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CMC
Lahore, Pakistan

Bonkovsky H.L.,CMC | Bonkovsky H.L.,Universities of and
Liver International | Year: 2012

Porphyria cutanea tarda (PCT) is the most common form of porphyria across the world. Unlike other forms of porphyria, which are inborn errors of metabolism, PCT is usually an acquired liver disease caused by exogenous factors, chief among which are excess alcohol intake, iron overload, chronic hepatitis C, oestrogen therapy and cigarette smoking. The pathogenesis of PCT is complex and varied, but hereditary or acquired factors that lead to hepatic iron loading and increased oxidative stress are of central importance. Iron loading is usually only mild or moderate in degree [less than that associated with full-blown haemochromatosis (HFE)] and is usually acquired and/or mutations in HFE. Among acquired factors are excessive alcohol intake and chronic hepatitis C infection, which, like mutations in HFE, decrease hepcidin production by hepatocytes. The decrease in hepcidin leads to increased iron absorption from the gut. In the liver, iron loading and increased oxidative stress leads to the formation of non-porphyrin inhibitor(s) of uroporphyrinogen decarboxylase and to oxidation of porphyrinogens to porphyrins. The treatment of choice of active PCT is iron reduction by phlebotomy and maintenance of a mildly iron-reduced state without anaemia. Low-dose antimalarials (cinchona alkaloids) are also useful as additional therapy or as alternative therapy for active PCT in those without haemochromatosis or chronic hepatitis C. In this review, we provide an update of PCT with special emphasis upon the important role often played by the hepatitis C virus. © 2012 John Wiley & Sons A/S. Source


News Article
Site: http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2F%28ISSN%291616-3028

The regeneration of artificial bone substitutes is a potential strategy for repairing bone defects. However, the development of substitutes with appropriate osteoinductivity and physiochemical properties, such as water uptake and retention, mechanical properties, and biodegradation, remains challenging. Therefore, there is a motivation to develop new synthetic grafts that possess good biocompatibility, physiochemical properties, and osteoinductivity. Here, we fabricate a biocompatible scaffold through the covalent crosslinking of graphene oxide (GO) and carboxymethyl chitosan (CMC). The resulting GO-CMC scaffold shows significant high water retention (44% water loss) compared with unmodified CMC scaffolds (120% water loss) due to a steric hindrance effect. The modulus and hardness of the GO-CMC scaffold are 2.75- and 3.51-fold higher, respectively, than those of the CMC scaffold. Furthermore, the osteoinductivity of the GO-CMC scaffold is enhanced due to the π–π stacking interactions of the GO sheets, which result in striking upregulation of osteogenesis-related genes, including osteopontin, bone sialoprotein, osterix, osteocalcin, and alkaline phosphatase. Finally, the GO-CMC scaffold exhibits excellent reparative effects in repairing rat calvarial defects via the synergistic effects of GO and bone morphogenetic protein-2. This study provides new insights for developing bone substitutes for tissue engineering and regenerative medicine.


