Mammalian Target of Rapamycin (mTOR) and S6 Kinase Down-regulate Phospholipase D2 Basal Expression and Function

The mammalian target of rapamycin (mTOR) and S6 kinase (S6K) pathway is essential for cell differentiation, growth, and survival. Phospholipase D2 (PLD2) plays a key role in mTOR/S6K mitogenic signaling. However, the impact of PLD on mTOR/S6K gene expression is not known. Here we show that interleukin-8 (IL-8) increases mRNA expression levels for PLD2, mTOR, and S6K, with PLD2 preceding mTOR/S6K in time. Silencing of PLD2 gene expression abrogated IL-8-induced mTOR/S6K mRNA expression, whereas silencing of mTOR or S6K gene expression resulted in large (>3-fold and >5-fold, respectively) increased levels of PLD2 RNA, which was paralleled by increases in protein expression and lipase activity. Treatment of cells with 0.5 nm rapamycin induced a similar trend. These results suggest that, under basal conditions, PLD2 expression and concomitant activity is negatively regulated by the mTOR/S6K signaling pathway. Down-regulation of PLD2 was confirmed in differentiated HL-60 leukocytes overexpressing an mTOR-wild type, but not an mTOR kinase-dead construct. At the cellular level, overexpression of mTOR-wild type resulted in lower basal cell migration, which was reversed by treatment with IL-8. We propose that IL-8 reverses an mTOR/S6K-led down-regulation of PLD2 expression and enables PLD2 to fully function as a facilitator for cell migration.     

Design and Optimization of Sustained-Release Divalproex Sodium Tablets with Response Surface Methodology

Response surface methodology is defined as a collection of mathematical and statistical methods that are used to develop, improve, or optimize a product or process. In the present study, a statistical design (Mixture Design) was employed for formulation and optimization of a sustained-release hydrophilic divalproex sodium matrix tablet. Different excipients were used to improve the drug’s poor flowability. The hardness of the prepared tablets and also their release pattern were tested. The formulation design was carried out employing mixture design using four excipients in three levels. The Carr’s index of formulations and tensile strength were determined and analyzed using Minitab software. The suitable formulations regarding flowability and tablet tensile strength were selected by this software for subsequent drug release studies. The dissolution tests were carried out in acidic and basic phases which were previously proved to be biomimetic. Samples were analyzed using HPLC, and release data were compared to Depakine® (sustained-release divalproex from Sanofi). Release kinetics was also determined for selected formulations. Selected formulations were subjected to dissolution test and showed similar dissolution profiles with Depakine® based on difference and similarity factor calculations. The software selected an optimized formulation which had a slightly different release pattern in vitro compared to innovator but of nearly zero-order kinetics. It can be concluded that application of Mixture Design is a shortcut method to design suitable formulations of sustained-release divalproex sodium containing hydrophilic matrix tablets by direct compression method.     

Hydrodynamic characteristics and overall volumetric oxygen transfer coefficient of a new multi-environment bioreactor

The hydrodynamic characteristics and the overall volumetric oxygen transfer coefficient of a new multi-environment bioreactor which is an integrated part of a wastewater treatment system, called BioCAST, were studied. This bioreactor contains several zones with different environmental conditions including aerobic, microaerophilic and anoxic, designed to increase the contaminant removal capacity of the treatment system. The multi-environment bioreactor is designed based on the concept of airlift reactors where liquid is circulated through the zones with different environmental conditions. The presence of openings between the aerobic zone and the adjacent oxygen-depleted microaerophilic zone changes the hydrodynamic properties of this bioreactor compared to the conventional airlift designs. The impact of operating and process parameters, notably the hydraulic retention time (HRT) and superficial gas velocity (U _G), on the hydrodynamics and mass transfer characteristics of the system was examined. The results showed that liquid circulation velocity (V _L), gas holdup (ε) and overall volumetric oxygen transfer coefficient ( $ k_{\text{L}} a_{\text{L}} $ ) increase with the increase of superficial gas velocity (U _G), while the mean circulation time (t _c) decreases with the increase of superficial gas velocity. The mean circulation time between the aerobic zone (riser) and microaerophilic zone (downcomer) is a stronger function of the superficial gas velocity for the smaller openings (1/2 in.) between the two zones, while for the larger opening (1 in.) the mean circulation time is almost independent of U _G for U _G ≥ 0.023 m/s. The smaller openings between the two zones provide higher mass transfer coefficient and better zone generation which will contribute to improved performance of the system during treatment operations.     

