Rhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11-cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear.We herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 4′-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108–135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees.Accessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III. G-protein-coupled receptors (GPCRs), the largest known family of cell surface receptors, mediate a wide variety of signal transduction processes. Upon recognition of diverse extracellular signals including hormones, neurotransmitters, olfactants, tastants, and light, GPCRs initiate intracellular signaling by interacting with heterotrimeric G proteins Rhodopsin is composed of the 40-kDa apoprotein opsin (348 amino acids) and its chromophore 11-cis-retinal, which acts as an inverse agonist in the rhodopsin ground state Several approaches, including electron paramagnetic resonance, nuclear magnetic resonance, and crystallization, have been used to obtain insight into the structure of rhodopsin and its conformational changes on light activation The aim of this study is to better understand GPCR activation by investigating the conformational changes of rhodopsin upon light activation. Cysteines introduced one at a time along the full length of the TM helices will be tested for their accessibility and consequent reactivity to sulfhydryl reagents in the dark and on light activation.To determine the conformational changes of rhodopsin on light activation, a basal rhodopsin mutant was constructed replacing the six naturally-occurring free cysteine residues with the neutral amino acid alanine, to avoid ambiguity caused by signals derived from natural cysteines in rhodopsin. As shown in trans-retinal, 4-PDS and phenylmethylsulfonylfluoride (PMSF) were purchased from Sigma . n-dodecyl-β-D-maltopyranoside (DM) was acquired from Anatrace . Antirhodopsin monoclonal antibody rho-1D4 was purified from myeloma cell lines and coupled to cyanogen bromide-activated Sepharose 4B (Sigma) as described elsewhere 2PO4, 10 mM Na2HPO4 (pH 7.2); buffer B, buffer A plus 1% DM (w/vol); buffer C, buffer A plus 0.05% DM; buffer D, 2 mM NaPi (pH 6.0), 0.05% DM; buffer E, 2 mM NaPi (pH6.0), 0.05% DM, 100 µM nonpeptide; and buffer F, 2 mM NaPi (pH8.0), 0.05% DM.All-A synthetic opsin construct containing the mutations C140A, C167A, C185A, C222A, C264A, and C316A was generated . All of the single-cysteine mutants in the sequence 107–135 were derivatives of this mutant, which are labeled as P107C–R135C/Basal mutant. As controls for the experiments, similar single-cysteine substituted mutants at amino acid positions 107–135 were generated on the background of wild type (WT) rhodopsin, containing the six natural cystein residues, which are labeled as P107C–R135C/WT.The mutants P107C, T108C, G109C, and N111C were prepared by fragment replacement mutagenesis in the synthetic gene for bovine opsin GAA TTC ATG AAC GGT ACC GAA GGC CCA5′ CTG CAA  (EcoRI); the primer AC TAG TGA CCA CAG TGC AAT TTC ACC GCC CAG GGT GGC AAA GAA GCC CTC GCA GTT5′ GAC T for L112C; and the primer AC TAG TGA CCA CAG TGC AAT TTC ACC GCC CAG GGT GGC AAA GAA GCC GCA GAG GTT5′ GAC T (speI) for E113C . The PCR products were digested to provide the EcoRI/speI fragments (nucleotides 2–387) containing the cysteine codons, which were then subcloned into the large fragment speI/EcoRI (nucleotides 388–6182) of the L119C/Basal mutant. The DNA sequences of the fragments containing the mutated regions in all constructs were confirmed by the dideoxynucleotide sequencing method.PCR mutagenesis was used for preparation of the mutants L112C/Basal mutant and E113C/Basal mutant. The first step involved PCR reactions with the A132C/Basal plasmid as the template, using the following primers containing the above single-cysteine codon, one at a time: the primer cis-retinal were performed as previously described cis-retinal  and 0.1 mM PMSF (2 mL/plate) at 4°C with end-over-end mixing for 4 h in the dark for generating the rhodopsin chromophore. Then the pellet cells were placed in the SORVALL RC-5  and solubilized in buffer B containing 1% DM and 0.1 mM PMSF for 1 h at 4°C. The suspension was centrifuged at 35,000 rpm for 30 min at 4°C in a Ti60 rotor to remove the insoluble material. Mutant rhodopsins were purified by using 1D4-sepharose 4B affinity chromatography as previously described Transient transfection of COS-1 cells and treatment of the harvested cells with 11-500) used for WT rhodopsin was 40,600 M−1cm−1The formation of the samples' chromophore was checked by the difference of UV-visible (UV/vis) absorption spectra before the rhodopsin was incubated with 1D4-antibody during the protein purification. UV-visible (UV-vis) absorption spectra of the purified proteins was recorded with a Perkin-Elmer λ-35 UV-vis spectrophotometer equipped with water-jacketed cuvette holders connected to a circulating water bath. All spectra were recorded with a bandwidth of 2 nm, a response time of 1 s, and a scan speed of 480 nm/min at 20°C either in the dark, after acidification, or after illuminating the samples for 30 sec with a fiber optic light equipped with a >495 nm long-pass filter. The molar extinction value  after illumination are shown in 1/2 was 14.7 min under the assay conditions   showed the fastest Meta II decay among rhodopsin mutants  . The Met mutants .The purified rhodopsin mutants P107C/, N111C/, G114C/, A124C/, L125C/, W126C/, L128C/, and E134C/Basal mutant did not form the rhodopsin-like chromophore. Immunoblotting analysis of the solubilized extracts of cells expressing each mutant showed that most of the mutants showed a similar level of opsin expression compared with that of the basal mutant (data not shown). The difference in UV-vis spectra confirmed that there was not the formation of chromophore in these cysteine mutants .max = 380 nm with a small second component absorbing at 505 nm. But mutant G114C/WT only formed a pigment with λmax = 380  in the dark Upon photoactivation, these mutants incorporated ∼1 mole of the reagent per mole of rhodopsin over time after illuminating the samples for 30 sec. After light activation, comparison of the rate of reactivity calculated from absorbance increase at 323 nm indicated that mutants T108C/, G109C/, E113C/, and L131C/Basal mutant showed rapid reactions with 4-PDS. Reaction rate of mutants L112C/, G121C/, and A132C/Basal mutant with 4-PDS was moderate, while mutants F115C/, T118C/, L119C/, V129C/, V130C/, and R135C/Basal mutant showed a slow rate of reaction with 4-PDS. The slower reactions were observed with F116C/, A117C/, G120C/, E122C/, I123C/, S127C/, and I133C/Basal mutant. The cysteine mutant E113C/Basal mutant showed