Saccharomyces cerevisiae is currently one of the most promising alternatives to conventional transport fuels. Lignocellulosic hemicelluloses obtained after hydrothermal pretreatment are important feedstock for bioethanol production. However, hemicellulosic materials cannot be directly fermented by yeast: xylan backbone of hemicelluloses must first be hydrolyzed by heterologous hemicellulases to release xylose, and the yeast must then ferment xylose in the presence of fermentation inhibitors generated during the pretreatment.Bioethanol produced by the yeast S. cerevisiae strains engineered with the xylose metabolic pathway. Antibiotic markers were efficiently recycled by a novel counter selection strategy using galactose-induced expression of both FLP recombinase gene and GIN11 flanked by FLP recombinase recognition target (FRT) sequences. Nine genes were functionally expressed in an industrial diploid strain of S. cerevisiae: endoxylanase gene from Trichoderma reesei, xylosidase gene from Aspergillus oryzae, β-glucosidase gene from Aspergillus aculeatus, xylose reductase and xylitol dehydrogenase genes from Scheffersomyces stipitis, and XKS1, TAL1, FDH1 and ADH1 variant from S. cerevisiae. The genes were introduced using the homozygous integration system and afforded hemicellulolytic, xylose-assimilating and inhibitor-tolerant abilities to the strain. The engineered yeast strain demonstrated 2.7-fold higher ethanol titer from hemicellulosic material than a xylose-assimilating yeast strain. Furthermore, hemicellulolytic enzymes displayed on the yeast cell surface hydrolyzed hemicelluloses that were not hydrolyzed by a commercial enzyme, leading to increased sugar utilization for improved ethanol production.A GIN11/FRT-based multiple-gene integration system was developed for introducing multiple functions into the recombinant S. cerevisiae strains.The multifunctional yeast strain, developed using a GIN11/FRT-based marker recycling system, achieved direct conversion of hemicellulosic biomass to ethanol without the addition of exogenous hemicellulolytic enzymes. No detoxification processes were required. The multiple-gene integration technique is a powerful approach for introducing and improving the biomass fermentation ability of industrial diploid The online version of this article (doi:10.1186/s12934-014-0145-9) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae and is currently one of the most promising alternatives to conventional transport fuels. S. cerevisiae is a superior ethanol producer with demonstrated fast sugar consumption, high ethanol yield from glucose, and high resistance to ethanol. Lignocellulosic biomass such as corn stover, rice and wheat straw, sugarcane bagasse, wood chips and other agricultural residues comprise mainly cellulose, hemicelluloses and lignin. Although the composition varies with these feedstock, hemicelluloses are the second most abundant constituent of lignocellulosic biomass. If the economic success of lignocellulosic ethanol is to be realized, both cellulose and hemicelluloses must be utilized for ethanol production.Environmental concerns and shrinking oil reserves have resulted in governmental incentives to develop environmentally benign and sustainable fuels. The utilization of lignocellulosic biomass for the production of fuels has received particular attention in recent years. Bioethanol is produced by the fermentation of biomass by the yeast S. cerevisiae cannot. Thus, many researchers have attempted to produce xylanolytic enzymes in S. cerevisiae strains  supplemented with antibiotics, and in YPD medium . PvuII-PciI site of pBluescriptSK + ® . All oligonucleotides used for plasmid construction and PCR are shown in Additional file GIN11 and GAL1 promoter were obtained by PCR with primer sets P3/P4 and P5/P6, respectively, using genomic DNA from S. cerevisiae X2180-1A [GIN11 and GAL1 promoter were inserted into the SpeI-EcoRV site of MCS linker 1. The geneticin resistance gene (G418r) and nourseothricin resistance gene (natMX) were obtained from pYC030 and pYC050 [AscI and inserted into the AscI site adjacent to GIN11, yielding pBGIN11-03 and pBGIN11-05, respectively. GIN11 is on chromosome XIII 923146 to 923827, and identified as a gene that leads S. cerevisiae cells to die when overexpressed [GAL1 promoter, GIN11, and a marker gene were located between the FRT sequences in pBGIN11-03 and pBGIN11-05. The expression units X1X2XKN2, XYNII, BGL1XYLA, and m6ADH1FDH1TAL1 were inserted into the MluI and NotI site of pBGIN11-03 and pBGIN11-05, yielding expression unit vector-03 and vector-05, respectively. Each expression unit was derived as follows. PYK1 terminator was obtained by PCR using primer set P7/P8, then was replaced with TDH3 terminator adjacent to the XYL2 of pIUX1X2XKN [BssHII digestion. The expression unit XYNII was obtained from pdU-GPAGXynII [NotI digestion. The expression unit BGL1XYLA was obtained from pIHPGBGGPXylA [BssHII digestion. Three expression cassettes, m6ADH1, FDH1, and TAL1, were obtained by NotI and PspOMI digestion from pEWm6ADH1 [FDH1 [NotI of pBGIN11-03 and pBGIN11-05 in tandem.Multiple cloning site (MCS) linker 1, made with oligonucleotides P1 and P2, was inserted into the X2180-1A  as a temd pYC050  by digesxpressed . GAL1 prUX1X2XKN , yieldinPvuII site of pBluescriptSK + ®. Four inter-ORF regions on genomic DNA, I2, I6, I7S, and I10, were obtained by PCR using X2180-1A as template DNA and the primer sets P11/P12, P13/P14, P15/P16, and P17/P18, respectively, and inserted into the NotI site of MCS linker 2. The I2, I6, I7S, and I10 regions are located between YFL021W and YFL020C, YGR249W and YGR250C, YKL219W and YKL218C, and YPL257W and YPL256C, respectively. The inter-ORF regions are situated downstream of both adjacent ORFs. Linker 1 consisting of P19 and P20 was introduced into the Xba I site of I2 region, the blunt ended BamHI-EcoRV site of I6 region, and the BsaAI site of I7S region. Linker 2 consisting of P21 and P22 was inserted into the Xba I site of the I10 region. The four resulting pIX + Linker plasmids were digested by ApaI and ligated with expression unit vector-03 (or −05) digested by ApaI or PciI to generate the integration plasmids. The vectors prepared for integration into the genome of S. cerevisiae were referred to as pI2X1X2XKN2-03, pI2X1X2XKN2-05, pI6XYNII-03, pI6XYNII-05, pI7SBGL1XYLA-03, pI7SBGL1XYLA-05, pI10m6ADH1FDH1TAL1-03, and pI10m6ADH1FDH1TAL1-05.MCS linker 2, made with oligonucleotides P9 and P10, was inserted into the ampr and ori was obtained by PCR using the primer set P23/P24, which was subsequently self-ligated. Hygromycin resistant gene (hphMX) was obtained by AscI digestion of pYC040 [EcoRI-HindIII digestion of YRp7 [hphMX and ARS1 were inserted into the AscI site and EcoRI-HindIII site, respectively, of the self-ligated fragment. The resultant plasmid was digested with SpeI and HindIII. GAL1 promoter (SpeI-BamHI), FLP gene  and TDH3 terminator  were introduced in tandem, yielding pYR-HFLPG. GAL1 promoter and FLP gene were obtained by PCR using X2180-1A as template DNA and primer sets P25/P26 and P27/P28, respectively. TDH3 terminator was obtained from pUP3GLP [SalI-HindIII digestion.To make pYR-HFLPG, a partial fragment of pBR322  containif pYC040  and ARS1 of YRp7 ; hphMX a pUP3GLP  by SalI-S. cerevisiae strain Sun049 was obtained from Suntory Limited  [FseI. Both ends of the resultant linear fragment contained an inter-ORF region I2; this fragment was introduced into sun049 as the first transformation. The first transformants were selected on YPD containing 300 μg/mL geneticin. The integration of the expression unit into the I2 region of the S. cerevisiae genome was confirmed by PCR using the primers P29, P30, P32 and P33. P29 and P32 are the sequences located upstream and downstream of I2 region, respectively, and P30 and P33 are the sequences within the transformed linear fragment, G418r and X1X2XKN, respectively. The second transformation, with pI2X1X2XKN2-05, was performed using the first transformant as the host. Selection was conducted on YPD medium containing 300 μg/mL geneticin and 50 μg/mL nourseothricin. Homozygous integration into the I2 region was confirmed by PCR using a primer set, P29/P31. pYR-HFLPG was transformed into the second transformant to eliminate the antibiotic marker genes in the resulting third transformant. The third transformants were selected on YP medium  containing 20 g/L galactose and 200 μg/mL hygromycin. As the expression of GIN11 leads cells to die, only cells in which homologous recombination between the two FRT sequences occurred should be able to grow on medium containing galactose. The expression of FLP recombinase promotes homologous recombination. Introduction of pYR-HFLPG should confer marker recycling in strain sun049, which lacks a 2-μm plasmid carrying FLP recombinase. Marker elimination was verified by colonies being sensitive to geneticin and nourseothricin, and by PCR using the primer set P34/P35. pYR-HFLPG has a chromosomally-derived ARS and is an unstable plasmid; consequently, it can be easily cured from the host when the yeast is grown on YPD without hygromycin. This procedure provided Sun49-1. Similarly, Sun49-5 was constructed from Sun49-1 using plasmids pI6XYNII-03/-05 and pI7SBGL1XYLA-03/-05, Sun49-7 was constructed from Sun49-5 by transformation with pI10m6ADH1FDH1TAL1-03/-05, and Sun49-24 was constructed from Sun49-1 by transformation with pI10m6ADH1FDH1TAL1-03/-05. P36 to P50 were used in order to verify the integration of the expression unit or elimination of the marker gene during the construction of Sun49-5, Sun49-7 and Sun49-24.The industrial , Japan) . The vecg for 5 min at 4°C and washed with 10 mM potassium phosphate buffer (pH7.5) containing 2 mM EDTA. Then, the cells were resuspended in 100 mM potassium phosphate buffer (pH7.5) containing 2 mM MgCl2 and 2 mM dithiothreitol. The suspended cells were mixed with glass beads (0.5 mm diameter), disrupted by shaking at 2500 rpm at 4°C for 5 min with a Multi-beads shocker . The cell extract, collected after centrifugation at 15000 × g for 5 min at 4°C, was used for the enzyme assay. XR activity was measured spectrophotometrically by monitoring NADPH oxidation at 340 nm in a reaction mixture with the following composition: 100 mM sodium phosphate buffer (pH7.0) at 30°C, 200 mM xylose, and 0.24 mM NADPH. XDH activity was measured spectrophotometrically by monitoring NAD+ reduction at 340 nm in a reaction mixture with the following composition: 100 mM Tris–HCl (pH7.0) at 30°C, 1 mM MgCl2, 50 mM xylitol, and 2 mM NAD+. The activities of TAL and FDH were measured as previously described [+ per minute. The activities of endoxylanase, xylosidase and BGL on the yeast cell surface were determined as described previously [After cultivation in YPD medium for 48 h at 30°C, cells were harvested by centrifugation at 6000 × escribed . One unig for 5 min at 4°C, washed twice with distilled water, and then inoculated into YPX medium  with or without addition of an inhibitor mixture consisting of 30 mM acetate, 10 mM formate and 10 mM furfural. The initial cell concentration was adjusted to 50 g wet cells/L. Wet cell weight was determined by weighing a cell pellet that was harvested by centrifugation at 1,000 × g for 5 min. The estimated dry cell weight (DCW) was approximately 0.15-fold the wet cell weight. All fermentations were performed at 30°C under oxygen limited conditions at an agitation speed of 500 rpm in 100-mL closed bottles equipped with a bubbling CO2 outlet and a stir bar, as described previously [g for 5 min. The concentrations of acetate, ethanol, formate, glycerol, xylitol, and xylose in the fermentation medium were determined as described previously [Following cultivation under aerobic conditions for 48 h at 30°C in YPD medium, yeast cells were collected by centrifugation at 1000 × eviously . Cell greviously .A hemicellulosic fraction obtained by liquid hot water treatment of rice straw at 130-300°C under the pressure of less than 10 MPa was purchased from Mitsubishi Heavy Industries, Ltd. (Tokyo Japan). The hemicellulosic fraction was separated from solid cellulose-enriched fraction by filtration and adjusted to pH 5 using NaOH as described previously [