News Article
Site: http://www.nature.com/nature/current_issue/

Expression plasmid pJH114 containing the five E. coli bamABCDE genes which were under the control of a trc promoter, and with an octa-histidine (8 × His) tag at the C terminus of bamE was initially used for overexpression of BamABCDE complex in E. coli HDB150 cells16. Expression of the native BamABCDE complex was induced with 100 μmol l−1 isopropyl-β-D-1-thiogalactopyranoside (IPTG; Formedium) at 20 °C overnight when the absorbance of the cell culture at 600 nm reached 0.5–0.8. The selenomethionine-labelled BAM complexes were expressed in M9 medium supplemented with selenomethionine Medium Nutrient Mix (Molecular Dimensions) and 100 mg l−1 L-(+)-selenomethionine (Generon) using the similar conditions as the native BamABCDE. Both native and selenomethionine-labelled BamABCDE complexes were purified using a similar protocol. In brief, the cells were pelleted and resuspended in lysis buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 μg ml−1 DNase I and 100 μg  ml−1 lysozyme and lysed by passing through a cell disruptor (Constant Systems) at 206 MPa. The lysate was centrifuged to remove the cell debris and unbroken cells, and the supernatant was ultracentrifuged to pellet the membranes at 100,000g for 1 h. The cell membranes were resuspended in solubilization buffer containing 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole and 1–2% n-dodecyl-β-D-maltopyranoside (DDM; all detergents were purchased from Anatrace) and rocked for 1 h at room temperature or overnight at 4 °C. The suspension was ultracentrifuged and the supernatant was applied to a 5-ml pre-equilibrated HisTrap HP column (GE Healthcare). The column was washed with wash buffer containing 20 mM Tris-HCl, pH 8.0, 300 mM NaCl and 35 mM imidazole and eluted with elution buffer containing 300 mM imidazole. The eluent was applied to HiLoad 16/600 Superdex 200 prep grade column (GE healthcare) pre-equilibrated with gel filtration buffer containing 20 mM Tris-HCl, pH 7.8, 300 mM NaCl and detergents. Different detergents were used in protein purification procedures. The purified BamABCDE complex was analysed by SDS–PAGE (Extended Data Fig. 1 and Supplementary Fig. 1), which indicated that BamB is not enough in the complex, and BamB is absent in the determined structure. We therefore decided to generate a new plasmid to express the BamABCDE complex. Additional copy of the E. coli bamB gene was introduced into pJH114 (ref. 16) after the 8 × His tag to generate a new expression plasmid pYG120 using a modified sequence and ligation-independent cloning (SLIC) method42. In brief, vector backbone and bamB gene fragments were amplified by PCR using Q5 Hot Start High-Fidelity DNA Polymerase (New England BioLabs), and plasmid pJH114 as template and primers PF_pJH114_SLIC (5′-GTTAATCGACCTGCAGGCATGCAAG-3′) and PR_pJH114_SLIC (5′-CTCTAGAGGATCTTAGTGGTGATGATGGTG-3′), and PF_EBB_SLIC (5′-TCATCACCACTAAGATCCTCTAGAGAGGGACCCGATGCAATTGC-3′) and PR_EBB_SLIC (5′-CTTGCATGCCTGCAGGTCGATTAACGTGTAATAGAGTACACGGTTCC-3′), respectively. Gel-extracted fragments were digested by T4 DNA polymerase (Fermentas) at 22 °C for 35 min followed by 70 °C for 10 min, and then placed on ice immediately. The digested fragments were annealed in an annealing buffer (10 mM Tris, pH 8.0, 100 mM NaCl and 1 mM EDTA) by incubating at 75 °C for 10 min and decreasing by 0.1 °C every 8 s to 20 °C. The mixture was transformed into E. coli DH5α for plasmid preparation. The DNA sequences were confirmed by sequencing. For the purification of the BamABCDE complex from the pYG120 construct, the wash buffer, elution buffer and gel filtration buffer were supplemented with different detergent combinations. A second gel filtration was performed to change detergents with gel filtration buffer containing 1 CMC N-octyl-β-D-glucopyranoside (OG) and 1 CMC N-dodecyl-N,N-dimethylamine-N-oxide (LDAO). For BamABCDE complex purification from construct pJH114, the wash buffer, elution buffer and gel filtration buffer were supplemented with 2 CMC N-nonyl-β-D-glucoside (β-NG) and 1 CMC tetraethylene glycol monooctyl ether (C8E4). The peak fraction was pooled and concentrated using Vivaspin 20 centrifugal concentrator (Sartorius, molecular mass cut off: 100 kDa). The selenomethionine-labelled proteins were purified in the same way as the native proteins of BamABCDE complex. The purified proteins were concentrated to 8–12 mg ml−1 for crystallization. For NaI co-crystallization, NaCl was replaced by NaI in the gel filtration buffer. All crystallizations were carried out by sitting-drop vapour diffusion method in the MRC 96-well crystallization plates (Molecular Dimensions) at 22 °C. The protein solution was mixed in a 1:1 ratio with the reservoir solution using the Gryphon crystallization robot (Art Robbins Instruments). The best NaI co-crystallized crystals were grown from 150 mM HEPES, pH 7.5, 30% PEG6000 and CYMAL-4 in MemAdvantage (Molecular Dimensions) as additive. The best native crystals were grown from 150 mM HEPES, pH 7.5 and 27.5% PEG6000. The best selenomethionine-labelled crystals were grown from 100 mM Tris, pH 8.0, 200 mM MgCl  . 