the fastest reaction rate with 4-PDS among all of the mutants at helix-III. The time courses for the reactions and exponential fits for selected mutants are shown in The big group including L112C-A132C/Basal mutant showed no reaction at all using prolonged times, but G109C/Basal and I133C showed completely reaction in the dark, which confirmed that hydrophobic/hydrophilic phase boundaries exist at positions of G109 and I133 of Helix-III. To determine whether the cysteine accessibility data agree with the accepted rhodopsin 3-dimensional structural model, two mutants GI09C/Basal mutant and I133C/Basal mutant were analyzed by vacuum electrostatics of rhodopsin surface charge changes in the inactive and partially active states . The anaThe crystal structure of rhodopsin in the ground state provided the first detailed three-dimensional structural model for a GPCR 5,6F (D/E)R(Y/W) motif provides structural constraints in rhodopsin that rearrange in response to photoisomerization during formation of the G protein-activating Meta II Light-catalyzed retinal isomerization causes specific movements in the TM helices, resulting in a conformational change in the CP domain preparing the molecule for the protein-protein interactions involved in signal transduction 280/A500 ratio of 1.7 and an absorption visible maximum slightly blue-shifted to 495 nm. Thus, 11-cis-retinal binding to the basal mutant is near normal, the structure of the basal mutant is similar to that of WT rhodopsin and the substituted alanines lie in a similar orientation to that of the WT cysteine residues As shown in 280/A500 ratio was significantly increased to ∼2.5, indicating some degree of misfolding for these two mutants. This concurs with the previously reported spectrum of R135L 280/A500 ratio indicates that a cysteine substitution at position 109 may affect the correction matching of the disulfide bond. In the control experiment, G109C/WT did not form the rhodopsin-like chromophore, but G109C/Basal mutant formed the chromophore. This result suggests that the amino acid cysteine at position 109 may form an erroneous disulfide bond with cysteine at either position 185 or 187 and results in the misfolding of the protein.Two of the studied mutants, G109C/Basal mutant and R135C/Basal mutant, regenerated with 11-cis-retinal to different levels when compared to the WT . The A28280/A500 ratios from 1.8 to 6.4  max values (500 nm→485 nm) and decreased retinal binding that corresponded to the bulk of the substituted side chain. G121 also is a specific component of the 11-cis-retinal binding pocket in rhodopsin The rate of Meta II decay of mutant G121C/Basal mutant (1.8 min) was the fastest among the cysteine mutants . Acidifi8 to 6.4 . G121 ismax = 380 nm) and a slightly red-shifted pigment (λmax = 505 nm) (max = 505 nm) of the mutant E113C/Basal mutant completely converted to λmax = 380 nm . After acidification, the chromophore changed to 390 nm  also formed a markedly blue-shifted pigment (λ 505 nm) . The pig= 380 nm . This iso 440 nm . Mutant o 390 nm . The E11max = 380 nm)  . The amomax = 484 nm. Increasing illumination time resulted in more Meta II formation. UV-absorption spectroscopy has suggested that the local protein environment around Trp residues changes during the conversion of rhodopsin to Meta II, and a linear dichroism study of UV-difference bands indicated a reorientation of an indole side chain of Trp during the Meta I to Meta II conversion Mutants A117C/WT and W126C/WT formed the complex of Meta I and Meta II after illuminating the samples for 30 sec . The chrMutants P107C, N111C, G114C, A124C, L125C, W126C, L128C, and E134C on the background of the basal mutant failed to regenerate with 11-cis-retinal. The absence of chromophore regeneration for these mutants was confirmed by western blotting and UV-vis absorption spectra . HoweverA number of factors could affect the reactivity of the sulfhydryl groups in different cysteines, including solvent accessibility, disposition within the structure, and the dielectric constant prevailing in the immediate environment Another group, which showed markedly different reaction rates with 4-PDS, includes T108C/, G109C/, E113C/, and L131C/Basal mutant. The high accessibility of cysteine in mutant E113C/Basal mutant with 4-PDS in the dark demonstrated that the internal salt bridge with the Schiff base was broken by a cysteine substitution at E113, which opened the opsin structure already in the dark. The rates of reaction with 4-PDS upon illumination are shown in 1/2∼16 min), which is similar to that of WT , whereas most of mutants involving amino acids near the TM C-terminus, including L119C-R135C/Basal mutant, showed a long Meta II decay (t1/2∼40 min), which is similar to that of basal mutant   . However 41 min) . Thus, iWater reorganization following photoactivation releases the constraints mediated by ionic lock (ERY motif), and a secondary internal hydrogen bonding network (Y206-H211-M163-E122), which may imply movement of water molecules and/or side chains and structural alterations of the helices In summary, our data show that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The chromophore-facing residues of Helix-III become water-accessible after photoactivation.Figure S1Schematic representation of the rhodopsin cysteine mutants with 4-PDS. The reaction product thiopyridone is maximum at 323 nm. Thus, the reaction kinetics and the number of reactive cysteines can be determined by monitoring the absorption at this wavelength.(TIF)Click here for additional data file.Figure S210% SDS-PAGE analysis of the purified cysteine mutants on the background of basal mutant.(TIF)Click here for additional data file.Figure S3Meta II decay of wild type rhodopsin and basal mutant. The changes in fluorescence were measured in a buffer containing 2 mM Na-Pi (pH 6.0) and 0.05% DM after illuminating the samples for 30 sec at 20°C.(TIF)Click here for additional data file.Figure S4UV-Vis spectra of rhodopsin cysteine mutant L125C/Basal mutant and E134C/Basal mutant. Mutants were purified from COS cells after regeneration with 25 µM 11-cis-retinal. UV-Vis spectra were recorded in the dark with rhodopsin eluted in a buffer containing 2 mM NaPi (pH6.0), 0.05%DM, 100 µM C' 1–9 peptide and 100 mM NaCl.(TIF)Click here for additional data file.Figure S5Comparison of PDS labeling rate among cysteine mutants. These mutants include T108/, G109/, L112/, E113/, L119/, G120/, G121/, E122/, and I123/Basal mutant. The reaction was carried out with 0.5 µM of rhodopsin mutant and 25 µM 4-PDS in phosphate buffer (pH8.0) and 0.05% DM at 20°C. Time-dependent changes in absorbance at 323 nm after photoactivation were plotted.(TIF)Click here for additional data file.


