificity of the antibodies towards HABP was confirmed by Western blot analysis of tissue extracts. Immunoblot analysis has elucidated the occurrence of this glycoprotein in various tissues. Moreover, HABP present in these tissues are shown to be structurally and immunologically identical. However, this glycoprotein is antigenically distinct from other well characterized extracellular proteins, e.g., fibronectin, laminin and collagen type IV. With the help of enzyme-linked immunosorbent assay (ELISA) and iodinated [125I]HABP, it has been shown that kidney HABP binds specifically to hyaluronic acid (HA) amongst all the glycosaminoglycans (GAGs), however, HABP can interact with other matrix proteins, e.g., laminin, fibronectin, and collagen type IV. The apparent dissociation constants of HABP for HA, laminin, fibronectin, and collagen type IV were approximately in the range of 10(-9) M, and kinetic analysis showed that these binding interactions were complex and of positive cooperative nature. Indirect immunofluorescence staining demonstrated its localization on human fetus lung fibroblast cell surface. Detection of 34 kDa HABP in the serum-free supernatant culture medium of fibroblasts was further evident by immunoblot analysis, thus confirming the secretory nature of HABP and its occurrence in the extracellular matrix

Tuning Solvent Miscibility: A Fundamental Assessment on the Example of Induced Methanol/n-Dodecane Phase Separation

In this work, we assess the fundamental aspects of mutual miscibility of solvents by studying the mixing of two potential candidates, methanol and n-dodecane, for nonaqueous solvent extraction. To do so, H-1 NMR spectroscopy and molecular dynamics simulations are used jointly. The NMR spectra show that good phase separation can be obtained by adding LiCl and that the addition of a popular extractant (tri-n-butyl phosphate) yields the opposite effect. It is also demonstrated that in a specific case the poor phase separation is not due to the migration of n-dodecane into the more polar phase, but due to the transfer of the extractant into it, which is especially relevant when considering industrial applications of solvent extraction. With the aid of molecular dynamics simulations, explanations of this behavior are given. Specifically, an increase of all hydrogen-bond lifetimes is found to be consequent to the addition of LiCl which implies an indirect influence on the methanol liquid structure, by favoring a stronger hydrogen-bond network. Therefore, we found that better phase separation is not directly due to the presence of LiCl, but due to the "hardening" of the hydrogen-bond network

Effect of corrosion on the natural and whirl frequencies of a functionally graded rotor-bearing system subjected to thermal gradients

Corrosion causes a loss of material resulting in the reduction of mass and stiffness of a component, which consequently affects the dynamic characteristics of any system. Fundamental frequency analysis of a corroded functionally graded (FG) rotor system, using the finite element method based on the Timoshenko beam theory, was investigated in the present paper. The functionally graded shaft consisting of an inner metallic core and an outer ceramic layer was considered with the radial gradation of material properties based on the power law. Nonlinear temperature distribution (NLTD) based on the Fourier law of heat conduction was used to simulate the thermal gradient through the cross-section of the FG rotor. The finite element formulation for a functionally graded shaft with a corrosion defect was developed and the dynamic characteristics were investigated, which is the novelty of the present work. The corrosion parameters such as length, depth and position of the corrosion defect in the shaft were varied and a parametric study was performed to investigate changes in the natural and whirl frequencies. An analysis was carried out for different power indexes and temperature gradients of the functionally graded shaft. The effects of corrosion were analysed and important conclusions are drawn from the investigations

Biological pathways and in vivo antitumor activity induced by Atiprimod in myeloma

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Photodynamic vaccination: Novel strategies of vaccine delivery by intraphagolysosomal parasites and their oxidative inactivation for safe and effective applications.

Leishmania (trypanosomatid protozoa) have a number of advantages, which distinguish them favorably from other microbes/particles to serve as a universal vaccine carrier. Biotechnology is well-established to produce transgenic Leishmania for expressing peptide vaccines. Multiple vaccines can be expressed episomally at high capacity via its eukaryotic mechanisms of protein translation and post-translational modifications, e. g. N-glycosylation. Technology is also available to grow Leishmania in serum-free/autoclavable or chemically defined media that is expandable to industrial scale cost-effectively. The major attributes of Leishmania for vaccine delivery are their surface glyco-conjugates responsible for the following properties: Protection of Leishmania, hence their expressed vaccines from losses to the activities of humoral factors in the mammalian hosts; Targeting the vaccines to the phagosome-lysosome vacuolar system of macrophages and dendritic cells - a desirable destination for effective vaccine processing and presentation; and Serving as potential adjuvants for the natural vaccines to elicit the lasting immunity seen after spontaneous/therapeutic cure of human leishmaniasis. Significantly, the surface glycoconjugates retain these functional activities when Leishmania are photo-inactivated with reactive oxygen species (ROS). Photo-inactivation of Leishmania with ROS eliminates their immunosuppressive activities and releases vaccines in macrophages. A complete loss of Leishmania viability in vitro and in vivo ensues, especially when doubly photo-inactivated for cytosolic accumulation of uroporphyrin and endosomal uptake of exogenous cationic phthalocyanines. The delivery of ovalbumin (OVA) as a surrogate vaccine by such photo-inactivated Leishmania is more effective than by conventional methods in activation of OVA peptide-specific CD4+ and CD8+ T cells. Previously, evidence has been presented, showing that photodynamic vaccination of animals with Leishmania so inactivated produced effective immunity that is adaptively transferable to naïve individuals against experimental leishmaniasis. Immunotherapy of canine leishmaniasis with photo-inactivated Leishmania produced encouraging preliminary results. Work is also underway to use such carrier to deliver add-on vaccines against other infectious and malignant disease