6H O, 24% PEG1000 MME and OGNG in MemAdvantage as additive. The crystals were harvested, flash-cooled and stored in liquid nitrogen for data collection. The data sets of selenomethionine labelled BAM complex were collected on the I03 beamline at Diamond Light Resources (DLS) at a wavelength of 0.9795 Å. All data were indexed, integrated and scaled using XDS43. The crystals belong to space group of P4 2 2, with the cell dimensions a = b = 254.16 Å, c = 179.22, α = β = γ = 90°. There are two complexes in the asymmetric unit. The structure was determined to 3.9 Å resolution (Extended Data Table 1) using ShelxD44, 45. Fifty-six selenium sites were found, which gave a figure of merit (FOM) of 0.32. After density modification using DM46, the BamACDE complex was clearly visible in the electron density map, but without BamB. Using the individual high-resolution models, the BamACDE complex was built using Coot47 by skeletonizing the electron density map and docking the BAM subunits in the electron density map with selenomethionine sites used as guides. Rigid body refinement was performed following manual docking. NCS refinement was used along with TLS refinement against groups automatically determined using PHENIX48. Restrained refinement was performed with group B-factors alongside reference model secondary structure restraints from higher resolution models. Weights were automatically optimised by PHENIX48. To obtain the BamABCDE complex structure, the new construct was used to produce sufficient BamB to form the BamABCDE complex. The data sets of BamABCDE complex were collected on the I02 beamline at DLS. The crystals belong to space group P4 2 2, with the cell dimensions a = b = 116.69 Å, c = 435.19 Å, α = β = γ = 90°. There is one complex molecule in the asymmetric unit. Although the crystals diffracted to 2.90 Å, the crystal structure of BamABCDE could not be determined by molecular replacement. BamABCDE complex was crystallized in presence of 0.2 M sodium iodide, and SAD data sets were collected at a wavelength of 1.8233 Å. Four 360° data sets were collected on different regions of the same crystal of NaI co-crystallization then combined. The phases were determined by ShelxD44, 45 at 4 Å resolution. Eleven iodide sites were found, which gave a FOM of 0.28. The phases were extended to 2.90 Å by DM46, and the model was built using Coot47 by skeletonizing the electron density map and docking the individual high-resolution subunits in the electron density map and rigid body fit this model into the higher resolution native data set while retaining and extending the free R set from the iodide data set. The BamABCDE complex was refined using PHENIX48. TLS groups were automatically determined using PHENIX48 and used for refinement along with individual B-factors. Weights were automatically optimised and secondary structure restraints were used. An E. coli bamA expression plasmid was constructed for functional assays using SLIC method as described above. An N-terminal 10 × His tag fused with bamA starting from residue 22 was amplified by PCR using Q5 Hot Start High-Fidelity DNA Polymerase (New England BioLabs), and plasmid pJH114 as template and primers PF_bamA_SLIC (5′-CCATCATCATCATCATCATCATCATGAAGGGTTCGTAGTGAAAGATATTCATTTCGAAG-3′) and PR_bamA_SLIC (5′-AGACTCGAGTTACCAGGTTTTACCGATGTTAAACTGGAAC-3′). Vector backbone was amplified from a modified pRSFDuet-1 vector (Novagen, Merck Millipore) containing an N-terminal pelB signal peptide coding sequence with primers PF_RSFM_SLIC (5′-CGGTAAAACCTGGTAACTCGAGTCTGGTAAAGAAACCGCTGC-3′) and PR_RSFM_SLIC (5′-ATGATGATGATGATGATGATGATGGTGATGGGCCATCGCCGGCTG-3′). Plasmids were prepared using GeneJET Plasmid Miniprep Kit (Thermo Scientific). Site-directed mutagenesis was performed according to a previously described protocol49 with slight modification (PCR conditions and the sequences of the primers are available on request). The sequences of the wild type and all mutant constructs of BamA were confirmed by sequencing. E. coli JCM166 cells3 transformed with the wild-type BamA or its mutants were plated on LB agar plates supplemented with 50 μg ml−1 kanamycin and 100 μg ml−1 carbenicillin in the presence or absence of 0.05% L-(+)-arabinose and grown overnight at 37 °C. Single colonies grown on arabinose-containing plates were inoculated in 10 ml LB medium supplemented with 50 μg ml−1 kanamycin, 100 μg ml−1 carbenicillin and 0.025% L-(+)-arabinose, and incubated at 200 r.p.m. at 37 °C for 16 h. For plate assays, the cells were pelleted and resuspended in fresh LB medium supplemented with 50 μg ml−1 kanamycin and 100 μg ml−1 carbenicillin, and diluted to an A of ~0.3 and streaked onto LB agar plates supplemented with 50 μg ml−1 kanamycin, 100 μg ml−1 carbenicillin in the presence or absence of 0.05% L-(+)-arabinose and cultured at 37 °C for 12–14 h. Western blotting was performed to examine protein expression levels of BamA in the membrane. 