S100 proteins are a large family of calcium binding proteins present only in vertebrates. They function intra- and extracellularly both as regulators of homeostatic processes and as potent effectors during inflammation. Among these, S100A8 and S100A9 are two major constituents of neutrophils that can assemble into homodimers, heterodimers and higher oligomeric species, including fibrillary structures found in the ageing prostate. Each of these forms assumes specific functions and their formation is dependent on divalent cations, notably calcium and zinc. In particular, zinc appears as a major regulator of S100 protein function in a disease context. Despite this central role, no structural information on how zinc bind to S100A8/S100A9 and regulates their quaternary structure is yet available.2-cacodylate complex in our second crystal form induces ligand swapping within the canonical His4 zinc binding motif, thereby creating two new Zn-sites, one of which involves residues from symmetry-related molecules. Finally, we describe the calcium-induced S100A8 tetramer and reveal how zinc stabilizes this tetramer by tightening the dimer-dimer interface.Here we report two crystallographic structures of calcium and zinc-loaded human S100A8. S100A8 binds two zinc ions per homodimer, through two symmetrical, all-His tetracoordination sites, revealing a classical His-Zn binding mode for the protein. Furthermore, the presence of a (Zn)2+/Ca2+-bound hS100A8 demonstrate that S100A8 is a genuine His-Zn S100 protein. Furthermore, they show how zinc stabilizes S100A8 tetramerization and potentially mediates the formation of novel interdimer interactions. We propose that these zinc-mediated interactions may serve as a basis for the generation of larger oligomers in vivo.Our structures of ZnThe online version of this article (doi:10.1186/s12900-016-0058-4) contains supplementary material, which is available to authorized users. S100 proteins belong to the EF-hand calcium-binding protein superfamily and count more than 20 members that are expressed exclusively in vertebrates, in a tissue- and cell-specific manner –6, thus S100A8 and S100A9 constitute two of the most potent pro-inflammatory molecules among the S100 protein family , 12. Alt2+, Mn2+, Cu2+, and Fe2+ and, by efficiently sequestrating these divalent cations in the extracellular environment, it acts as a potent antimicrobial agent [2+-coordinating residues are conserved in S100A8 and S100A9, suggesting that both proteins could bind zinc in a similar fashion. However, no structural data are yet available to precisely characterize the zinc binding mode of either S100A8 and S100A9 homodimers or S100A8/A9 heterodimer. Zinc was furthermore shown to enhance the oligomerization of these proteins, either as homo- and heterotetramers or as much more complex amyloid structures [2+, since the two EF-hand Ca2+-binding sites would be fully saturated at all time in the extracellular compartment due to the high calcium concentrations [As for all S100 proteins, S100A8, S100A9, and their heterodimer bind two calcium ions per monomer, one in each EF-hand motif , 15, 17.al agent –28. Inteal agent  althoughal agent . Indeed,al agent  and the al agent –34. Thesructures , 36. Fintrations . Zinc ap2-cacodylate complex in one of the two Zn-sites per homodimer, leading to ligand swapping and creation of two novel Zn-sites. Analysis of the quaternary arrangement of hS100A8 in both crystal forms suggests that zinc enhances tetramer stabilization and could further participate in the formation of higher oligomeric species and/or in the interaction with S100A8 binding partners.To better understand how zinc modulates the structure-function relationship of S100A8 and S100A9, we have undertaken the structural studies of these S100 proteins in the presence of zinc. Here we report the crystallographic structures of human S100A8 (hS100A8) bound to zinc and calcium at 2.2 and 2.1 Å, respectively. The first structure reveals a classical, all-histidine zinc-binding motif, thereby classifying S100A8 as a genuine His-Zn S100 protein. The second structure displays an atypical (Zn)12121 and a C2221 symmetry, as compared to Ca2+-bound hS100A8 which crystallized in P3121 [2+-hS100A8 homodimer structure. For crystal form 2 (C2221), structure determination was also successful using SAD-phasing in PHENIX.AUTOSOLVE [2+ ions and four of the eight Ca2+ ions  from the (Zn)2-cacodylate complex which is further held in place by His91 from monomer B, by a second chloride ion and by the second hydroxyl group of the cacodylate molecule. Finally, the new position of His87 leads to the creation of a third Zn-site (Zn4) which is completed by Glu93 from the very C-terminal end of helix H4 (monomer B), Glu57 from a symmetry-related molecule, and a third chloride ion  while Gln69 carbonyl group (monomer B) connects with the side chain of Asn61 . Interestingly, in Ca2+-hS100A8, the second hS100A8 homodimer is pulled away from the interface on one side, due to the slightly different orientation of helix H4 2-cacodylate complex can insert into one of the canonical Zn-sites and disrupt the classical His4 tetracoordination sphere, inducing ligand swapping and the formation of two new Zn-sites. This is not unique to S100A8 as a pH-dependent ligand swapping for zinc coordination has also been described for S100B [2+-bound calprotectin [in vivo under specific conditions. Ligand swapping in these flexible C-terminal regions could therefore potentially contribute to the formation of novel interdimer interactions, through ions coordination, that would lead to the formation of higher oligomeric species. Similarly, reorganization of the flexible C-terminus of both S100A8 and S100A9 by ligand swapping could induce the formation of cation binding sites at the interface between these S100 proteins and specific binding targets, the metal coordination being provided by both interacting partners. Such mechanisms would provide a structural rationale for the influence of zinc on S100:target recognition, as well as on the formation of S100A8/A9 amyloid-like structures which is known to be regulated by both calcium and zinc [Here we report the crystal structure of human S100A8 in the presence of both calcium and zinc. The structure reveals an all-histidine Zn-binding site and demonstrates that hS100A8 belongs to the His-Zn group of S100 proteins, as expected from sequence comparisons. The structure further reveals that zinc allows tightening of the dimer-dimer interface within hS100A8 homotetramer, thus pointing towards an enhancing role of zinc in S100A8 tetramerization. Our data therefore suggest that, as for several other S100 proteins, divalent cations, and notably zinc, influence S100A8 oligomerization. The second Znor S100B . Furtherrotectin , 44. Takand zinc , 40.The gene coding for hS100A8, codon-optimized for bacterial expression, was purchased from Genscript (GenScript USA Inc.) and recloned into the Nco I – Hind III fragment of vector pETM13 (EMBL vector collection) to allow expression of the protein without any tags. To be in frame with the start codon in pETM13, two additional nucleotides (C + A) were introduced in between the Nco I site and the beginning of the hS100A8 sequence, yielding a protein with an additional alanine residue in between the initial methionine and the following leucine residue.Escherichia coli BL21(DE3) cells and purified in a three-step procedure including affinity chromatography on a 5 ml HisTrap FF Ni-column , anion exchange on a 9 ml Source 15Q column  and size exclusion chromatography (SEC) on a 120 ml home-packed Superdex 75 column. BL21(DE3) cells transformed with the hS100A8-pETM13 plasmid were grown at 310 K until their absorbance at 600 nm reached 0.6–0.8. Protein expression was then induced overnight at 291 K by addition of 1 mM IPTG. The cells were harvested by centrifugation, resuspended in a buffer containing 50 mM HEPES pH 7.5, 200 mM NaCl, 30 mM imidazole, 1 mM PMSF (Buffer A) and disrupted by sonication. After clarification by centrifugation, the supernatant was loaded onto the 5 ml Ni-column equilibrated in Buffer A and the non-specifically bound contaminants were washed off with high salt (Buffer A containing 1 M NaCl). As hS100A8 contains several histidine residues on its C-terminus, it binds relatively strongly to the Ni-column, even in the absence of a His-tag, and could be eluted with a buffer consisting of 50 mM HEPES pH 7.5, 200 mM NaCl, 250 mM imidazole, 1 mM PMSF. The protein was then dialyzed overnight at 277 K against 50 mM Tris-HCl pH 8.8, 50 mM NaCl and loaded onto the anion-exchange column. Elution was performed with a 100 ml gradient from 50 to 300 mM NaCl. hS100A8 appeared in the run-through fraction and in the first fractions of the gradient while most of the contaminants bound more strongly to the Source 15Q column. The hS100A8-containing fractions were pooled and concentrated to 5 ml before loading onto the SEC column equilibrated in 20 mM HEPES pH 7.5, 100 mM NaCl, 5 mM CaCl2. The protein eluted as two separate peaks . Calibration of the SEC column was performed in the same buffer as for the S100A8 run using the Gel Filtration Markers Kit for Protein Molecular Weights 6500–66000 Da (Sigma-Aldrich).To determine the molecular weight of the two oligomeric forms of hS100A8 obtained during SEC  and the tetrameric (3.2 mg ml−1) forms of the protein after adding 3 mM ZnCl2 to the protein solution. Crystallization was carried