Bufferless lysis of erythrocytes for isolation of hemoglobin using modified cellulose acetate membranes

This paper presents a modified cellulose acetate membrane prepared using a dry casting technique that can be used to perform lysis of erythrocytes and isolation of hemoglobin. Isolation of hemoglobin is thus achieved without the use of lysis buffers. Cellulose acetate (CA) membranes are embedded with ammonium chloride (NH4Cl) and potassium bicarbonate (KHCO3), which act as lysing agents. The presence of embedded salts is confirmed using EDS analysis. The pores in the CA membrane act as filters. The average pore size in these membranes is designed to be 1.5 mu M, as characterized by SEM analysis, so that they allow hemoglobin to pass through and block all other cells and unlysed erythrocytes present in blood. When a drop of blood is added to the membrane, the NH4Cl and KHCO3 embedded in the membrane dissolve in plasma and lyse the erythrocytes. The filtered hemoglobin is characterized using UV-Vis Spectroscopy. The results indicate extraction of higher concentration of hemoglobin compared with conventional methods.

2,3-Unsaturated enoses. A Pummerer rearrangement route to sugar vinyl sulfides and synthesis of 3-deoxy-3-alkyl/arylsulfinyl pyranosides

A Pummerer rearrangement of 2,3-dideoxy-3-alkyl/arylsulfinyl-arabino-hexopyranosides is reported. Treatment of sulfinyl-arabino-hexopyranoside derivatives, obtained through oxidation of the corresponding thio-derivatives, with trifluoroacetic anhydride (TFAA)/pyridine led to a facile formation of 2,3-dideoxy-3-alkyl/arylthio-hex-2-enopyranosides. Upon conversion of sugar vinyl sulfides to vinyl sulfoxides, conjugate addition reactions were conducted with alkoxides, to afford 3-deoxy-3-alkyl/arylsulfinyl pyranosides, in the manno-configuration exclusively. Whereas the conjugate addition reaction did not proceed with ether protecting groups, ester protecting groups and free hydroxyl groups in the sugar vinyl sulfoxide permitted the reaction. (C) 2012 Elsevier Ltd. All rights reserved.

Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface

Background: Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism. Results: Here we report kinetic characterization of S. typhimurium AckA (StAckA) and structures of its unliganded (Form-I, 2.70 angstrom resolution) and citrate-bound (Form-II, 1.90 angstrom resolution) forms. The enzyme showed broad substrate specificity with k(cat)/K-m in the order of acetate > propionate > formate. Further, the K-m for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis) more efficiently. ATP and Mg2+ could be substituted by other nucleoside 5'-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic beta beta beta alpha beta alpha beta alpha topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes. Conclusions: The biochemical and structural characterization of StAckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

Stiffness- and wettability-dependent myoblast cell compatibility of transparent poly(vinyl alcohol) hydrogels

This study reports the in vitro compatibility of muscle cells (C2C12 mouse myoblast cell line) with the transparent poly(vinyl alcohol) (PVA) hydrogels and the results are explained on the basis of surface wettability, crystallinity, and nanoscale elastic stiffness property. Nanoindentation was carried out with a maximum load of 100 mu N for all the hydrogel compositions and the properties such as elastic stiffness, hardness and total work done during indentation were computed. The difference in cell viability as well as adhesion of cultured myoblast cells on the investigated hydrogel substrates were discussed in reference to the difference in the nanoscale elastic properties, crystallinity, and surface wettability. An important result has been that both elastic stiffness and surface wettability synergistically influence myoblast viability/adhesion on PVA hydrogels. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.