YN-2011 is a highly pathogenic North American porcine reproductive and respiratory syndrome virus (PRRSV). Unlike previously described PRRSVs, which contained a 30-amino-acid deletion in NS2, YN-2011 had no amino acid deletions or insertions but had several new mutations in NS2. Here, we announce the complete genome sequence of YN-2011. Arterivirus of the family Arteriviridae in the order Nidovirales (Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of an economically important swine disease that has been devastating the swine industry since the late 1980s . The PCR products were cloned into pMD19T vector (TaKaRa), sequenced three times using an ABI 3730 Sanger-based genetic analyzer, and assembled using DNAStar version 7.1 to obtain the complete genome sequence. The completed sequence showed that, including the poly(A) tail, the genomic sequence of YN-2011 was 15,432 nucleotides in length. Genetic analyses demonstrated that YN-2011 shared 99.5% genome similarity with strain VR-2332, but a dramatically low degree of genetic homology (37.2%) with the representative highly pathogenic PRRSV JXA1 strain was observed; the phylogenetic tree showed that YN-2011 and VR-2332 are the same subgroup. The NS2 of YN-2011 and that of the VR-2332 strain show a genetic homology of 99.4%. There is no amino acid deletion or insertion in NS2 protein sequence, but there are new several substitutions, such as T-I (111), S–F (285), EV-QL , P–S (475), T-A (491), Y–H (566), D–N (568), H–R (600), R–H (684), M-K (768), and F-L (926) (amino acid positions are given in parentheses).In contrast with the recombinant or overattenuated HP–PRRSV strain as has been reported previously , 9, 10, JX857698.The virus genome sequence of strain YN-2011 is available in GenBank under accession no.


Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections. Lantibiotic biosynthetic machinery is encoded by gene cluster composed by a structural gene that codes for a pre-lantibiotic peptide and other genes involved in pre-lantibiotic modifications, regulation, export and immunity.Lantibiotics are heat-stable peptides characterized by the presence of thioether amino acid lanthionine and methyllanthionine. They are capable to inhibit the growth of Gram-positive bacteria, including Bacillus amyloliquefaciens GA1 was found to produce an antimicrobial peptide, named amylolysin, active on an array of Gram-positive bacteria, including methicillin resistant S. aureus. Genome characterization led to the identification of a putative lantibiotic gene cluster that comprises a structural gene (amlA) and genes involved in modification (amlM), transport (amlT), regulation (amlKR) and immunity (amlFE). Disruption of amlA led to loss of biological activity, confirming thus that the identified gene cluster is related to amylolysin synthesis. MALDI-TOF and LC-MS analysis on purified amylolysin demonstrated that this latter corresponds to a novel lantibiotic not described to date. The ability of amylolysin to interact invitro with the lipid II, the carrier of peptidoglycan monomers across the cytoplasmic membrane and the presence of a unique modification gene suggest that the identified peptide belongs to the group B lantibiotic. Amylolysin immunity seems to be driven by only two AmlF and AmlE proteins, which is uncommon within the Bacillus genus.Bacillus amyloliquefaciens strains Y2 and HIL Y-85,544728, reports on the synthesis of type B-lantibiotic in this species are scarce. This study reports on a genetic and structural characterization of another representative of the type B lantibiotic in B. amyloliquefaciens. Apart from mersacidin produced by  Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections  lipid II, was prepared in vitro using E. coli K12 purified membrane from UDP-GlcNac and UDP-MurNAc-L-Ala-D-Glu-meso-DAP-D-[14C]Ala-D-[14C]Ala and subsequently purified as previously described [14C] lipid II , amylolysin (0.2 µM), His-tag PBP1bγ from E. coli (15 nM) were incubated in 25mM Tris-HCl , 0,5 % decyl PEG, 10 mM MgCl2, 12% 1-octanol, 25 % DMSO for 1 hour at 30 °C. The reaction products were separated by thin layer chromatography (TLC) on silica plate  using a mixture of methanol/chloroform/ammoniac/water  as mobile phase. For interaction experiment, [14C] lipid II  and amylolysin (0.2 µM) were incubated in DMSO/1-octanol mixture (60/40 v/v) for 1 hour at room temperature before being subjected to TCL using a butanol/acetic acid/pyridine/water mixture  as mobile phase. The radioactive compounds were detected and analyzed with a Molecular Imager FX system (Biorad Laboratories).Radiolabelled UDP-MurNac-pentapeptide, thereafter [escribed . Inhibitescribed  with somFigure S1Sequence alignment of the N-terminus of type B lantibiotic modification enzymes.(DOCX)Click here for additional data file.Table S1Strains and plasmids used in this study.(DOCX)Click here for additional data file.Text S1Construction of strain RFB136.(DOCX)Click here for additional data file.


