Structure and water permeability of fully hydrated diphytanoylPC

Diphytanoylphosphatidylcholine (DPhyPC) is a branched chain lipid often used for model membrane studies, including peptide/lipid interactions, ion channels and lipid rafts. This work reports results of volume measurements, water permeability measurements P_f, X-ray scattering from oriented samples, and X-ray and neutron scattering from unilamellar vesicles at T = 30 °C. We measured the volume/lipid V_L = 1426 ± 1 Å³. The area/lipid was found to be 80.5 ± 1.5 Å² when both X-ray and neutron data were combined with the SDP model analysis (Ku?erka, N., Nagle, J.F., Sachs, J.N., Feller, S.E., Pencer, J., Jackson, A., Katsaras, J., 2008. Lipid bilayer structure determined by the simultaneous analysis of neutron and X-ray scattering data. Biophys. J. 95, 2356–2367); this is substantially larger than the area of DOPC which has the largest area of the common linear chain lipids. P_f was measured to be (7.0 ± 1.0) × 10^?3 cm/s; this is considerably smaller than predicted by the recently proposed 3-slab model (Nagle, J.F., Mathai, J.C., Zeidel, M.L., Tristram-Nagle, S., 2008. Theory of passive permeability through lipid bilayers. J. Gen. Physiol. 131, 77–85). This disagreement can be understood if there is a diminished diffusion coefficient in the hydrocarbon core of DPhyPC and that is supported by previous molecular dynamics simulations (Shinoda, W., Mikami, M., Baba, T., Hato, M., 2004. Molecular dynamics study on the effects of chain branching on the physical properties of lipid bilayers. 2. Permeability. J. Phys. Chem. B 108, 9346–9356). While the DPhyPC head–head thickness (D_HH = 36.4 Å), and Hamaker parameter (H = 4.5 × 10^?21 J) were similar to the linear chain lipid DOPC, the bending modulus (K_C = 5.2 ± 0.5 × 10^?21 J) was 30% smaller. Our results suggest that, from the biophysical perspective, DPhyPC belongs to a different family of lipids than phosphatidylcholines that have linear chain hydrocarbon chains.     

Development of new acetylcholinesterase reactivators: Molecular modeling versus in vitro data

In this work a theoretical methodology for evaluation of the association and kinetic reactivation constants of oximes using the Molegro^® and Spartan^® softwares was proposed and validated facing in vitro data previously reported in the literature. Results showed a very good agreement between the theoretical binding free energies of the reactivators and experimental data, suggesting that the proposed methodology could work well in the prediction of kinetic and thermodynamics parameters for oximes that might be helpful for the design and selection of new and more effective oximes.     

Redirecting Lentiviral Vectors Pseudotyped with Sindbis Virus-Derived Envelope Proteins to DC-SIGN by Modification of N-Linked Glycans of Envelope Proteins