out using the sitting-drop vapor-diffusion technique in 96-well Swissci MRC crystallization plates with the help of a MOSQUITO robot (TTP LabTech) and commercial screens from Hampton Research (INDEX screen) and Molecular Dimensions Ltd . Several hits were obtained for the dimeric form whereas only a few small crystals appeared for the tetramer, probably due to the much lower protein concentration imposed by precipitation at higher concentrations. Two conditions were further optimized by varying the precipitant concentration, pH, ionic strength, and protein concentration, and by screening with an additive collection (Hampton Research). The final optimal conditions were 0.2 M ammonium acetate, 0.1 M Na acetate pH 4.0, 15 % PEG 3350, 0.4 % 2,2,2-trifluoroethanol for crystal form 1 and 5 mM ZnCl2, 0.1 M Na cacodylate pH 6.5, 8 % PEG 8000 for crystal form 2.Crystallization of hS100A8 in the presence of zinc was performed with both the dimeric  or with 25 % glycerol added . Diffraction data were collected at 100 K on the I911-3 beamline at MAX-lab  (Table 12121 symmetry with eight molecules of hS100A8 per asymmetric unit and diffracted to a maximal resolution of 2.2 Å. Crystal form 2 displayed a C2221 symmetry with four molecules of hS100A8 per asymmetric unit and the data could be processed to a maximum resolution of 2.1 Å. Both structures were solved by molecular replacement with PHASER [Crystal form 1 displayed a P2h PHASER  from theh PHASER  using thh PHASER  as a seah PHASER  and cyclh PHASER  using inh PHASER . All figEthics was not requested for this study since it does not involved human participants or animals, and the human protein we crystallized was expressed recombinantly in a bacterial system from a synthetic DNA construct.Not applicable.2+/Ca2+-hS100A8 from crystal forms 1 and 2 have been deposited in the Protein Data Bank with accession numbers 5HLV and 5HLO, respectively. Additional files can be found on the online version of this paper and contain Additional file The coordinates and structure factors for the structures of Zn


LC3  to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates),  p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes.We characterized the dynamics of autophagy  Specifications TableValue of the data•This data provides characterization of basal macroautophagy flux and response to classical inducers (EBSS) and inhibitors (Baf. A1) of this mechanism in MEFs, HFs, SH-SY5Y and N27 cell lines.•The data would be valuable for further studies of autophagy dynamics in these four cell lines.•This data could give a basis for further experiments on revealing the underlying mechanism of autophagy in these cell lines.•The data support the development of transcription analysis of autophagy markers.p62 mRNA expression was performed in all cell lines  supplemented with 10% fetal bovine serum (FBS) , 1% L-glutamine  and penicillin-streptomycin . For N27 cell line, the culture medium was made of RPMI 1640 medium (1X)  supplemented with 10% FBS, L-glutamine and penicillin-streptomycin.5 (MEFs), 1×106 (HFs), 2×106 (SH-SY5Y) and 4×105 (N27) in 75-cm2 tissue culture flasks and incubated at 37 °C under saturating humidity in 5% CO2/95% air. Confluent cells (~80%) were trypsinized and plated into a 6 or 24-well plates at densities of 3×104 cells/mL (MEFs and HFs), 1×105 cells/mL (SH-SY5Y) and 3.5×104 cells/mL (N27).Cells were seeded at densities of 3×10After 24 h , the culture medium was replaced with different treatments . To induce autophagy by starvation conditions, the culture medium was replaced with EBSS . To block fusion between autophagosomes and lysosomes, cells were incubated with 100 nM Baf. A1 . For combined treatment, cells were preincubated with 100 nM Baf. A1 for 1 h and then they were washed with phosphate-buffered saline (PBS) 1X and treated with 100 nM Baf. A1 and EBSS. All treatments lasted 4 h.Following treatments, cells were processed as described previously Equal amounts of protein (25–40 µg/condition) were resolved by 12% SDS-gel electrophoresis and transferred to polyvinylidene fluoride membranes, according to a partially modified conventional protocol For the detection of endogenous p62 and LC3B, cells were seeded on coverslips, fixed with paraformaldehyde (4% in PBS 1X) and permeabilized with Triton X-100 solution (0.1% in PBS 1X) for 10 min. To block non-specific binding, cells were incubated with 10% FBS in PBS 1X for 20 min, followed by incubation with primary antibodies anti-p62 (1:500) and anti-LC3B (1:500) for 1 h at room temperature. After that, cells were incubated with Alexa Fluor 488 anti-rabbit (1:1000)  and 568 anti-mouse (1:1000)  secondary antibodies for LC3 and p62, respectively. Finally, coverslips were mounted on microscope slides, by using fluoromount-G  medium. Images were taken by using an inverted fluorescence microscope  equipped with a camera . The quantitative measurement of the fluorescence signal was performed using ImageJ software analyzing at least 200 cells per condition. Immunofluorescence procedure was developed as previously described p62 mRNA expression was measured by qPCR with KAPA SYBR Fast reagents (KK4601), by using the primers described above. GAPDH gene expression was used as an endogenous control, and the expression level was calculated by using the (2-∆∆Ct) ratio Total RNA was extracted by RNeasy Mini Kit ; 500 ng of total RNA were reverse-transcribed into complementary DNA by using a QuantiTect Reverse Transcription Kit , both according to the manufacturer׳s protocol. p<0.05) were considered statistically significant . Non-significant results are not indicated in the figures. The data are expressed as the mean±the standard error of the mean (SEM).Each experiment was repeated at least three times. The data were analyzed by two-tailed unpaired Student׳s t-test and ANOVA test where applicable, and all comparisons with a p value less than 0.05 (


In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the  The solution was replaced after every 10 minutes. Subsequently, the gel was incubated in 50 mL of renaturation buffer for another 3 hours at 4°C without agitation. The solution was replaced, and the incubation was continued for 16 hours with the same conditions.In-gel kinase assays were performed as described by Wooten et al. . Two mil2, 2 mM β-mercaptoethanol, 0.1 mM EGTA) for 1 hour with gentle agitation. For the kinase reaction, the gel was incubated with 50 μM of 5 μCi/mL [γ_32P]ATP in 3 mL of kinase buffer for 4 hours with gentle agitation. For inhibition assays, 15 μg/mL of PKI (Promega) were included in the kinase buffer. The reaction was terminated by washing the gel five times with 50 mL of stop buffer  for 75 minutes. The gel was dried on cellophane membrane for 16 hours at room temperature, and finally the radioactive signal was detected using X-ray film (Kodak).Prior to the kinase reaction, the gel was washed twice with 50 mL of kinase buffer  or 20 μg of cell lysate in 50 mM KH2PO4, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 20 mM MgCl2, 10 mM β-mercaptoethanol, 3 mM of [γ_32P]ATP (1 μCi) or unlabeled ATP, 1× complete protease (Roche) and 1× phosphatase inhibitor cocktail  in a total volume of 40 μL at 30°C for 1 hour. The reaction was terminated by heating at 95°C for 5 minutes in 2× SDS loading buffer. In vitro phosphorylation of other human and yeast CKs was performed as described above using 80 U of PKA. Samples containing 5 or 10 μg of phosphorylated protein per well were analyzed by 10% SDS-PAGE. The phosphoprotein was detected by either autoradiography or Pro-Q Diamond phosphoprotein gel stain (Invitrogen). The phosphoprotein gel staining was performed according to the manufacturer’s protocol. The level of protein loading was determined by Coomassie blue stain or SYPRO® Ruby stain (Invitrogen). For inhibition assays, 15 to 45 μg/mL of PKI (Promega) were added to the reaction mixture. To investigate the effect of PKA and ATP concentrations on the level of CKβ phosphorylation, 20 to 120 U of PKA and 25 to 100 μM of ATP were used in the in vitro CKβ phosphorylation reactions.2 chromatography procedure for phosphopeptide enrichment . GFP-CKβ protein was immunoprecipitated with CKβ antibody : Here, 0The phosphoprotein was detected by phospho-(Ser/Thr) PKA substrate (PhosphoPKAS) antibody  diluted at 1:1000 ratio with blocking buffer . The primary antibody was incubated with the membrane at 4°C for 16 hours followed by detection with rabbit IgG-horseradish peroxidase (HRP)-conjugated secondary antibody . The signal was developed by Supersignal West Pico chemiluminescent substrate (Thermo Scientific) and detected by using the FUSION-FX chemiluminescence documentation system (Vilber Lourmat). After imaging, the antibody bound on the membrane was removed with stripping buffer , and the membrane was reprobed with rabbit anti-GFP antibody (Abcam) at 1:1000 dilution.For the detection of CKβ-phosphorylating kinase in the in-gel kinase assay, a replicated gel was blotted onto nitrocellulose membrane followed by Western detection with 1:5,000 dilution of PKA catalytic subunit-recognizing polyclonal antibody (Abcam) according to similar procedures as described above.2, 0.5 mM phosphoenolpyruvate, 0.25 mM NADH, 2 mM ATP, 4 U of pyruvate kinase, 5 U of lactate dehydrogenase, 1 to 2 μg of purified CK protein and 4 mM choline chloride or 4 mM ethanolamine in a final volume of 1 mL. The reaction was performed at 37°C and initiated by adding choline or ethanolamine. Any background activity before the addition of choline or ethanolamine was subtracted from the total activity. The purified CK protein used in such assays was either unphosphorylated, in vitro phosphorylated (as described above), or the S39D/S40D phosphorylation mimic mutant. The in vitro phosphorylated CKβ was used immediately for enzymatic assay after the 1 hour PKA treatment. The unphosphorylated CKβ wild type and the S39D/S40D CKβ variant were either freshly purified proteins or purified proteins stored at −80°C. All the kinetic parameters were obtained from nonlinear regression curve fitting of the Michaelis-Menten plot by using GraphPad Prism version 5.0.Choline kinase activity was determined by using the pyruvate kinase (PK)-lactate dehydrogenase (LDH) coupled-enzyme assay as previously described  using a 50) values of the unphosphorylated, PKA- phosphorylated, and the S39D/S40D phosphorylation-mimic mutant CKβ. IC50 was determined by the standard CK assay as described above in the presence of saturating substrate concentration (4 mM choline) and increasing HC-3 concentrations, and fitting the data plotted in a dose-response curve by using GraphPad Prism 5.0.The effect of PKA-induced phosphorylation on HC-3 inhibition of CKβ was investigated by comparing the half-maximal inhibitory concentration Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.