Eukaryotic translation initiation factor 4G (eIF4G) is thought to influence the translational efficiencies of cellular mRNAs by its roles in forming an eIF4F-mRNA-PABP mRNP that is competent for attachment of the 43S preinitiation complex, and in scanning through structured 5' UTR sequences. We have tested this hypothesis by determining the effects of genetically depleting eIF4G from yeast cells on global translational efficiencies (TEs), using gene expression microarrays to measure the abundance of mRNA in polysomes relative to total mRNA for ~5900 genes.Although depletion of eIF4G is lethal and reduces protein synthesis by ~75%, it had small effects (less than a factor of 1.5) on the relative TE of most genes. Within these limits, however, depleting eIF4G narrowed the range of translational efficiencies genome-wide, with mRNAs of better than average TE being translated relatively worse, and mRNAs with lower than average TE being translated relatively better. Surprisingly, the fraction of mRNAs most dependent on eIF4G display an average 5' UTR length at or below the mean for all yeast genes.7 G cap- and poly(A)-binding factors (eIF4E and PABP), is not essential for translation of most  mRNAs but enhances the differentiation of translational efficiencies genome-wide.This finding suggests that eIF4G is more critical for ribosome attachment to mRNAs than for scanning long, structured 5' UTRs. Our results also indicate that eIF4G, and the closed-loop mRNP it assembles with the m Attachment of the 43S complex at the mRNA 5' end is stimulated by the eIF4F complex, comprised of cap-binding protein eIF4E, the scaffold subunit eIF4G, and the DExD/H-box helicase eIF4A, which is thought to provide a single-stranded region in the mRNA for recruiting the ribosome. Binding sites in eIF4G for either eIF3  or eIF5 and eIF1 (in yeast) are thought to facilitate recruitment of the 43S PIC to eIF4F bound at the cap structure. eIF4G also harbors a binding site for the poly(A)-binding protein (PABP) that, together with an RNA binding domain in the middle region of mammalian eIF4G, increases the stability of eIF4F binding to the mRNA 5' end and also mediates circularization of mRNA in the activated eIF4F·mRNA·PABP mRNP -Met incorporation under these conditions  by a factor > 5 compared to the P/M ratio for the WT strain under the same conditions Figure . This iss Figure . Thus, iAlthough a significant level of translation continues following the extensive depletion of eIF4G in the degron mutant Figure , it was RPL41A and RPL41B mRNAs were examined because their coding sequences, of only 78 nt , and broken by vortexing with glass beads. Polysomes were separated by loading whole cell extracts (WCEs) onto 4.5-45% sucrose gradients and centrifuged in a SW41Ti rotor (Beckman) at 39,000 rpm for 2.5 h at 4°C as described previously  4G/2-[ΔΔCt(HP-T)] WT)(0.114/0.308), where 0.114 and 0.308 are the fractions of 18S rRNA present in heavy polysomes in the mutant and WT cells, respectively, ΔΔCt(HP-T) = ΔCt(HP)norm -ΔCt(T)norm, ΔCt(HP)norm = Ct(HPGOI)-Ct(HP18S), and ΔCt(T)norm = Ct(TGOI)-Ct(T18S). The primers employed for qRT-PCR analysis of the mRNAs for these genes are listed in Table S3  and total RNA isolated from WT and eIF4G-depleted cells. Spreadsheet 1 "microarray of 5869 ORFs" contains the data from microarray analysis of 5869 ORFs using HP and total RNA samples from WT and eIF4G mutant strains, from projects  I-III. Columns A-C contain the Nimblegen sequence identification number, SGD ORF name, and functional annotation, respectively. Columns D-F contain the log2 of signal intensity for the HP RNA sample from the eIF4G mutant in project I (log4G-HP_I), the cognate value for total RNA (log4G-T_I), and the corresponding TE value (TE 4G_I) calculated as the ratio of these two intensities (HP/T), respectively. Columns G-I contain the corresponding data found in D-F for the HP and T RNA samples but from the WT strain in project I. Columns J-L and M-O contain the corresponding data found in D-F and G-I but from project II, and columns P-R and S-U contain the cognate data from project III. Columns V and W contain the mean TE4G and mean TEWT values, respectively, obtained by averaging the TE values from projects I-III. The log2 of the values in columns V and W were plotted in Figure 4G to TEWT values from projects I, II, and III, respectively, contained in columns F and I, L and O, and R and U, respectively. Column AA contains the average of columns X-Z, AB contains the S.E.M. calculated from the data in X-Z, and column AC contains the results of a two-tailed Students t-test conducted to determine if the set of TE4G values in columns F, L, and R are significantly different from the set of TEWT values in columns I, O, and U. Spreadsheet 2 "t-test calcs" contains these last calculations. Spreadsheet 3 "" contains the 94 genes with mean TE4G/TEWT values (in column AA) ≤ 0.71, and also includes in columns AD-AI the lengths and MFEs of 5' UTRs, and numbers of uORFs, all from [4G/TEWT values (in column AA) ≥ 1.4, with the identical column definitions described above for sheet 3. Spreadsheet 5 "Lawless UTRs" contains the data from Additional file 9 of Lawless et al [all from , and 3' all from . Spreadsss et al . Spreadsss et al .Click here for fileComparison of translational efficiencies between WT and eIF4G degron mutant. -Figure S1: Comparison of the ratios of TE values between the WT and eIF4G degron mutant determined by qRT-PCR and microarray analysis of selected mRNAs. -Figure S2: Comparison of translational efficiencies for all genes with ORF lengths < 625 nt between WT and eIF4G degron mutant cells. -Figure S3: Inverse correlation between translational efficiency and coding sequence length is dampened by eIF4G depletion, especially for genes with short ORFs. -Table S1: Variances from MA plots of microarray data. -Table S2: PCR Primers for measuring polysome distributions of selected mRNAs. -Table S3: Primers for qRT-PCR determination of TE4G/TEWT ratios and comparison of mean TE4G/TEWT ratios determined by microarray versus qRT-PCR analysis.Click here for fileExcel file containing results of microarray