Redirecting the tropism of viral vectors enables specific transduction of selected cells by direct administration of vectors. We previously developed targeting lentiviral vectors by pseudotyping with modified Sindbis virus envelope proteins. These modified Sindbis virus envelope proteins have mutations in their original receptor-binding regions to eliminate their natural tropisms, and they are conjugated with targeting proteins, including antibodies and peptides, to confer their tropisms on target cells. We investigated whether our targeting vectors interact with DC-SIGN, which traps many types of viruses and gene therapy vectors by binding to the N-glycans of their envelope proteins. We found that these vectors do not interact with DC-SIGN. When these vectors were produced in the presence of deoxymannojirimycin, which alters the structures of N-glycans from complex to high mannose, these vectors used DC-SIGN as their receptor. Genetic analysis demonstrated that the N-glycans at E2 amino acid (aa) 196 and E1 aa 139 mediate binding to DC-SIGN, which supports the results of a previous report of cryoelectron microscopy analysis. In addition, we investigated whether modification of the N-glycan structures could activate serum complement activity, possibly by the lectin pathway of complement activation. DC-SIGN-targeted transduction occurs in the presence of human serum complement, demonstrating that high-mannose structure N-glycans of the envelope proteins do not activate human serum complement. These results indicate that the strategy of redirecting viral vectors according to alterations of their N-glycan structures would enable the vectors to target specific cells types expressing particular types of lectins.The ultimate goal of gene therapy is cell- and tissue-specific targeted delivery of therapeutic genes. A targeted system increases the therapeutic effects of transgenes at the site of action while reducing adverse effects in surrounding cells and tissues that commonly occur through nonspecific modes of gene delivery (5-8). Gene therapy vectors that can home to specific cells and tissues after intravenous administration, also known as targeting vectors, are ideal for targeted delivery (62). In the past, many attempts have been made to develop targeting viral vectors by using adenovirus, adeno-associated virus, oncoretrovirus, lentivirus, measles virus, and alphavirus (70, 89).To create targeting viral vectors, the natural tropisms of the viruses must first be eliminated and new binding specificities conferred (89). The binding of envelope viruses, such as oncoretrovirus, lentivirus, measles virus, and alphavirus, is mediated by envelope proteins. To redirect the tropisms of these viruses, the original receptor-binding regions of their envelope proteins must be eliminated. We have developed targeting oncoretroviral and lentiviral vectors by pseudotyping them with modified Sindbis virus envelope proteins and by mutating the receptor-binding regions of the envelope proteins, thereby reducing the nonspecific transduction of untargeted cells (61, 63-66). The mutated regions of the envelope protein originally interact directly with other receptors, including heparan sulfate, laminin receptor, and/or unknown molecules (10, 46, 67, 90). These mutations reduced the nonspecific transduction of the liver and spleen when the vectors were administered intravenously (66). By conjugating the virus with targeting ligands, including antibodies and peptides, the virus can transduce specific cells and tissues both in vitro and in vivo (53, 61, 63-66, 71, 72). These results demonstrated that we can eliminate the natural tropism of the Sindbis virus envelope protein while maintaining its fusion activity.However, the N-glycans of the envelope proteins are still intact and possibly interact with cell surface lectins. DC-SIGN is the best-known cell surface lectin expressed on dendritic cells, certain macrophages, and activated B cells (27, 29, 30).Structural and biochemical studies show flexible modes of DC-SIGN binding to cognate saccharides. The trimannose core unit of high-mannose N-glycans is the primary binding site for DC-SIGN (23), while nonreducing alpha1-2-linked terminal mannose moieties contribute to the high avidity seen when DC-SIGN binds the Man8 or Man9 structures common to many viral envelope glycoproteins (22). DC-SIGN traps a wide variety of viruses and viral vectors (HIV [29, 30], simian immunodeficiency virus [50], human T-cell leukemia virus type 1 [12], measles virus [17, 18], dengue virus [86], feline corona virus [77], herpes simplex virus type 1 [16], human cytomegalovirus [36], human herpesvirus type 8 [76], Ebola virus [1], West Nile virus [15], influenza virus [91], Marburg virus [57], and severe acute respiratory syndrome virus [93]) by binding to the N-glycans of the viruses and viral vectors. Binding of DC-SIGN with virus and viral vectors results in enhanced infection and/or transduction of DC-SIGN-positive cells (cis infection/transduction) and/or neighboring cells (trans infection/transduction).If any targeting vector can be trapped by