Corydalis yanhusuo W.T. Wang; YHS), is a well-known traditional Chinese herbal medicine, has been used in China for treating pain including chest pain, epigastric pain, and dysmenorrhea. Its alkaloid ingredients including tetrahydropalmatine are reported to inhibit cytochromes P450 (CYPs) activity in vitro. The present study is aimed to assess the potential of total alkaloid extract (TAE) from YHS to effect the activity and mRNA levels of five cytochromes P450 (CYPs) in rat.Yanhusuo  for 14 days, alanine aminotransferase (ALT) levels in serum were assayed, and hematoxylin and eosin-stained sections of the liver were prepared for light microscopy. The effects of TAE on five CYPs activity and mRNA levels were quantitated by cocktail probe drugs using a rapid chromatography/tandem mass spectrometry (LC-MS/MS) method and reverse transcription-polymerase chain reaction (RT-PCR), respectively.In general, serum ALT levels showed no significant changes, and the histopathology appeared largely normal compared with that in the control rats. At 30 and 150 mg/kg TAE dosages, an increase in liver CYP2E1 and CYP3A1 enzyme activity were observed. Moreover, the mRNA levels of CYP2E1 and CYP3A1 in the rat liver, lung, and intestine were significantly up-regulated with TAE from 6 and 30 mg/kg, respectively. Furthermore, treatment with TAE (150 mg/kg) enhanced the activities and the mRNA levels of CYP1A2 and CYP2C11 in rats. However, the activity or mRNA level of CYP2D1 remained unchanged.These results suggest that TAE-induced CYPs activity in the rat liver results from the elevated mRNA levels of CYPs. Co-administration of prescriptions containing YHS should consider a potential herb (drug)–drug interaction mediated by the induction of CYP2E1 and CYP3A1 enzymes. Corydalis yanhusuo W.T. Wang, belonging to the Papaveraceae family, is a well-known traditional Chinese herbal medicine. The dried and pulverized tubers of C. yanhusuo are called Yanhusuo (YHS). It is among the 50 fundamental herbs in Chinese herbology and has been used traditionally for its sedative, neuroleptic, and analgesic properties[levo-Tetrahydropalmatine (l-THP) is the main active ingredient of YHS[l-THP is approved for use and available as Rotundine or Rotundin in China[l-THP has been reported to show toxic effects such as depression of neurological, respiratory, and cardiac function in pediatric poisonings, as well as acute or chronic hepatitis after regular use in adults[operties. Alkaloioperties, and stuoperties, antinocoperties, antihypoperties. levo-Tet of YHS, and purin China. Recentln adults, 10.50 = 3.04 μM ± 0.26 μM) and recombinant human CYP3A4 (IC50 = 41.5 μM ± 3.8 μM) activities[l-THP significantly inhibited CYP2D6 activity[The effects of YHS on drug-metabolizing enzymes such as cytochromes P450 (CYPs), which are important in controlling xenobiotic metabolism, have occasionally been reported. CYPs is a group of hemoproteins that are important for oxidative metabolism (phase I) of clinically used drugs and other xenobiotics. Co-admitivities; THP enaactivity; protopiactivity.in vivo, especially after repeated administration, is limited. In the present study, a range of dosages of TAE from YHS  were orally administered to male Sprague–Dawley rats. The effects of TAE on the expression of five drug-processing genes including CYP1A2, CYP2C11, CYP2D1, CYP2E1, and CYP3A1 were investigated. Moreover, CYPs activities and hepatotoxicity were characterized.However, information about whether total alkaloid extract (TAE) from YHS influences CYPs yanhusuo were purchased from Tianjin Tongrentang pharmacy  in October by M. Li . The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM (No. 2012).The dried and pulverized tubers of Phenacetin (PHE), paracetamol (PAR), tolbutamide (TOL), 4-hydroxytolbutamide (OHTOL), dextromethorphan (DEXM), dextrorphan (DEXP), midazolam (MDZ), 1-hydroxymidazolam (OHMDZ), chlorzoxazone (CHL), 6-hydroxychlorzoxazone (OHCHL), β-nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma Chemical Co. ; carbamazepine , phenobarbital (PB) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products . Methanol and acetonitrile were high performance liquid chromatography-grate from Concord Corporation . Ultra-pure water was obtained from a Milli-Q Pluswater purification system . All other regents were of analytical grade.ad libitum. All procedures involving animals were conducted in conformity with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines, and were approved by the Academy of Military Medical Science Institutional Animal Care and Use Committee (Certificate No. SCXK620076004).Male Sprague-Dawley rats  were purchased from the Laboratory Animal Science and Technology of Tianjin Shanchuanhong Co., Ltd . All rats were kept under a standard 12-h dark/light cycle with water and food provided yanhusuo (2 Kg) were refluxed with 70% alcohol for three times. The pooled extract was concentrated under vacuum drying at 50°C. The dry powder was then dissolved in 200 mL water. The extracted solution was purified using 732 strong acid cation exchange resin, and the following elution program was used: six times water, and six times 70% ethanol:water containing 5% ammonia. A solution of 70% ethanol:water containing 5% ammonia was obtained, and the solvent was evaporated under reduced pressure. Preliminary analysis of the extracts was performed using an Agilent 1200 series high performance liquid chromatography (HPLC) instrument . Chromatographic separation was performed using an Agilent Zorbax HC-C18 (4.6 mm × 250 mm × 5 μm) column . The mobile phase were (A) 0.5% phosphate buffer (pH 5.0) and (B) methanol and the elution system was as follows: 0 - 25 min, from 85% A to 70% A; 25 - 75 min, from 70% A to 65% A; 75 -103 min, from 65% A to 25% A; 103 - 105 min, from 25% A to 20% A; 105 - 115 min, from 20% A to 85% A;115 - 125 min, 85% A. The flow rate was 1.0 mL/min, and the UV wavelength was 280 nm. The major alkaloids in YHS extract were quantified, and HPLC : three TAE-treated groups, the control group, and the positive control group, respectively. In 14 consecutive days, TAE-treated groups were given TAE orally at doses of 6, 30, 150 mg/kg once daily, the positive control group was intraperitoneal injection with phenobarbital (80 mg/kg) once daily, whereasFollowing the 14-day treatment, livers were removed from the rats, fixed in 10% neural buffered formalin for 48 h, processed, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin for histological evaluation. Incidence of hepatic lesions was determined by histologically examining the same liver section from each animal. Serum aMicrosomes were isolated from the rats livers (approximately 2 g) as described. A rapidIncubation mixtures (200 μL) containing 0.1 M potassium phosphate buffer (pH 7.4), 0.25 mg/mL rat microsomal protein, and 10/100/2.5/20/5 μM PHE/TOL/DEXM/CHL/MDZ were preincubated for 5 min at 37°C in a water bath with gentle shaking. The reaction was initiated by addition of 1 mM NADPH and terminated with 400 μL chilled methanol  after 20 min incubation. After shaking (2 min) and centrifugation , the organic phase was separated and dried. The residues were dissolved in 200 μL of methanol and water  and centrifuged at 10000 g for 5 min. A 10 μL aliquot of supernatant was injected for determination of the metabolites of five CYPs probe substrates (PAR/OHTOL/DEXP/OHCHL/ OHMDZ) by LC-MS/MS.,s recommendations. Quantification of mRNA was performed on the Applied Biosystem 7500 Real-Time PCR System  using SYBR Select Master Mix  according to the manufacture,s recommendations. PCR reactions contains an initial denaturating cycle at 95°C for 10 min , followed by 40 amplification cycles: 95°C for 15 s and 60°C for 1 min. The primer pairs for five CYPs and β-actin genes and predicted size of PCR products are shown in Table Total RNA were extracted from the samples of liver (approximately 50 mg), kidney (approximately 50 mg), lung (approximately 50 mg) and intestine (approximately 50 mg) were extracted using Trizol reagent  according to the protocol provided by the manufacturer. The concentration of RNA was determined by spectrophotometry at 260 nm. RNA samples (2 μg) were reverse transcribed using the High Capacity cDNA Reverse Transcription Kit  to obtain cDNA according to the manufacturemetabolite × Lincubation/Tincubation /(CHLMs × Lincubtiaon), in which Cmetabolites represents the concentration of metabolites of CYPs probe substrates, Lincubation represents the volume of incubation system, Tincubation represents the incubation time, and CHLMs represents the concentration of HLMs. Values were expressed in units of pmol · min-1 · mg-1. Independent-sample t-test was used to analyze the differences between the enzyme activities, gene expression values with control values by SPSS 13.0. Differences were considered significant at P < 0.05.Values are expressed as mean ± SD. The enzyme activity was calculated as follows: enzyme activity = CNo significant change was observed in serum ALT levels in the TAE-treated rats (data not shown). Figure P > 0.05; Table After TAE treatment, CYPs activities in the rat liver were quantified using a cocktail of probe drugs and LC-MS/MS. TAE significantly increased the enzyme activities of CYP2E1 and CYP3A1. In the treated rats, the three TAE dosages  significantly increased the enzyme activity of CYP2E1 by 20%, 68.2%, and 146.1%, respectively, and the three TAE dosages increased the levels of CYP3A1 by 27.84%, 40.3% and 51.9%, respectively, compared with those in the control rats. The highest TAE dosage  increased the enzyme activity of CYP1A2 by 44.9% and the enzyme activity of CYP2C11 by 35.4%. The three TAE-treated groups were not significantly different from the control groups in terms of CYP2D1 expression , and only up-regulation of the gene expression levels of CYP2C11 at the highest dosage was significant in the rat liver, which showed 2.3-fold increase in the CYP1A2/β-actin mRNA levels. We were unable to detect changes in the mRNA levels of CYP2C11 in the rat kidney, lung, and intestines because of the low gene expression levels of CYP2C11 in these organs. The mRNA levels of CYP2D1 did not change significantly among the three TAE dosages.Changes in the mRNA levels of CYP1A2, CYP2C11, and CYP2D1 were evaluated by