analysis of translational efficiencies using RNA from "light polysomes" (LP) and total RNA isolated from WT and eIF4G-depleted cells. Spreadsheet 1 "Project averages LP over T_4G,WT" contains our data from microarray analysis of 5868 ORFs using LP and total RNA samples from WT and eIF4G mutant strains, from all three projects (I-III). Columns A-B contain the Nimblegen sequence identification number and SGD ORF name, respectively. Columns C-F contain the mean signal intensities, averaged over all three technical replicates and all three projects  for the LP and total RNA samples from the eIF4G mutant (C-D) and from WT (E-F), respectively. Columns G and H contain the mean TE4G and mean TEWT values, respectively, calculated as the ratio of column C to column D (column G), or the the ratio of column E to column F (column H). Column I is the ratio of column G to column H. Spreadsheet 2 "Arava et al, 1-3 ribos per mRNA" contains the list of 564 genes whose polysomal mRNAs contain predominately 1-3 ribosomes, from Arava et al [va et al . SpreadsClick here for file


Cyprinus carpio) involves cyclical seasonal gene reprogramming as an adaptation response to its natural environment, when rRNA synthesis and processing are profoundly affected. Epigenetic mechanisms primarily contribute to the transcriptional modulation of ribosomal genes concomitant with the acclimatization process, thus significantly regulating this process. The aim of this study was to describe the presence of several H2A.Z subtypes in carp, and assess the role of H2A.Z on the ribosomal cistron in summer- and winter-acclimatized carp.The specific deposition of histone variants into chromatin is an important epigenetic mechanism that contributes to gene regulation through chromatin architectural changes. The histone variant H2A.Z is essential in higher eukaryotes, and its incorporation within chromatin is a relevant process for gene expression and genome stability. However, the dual positive and negative roles of H2A.Z in gene regulation still remain unclear. We previously reported that acclimatization in common carp fish  was strongly enriched on regulatory regions of the ribosomal cistron in summer-acclimatized carp. Additionally, H2A.Z was present in both heterochromatin and euchromatin states on ribosomal cistron and RNA polymerase II promoters.This paper reports for the first time about the transcriptional expression of four different H2A.Z subtypes belonging to the same organism. Remarkably, a novel H2A.Z.7 was found, which corresponds to a tissue-specific histone subtype that contains seven amino acid residues longer than the canonical H2A.Z. Moreover, H2A.Z enrichment through the ribosomal cistron was significantly higher during summer, when rRNA transcription and processing are highly active, than it was in winter. Similar patterns of H2A.Z enrichment are found in two seasonally active promoters for genes transcribed by RNA polymerase II, the Our study revealed seasonally-dependent H2A.Z enrichment for active ribosomal cistron and RNA polymerase II promoters during the carp environmental adaptation. Moreover, seasonal H2A.Zub enrichment appears as a specific mechanism contributing to the regulation of chromatin architecture under natural conditions. The existence of several H2A.Z subtypes in carp suggests that the epigenetic regulation in this species constitutes a complex and finely tuned mechanism developed to cope with seasonal environmental changes that occur in its habitat. Living organisms are constantly exposed to environmental fluctuations. In order to survive, they must adapt by inducing compensatory responses . In partIt is well known that eukaryotic gene regulation and expression are connected to the structural organization of chromatin. Chromatin, through its capability to transiently pack and unpack the genome, plays a key role in controlling access to genetic information . The basH2A.Z genes are essential for the development and viability of many organisms, such as Drosophila melanogaster were deduced as the following oligonucleotides: Z-380pb, F 5′-GCAGGTGGAAAAGCAGGT AAAG-3′ and Z-380pb, R 5′-GCGGTTTTCTGCTGGCCCTTCTT-3′. A 380 bp fragment corresponding to a partial mRNA region of H2A.Z was amplified by RT-PCR using carp liver tissue as a template. The product was cloned in the pGEM-T vector  and sequenced. Full mRNA of different carp H2A.Z subtypes were isolated using FirstChoice RLM-RACE  with specific oligonucleotides deduced from the fragments described earlier. The 5′UTR region was completed with Z-380pb, R and 5′Z inner 5′-CCAGCTGTAGATGA CGAG-3′, whereas the 3′UTR region was completed with Z-380 pb, F and 3′Z inner 5′-GTCTACAGTGCAGCCATC-3′. All PCR reactions were performed with Phusion High-Fidelity DNA Polymerase  and DNA sequencing services were performed by Macrogen .From A total of 100 mg of pituitary tissue from summer- and winter-acclimatized carp were fixed using 4% paraformaldehyde (w/v). Sections were embedded in Paraplast and cut at 5 μm in a cryostat Leica CM1850 . The primary antibody used was anti-H2A.Z  incubated at 4°C for 16 hours and then washed three times with PBS. The secondary antibody was anti-rabbit IgG (1:400) conjugated to Alexa Fluor 647  and 4',6-diamidino-2-phenylindole (DAPI) (1:1000), which was incubated for 1 hour at 25°C. Confocal microscopy was carried out in Axiovert 100 M  and analyzed with ImageJ software , Bethesda, MD, USA).260nm. The cDNA was synthesized from 2 μg of total RNA with M-MLV Reverse Transcriptase (Promega) and random primers according to the manufacturer’s instructions. The resulting cDNAs were PCR-amplified in GoTaq Green Master Mix (Promega) using oligonucleotides, showed in Additional file Total RNA was purified from tissues, such as the liver, brain, pituitary, testis, and gills using TRIzol reagent . The quality of RNA was evaluated on 1.2% agarose gel electrophoresis and quantified spectrophotometrically at ART-qPCR was performed with the appropriate oligonucleotides  and anti-H3 . The ChIP-reChIP assays were performed immunoprecipitating first with anti-H2A.Z antibodies, and then an additional immunoprecipitation with polyclonal histone