50 ml of overnight cultures of transformed JCM166 cells with respective wild-type or each mutant of BamA were pelleted. The cells were resuspended in 25 ml 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and sonicated. The cell debris and unbroken cells were removed by centrifugation at 7,000g for 30 min. The supernatant was centrifuged at 100,000g for 60 min and the membrane fraction was collected. The membrane fraction was suspended in 5 ml buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 1% 3-(N,N-dimethylmyristylammonio)-propanesulfonate (Sigma) and solubilized for 30 min at room temperature. Samples were mixed with 5 × SDS–PAGE loading buffer, heated for 5 min at 90 °C, cooled for 2 min on ice and centrifuged. Ten microlitres of each sample was loaded onto 4–20% Mini-PROTEAN TGX Gel (Bio-Rad) for SDS–PAGE and then subjected to immunoblot analysis. The proteins were transferred to PVDF membrane using Trans-Blot Turbo Transfer Starter System (Bio-Rad) according to the manufacturer’s instructions. The PVDF membranes were blocked in 10 ml protein-free T20 (TBS) blocking buffer (Fisher) overnight at 4 °C. The membranes were incubated with 10 mL His-Tag monoclonal antibody (diluted, 1:1,000) (Millipore) for 1 h at room temperature followed by washed with PBST four times and incubated with IRDye 800CW goat anti-mouse IgG (diluted, 1:5,000) (LI-COR) for 1 h. The membrane was washed with PBST four times and PBS twice. Images were acquired using LI-COR Odyssey (LI-COR). The JCM166 cells containing the double cysteine mutants Gly393Cys/Gly584Cys, Glu435Cys/Ser665Cys and Glu435Cys/Ser658Cys of BamA were cultured overnight in LB medium with 50 μg ml−1 kanamycin, 100 μg ml−1 carbenicillin and 0.025% L-(+)-arabinose, respectively. The membrane fraction from 50 ml cells was isolated and solubilized as described above. The samples were mixed with SDS loading buffer and then boiled for 5 min or kept at room temperature for 5–10 min. SDS–PAGE was performed at 4 °C by running the gel for 60 min at 150 V. The proteins were transferred to PVDF membrane as described above and the BamA mutants were detected by western blotting. All molecular dynamics simulations were performed using GROMACS v5.0.2 (ref. 50). The Martini 2.2 force field51 was used to run an initial 1 μs Coarse Grained (CG) molecular dynamics simulation to permit the assembly and equilibration of a 1-palmitoly, 2-cis-vaccenyl, phosphatidylglycerol (PVPG): 1-palmitoly, 2-cis-vaccenyl, phosphatidylethanolamine (PVPE) bilayers around the BamABCDE complexes52. Using the self-assembled system as a guide the coordinates of the BAM complexes were inserted into an asymmetric model E. coli OM, comprised of PVPE, PVPG, cardiolipin in the periplasmic leaflet and the inner core of Rd1 LPS lipids in the outer leaflet53, using Alchembed54. This equated to a total system size of ~500,000 atoms. The systems were then equilibrated for 1 ns with the protein restrained before 100 ns of unrestrained atomistic molecular dynamics using the Gromos53a6 force field55. The lipid-modified cysteine parameters were created from lipid parameters for diacylglycerol and palmitoyl and appended to the parameters of the N-terminal cysteines56. Systems were neutralised with Mg2+ ions, to preserve the integrity of the outer leaflet of the OM, and a 150 mM concentration of NaCl. All ~500,000 atom systems were all run for 100 ns, with box dimensions in the region of 200 × 200 × 150 Å3. To assess the stability of the subunit stoichiometry we assessed various combinations of BAM assemblies. For both BamACDE and BamABCDE crystal structures, we investigated ABCDE, AD and A alone, with three repeats each; while single simulations were also performed for BamABD, ACD, ADE, ABDE and ACDE, with a total simulation time equating to 2.8 μs. In cases where domains or subunits were missing these were added to the complex by structurally aligning the resolved units from the companion structure. For BamB, this was added to the BamACDE complex by structurally aligning POTRA 3. For the full BamC, this was added to the BamABCDE by aligning the resolved N-terminal domains. Individual protein complexes were configured and built using Modeller57 and PyMOL (The PyMOL Molecular Graphics System, version 1.8, Schrödinger, LLC). All simulations were performed at 37 °C, with protein, lipids and solvent separately coupled to an external bath, using the velocity-rescale thermostat58. Pressure was maintained at 1 bar, with a semi-isotropic compressibility of 4 × 10−5 using the Parinello–Rahman barostat59. All bonds were constrained with the LINCS algorithm60, 61. Electrostatics was measured using the Particle Mesh Ewald (PME) method62, while a cut-off was used for Lennard–Jones parameters, with a Verlet cut-off scheme to permit GPU calculation of non-bonded contacts. Simulations were performed with an integration time-step of 2 fs. The linear interpolation between the three structures was performed using the morph operation in Gromacs tools50. Analysis of the molecular simulations was performed using Gromacs tools50, MDAnalysis63 and locally written scripts. Conservation analysis was performed using Consurf64. For each subunit, 150 homologues were collected from UNIREF9065 using three iterations of CSI-Blast66, with an E-value of 0.0001. The Consurf scores were then mapped into the B-factor column for each of the subunits.