real-time PCR. As shown in Figure qi, reinforce vital energy, and relieve painful conditions such as headache, chest pain, and abdominal pain[In traditional Chinese herbal medicine, YHS is generally used to dispel stasis and move nal pain. The maiTo date, some compounds in YHS have been found to exert the inhibitory or inductive effects on CYPs, 21, butCYP2E1 catalyzes the biotransformation of almost 2% of all clinically used drugs in humans. Rat CYPl-THP, and the plant alkaloid responsible for its toxicity[Cases of human poisoning have occurred because of unregulated use of proprietary biopharmaceuticals containing purified THP. Several case reports associate the development of acute or chronic hepatitis with the chronic ingestion of a Chinese herbal medication “Jin Bu Huan Anodyne Tablets”, 30, whitoxicity, 30. In The CYP3A subfamily is the most important hepatic metabolic enzyme in the metabolism of 40% to 60% of all drugs. CYP3A4 After the treatment of rats for 14 days with different dosages of TAE, significant increases were observed in the mRNA expression and enzyme activities of CYP1A2 and CYP2C11 at 150 mg/kg TAE, but the mRNA levels and enzyme activities of CYP2D1 did not change significantly among the three TAE dosages. In the human liver, CYP1A2, CYP2C and CYP2D6 are involved in the metabolism of 4%, 16%, and 30%, respectively, of drugs on the market. AhR actTAE from YHS significantly induced the mRNA expression and enzyme activity of CYP2E1 and CYP3A1 in the rat liver, lung, and intestine. Furthermore, enzyme activity correlated well with mRNA expression. The results of the present dose–response study in rats suggest that potential CYP2E1 and CYP3A drug-drug interactions are unlikely at clinical dosages of TAE, but need to be considered when high dosages of TAE or TAE-containing products are co-administered with substrates of CYP1A2 or CYP2C11. Complex herb (drug)-drug interactions may ensue from the co-administration of YHS with other drugs, which is mediated by CYP2E1 and CYP3A1 enzymes.


The keratin-associated proteins (KAPs) are fundamental components of hair and wool fibres, and are believed to in part be responsible for some of the properties of these fibres. KAPs can be divided into three groups: the high sulphur (HS) KAPs, the ultra-high sulphur (UHS) KAPs and the high glycine-tyrosine (HGT) KAPs. KAP8 is a HGT-KAP family and was believed to be coded for by a single gene in both humans and sheep. However, the recent identification of a KAP8-2 gene in goats led us to investigate whether a KAP8-2 gene exists in sheep.KRTAP8-2 identified a homologous region on sheep chromosome 1 . This region was clustered with a number of previously identified KAP genes including (in order from the centromere) KRTAP11-1, KRTAP7-1, KRTAP8-1, KRTAP6-2, KRTAP6-1, KRTAP13-3 and KRTAP24-1. PCR-SSCP analysis of the notional gene revealed two dissimilar PCR-SSCP banding patterns, representing two DNA sequences. A single nucleotide difference 21 bp upstream of the TATA box was identified. The two sequences did not have great homology with known ovine KRTAP sequences, but high sequence identity was found with KRTAP8-2 from goats and reindeer. These results suggest that sheep possess a KAP8-2 gene and that this gene is polymorphic.A BLAST search of the Ovine Genome Assembly v2.0 using the coding sequence of caprine The notional KAP8-2 protein is comprised of 63 amino acid residues and is rich in glycine and tyrosine, but has a low cysteine content. In contrast to other HGT-KAPs, ovine KAP8-2 contains more acidic amino acid residues, and this would likely result in a lower isoelectric point (pI) of 6.3.The online version of this article (doi:10.1186/2193-1801-3-528) contains supplementary material, which is available to authorized users. The keratin-associated proteins (KAPs) are part of the matrix of wool fibres and form a cross-linked network with the keratin intermediate filaments , KAP7 (type II) and KAP8 (type II)  Romney-cross sheep were investigated. The NZ Romney and its crosses are the most common dual-purpose sheep New Zealand and include the Perendale and Coopworth breeds. Samples of blood from these sheep were collected directly onto FTA cards  and DNA for analysis was purified from 1.2 mm punches from the cards, using a procedure described by Zhou et al. .http://www.livestockgenomics.csiro.au/sheep). The sequence that showed the most homology with the caprine sequence was presumed to be the notional ovine KAP8-2 gene.The coding sequence of the caprine KAP8-2 gene (GenBank AY510123) was used to BLAST search the Ovine Genome Assembly v2.0 .Two PCR primers (5’-taggcagtcagtcatcctg-3’ and 5’-atagagaatatgaagtccacg-3’) were designed based on the sequence homologous to caprine 2+, 0.5 U of Taq DNA polymerase  and 1× reaction buffer supplied in a 20-μL reaction. The thermal profile for amplification consisted of 2 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 60°C and 30 s at 72°C, with a final extension of 5 min at 72°C. This was done in S1000 thermal cyclers .PCR amplification was undertaken using the purified genomic DNA on one punch of the FTA paper, 0.25 μM of each primer, 150 μM of each dNTP , 2.5 mM of MgAmplicons were visualized by electrophoresis in 1% agarose (Bioline) gels, using 1 × TBE buffer containing 200 ng/mL of ethidium bromide.PCR amplicons were subject to SSCP analysis. A 0.7-μL aliquot of each amplicon was mixed with 7 μL of loading dye  and after denaturation at 95°C for 5 min, the samples were cooled rapidly on wet ice and loaded on 16 cm × 18 cm, 14% acrylamide:bisacrylamide (37.5:1) (Bio-Rad) gels. Electrophoresis was performed using Protean II xi cells (Bio-Rad), at 200 V for 18 h at 25°C in 0.5 × TBE buffer. The gels were silver-stained by the method of Byun et al. .PCR amplicons representing individual SSCP patterns were purified using a MinElute PCR Purification kit (Qiagen) and then directly sequenced in both directions.http://www.ncbi.nlm.nih.gov/) databases using the sequences identified, to find homologous sequences.DNA sequence analyses were carried out using DNAMAN  and a BLAST search was undertaken of the NCBI GenBank (KRTAP8-2 coding sequence (AY510123) revealed a region on sheep chromosome 1  that contained a 192-bp open reading frame and that had 99% homology with the caprine gene. Near this region, seven previously described ovine KAP genes were also identified and these (including KRTAP8-2) were KRTAP11-1, KRTAP7-1, KRTAP8-1,KRTAP8-2, KRTAP6-2, KRTAP6-1, KRTAP13-3 and KRTAP24-1(in order from the centromere)  and reindeer (EF407854), with 99% and 95% similarity respectively in the coding region. These sequences were assumed to represent allelic variants of ovine KRTAP8-2 and were named variants A and B. They were placed in GenBank under the accession numbers KF220646 and KF220646, respectively.The sheep sequences identified here did not share great homology with any other known ovine A was found most frequently (at a frequency of 90.5%), while variant B was less common (at a frequency of 9.5%) in the Romney-cross sheep investigated. Three genotypes were observed, with frequencies of 83%, 15% and 2% for AA, AB and BB respectively.Variant KRTAP8-2 sequence would encode a 63 amino acid polypeptide, that contained a high level of glycine (23.8 mol%) and tyrosine (20.6 mol%), accounting for 44.4 mol% in total of the amino acid content. It had a moderate amount of phenylalanine (9.5 mol%) and serine (7.9 mol%), but a relatively low cysteine content (3.2 mol%). This polypeptide also possessed 3.2 mol% aspartic acid and 1.6 mol% glutamic acid, amino acids that are absent in other HGT-KAPs. The calculated isoelectric point (pI) of the protein was 6.3 , while the type II keratins tend to be more basic (pI 6.5-8.5)


Runx1 is expressed at much lower levels in E8.5 intra-embryonic progenitors, while Bmi1 expression is increased. Remarkably, the ectopic expression of Runx1 in these progenitors fully restores their hemogenic potential, as does the suppression of BMI1 function. Altogether, our data demonstrate that hemogenic competency in recently specified endothelial progenitors is restrained through the active silencing of Runx1 expression.It is now well-established that hematopoietic stem cells (HSCs) and progenitor cells originate from a specialized subset of endothelium, termed hemogenic endothelium (HE), via an endothelial-to-hematopoietic transition. However, the molecular mechanisms determining which endothelial progenitors possess this hemogenic potential are currently unknown. Here, we investigated the changes in hemogenic potential in endothelial progenitors at the early stages of embryonic development. Using an ETV2::GFP reporter mouse to isolate emerging endothelial progenitors, we observed a dramatic decrease in hemogenic potential between embryonic day (E)7.5 and E8.5. At the molecular level,   •+ progenitors from E8.5 embryos have very low hemogenic potentialETV2•+ progenitors specifically lack Runx1 expressionE8.5 ETV2•Runx1 confers hemogenic potential to E8.5 ETV2+ progenitorsEctopic expression of •Runx1 silencing in ETV2+ endothelial progenitors is mediated by BMI1 Runx1 transcription by a BMI1-mediated mechanism.Hematopoiesis originates from specialized HE at specific stages of embryonic development. Eliades et al. demonstrate that RUNX1 repression is critical for suppressing hemogenic competency in endothelial progenitors at the E8.5 developmental stage and that this is achieved through the silencing of  Hematopoiesis emerges early in the vertebrate embryo and occurs in three major distinct waves . The twoT) and the fetal liver kinase receptor (Flk1)  family of transcription factors, marks a subset of mesoderm FLK1+ cells as well as the next stage of blood specification, the HE. Additionally using a knockout approach, our study revealed the essential role of Etv2 for the generation of HE  . Cell-far (Flk1) . Additior (Flk1) . This HEr (Flk1) . We receon of HE . We and on of HE . ETV2 exnt of HE . Furthernt of HE . The tragenitors . Moreove program .