H4 (acetyl K12), anti-histone H3 , and anti-ubiquitin . In the last case, although we did not have antibodies for carp H2A.Zub, our results were in agreement with H2A.Zub form because all data values obtained were higher than the unspecific IgG IP . The immunoprecipitated DNA was amplified by qPCR on an Mx3000P Real-Time PCR System (Stratagene) using oligonucleotides corresponding to IGS, T0`, T0, CP and 5.8S regions of carp rDNA  assays were undertaken as previously described . For eact method [ChIP-qPCR was performed using SYBR Master Mix (Stratagene). The specificity of the PCR reaction was assessed by melting-temperature profiles. The fold enrichment was calculated using the ΔΔCt method :tx corresponds to the Ct value for each specific antibody used. All assays were performed in triplicate and repeated with three different animal samples for each season. Results represent the mean (± SD) of three experiments.Where CH2A.Z were retrieved from the GenBank database (Additional file The CDS from different eukaryotes corresponding to bp: base pairs; CDS: Coding DNA sequences; ChIP: Chromatin immunoprecipitation, ChIP-reChIP, sequential chromatin immunoprecipitation; CNI: Close-Neighbor-Interchange; CP: Core promoter; DAPI: 4',6-diamidino-2-phenylindole; ECL: Enhanced chemiluminescence; H2A.Zub: ubiquitylated-H2A.Z; H3K9me3: Trimethylated histone H3 at lysine 9; H4K12ac: acetylated histone H4 at lysine 12; IgG: Immunoglobulin G; IGS: Intergenic spacer; ME: Minimum evolution; PBS: Phosphate buffered saline; PCR: Polymerase chain reaction; PTMs: Post-translational modifications; qPCR: quantitative PCR; RT: Reverse transcription; RT-PCR: Reverse transcription polymerase chain reaction; SD: Standard deviation; UCD: University College Dublin; WGD: Whole genome duplication.The authors declare that they have no competing interests.NS carried out the experiments and wrote the manuscript. GN carried out some ChIP assays. MR carried out the immunofluorescence experiment. * MR is currently working at Departamento Ciencias Fundantes, Universidad Central de Chile. MA directed the research and co-wrote the manuscript. AM assisted with project design and development, and drafted the manuscript. All authors read and approved the final manuscript.Carp fish exposed to a wide range of temperature and photoperiod conditions. Graph of temperature and photoperiod changes during a seasonal annual cycle.Click here for fileIdentification and analysis of H2A.Z subtypes isolated from carp. Multiple nucleotide and amino acid sequence alignment of H2A.Z subtypes isolated from carp are shown. Black boxes represent the precise sites of nucleotide differences between CDS from H2A.Z.2.1, H2A.Z.3.1, H2A.Z.3.2, and H2A.Z.7. The deduced amino acid sequences of each H2A.Z subtype are shown in colors. The additional seven amino acids found in carp H2A.Z.7 are denoted with a red rectangle. CDS, coding DNA sequences.Click here for fileJAK1 during seasonal acclimatization of carp. Transcriptional expression analysis of JAK1 in liver tissues from summer- and winter-acclimatized carp. The mRNA levels of JAK1 were calculated using the Pfaffl method [Constitutive gene expression of l method . Three dClick here for fileList of oligonucleotides used for RT-PCR, RT-qPCR, and ChIP assays. ChIP, chromatin immunoprecipitation; RT-PCR, reverse transcription polymerase chain reaction; RT-qPCR, reverse transcription quantitative PCR.Click here for fileGenBank Accession Numbers of CDS used for the phylogenetic analysis. The list depicts histone H2A.Z in eukaryotic organisms used in the phylogenetic analysis. CDS, coding DNA sequences.Click here for file


To study the use of size, maturation and CYP2D6 genotype score as predictors of i.v. tramadol (M) disposition throughout human life, published observations were pooled -6.M and O-demethyltramadol (M1) observations in 295 subjects  were available for population PK analysis . Covariates were weight, age, sex, disease  and CYP2D6 genotype score. A sigmoid maturation model was used to describe changes in M (CLPO + CLPM), M1 formation (CLPM) and M1 elimination (CLMO) clearance. Phenotype-based and genotype-based models were used to distinguish poor CLPM subjects.Differences in M disposition between children and adults were largely accounted for by maturation and size. CKLPM  and CLPO  display fast maturation, while CLMO matures slower. The phenotype-based mixture model estimated that 8.6 were slow metabolizers . Genotype-based estimates were also lower (68%) but not all subjects with a low CYP2D6 score were in the poor metabolizer group.Maturation of M elimination occurs early with 50% of adult values at full-term age. Maturation and age are key predictors, while CYP2D6 genotype score only explains some of the variability in M disposition.


Background: The corazonin system is of importance for insects to control multiple physiological processes.Results:Bombyx BNGR-A21 was activated in signaling and internalization by direct interaction with corazonin.Conclusion:Bombyx corazonin was a specific ligand for dual Gq- and Gs-coupled BmCrzR.Significance: Our findings will lead to a better understanding of the corazonin system in regulation of fundamental physiological processes. Drosophila melanogaster, Manduca sexta, and Musca domestica. However, detailed information on the signaling and major physiological functions of corazonin and its receptor is largely unknown. In the current study, using both the mammalian cell line HEK293 and insect cell lines BmN and Sf21, we paired the Bombyx corazonin neuropeptide as a specific endogenous ligand for the Bombyx neuropeptide G protein-coupled receptor A21 (BNGR-A21), and we therefore designated this receptor as BmCrzR. Further characterization indicated that synthetic BmCrz demonstrated a high affinity for and activated BmCrzR, resulting in intracellular cAMP accumulation, Ca2+ mobilization, and ERK1/2 phosphorylation via the Gq- and Gs-coupled signaling pathways. The direct interaction of BmCrzR with BmCrz was confirmed by a rhodamine-labeled BmCrz peptide. Moreover, experiments with double-stranded RNA and synthetic peptide injection suggested a