Dual Role of Acetate as a Nucleophile and as an Internal Base in Cycloplatination Reaction of sym-N,N `,N `'-Triarylguanidines

Reaction of cis-Cl2Pt(S(O)Me-2)(2)] with 1 equiv of sym-N,N',N `'-triarylguanidines, ArN=C(NHAr)(2) (sym = symmetrical; Ar = 2-MeC6H4 (LH22-tolyl), 2-(MeO)C6H4 (LH22-anisyl), 4-MeC6H4 (LH24-tolyl), 2,5-Me2C6H3 (LH22,5-xylyl), and 2,6-Me2C6H3 (LH22,6-xylyl)) in toluene under reflux condition for 3 h afforded cis- or trans-Cl2Pt(S(O)Me-2)(ArN=C(NHAr)(2))] (Ar = 2-MeC6H4 (1), 2-(MeO)C6H4 (2), 4-MeC6H4 (3), 2,5-h Me2C6H3 (4), and 2,6-Me2C6H3 (5), respectively) in 83-96% yield. Reaction of cis-Cl2Pt(S(O)Me-2)(2)] with 1 equiv of LH22-tolyl and LH24-tolyl in the presence of 1 equiv of NaOAc in methanol under reflux condition for 3 h afforded acetate-substituted products, cis-(AcO)ClPt(S(O)Me-2)(ArN=C(NHAr)(2))] (Ar = 2-MeC6H4 (6) and 4-MeC6H4 (7)) in 83% and 84% yields, respectively. Reaction of cis-Cl2Pt(S(O)Me-2)(2)] with 1 equiv of LH22-anisyl and LH22-tolyl in the presence of 1 equiv of NaOAc in methanol under reflux condition for 3 and 12 h afforded six-membered C,N] platinacycles, Pt{kappa(2)(C,N)-C6H3R-3(NHC(NHAr)(=NAr))-2}Cl(S(O)Me-2)] (Ar = 2-RC6H4; R = OMe (8) and Me (9)), in 92% and 79% yields, respectively. The new complexes have been characterized by analytical and spectroscopic techniques, and further the molecular structures of 1, 2, 4, 5, 6, and 8 have been determined by single-crystal X-ray diffraction. The platinum atom in 1, 4, and 5 exhibited the trans configuration, while that in 2, 6, and 8 exhibited the cis configuration. Complex 6 is shown to be the precursor for 9, and the former is suggested to transform to the latter possibly via an intramolecular C-H activation followed by elimination of AcOH. The solution behavior of new complexes has been studied by multinuclear NMR (H-1, Pt-195, and C-13) spectroscopy. The new complexes exist exclusively as a single isomer (trans (1 and 5) and cis (6 and 7)), a mixture of cis and trans isomers with the former isomer being predominant in the case of 2 and the latter isomer being predominant in the case of 3. Complex 5 in the trans form revealed the presence of one isomer at 0.007 mM concentration and two isomers in about 1.00:0.12 ratio at 0.154 mM concentration as revealed by H-1 NMR spectroscopy, and this has been ascribed to the restricted Pt-S bond rotation at higher concentration. Platinacycle 8 exists as one isomer, while 9 exists as a mixture of seven isomers in solution. The influence of steric factor, pi-acceptor property of the guanidine, subtle solid-state packing forces upon the configuration of the platinum atom, and the number of isomers in solution have been outlined. Factors that accelerate or slow down the cycloplatination reaction, the role of NaOAc, and a plausible mechanism of this reaction have been discussed.

Flexible poly(vinyl alcohol-co-ethylene)/modified MMT moisture barrier composite for encapsulating organic devices

Flexible, nano-composite moisture barrier films of poly(vinyl alcohol-co-ethylene) with surface modified montmorillonite fabricated by solution casting were used to encapsulate organic devices. The composite films were characterized by FTIR, UV-visible spectroscopy and SEM imaging. Thermal and mechanical properties of the composite films were studied by DSC and UTM. Calcium degradation test was used to determine the transmission rate of water vapour through the composite films, which showed a gradual reduction from similar to 0.1 g m(-2) day(-1) to 0.0001 g m(-2) day(-1) with increasing modified montmorillonite loading in the neat copolymer. The increase in moisture barrier performance is attributed to the decreased water vapour diffusivity due to matrix-filler interactions in the composite. The accelerated aging test was carried out for non-encapsulated and encapsulated devices to evaluate the efficiency of the encapsulants. The encapsulated devices exhibited longer lifetimes indicating the efficacy of the encapsulant.