+ cells, giving rise to all endothelial and hematopoietic derivatives  at E8.5 also co-expressed these markers, suggesting that this previously uncharacterized ETV2+ population could share similar hemogenic potential as the earlier E7.5 ETV2::GFP+ population  and E8.5 (YS) counterparts  ETV2::GFP+FLK1+CD41− populations both express FLK1, TIE2, and cKIT, markers also expressed in HE, they differ significantly in terms of their hemogenic potential.We previously generated an ETV2::GFP transgenic mouse line in which GFP marks ve of HE . A largepulation A. ETV2:: of Etv2 A. To comturation A. After turation B. Only aterparts D and 1E.+FLK1+ population still remains unclear. Previous work has shown that RUNX1+GATA1− cells from E7.5 extra-embryonic tissue are able to migrate to the EP in a circulation-independent manner  confirmed close association among biological replicates but also clear differences between the two populations , an early mesoderm marker, and Etv2 were expressed at lower levels in the E8.5 (EP) population  population. Runx1 followed a similar pattern of expression and was, in fact, one of the few hematopoietic transcription factors differentially expressed between the two ETV2::GFP+FLK1+ populations, along with Gata1 and Gata2  was absent in both populations  ETV2::GFPulations A. In addpulation  , and E8.5 (YS) ETV2::GFP+ embryos using a nanofluidic platform. A total of 96 genes were analyzed, including housekeeping genes, cell-cycle genes, and transcription factors involved in mesoderm, endothelial, and hematopoietic development. Unsupervised hierarchical clustering and PCA analysis showed that the E8.5 (EP) and E8.5 (YS) populations cluster closely together and are distinct from the E7.5 cells  expression was also high in all populations, while Bmp4 was downregulated in E8.5, a finding consistent with the global expression data. Similarly, while Runx1b was expressed at high levels in most E7.5 single cells (72 out of 80), only 24 out of the 103 E8.5 (EP) and 20 out of the 79 E8.5 (YS) single cells expressed this transcription factor  population, while E7.5 displayed an overall high expression pattern for a number of hematopoietic transcription factors. In contrast, the majority of the E8.5 cells analyzed had low or undetectable expression levels of hematopoietic genes such as Myb or Spi1 (Runx1 (13 out of 17 cells), the majority of which (11 out of 13 Runx1+ cells) were Gata1− and could represent the migrating RUNX1+GATA1− cell population previously described  and E8.5 (YS) cells did not express hematopoietic genes. A subset of Etv2− cells, including E8.5 (EP) and E8.5 (YS) cells, expressed a strong endothelial signature and could represent cells further committed to the endothelial lineage. A third subset of Etv2− cells only detected in E8.5 (EP) had low or undetectable expression of either endothelial or hematopoietic genes. These cells, only found in the EP, could represent progenitors differentiating toward cardiac fate, in line with a previous report demonstrating the necessity for Etv2 silencing for cardiac differentiation  did not express BMP4 or active Smad1/5 and lacked detectable expression of RUNX1b.In the gastrulating embryo, Indian hedgehog is secreted from visceral endoderm and stimulates the expression of BMP4 from the extraembryonic mesoderm. BMP4, in turn, feeds back to mesodermal cells (in an autocrine or paracrine manner) to induce the hematopoietic and endothelial programs . It was  pathway . Therefollantois A. Accord SMAD1/5 B. In conpSMAD1/5 C and 5D.Runx1 in ETV2::GFP+FLK1+CD41− cells correlated with higher hemogenic potential at E7.5 when compared to the E8.5 stage, we hypothesized that the expression of Runx1b might potentially restore the ability of E8.5 endothelial progenitor cells to produce hematopoietic cells. Therefore, we sorted E8.5 (EP) ETV2::GFP+FLK1+CD41− cells, transduced them with Runx1b or control empty lentivirus, and tested their hemogenic potential , but no change in PCNA protein levels was detected  on control and Runx1-treated cells. Runx1-transduced ETV2::GFP+FLK1+CD41− cultures had a far greater frequency of cells giving rise to hematopoietic cells as compared to control , an effect that is independent of the proliferative state of these cells. Altogether, these data suggest that the ability of ETV2::GFP+FLK1+CD41− endothelium progenitors in the developing embryo to give rise to hematopoietic cells is mainly dependent on Runx1 expression.The effect of detected B. Cell-cdetected C. These  control E. In conBmi1 was highly expressed in E8.5 intra-embryonic ETV2::GFP cells but was absent or expressed at low levels in the E7.5 ETV2::GFP population  analysis and May-Grünwald-Giemsa staining showed a strong increase in CD45+ and CD11b+ hematopoietic cells upon BMI1 inhibition -sequencing study has shown the importance of RUNX1 in orchestrating the formation of hematopoietic-specific transcription binding pattern at the onset of hematopoiesis  containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 50 U/ml penicillin/streptomycin (Pen/Strep), 0.6% transferrin, 2% leukemia inhibitory factor (LIF), 48.75 μg/ml monothiolglycerol, 25 μg/ml ascorbic acid, 2% LIF, 1% kit ligand (KL) supernatant, 10 ng/ml oncostatin M, 1 ng/ml basic fibroblast growth factor (bFGF), and 1 μg/ml osteopontin. Where indicated, cells were cultured in semisolid clonogenic assay . BMI1/RIRunx1b and Tomato cDNAs separated by a 2A sequence . Cells were sorted using Aria II, Aria III, or Influx and analyzed with an LSRII cytometer (BD Biosciences).∗p < 0.05 and ∗∗p ≤ 0.001.Data were analyzed using a Student’s t test. Significant differences are Additional information is available in the A.E. designed and performed experiments, analyzed the data, and wrote the manuscript. S.W. and R.P. performed experiments; E.M. and M.Z.H.F. performed bioinformatics analysis, J.B.G. and B.P. assisted with microscopy; V.K. and G.L. designed and supervised the project, analyzed the data, and wrote the manuscript.


A number of pre-treatment resistance associated amino acid variants (RAVs) and polymorphisms have been associated with reduced response to treatment.We measured the prevalence of RAVs/polymorphisms in a PI treatment-naïve HCV genotype 1 Scottish cohort using Sanger sequencing.n = 146) attending NHS Greater Glasgow and Clyde clinics were investigated for RAVs/polymorphisms to the PIs boceprevir, telaprevir and simeprevir. The NS3/4A region was amplified by nested polymerase chain reaction. The 1.4 kb amplified product was sequenced using an ABI 3710XL DNA sequencer. Sequence analysis was performed using web-based ReCall (beta 2.10). Amino acid positions 36, 41, 43, 54, 55, 80, 109, 122, 155, 156, 168 and 170 were analysed for RAVs/polymorphisms.Chronically infected, treatment-naïve, HCV genotype 1 patients  of patients had an RAV or polymorphism detected. Overall, 13.69% (20/146) of patients had HCV virus that contained the Q8 K polymorphism. Other RAVs detected were: V36 M 0.70% (1/146), V36L 0.70% (1/146), T54S 6.85% (10/146), V55A 3.42% (5/146) and V/I170A 0.68% (1/146). Four patients had dual combinations of mutations .Q80K was the most prevalent baseline polymorphism detected in the Scottish cohort. Simeprevir treatment is not recommended in patients infected with the Q80K genotype 1a variant. This highlights the need for baseline sequencing prior to administration of this drug in this population. For example, RAVs at position 156 (A156T/V) and R155K have been shown to reduce the effectiveness of all current PIs 2We measured the prevalence of natural resistance polymorphisms in a protease inhibitor treatment-naïve HCV genotype 1 Scottish cohort using Sanger sequencing.33.1n = 140) were subtype 1a and six subtype 1b. All patients had a detectable HCV RNA tested by Abbott RealTime HCV (detection limit 12 IU/ml).Stored plasma samples, taken between August 2013 and March 2014 for 146 chronically infected HCV genotype 1 patients attending clinics within NHS Greater Glasgow & Clyde, were used in this study. The patients consisted of 141 treatment naïve patients and 5 treatment relapsers who had previously been treated with pegylated IFN and RBV. The majority of the patients (3.2RNA was extracted using the NucliSens easyMag (BioMerieux). Using the on-board lysis protocol, 1000 μl of sample was eluted to 60 μl.3.3http://pssm.cfenet.ubc.ca/home/index). Using the amino acid feature on ReCall, the following amino acid positions were analysed: 36, 41, 43, 54, 55, 80, 109, 122, 155, 156, 168 and 170.The NS3/4A region was amplified by nested polymerase chain reaction (PCR) using a method and primers supplied by Dr Richard Harrigan (British Columbia Centre for Excellence in HIV/AIDS). The 1st round primer sequences were: 5′ TTCAGCCTGGACCCTACCTTTACCAT 3′ (position 4731–4756), 5′ ATGGAGATCAAGGTCATCACGTGGGG 3′ (position 3276–3301) and 5′ GTGGCCGTAGAGCCTGTCGTCTTC 3′ (position 3246–3269). The 2nd round primer sequences were: 5′ GACTTCGACTCTGTGATAGACTGCAAC 3′ (position 4680–4706), 5′ TCAAGGTCATCACGTGGGGGGCGGA 3′ (position 3283–3307) and 5′ TACCGGCGACTTCGACTCGGTGAT 3′ (position 4673–4696). The 1st round PCR amplification was carried out using a Qiagen OneStep RT-PCR kit and the 2nd round with the Expand High Fidelity PCR system (Roche Diagnostics GmbH). Sanger sequencing was performed on the ABI 3710XL DNA sequencer with Big Dye v3.1. The 1.4 kb sequence was analysed using web-based ReCall beta v2.10 . The low level of subtype 1b patients in this study is a reflection of the Scottish population, where subtype 1a predominates. This is also a reflection of the UK population as a whole The V36L/M, T54S, V55A and V/I170A mutations detected in this study are low level resistance RAVs that have little effect on SVR rates in patients treated with triple therapy In this study four (2.74%) subtype 1a patients were found to have RAV combinations, which all contained T54S with another mutation . Bae et al. This study examined the frequency of NS3 variants detected by Sanger sequencing. Sanger sequencing will only detect these variants at a frequency of >20%. Next generation sequencing (NGS) can detect lower frequency variants by measuring population variants that occur at <1% Currently, European guidelines have six suggested treatment options for genotype 1 patients with simeprevir recommended within two of these treatment protocols Janssen Pharmaceutical funded the implementation of Q80K testing in the West of Scotland Specialist Virology Centre. TA is funded by the MRC (G0801822) and ECT by the Welcome Trust (WT102789).None declared.Not required.