DC-SIGN, it is necessary to eliminate its binding to DC-SIGN to increase the targeting specificity of the virus in vivo (28, 49, 73). In addition to enhanced infection/transduction, binding to DC-SIGN causes signaling that can activate DC-SIGN-expressing antigen-presenting cells (32, 38). Activation of antigen-presenting cells can lead to adverse effects, including systemic inflammation and immune reactions to viral vectors and their transgene products (7, 8, 32, 59, 88). Therefore, investigation of the interactions between viral vectors and DC-SIGN, identification of N-glycans that mediate binding to DC-SIGN, and elimination of interactions with DC-SIGN are important aspects of reducing adverse effects of vector administration and prolonging transgene expression.The envelope protein of our targeting lentiviral vectors, the Sindbis virus envelope protein, contains four N-linked glycans (9, 48). Sindbis virus can replicate in insect and mammalian cells, which have different types of enzymes to process N-glycans (3). Therefore, the structures of N-glycans differ between the virus produced in insect cells and that produced in mammalian cells (40, 58). The N-glycans of the virus produced in insect cells have either the high-mannose or the paucimannosidic structure. Paucimannosidic structure N-glycans, as well as high-mannose structure N-glycans, have terminal mannose residues, and all N-glycans produced in insect cells are predicted to be able to bind DC-SIGN (Fig. ​(Fig.11 a) (39, 47). On the other hand, two N-glycans of the virus produced in mammalian cells have the high-mannose structure, while two others have the complex structure (40, 58). The two complex structure N-glycans have been shown to be exposed on the surface of the envelope protein, while the two high-mannose structure N-glycans are buried within the center of the trimer of the envelope proteins (74, 94). Therefore, the virus produced in insect cells can access DC-SIGN as its receptor while the virus produced in mammalian cells cannot (47). Because our targeting vectors are produced in mammalian cells, they should not bind DC-SIGN efficiently. However, one group demonstrated that lentiviral vectors pseudotyped with a modified Sindbis virus envelope protein bind to DC-SIGN and target DC-SIGN-positive cells (92), in contrast to the results seen with replication-competent Sindbis virus. Both Sindbis virus and the pseudotyped lentiviral vectors were produced in mammalian cells; Sindbis virus was produced in baby hamster kidney (BHK) cells, chicken embryonic fibroblasts, and hamster fibroblast cells; and the pseudotyped vector was produced in human embryonic kidney fibroblast (293T) cells (69). Because it is known that the N-glycans of the HIV envelope protein produced in lymphocytes have structures different from those produced in macrophages, the different producer cells may account for the differences between the N-glycan structures of the virus and Sindbis virus envelope-pseudotyped lentivectors (54, 55). It is also known that the N-glycan structure of dengue virus can be altered by the presence of viral capsid (35). Thus, the capsid of Sindbis virus and HIV could also affect the structures of the N-glycans of envelope proteins differently.Open in a separate windowFIG. 1.(a) N-glycan structures and processing pathway. All N-glycans are first produced as the high-mannose structure in both mammalian cells and insect cells. In mammalian cells, certain N-glycans are further processed to the complex structure. In insect cells, certain N-glycans are further processed to the paucimannosidic structure. DMNJ inhibits mannosidase I, which is necessary for the formation of the complex structure; thus, all N-glycans have the high-mannose structure when generated in the presence of DMNJ. One representative structure of each N-glycan is shown. Man, mannose; GlcNAc, N-acetylglucosamine; SA, sialic acid; Gal, galactose. (b) Schematic representation of chimeric Sindbis virus envelope proteins. The Sindbis virus envelope protein is first synthesized as a polypeptide and subsequently cleaved by cellular proteases to generate the E3, E2, 6K, and E1 proteins. E1 and E2 are incorporated into the viral envelope, and E3 and 6K are leader sequences for E2 and E1, respectively. The N-linked glycosylation sites of the envelope proteins are shown. 2.2 is a modified Sindbis virus envelope protein in which the IgG-binding domain of protein A (ZZ) was inserted into the E2 region at aa 70. 2.2 1L1L has two flexible linkers (Gly-Gly-Gly-Gly-Ser) at aa 70 of the E2 protein. 2.2 ΔE2-196N does not have the N-glycan at E2 aa 196, 2.2 ΔE1-139N does not have the N-glycan at E1 aa 139, and 2.2 ΔE2-196N E1-139N does not have the N-glycans at either E2 aa 196 or E1 aa 139.In this study, we investigated whether our targeting vector binds DC-SIGN. We found that DC-SIGN does not mediate the transduction of our targeting vectors efficiently. The vectors can be redirected to DC-SIGN by modifying the structures of the N-glycans of the envelope proteins by using the mannosidase I inhibitor deoxymannojirimycin (DMNJ) (25, 47, 51).     