Nanolipodendrosome-loaded glatiramer acetate and myogenic differentiation I as augmentation therapeutic strategy approaches in muscular dystrophy

Backgrond: Muscular dystrophies consist of a number of juvenile and adult forms of complex disorders which generally cause weakness or efficiency defects affecting skeletal muscles or, in some kinds, other types of tissues in all parts of the body are vastly affected. In previous studies, it was observed that along with muscular dystrophy, immune inflammation was caused by inflammatory cells invasion - like T lymphocyte markers (CD8+/CD4+). Inflammatory processes play a major part in muscular fibrosis in muscular dystrophy patients. Additionally, a significant decrease in amounts of two myogenic recovery factors (myogenic differentation 1 MyoD] and myogenin) in animal models was observed. The drug glatiramer acetate causes anti-inflammatory cytokines to increase and T helper (Th) cells to induce, in an as yet unknown mechanism. MyoD recovery activity in muscular cells justifies using it alongside this drug. Methods: In this study, a nanolipodendrosome carrier as a drug delivery system was designed. The purpose of the system was to maximize the delivery and efficiency of the two drug factors, MyoD and myogenin, and introduce them as novel therapeutic agents in muscular dystrophy phenotypic mice. The generation of new muscular cells was analyzed in SW1 mice. Then, immune system changes and probable side effects after injecting the nanodrug formulations were investigated. Results: The loaded lipodendrimer nanocarrier with the candidate drug, in comparison with the nandrolone control drug, caused a significant increase in muscular mass, a reduction in CD4+/CD8+ inflammation markers, and no significant toxicity was observed. The results support the hypothesis that the nanolipodendrimer containing the two candidate drugs will probably be an efficient means to ameliorate muscular degeneration, and warrants further investigation.

Propyl-2-(8-(3,4-Difluorobenzyl)-2 `,5 `-Dioxo-8-AzaspiroBicyclo3.2.1] Octane-3,4 `-Imidazolidine]-1 `-yl) Acetate Induces Apoptosis in Human Leukemia Cells through Mitochondrial Pathway following Cell Cycle Arrest

Background: Due to the functional defects in apoptosis signaling molecules or deficient activation of apoptosis pathways, leukemia has become an aggressive disease with poor prognosis. Although the majority of leukemia patients initially respond to chemotherapy, relapse is still the leading cause of death. Hence targeting apoptosis pathway would be a promising strategy for the improved treatment of leukemia. Hydantoin derivatives possess a wide range of important biological and pharmacological properties including anticancer properties. Here we investigated the antileukemic activity and mechanism of action of one of the potent azaspiro hydantoin derivative, (ASHD). Materials and Methods: To investigate the antileukemic efficacy of ASHD, we have used MTT assay, cell cycle analysis by FACS, tritiated thymidine incorporation assay, Annexin V staining, JC1 staining and western blot analysis. Results: Results showed that ASHD was approximately 3-fold more potent than the parent compounds in inducing cytotoxicity. Tritiated thymidine assay in conjunction with cell cycle analysis suggests that ASHD inhibited the growth of leukemic cells. The limited effect of ASHD on cell viability of normal cells indicated that it may be specifically directed to cancer cells. Translocation of phosphatidyl serine, activation of caspase 3, caspase 9, PARP, alteration in the ratio of BCL2/BAD protein expression as well as the loss of mitochondrial membrane potential suggests activation of the intrinsic pathway of apoptosis. Conclusion: These results could facilitate the future development of novel hydantoin derivatives as chemotherapeutic agents for leukemia.

Graphene based E. coli sensor on flexible acetate sheet

A low cost, reagent free, Escherichia coli sensor is demonstrated with graphene, on transparent flexible acetate substrate. Graphene is grown on 100 mu m thick Cu foil, using CVD process and subsequently transferred on to a flexible acetate substrate. Gold electrodes are deposited on graphene to form a two terminal, interdigitated capacitor structure. Impedance spectroscopy (10 Hz to 100 kHz) is performed to characterize the change in impedance, as a function of E. coli concentration on graphene surface. The residual methyl groups on graphene, resulting from the transfer process, act as binding sites for E. coli. It has been observed that the resistance of graphene decreases with increasing E. coli concentration. This is due to the increased hole doping induced by negatively charged E. coli. A sensitivity of 60% is achieved for an E. coli concentration of 4.5 x 10(7) cfu/ml. An equivalent RC model is proposed to explain the sensing mechanism. (C) 2013 Elsevier B.V. All rights reserved.