Pck2 expression prevented Gad8–TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress.Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKC In all Fission yeast contains two TOR kinases; Tor2 is the main TORC1 kinase, whereas the majority of TORC2 incorporates the non-essential Tor1 kinase –9. In geKsg1 activates the kinase; however, full Gad8 activation requires additional TORC2-dependent phosphorylation within its carboxy-terminus . Nitrogen starvation was applied as follows. Cells were cultured at 28°C in minimal sporulating liquid media (MSL) [6 cells ml−1 and filtered into MSL minus nitrogen source.To assay sexual differentiation, 4 × 10ia (MSL)  to a den6 cells ml−1. Ten-fold dilution series starting with 5 × 104 cells were spotted on media indicated.For stress sensitivity spot test assays, cells were grown in yeast extract media (YES) to a cell density of 1.5 × 104.2.6 cells ml−1 and filtered into media using 20 mM l-proline as nitrogen source (EMMP). Cells were harvested at indicated time point, fixed with 3% formaldehyde, washed with PBS and stained by calcofluor. Images of cells were obtained using a CoolSNAP HQ2 CCD camera and processed with ImageJ. The ratio of cells undergoing NETO was estimated from calcofluor-stained cells . Cell length at division was measured .Cells were grown at 28°C in EMMG to 1.5 × 104.3.6 cells ml−1 before FM4-64 dye 5 mg ml−1 was added to the culture in the ratio 1 : 10 000  and cells were incubated for a further 30 min before images of the living cells were captured with a CoolSNAP HQ2 CCD camera and processed with ImageJ. For each culture, at least 500 vacuoles were measured.In order to analyse vacuolar morphology, cells were grown to a density of 1.8 × 104.4.gad8 point mutations, and the mutated gad8 allele was introduced into cells through transformation. The recombinant gene was then used to replace the +ura4 gene in the +.gad8::ura4 The resulting strains were back-crossed, and prototroph progeny was selected. The presence of the mutation was verified by PCR. Thus, all gad8 point mutations used in this study are single point mutants integrated into the gad8 locus, and they are all prototrophic strains.Standard site-directed mutagenesis was used to generate E. coli BL-21 cells. Pck2 was amplified by PCR from genomic wt DNA, sequenced and cloned into pRep42 plasmid to facilitate Pck2 expression from the nmt42 promoter [For generation of the Tor1–GST fusion substrate in the Gad8 kinase assay, Tor1 was amplified using the following primers, 5′-CTCGGATCCATCTCGCATTTCCATCACACTTTCGAAG-3′ and 5′-ACGCGTCGACAAGTCTCCAATTAATCAAAGGGTCATAG-3′, and cloned into pET-41a(+) vector. Tor1–GST was expressed in 4.5.TCA precipitation protocol was followed for total protein extracts . The fol4.6.6 cells ml−1 were harvested and re-suspended in 20% TCA. Cells were disrupted using a Spex6870 freezer mill in liquid nitrogen. After washing with 0.1% TCA, the sample was re-suspended in sample buffer  plus 2% SDS with 3 min boiling. About 4.5 volumes of sample buffer plus 1% triton X-100 were added to the supernatant. The mix was centrifuged at 10 000g for 5 min. IP buffer  was added to the supernatant. The Gad8 kinase was immunoprecipitated on protein G dynal beads for 30 min at 4°C. The beads were then washed six times with sample buffer plus inhibitors, then heated to 80°C for 10 min prior to electrophoreses.A 20 l of fission yeast culture at 3 × 104.7.−1. The LTQ-Orbitrap XL mass spectrometer was operated in parallel data-dependent mode where the MS survey scan was performed at a nominal resolution of 60 000 (at m/z 400) resolution in the Orbitrap analyser between m/z range of 400–2000. The top six precursors were selected for CID in the LTQ at normalized collision energy of 35% using multi-stage activation at m/z 98.0, 49.0 and 32.7 Da.Large-scale Gad8 immunoprecipitations were run on a Nupage Bis–Tris 4–12% SDS–PAGE gel (Invitrogen), The Gad8 Coomassie-stained band was excised and digested with either 20 ng sequencing-grade trypsin (Sigma-Aldrich), 400 ng LysN (Associates of Cape Cod) or 350 ng Elastase  in 100 µl 40 mM ammonium bicarbonate, 9% (v/v) acetonitrile at 37°C for 18 h. The peptides were separated using a Nano-Acquity UPLC system (Waters) using a Waters NanoAcquity BEH C18 column  with a gradient of 1–25% (v/v) of acetonitrile, 0.1% formic acid over 30 min at a flow rate of 400 nl min4.8.−1 glutathione). The in vitro kinase assay was performed as previously described [3VO4, 15 mM PNPP, 1 mM PMSF and protease inhibitors) and resuspended in kinase assay buffer . It was mixed with 3 µg of Tor1–GST and 25 µM of ATP, incubated at 32°C for 30 min. The reaction was stopped by 5 min boiling with 2× loading buffer.Tor1–GST was purified as follows. BL-21 cell expression of Tor1–GST was disrupted with lysis buffer . The supernatant was incubated with glutathione sepharose for 2 h at 4°C. The sepharose was washed 10× with lysis buffer and Tor1–GST was eluted with elution buffer . Following cell lysis, Tor1 was immunoprecipitated on protein G Dynal beads and used directly in in vitro Tor1 kinase assays. The kinase assays were performed using the K-LISA mTOR activity kit that includes substrate according to Ikai et al. [i et al. .4.10.p < 0.05) as determined by Student's t-test.Asterisks represent statistical significance (


Copper nitrate and thiamine hydrochloride were selected as the starting materials in the water phase under hydrothermal conditions. No addition of a surfactant or a complex reagent was required for the synthesis of the Cu1.8S dendrite structures. Thiamine hydrochloride was employed as a sulfur source and structure-directing agent. The growth mechanism of Cu1.8S is tentatively discussed based on the experimental and computational results.A facile, green and environmental-friendly method for preparing Cu Finally, the resulting mixture was sealed into a 100 mL Teflon-lined stainless-steal autoclave, and kept at 180 °C for 24 h. After the solution cooled down to room temperature, the obtained material was washed with water and alcohol for several times, and dried at 60 °C in vacuum.The chemicals, including copper nitrate and thiamine hydrochloride  were of analytical grade and used without further purification. In a typical procedure, 0.5 g thiamine hydrochloride was dissolved in 60 ml distilled water, then 0.073 g Cu(NO2 F20 system operated at 200 kV.X-ray diffraction (XRD) analysis was performed with a Rigaku D/MAX-2500 V/PV spectrometer by using Cu-Kα radiation (40 kV and 200 mA) at a scanning speed of 4°/min over the 2θ range of 20–70°. Scanning electron microscopy (SEM) images were recorded with the Hitachi Field Emission Scanning electron microscope S4800. TEM analyses were performed on a Philips Tecnai G


During sepsis a leading symptom is capillary leakage caused by endothelial damage, followed by multiorgan dysfunction. Sphingosine-1-phosphate (S1P) is a bioactive lipid with multiple functions. Cellular reactions depend on the S1P concentration in the blood and its binding to five specific G-protein coupled receptors. S1P-regulated functions include: control of endothelial permeability; lymphocyte migration across microvessels depending on an S1P gradient; and control of vascular tone [®). The SOFA score was used to describe disease severity.Following ethical approval we enrolled patients fulfilling the ACCP/SCCM sepsis criteria into three groups . A group of 20 healthy donors served as controls. Serum blood samples, laboratory data and clinical parameters are presented for day 1. The primary outcome variable was serum S1P concentration (μg/l) quantified by mass spectrometry . The median SOFA score in our cohort was 6. We divided the cohort into two groups: SOFA score <6; and SOFA score >6. We tested the sensitivity and specificity of S1P to indicate disease severity by ROC analysis. In our cohort the area under the curve (AUC) for S1P was 0.77 (CI 0.670 to 0.870) and therefore higher when compared with common markers of inflammation , 0.68 (0.554 to 0.786) and 0.67 (0.571 to 0.794), resp.).We included 87 patients . The serum concentration of S1P (mean ± SD) in the control group was 484.6 ± 152.6 μg/l and significantly higher compared with Group A 239.4 ± 61.3 μg/l, Group B 248.6 ± 93.7 μg/l and Group C 141.6 ± 46.3 μg/l. We observed a negative correlation between S1P and SOFA score (Pearson Our findings suggest that S1P is a novel marker for severity of sepsis with severe sepsis and septic shock being associated with low levels of S1P. Moreover, blood concentrations of S1P might play a key role in sepsis pathophysiology.MSW and AN are equal contributors.