Isolation of a novel freshwater agarolytic Cellvibrio sp. KY-YJ-3 and characterization of its extracellular beta-agarase

A novel agarolytic bacterium KY-YJ-3, producing extracellular agarase, was isolated from the freshwater sediment of the Sincheon River in Daegu, Korea. On the basis of gram-staining data, morphology, and phylogenetic analysis of the 16S rDNA sequence, the isolate was identified as Cellvibrio sp. By ammonium sulfate precipitation followed by Toyopearl QAE-550C, Toyopearl HW-55F, and Mono-Q column chromatography, the extracellular agarase in the culture fluid could be purified 120.2-fold with yield of 8.1%. The specific activity of the purified agarase was 84.2 U/mg. The molecular mass of the purified agarase was 70 kDa as determined by dodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified agarase were 35 degrees C and pH 7.0, respectively. The purified agarase failed to hydrolyze the other polysaccharide substrates, including carboxymethyl (CM)-cellulose, dextran, soluble starch, pectin, and polygalacturonic acid. Kinetic analysis of the agarose-hydrolysis catalyzed by the purified agarase using thin layer chromatography (TLC) exhibited that the main products were neoagarobiose, neoagarotetraose, and neoagarohexaose. These results demonstrated that the newly isolated freshwater agarolytic bacterium KY-YJ-3 was a Cellvibrio sp., and could produce an extracellular beta-agarase, which hydrolyzed agarose to yield neoagarobiose, neoagarotetraose, and neoagarohexaose as the main products.     

Design,synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binding and intercalating functionalities, have the potential for increased DNA binding affinity and increased selectivity due to their dual mode of DNA binding. This study describes the synthesis of DNA minor groove binder netropsin analogs containing either one or two N-methylpyrrole carboxamide groups linked to DNA-intercalating anthrapyrazoles. Those hybrid molecules which had both two N-methylpyrrole groups and terminal (dimethylamino)alkyl side chains displayed submicromolar cytotoxicity towards K562 human leukemia cells. The combilexins were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIα-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIα decatenation activity, and for inhibition of DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA–topoisomerase IIα covalent complex indicating that they acted as topoisomerase IIα poisons. Some of the combilexins had higher affinity for DNA than their parent anthrapyrazoles. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazole groups and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents.     

Spectroscopic characterization and photobleaching kinetics of hypericin-N-methyl pyrrolidone formulations.

Hypericin (HY) is a promising photosensitizer in photodynamic therapy (PDT). It was recently reported that appropriate use of N-methyl pyrrolidone (NMP) enhanced in vivo PDT efficacy of HY and enhanced in vivo delivery of HY. This present study further investigates the use of NMP and other known non-toxic pharmaceutical additives, polyvinylpyrrolidone (PVP, K29/32) and copolyvidonum (S630), for formulating HY to enhance its delivery with photodynamic activity as a goal in mind. Hence, the first objective of this study was to characterize the solubilization of HY by NMP, K29/32 and S630. Thermodynamic considerations were used to explain the solvation process. Photobleaching is another important property of photosensitizers. There is no report on the photostability of HY in pharmaceutical formulations used for PDT. Therefore, the second objective of this study was to investigate the photobleaching of HY in these formulations. The fluorescence of HY was found to increase significantly in higher concentrations of NMP or when 5% of polymer was co-mixed with 5% of NMP solution. The photobleaching of HY in these formulations followed first-order kinetics. The loss of fluorescence paralleled to the loss of absorption of HY. The formulation of HY with 40% NMP was found to be the most stable.     

Morphological and molecular identity of Anopheles (Cellia) sundaicus (Diptera: Culicidae), the nominotypical member of a malaria vector species complex in Southeast Asia

A neotype for Anopheles (Cellia) sundaicus (Rodenwaldt), the nominotypical member of a malaria vector species complex, is selected from the progeny of a female collected at Pandan Beach near Lundu, Sarawak, Malaysia. Siblings of the neotype and other topotypic specimens are used to characterize and fix the morphological and molecular identity of the species as a foundation for systematic studies of the species complex. The species is described and illustrated in the adult, pupal and larval stages, and DNA sequence data are included for the mitochondrial cytochrome c oxidase I (COI) gene and the nuclear ITS‐2 region.     