possible role of BmCrz/BmCrzR in the regulation of larval growth and spinning rate. Our present results provide the first in-depth information on BmCrzR-mediated signaling for further elucidation of the BmCrz/BmCrzR system in the regulation of fundamental physiological processes.Corazonin, an undecapeptide neurohormone sharing a highly conserved amino acid sequence across Insecta, plays different physiological roles in the regulation of heart contraction rates, silk spinning rates, the induction of dark color and morphometric phase changes, and ecdysis. Corazonin receptors have been identified in  Periplaneta americana with a functional ability to accelerate the heartbeat (7]Corazonin (with Arg as the seventh residue from the N terminus) is the most universal in insect lineages eartbeat . The strlineages . Bombyx byx mori , and it byx mori . Studiesbyx mori .Manduca sexta  was identified from the fruit fly  in 2002 , followeM. sexta , Anophel gambiae , Apis meellifera , and Musomestica . These rignaling . The Crzmulation . In M. dg system .Bombyx neuropeptide G protein-coupled receptor BNGR-A21 has been identified as a corazonin-like receptor in B. mori . The pEGFP-N1 and pCMV-FLAG vectors were purchased from Clontech Laboratories, Inc. and Sigma, respectively. Monoclonal anti-FLAG® M2 antibody, monoclonal anti-FLAG M2-FITC antibody, horseradish peroxidase (HRP)-conjugated anti-mouse IgG, and Nifedipine were purchased from Sigma. Anti-phospho-ERK1/2 (Thr202/Tyr204) and anti-ERK1/2 antibodies were purchased from Cell Signaling Technology . H89, Go6983, U73122, U0126, cholera toxin (CTX), and pertussis toxin (PTX) were purchased from Tocris Bioscience. The membrane probe DiI , nuclear dye, RIPA lysis buffer, and horseradish peroxidase-conjugated secondary antibody were purchased from Beyotime . [Arg7]Corazonin was synthesized by GL Biochem Ltd. .Cell culture media and fetal bovine serum (FBS) were purchased from HyClone . G418, Lipofectamine 2000, and Opti-MEM® I reduced serum medium were purchased from Invitrogen. SuperFectinTMAB330442 as described below. The coding sequence of A21 was amplified for the pCMV-FLAG vector using the sense primer 5′-AAGCTTATGGACAACGAAGGCAACAGTAC-3′ and the antisense primer 5′-GGATCCTTATAACAAACTAATGTTCTGTCCAT-3′; an additional pair of primers was also designed for the pEGFP-N1 vector: the sense primer 5′-AAGCTTGCCACCATGGACAACGAAGGCAACAGTAC-3′ and the antisense primer 5′-GGATCCCGTAACAAACTAATGTTCTGTCCATTCG-3′. The corresponding PCR products were cloned into the pCMV-FLAG, pEGFP-N1, pBmIE1-FLAG, and pBmIE1-EGFP expression vectors using the Rapid DNA Ligation Kit  and we named these vectors Flag-A21, A21-EGFP, BmFlag-A21, and BmA21-EGFP, respectively. All constructs were sequenced to verify the correct sequences, orientations, and reading frames. Arrestin3-EGFP was generated as previously described . The BNGR-A21 cDNA plasmid constructs were transfected or cotransfected into HEK293, BmN, and Sf21 cells using Lipofectamine 2000 (Invitrogen) and SuperFectinTM II  according to the manufacturer's instructions. Selection for stable expression was initiated by the addition of G418 (800 μg/ml) 1–2 days after transfection.The human embryonic kidney cell line (HEK293) was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (HyClone) and 4 m5 cells were washed with phosphate-buffered saline (PBS) containing 0.5% BSA (FACS buffer) and incubated with 10 μg/ml of FITC-labeled anti-FLAG M2 monoclonal antibody (Sigma) in a total volume of 100 μl. After incubation for 60 min at 4 °C, the cells were pelleted and washed three times in FACS buffer. The cells were then fixed with 4% paraformaldehyde in FACS buffer and subjected to flow cytometry analysis on a FACScan flow cytometer .Approximately 2 × 10After seeding in a 48-well plate overnight, HEK293 or Sf21 cells transiently or stably cotransfected with BNGR-A21 and pCRE-Luc were grown to 90–95% confluence, stimulated with the corazonin peptides in DMEM without FBS, and incubated for 4–6 h at 37 °C. Luciferase activity was detected by a firefly luciferase assay kit . The cAMP concentration was assessed using a commercially available cAMP detection kit (R&D Systems).6 cells/ml in Hanks' balanced salt solution. The cells were detached by a Nonenzymatic Cell Dissociation Solution  or 0.02% EGTA and then loaded with 3 μm Fura 2-AM  for 30 min at 37 °C. Cells were washed twice in Hanks' solution and then resuspended in Hanks' solution at a concentration of 3 × 107 cells/ml. Calcium flux was measured using excitation wavelengths of 340 and 380 nm in a fluorescence spectrometer .The stable FLAG-A21- and BmFlag-A21-expressing HEK293 and Sf21 cells were washed twice with phosphate-buffered saline and resuspended at 5 × 10The HEK293 and Sf21 cells transiently or stably expressing BNGR-A21 were seeded in 24-well plates and starved for 60 min in serum-free medium to reduce background ERK1/2 activation and to eliminate the effects of the change of medium. After stimulation with the agonist, the cells were lysed by RIPA buffer . Equal amounts of total cell lysate were size-fractionated by SDS-PAGE (10–12%) and transferred to a PVDF membrane (Millipore). Membranes were blocked in blocking buffer (TBS containing 0.05–0.1% Tween 20 and 5% nonfat dry milk) for 1 h at room temperature and then incubated with rabbit monoclonal anti-phospho-ERK1/2 antibody  and anti-rabbit HRP-conjugated secondary antibody  according to the manufacturers' protocols. Total ERK1/2 was assessed as a loading control after p-ERK1/2 chemiluminescence detection using an HRP substrate purchased from Cell Signaling .l-lysine. The cells were stimulated with the indicated concentrations of the agonist at the indicated times the following day. The medium was aspirated, and the cells were washed once with Tris-buffered saline (TBS). After fixing the cells for 5 min at room temperature with 3.7% formaldehyde in TBS, the cells were washed three times with TBS and then blocked for 60 min with 1% bovine serum albumin/TBS followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:5000 in 1% BSA/TBS) for 60 min. To each well, 200 μl of HRP substrate (Sigma) was added, and the