Direct membrane association drives mitochondrial fission by the Parkinson disease-associated protein alpha-synuclein

The protein α-synuclein has a central role in Parkinson disease, but the mechanism by which it contributes to neural degeneration remains unknown. We now show that the expression of α-synuclein in mammalian cells, including neurons in vitro and in vivo, causes the fragmentation of mitochondria. The effect is specific for synuclein, with more fragmentation by α- than β- or γ-isoforms, and it is not accompanied by changes in the morphology of other organelles or in mitochondrial membrane potential. However, mitochondrial fragmentation is eventually followed by a decline in respiration and neuronal death. The fragmentation does not require the mitochondrial fission protein Drp1 and involves a direct interaction of synuclein with mitochondrial membranes. In vitro, synuclein fragments artificial membranes containing the mitochondrial lipid cardiolipin, and this effect is specific for the small oligomeric forms of synuclein. α-Synuclein thus exerts a primary and direct effect on the morphology of an organelle long implicated in the pathogenesis of Parkinson disease.     

Determination by liquid chromatography-mass spectrometry of clomiphene isomers in the plasma of patients undergoing treatment for the induction of ovulation

A rapid, sensitive and selective LC-MS method is described for the simultaneous determination of zuclomiphene and enclomiphene in plasma from patients undergoing treatment with clomiphene citrate for the induction of ovulation. Samples spiked with N-didesmethyltamoxifen, the internal standard, were extracted into methyl tertiary butyl ether. The compounds were separated on a Luna C(18) analytical column, and a mobile phase of methanol-water (70:30 v/v) containing 0.05% trifluoroacetic acid at a flow rate of 1ml/min. The limits of determination were 35pg/ml and 7pg/ml for zu- and enclomiphene, respectively. Within-day coefficients of variation ranged from 2.1% to 7.2%.     

Decoration of electrical conductive polyurethane-polyaniline/polyvinyl alcohol matrixes with mussel-inspired polydopamine for bone tissue engineering

Electrospinning is a versatile technology for the fabrication of nanofibrous matrixes to regenerate defects. This study aims to develop a functionalized and electroconductive polymeric matrix to improve rat bone marrow mesenchymal stem cell adhesion, proliferation, and differentiation. Herein, the influence of the chemical composition of the substrate on homogeneous modification of the surface with mussel-inspired polydopamine (PDA) is focused. Accordingly, the deposition of PDA on the surface was proved by Fourier transform infrared spectroscopy. Morphologies of the scaffolds demonstrated homogeneous decoration of the polyvinyl alcohol (PVA)/polyurethane (PU)-polyaniline (PANI) matrixes with PDA, while a lower density of mussel-inspired polymer was observed in bare PU-PANI constructs. Although uniform and dense precipitation of PDA reduced conductivity of scaffolds 1.2 times compared with the samples with a low density of the PDA, 1.1 and 1.2 times enhancement in tensile strength and Young's modulus, respectively, were the strength of the applied process, especially in bone tissue engineering area. Contact angle measurements demonstrated about two times reduction in measured values, which shows improvement in hydrophilicity of PDA-modified PVA/PU-PANI fibers compared with PDA-coated PU-PANI ones. Swelling ratio and mass loss ratio calculations revealed enhancement in measured values as a function of homogeneous and dense coating, which arise from hydrophilicity of the polymeric substrate. The bioactivity test indicated that a dense layer of PDA strongly supports formations of hydroxyapatite-like crystals. Moreover, homogeneous decoration of conductive matrixes with PDA showed suitable cell viability, adhesion, and spreading while cell-scaffolds interactions improved under electrical stimulation. Higher expression of alkaline phosphatase and secretion of Collagen I under the electrical field proved the applicability of modified electroconductive scaffolds for further preclinical and clinical studies to introduce as a reconstructive bone substitute.     

C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

Background

We have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects.

Methodology/Principal Findings

Three pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b^-CTE, pcDNA-cpa/b^-CTE, cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b^-CTE showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies.

Conclusions/Significance

This report demonstrates cSLN''s ability to boost immune response magnitude of cpa/cpb^-CTE cocktail vaccination against leishmaniasis so that the average parasite inhibition percent could be increased significantly. Hence, cSLNs can be considered as suitable adjuvant and/or delivery systems for designing third generation cocktail vaccines.