Thoracic pathologists are frequently faced with tissue specimens from intrathoracic/mediastinal tumors. Specifically the differentiation between thymic and pulmonary squamous cell carcinomas (SqCC) can be challenging. In order to clarify the differential diagnostic value of CD5 and CD117 in this setting, we performed a large scale expression study of both markers in 1465 non-small cell lung cancer (NSCLC) cases.Tissue microarrays of formalin-fixed paraffin-embedded resection specimens of 1465 NSCLC were stained with antibodies against CD117 and CD5. Positivity of both markers was correlated with clinicopathological variables.CD117 was positive in 145 out of 1457 evaluable cases (9.9 %) and CD5 was positive in 133 out of 1427 evaluable cases (9.3 %). 28 cases (1.9 %) showed coexpression of CD117 and CD5. Among the 145 cases that were positive for CD117, 97 (66.8 %) were adenocarcinomas (ADC), 34 (23.4 %) were SqCC, 5 (3.4 %) were adenosquamous carcinomas (ADSqCC), 8 (5.5 %) were large cell carcinomas (LC), and one (0.6 %) was a pleomorphic carcinoma (PC). In the CD5 positive group consisting of 133 cases, 123 (92.4 %) were ADC, 0 (0 %) were SqCC, 4 (3.0 %) were ADSqCC, 3 (2.2 %) LC and 3 (2.2 %) were PC. None of the 586 SqCC showed expression of CD5. No association of CD117- or CD5 positivity to patients’ age, pathological stages or to T-, N-, or M- categories was observed.A substantial subset of NSCLC exhibit positivity of CD117 and CD5. Since CD5 expression was not observed in pulmonary SqCC, but is expressed in the majority of thymic squamous cell carcinomas, the application of this immunomarker is a valuable tool in the differential diagnosis of thoracic neoplasms.The online version of this article (doi:10.1186/s13000-015-0441-7) contains supplementary material, which is available to authorized users. Comprehensive morphological and immunohistochemical subtyping of tumors is of growing importance for therapy selection and propelled the concept of a tumor-specific, individualized treatment. For non-small cell lung cancer (NSCLC) the current WHO Classification  thereforFormalin-fixed paraffin-embedded specimens of NSCLC resected from 2002 to 2010 at the Thoraxklinik at Heidelberg University were extracted from the archive of the Institute of Pathology, Heidelberg University with the support of the tissue bank of the National Center for Tumor Diseases (NCT: project: # 1283). Tissues were used in accordance with the ethical regulations of the NCT tissue bank established by the local ethics committee. A cohort of 1465 patients was identified for TMA construction. Diagnoses were made according to the recommendations of the World Health Organization classification for lung cancer 2015 [http://www.nordiqc.org). In brief, TMA slides were deparaffinized and pretreated with antigen retrieval buffer. Subsequent steps were performed on an immunostaining device . Pretreatment and dilution specifications are summarized in Additional file Immunohistochemistry (IHC) was performed with commercially available antibodies  and RStudio (v. 0.98.507). Barplots were created with the ggplot2-package (v. 1.0.1) and dependencies. p = 0.79 and p = 0.13), any T- (p = 0.07 and p = 0.02), N- (p = 0.16 and p = 0.03) and M-categories (p = 0.42 and p = 0.77) or clinical stage (p = 0.32 and p = 0.05).Clinicopathological data of 1465 NSCLC cases (stage IA-IV) were analyzed. The median age at diagnosis was 64 years (range: 30–89 years). The majority of patients were male (69.3 %). 711 patients were diagnosed with adenocarcinoma , 586 patients with squamous cell carcinoma , 55 patients with adenosquamous carcinoma , 71 patients with large cell carcinoma  and 42 patients with pleomorphic carcinoma , respectively. Neither CD117 positivity, nor CD5 positivity correlated to patients’ age (In 46 patients (3.1 %) CD117 and/or CD5 could not be evaluated because there were either no vital tumor cells or lost tissue cores during the staining process.Both, CD117 and CD5 showed diffuse membranous and cytoplasmic positivity  were ADC, 34 (23.4 %) were SqCC, 5 (3.4 %) were ADSqCC, 8 (5.5 %) were LC, and one (0.6 %) was a PC.p < 0.01).The frequency of CD117 positive tumors was higher in ADC than in any other histotype (p < 0.01). No SqCC showed expression of CD5.In the CD5 positive group consisting of 133 cases, 123 (92.5 %) were ADC, 0 (0 %) were SqCC, 4 (3.0 %) were ADSqCC, 3 (2.2 %) were LC and 3 (2.2 %) were PC. The frequency of CD5 positive tumors was higher in ADC than in any other histotype (p < 0.01). CD5 and CD5/CD117 positive patients were more often male (p < 0.01). Application of the immunoreactive score by Remmele et Stegner [In the 28 cases that exhibited coexpression of CD117 and CD5, 26 were ADC (92.8 %), one was ADSqCC (3.5 %) and one was a LC (3.5 %). Again, CD5/CD117 coexpression was significantly more frequent in ADC than in any other histotype  [CD5 has been investigated in NSCLC before and found to be present in 0–85 % of cases , 10, 21.n = 20) , 10, 21.In summary, we demonstrate on 1465 NSCLC cases that a substantial subset exhibit CD117 and CD5 positivity and that pulmonary SqCC are consistently negative for CD5. Thus, the application of both immunomarkers is a valuable tool in the differential diagnosis of mediastinal tumors.


The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition.Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNA  •Structures of eukaryotic translation initiation complexes in open and closed states•In the open complex the 40S head moves upward to open the mRNA entry channel latch•Transition to closed state locks initiator tRNA in the P site base-paired with AUG•The structures show how eIF3 contacts eIF2 and eIF1 on the 40S subunit interface The small ribosomal subunit latch has to open to allow mRNA loading. Llacer et al. report cryo-EM reconstructions of yeast translation pre-initiation complexes in both open and closed conformations, highlighting structural differences and elucidating key events in start codon recognition. The 43S preinitiation complex (PIC) thus formed binds to the capped 5′ end of mRNA in collaboration with the eIF4F complex, which consists of the cap-binding protein eIF4E, scaffolding protein eIF4G, and RNA helicase eIF4A. This 48S PIC, in an open conformation with tRNAi not fully engaged in the P site (POUT), then scans along mRNA. During the scanning process, GTP bound to eIF2 is hydrolyzed but the dissociated phosphate (Pi) is not released. Recognition of the start codon in the P site precipitates transition to a scanning-arrested, closed PIC with tRNAi accommodated in the P site (PIN). This rearrangement triggers release of eIF1 and resultant dissociation of Pi  and a sixth non-essential and substoichiometric subunit, eIF3j  from our groups shows details of the interaction of eIF1 and eIF1A with the ribosome and tRNA of eIF3 . eIF3 is of eIF3 . Mammalit, eIF3j . Crystalt, eIF3j , and a ct, eIF3j . Domainst, eIF3j . All knot, eIF3j . And yett, eIF3j . How eIFi not fully engaged in the P site, hereafter referred to as “py48S-open.” A second complex elucidated at 4.9 Å resolution contains mRNA with an AUG codon that presents a closed conformation with the latch closed, entry channel constricted, and tRNAi locked into the PIN state, hereafter referred to as “py48S-closed.” These structures show clear density for eIF1, eIF1A, mRNA, the entire TC, including previously unseen eIF2β, as well as segments of eIF3. The py48S-open complex shows an open latch conformation, expanded entry channel and widened P site, suggesting mechanisms for loading and scanning of mRNA and is markedly different from a pm48S reconstructed at 11.6 Å  that reveals an open, scanning-competent state with tRNAt 11.6 Å . CompariKluyveromyces lactis; and factors eIF1, eIF1A, eIF3, eIF5, and TC from S. cerevisiae. We used the U31:A39 variant of tRNAi that stabilizes the PIN state  was used instead of native eIF3 . We observe density for eIF2β in both complexes  and S2. The large data set and extensive classification also enabled us to determine structures of a 40S•eIF1•eIF1A complex at 3.5 Å resolution  and S4A S. cerevisiae numbering throughout the manuscript) lead to a Gcd− phenotype, indicating less stable TC binding to the PIC  of tRNAi are recognized by rRNA residues G1575 and A1576 in both py48S-closed and py48S-open  of eIF2β is positioned close to the GTP binding pocket of eIF2γ in both complexes C, similaognition , it remaIn our previously reported py48S structure , densityi and domains 1 and 2 of eIF2α in a concerted manner between the two complexes  mRNA encoding eIF1  using a defocus range of 1.8–3.2 μm. Images were recorded on a Falcon II detector at a calibrated magnification of 104,478 (yielding a pixel size of 1.34 Å). An in-house system was used to intercept the videos from the detector at a speed of 16 frames/s exposures. Micrographs that showed noticeable signs of astigmatism or drift were discarded.Data were collected on an FEI Titan Krios microscope operated at 300 kV under low-dose conditions  was placed into density by rigid-body fitting using Chimera . Wild-type tRNAi was used from PDB: 1YFG for initial rigid-body fitting into its corresponding density in the py48S-open complex. Model building and refinement were carried out using Coot and Refmac (The atomic model of py48S (PDB:  Chimera . Further Chimera . For py4d Refmac  (see Supd Refmac . All figd Refmac  or ChimeJ.L.L. and T.H. made the samples, collected and analyzed the data, determined the structures and wrote a first draft of the manuscript. L.M. and A.T. performed the genetic experiments. C.E.A. provided advice on eIF3 purification, helped characterize recombinant eIF3, and helped write the manuscript. J.R.L., A.G.H., and V.R. supervised the work and helped to write the manuscript.