Clot Permeability,Agonist Transport,and Platelet Binding Kinetics in Arterial Thrombosis

The formation of wall-adherent platelet aggregates is a critical process in arterial thrombosis. A growing aggregate experiences frictional drag forces exerted on it by fluid moving over or through the aggregate. The magnitude of these forces is strongly influenced by the permeability of the developing aggregate; the permeability depends on the aggregate’s porosity. Aggregation is mediated by formation of ensembles of molecular bonds; each bond involves a plasma protein bridging the gap between specific receptors on the surfaces of two different platelets. The ability of the bonds existing at any time to sustain the drag forces on the aggregate determines whether it remains intact or sheds individual platelets or larger fragments (emboli). We investigate platelet aggregation in coronary-sized arteries using both computational simulations and in vitro experiments. The computational model tracks the formation and breaking of bonds between platelets and treats the thrombus as an evolving porous, viscoelastic material, which moves differently from the background fluid. This relative motion generates drag forces which the fluid and thrombus exert on one another. These forces are computed from a permeability-porosity relation parameterized from experimental measurements. Basing this relation on measurements from occlusive thrombi formed in our flow chamber experiments, along with other physiological parameter values, the model produced stable dense thrombi on a similar timescale to the experiments. When we parameterized the permeability-porosity relation using lower permeabilities reported by others, bond formation was insufficient to balance drag forces on an early thrombus and keep it intact. Under high shear flow, soluble agonist released by platelets was limited to the thrombus and a boundary layer downstream, thus restricting thrombus growth into the vessel lumen. Adding to the model binding and activation of unactivated platelets through von Willebrand-factor-mediated processes allowed greater growth and made agonist-induced activation more effective.     

Utilizing Latent Multi‐Redox Activity of p‐Type Organic Cathode Materials toward High Energy Density Lithium‐Organic Batteries

Organic electrode materials hold great potential due to their cost‐efficiency, eco‐friendliness, and possibly high theoretical capacity. Nevertheless, most organic cathode materials exhibit a trade‐off relationship between the specific capacity and the voltage, failing to deliver high energy density. Herein, it is shown that the trade‐off can be mitigated by utilizing the multi‐redox capability of p‐type electrodes, which can significantly increase the specific capacity within a high‐voltage region. The molecular structure of 5,10‐dihydro‐5,10‐dimethylphenazine is modified to yield a series of phenoxazine and phenothiazine derivatives with elevated redox potentials by substitutions. Subsequently, the feasibility of the multi‐redox capability is scrutinized for these high‐voltage p‐type organic cathodes, achieving one of the highest energy densities. It is revealed that the seemingly impractical second redox reaction is indeed dependent on the choice of the electrolyte and can be reversibly realized by tailoring the donor number and the salt concentration of the electrolyte, which places the voltage of the multi‐redox reaction within the electrochemical stability window. The results demonstrate that high‐energy‐density organic cathodes can be practically achieved by rational design of multi‐redox p‐type organic electrode materials and the compatibility consideration of the electrolyte, opening up a new avenue toward advanced organic rechargeable batteries.     

Enterococcus faecium L-15 Extract Enhances the Self-Renewal and Proliferation of Mouse Skin-Derived Precursor Cells

Lactic acid bacteria (LAB) in the gastrointestinal tract have beneficial health effects. LAB activate the proliferation of intestinal stem cells and speed the recovery of damaged intestinal cells, but little is known about effect of LAB on other adult stem cells. In this study, a cell-free extract of Enterococcus faecium L-15 (L15) was exposed to mouse skin-derived precursor cells (SKPs), and the changes in characteristics associated with proliferation and self-renewal capacity were investigated. L15 increased the size of the spheres and the proliferation rate of SKPs. Cell cycle analysis revealed that cells in the S-phase increased after treatment with L15. In the L15-treated group, the total number of spheres significantly increased. The expression level of pluripotency marker genes also increased, while the mesenchymal lineage-related differentiation marker genes significantly decreased in the L15-treated group. The PI3K/Akt signaling pathway was activated by L15 in SKPs. These results indicate that L15 enhances proliferation and self-renewal of SKPs and may be used as a supplement for stem cell maintenance or application of stem cell therapy. This is the first report to investigate the functional effects of E. faecium on the proliferation and self-renewal capacity of SKPs.