Stereoselective geminal difunctionalization of vinyl arenes mediated by the bromonium ion

An anti-Markovnikov geminal oxyamination of styrenyl alkenes in an intermolecular fashion using the umpolung strategy mediated by the bromonium ion is reported. Isotope labeling studies confirm the migration of the phenyl group in the semipinacol rearrangement.

Nanoparticle-Driven Intermolecular Cooperativity and Miscibility in Polystyrene/Poly(vinyl methyl ether) Blends

The effect of silver nanoparticles (nAg) in PS/PVME polystyrene/poly(vinyl methyl ether)] blends was studied with respect to the evolution of morphology, demixing temperature, and segmental dynamics. In the early stage of demixing, PVME developed an interconnected network that coarsened in the late stage. The nAg induced miscibility in the blends as supported by shear rheological measurements. The physicochemical processes that drive phase separation in blends also led to migration of nAg to the PVME phase as supported by AFM. The segmental dynamics was greatly influenced by the presence of nAg due to the specific interaction of nAg with PVME. Slower dynamics and an increase in intermolecular cooperativity in the presence of nAg further supported the role of nAg in delaying the phase separation processes and augmenting the demixing temperature in the blends. Different theoretical models were assessed to gain insight into the dynamic heterogeneity in PS/PVME blends at different length scales.

Reduced graphene oxide induced phase miscibility in polystyrene-poly(vinyl methyl ether) blends

Graphene oxide and reduced graphene oxide (r-GO) were synthesized by wet chemistry and the effect of r-GO in PS-PVME blends was investigated here with respect to phase miscibility, intermolecular cooperativity in the glass transition region and concentration fluctuation variance by shear rheology and dielectric spectroscopy. The spinodal decomposition temperature (T-s) and correlation length were evaluated from isochronal temperature scans in shear rheology. The r-GO is shown to induce miscibility in the blends, which may lead to increased local heterogeneity in the blends, though the length of cooperatively re-arranged regions (xi) at T-g is more or less unaltered. The evolution of the phase morphology as a function of temperature was assessed using polarized optical microscopy (POM). In the case of the 60/40 PS-PVME blends with 0.25 wt% r-GO, apart from significant refinement in the morphology, retention of the interconnected ligaments of PVME was observed, even in the late stages of phase separation suggesting that the coarsening of the phase morphology has been slowed down in the presence of r-GO. This phenomenon was also supported by AFM. Surface enrichment of PVME, owing to its lower surface tension, in the demixed samples was supported by XPS scans. The interconnected network of PVME has resulted in significantly higher permittivity in the bi-phasic blends, although the concentration of r-GO is below the percolation threshold.

Aminosilane Functionalized Cenosphere in Poly(vinyl butyral) Composite Films: Moisture Resistant Encapsulated Schottky Devices

Functionalized cenosphere in PVB composite films were fabricated by melt processing. The composites show higher tensile strength with lower failure strain with increased filler ratio in the matrix. Fractographic images of the samples and DMA studies indicate brittle failure of the matrix. Moisture permeation and water contact angle studies reveal improved hydrophobicity of the matrix, while the factor of surface roughness increases the wettability at higher filler content. Schottky-structured devices encapsulated with functionalized cenosphere indicate enhanced resistance to moisture and increased life time for the devices.

Fabrication of Poly(Vinylidene Chloride-Co-Vinyl Chloride)/TiO2 Nanocomposite Films and Their Dielectric Properties

Flexible poly(vinylidene chloride-co-vinyl chloride)/TiO2 nanocomposite films were fabricated and their dielectric properties were studied. The structural characterization of the composites was carried out using various spectroscopic and electron microscopic techniques. From the thermal analysis of the composites, an improvement in the thermal properties was observed for the composites, as compared to the neat polymer. An increase in the DC conductivity was also observed in the composites, which was due to the tunneling of charge carriers. Furthermore, it was observed that the optimal loading of titania in the matrix was required, above which the properties of the composites showed deterioration. The study of the dielectric properties of the composites supports their use in microelectronic devices as separator in charge storage devices and in transistors.