News Article | March 30, 2015
Site: www.finsmes.com

SocialCar, a Barcelona, Spain-based private car renting platform, raised €800k in its first funding round. The round was led by Cabiedes & Partners with participation from angel investors including Pau Serracanta. The company intends to use the funds to continue to consolidate its market position in Spain and expand abroad. Founded in 2011 by Mar Alarcón, SocialCar provides a web and mobile platform for private people to rent cars from peers. The platform is currently used by 40,000 users in Barcelona, Madrid, Valencia, Mallorca, Menorca, Vigo, Sevilla, Bilbao and Ibiza.


News Article | November 5, 2015
Site: http://www.techtimes.com/rss/sections/auto.xml

After admitting in September that 11 million diesel-powered vehicles worldwide are fitted with defeat devices meant to lower nitrogen oxides tailpipe emissions, Volkswagen came up with a new disclosure that can add more woes to its yet unresolved scandal. Volkswagen said that the error now goes beyond what it has previously disclosed and estimated that around 800,000 more cars could also be affected. If this is true, it would mean an added cost of $2 billion for the German auto maker to handle. The company said that it "will immediately start a dialogue with the responsible type approval agencies regarding the consequences of these findings. This should lead to a reliable assessment of the legal, and the subsequent economic consequences of this not yet fully explained issue." The newly disclosed information came a day following new allegations made by the U.S. Environmental Protection Agency (EPA). According to the department, defeat devices are found on 3.0-liter diesel engines that are used in bigger SUVs and high-end vehicles from Volkswagen that reach around 10,000 in number. These include Porsche Cayenne, Audi Q5, Audi Q7, Audi A6, Audi A8 and Touareg. The vehicles allegedly emit up to nine times the legally allowable levels of nitrogen-oxide which Volkswagen consequently denied to be true. "The allegations are all the more serious given that VW's new CEO Matthias Müller came from Porsche and any hint of further deception could well see his position come under scrutiny," said Michael Hewson, a chief market analyst at brokerage company CMC Markets. "VW is leaving us speechless," said Arndt Ellinghorst, an automotive analyst at research firm Evercore ISI. It was the first time that an allegation was made against Porsche, the sports-car model by Volkswagen which is also one of its big money makers. Since it was previously run by Müller, the latest news had somehow created questions on the extent of his knowledge about Porsche's engines. Bipartisan leaders of the House Energy and Commerce Committee had reportedly sought additional documents in the wake of the new EPA findings from Michael Horn, President and CEO of Volkswagen Group of America and had set the date Nov. 16 as the deadline. "In light of yesterday's news, we ask that Volkswagen provide some basic facts and clarifications regarding the software installed in certain make and model year vehicles and how such devices affect the operation of the vehicles," wrote the committee. Likewise, it also sought a "detailed description of any software that served effectively to defeat emissions controls functions, including but not limited to what components of the engine it affects and how, for each of the make and model year vehicles."

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