samples were incubated at 37 °C for 20–30 min. The reactions were stopped by adding an equal volume of 1% SDS, and plates were read at 405 nm in a microplate reader .HEK293 cells stably transfected with the FLAG-A21 construct were seeded in 24-well dishes coated with poly-l-lysine and allowed to attach overnight under normal growth conditions. After 24 h, cells were stained with the membrane probe DiI  at 37 °C for 5–10 min, fixed with 4% paraformaldehyde for 15 min, and finally incubated with DAPI for 10 min. For the internalization assay, cells expressing A21-EGFP were treated with different concentrations of corazonin for different incubation times at 37 °C. For the β-arrestin and kurtz translocation assays, cells were cotransfected with the corresponding FLAG-A21 receptor constructs and β-arrestin1-EGFP, β-arrestin2-EGFP, or Bmkurtz-EGFP. After transfection, the cells were seeded onto glass coverslips, allowed to recover for 24–36 h in a 6-well plate, and incubated with 1 ml of DMEM without FBS, and if necessary, the corresponding ligands were added to stimulate the receptors for 5–15 min. The cells were visualized by fluorescence microscopy on a Zeiss LSM 510 laser scanning confocal microscope attached to a Zeiss Axiovert 200 microscope using a Zeiss Plan-Apochromat ×63, 1.40 NA oil immersion lens.For receptor surface expression analysis and internalization assays, HEK293 and BmN cells transiently or stably expressing receptor-EGFP were seeded onto glass coverslips coated with 0.1 mg/ml of poly-d-lysine-coated 96-well plates 1 day before the experiments. Cells were washed once with PBS, and 50 μl of PBS with 0.2% BSA was added to each well. Samples of labeled and unlabeled peptides were made in ligand buffer at a concentration four times the final concentration specified. An amount of 25 μl of labeled ligand and 25 μl of buffer or unlabeled ligand was added to each well. Cells were incubated at room temperature for 90 min and transferred onto ice. Cells were then washed three times with 100 μl of ice-cold PBS with 0.1% BSA as previously described methoxycarbonyl strategy on a 430A peptide synthesizer  and a 9050 Pepsynthesizer Plus . Crude peptides were purified by preparative reverse-phase high-performance liquid chromatography using a Dynamax-300 Å C18 25 cm × 21.4-mm inner diameter column with a flow rate of 9 ml/min and two solvent systems of 0.1% TFA/H2O and 0.1% TFA/acetonitrile. Fractions containing the appropriate peptide were pooled together and lyophilized. The purity of the final product was assessed by analytical reverse-phase high-performance liquid chromatography, capillary electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The corazonin peptides were prepared by solid-phase synthesis using the B. mori P50 were reared at 27 ± 3 °C and with a light:dark regime of 12 h:12 h on fresh mulberry leaves. Fourth instar larvae of similar size were divided into different treatment groups in the experiment. These larvae began molting on day 5 of the fourth instar and began spinning on day 7 of the fifth instar. Larvae were immobilized on ice before injection. B. mori CrzR dsRNA was generated in vitro by the MEGAscript® T7 Kit from Invitrogen according to the manufacturer's instructions using the sense primer 5′-TAATACGACTCACTATAGGGCTTCATTGGCAATGTGGCA-3′ and the antisense primer 5′-TAATACGACTCACTATAGGGGATTCTCGGTCACTATGCTGGT-3′. Corazonin and CrzR dsRNA were dissolved in PBS and diethyl pyrocarbonate/H2O, respectively, and injected into fourth and fifth instar larvae using a 10-μl microsyringe through the abdominal segments without injury of the silk glands. PBS without corazonin and pXef dsRNA (from MEGAscript® T7 Kit) were injected as the control. The weight of the larvae was recorded at an identical time each day. Bombycis and remnant mulberry leaves were weighed when completely dry. The cocoon layer and cocoon with pupa were weighed at the end of spinning.50 values. Statistical significance was determined using Student's t test. Probability values that were less than or equal to 0.05 were considered significant.All results are expressed as the mean ± S.E. Data were analyzed using nonlinear curve fitting (GraphPad Prism version 5.0) to obtain ECBombyx neuropeptide receptor BNGR-A21 has been proposed to have a close neighbor-joining phylogenetic tree with the Drosophila corazonin receptor by genomic and phylogenetic analysis (AB330442.1) was obtained by RT-PCR from the silk gland of silkworm larvae. As shown in Fig. 1A, the predicted open reading frame of BNGR-A21 encoding a protein with 437 amino acids contains a typical structure of seven putative transmembrane domains and shows 37.2, 36.7, 46.1, and 81.3% identity to D. melanogaster, A. gambiae, A. mellifera, and M. sexta corazonin receptors, respectively. To further assess its functional activity as a transmembrane receptor, BNGR-A21 with an N-terminal FLAG tag and BNGR-A21 with the enhanced green fluorescent protein (EGFP) fused to the C terminus were constructed and stably expressed in human embryonic kidney 293 (HEK293) cells and insect Spodoptera frugiperda (Sf21) or B. mori (BmN) cells. Significant cell surface expression was detected by FACS and ELISA analysis  and observed under fluorescence microscopy , suggesting that the N-terminal FLAG tag and the C-terminal GFP tag did not affect BNGR-A21 expression and orientation in the cell membrane in HEK293 and Sf21 cells.The analysis . The fulA. gambiae  and neuropeptide F-1 (NPF1) were unable to activate BNGR-A21 (D and E). As a control, no change in the CRE-driven luciferase activity was detected in parental HEK293 and Sf21 cells. Pretreatment with 100 ng/ml of PTX, an inhibitor of Gαi protein, exhibited no effect on cAMP generation in BNGR-A21-expressing cells stimulated by corazonin, whereas stimulation with CTX, which constitutively activates Gαs subunits, led to a remarkable increase in the cellular levels of cAMP (A–C), suggesting that Gs was likely involved in the BNGR-A21-mediated signaling in HEK293 and Sf21 cells.Previous studies have shown that the corazonin receptor was only activated by its endogenous corazonin, leading to cAMP accumulation, in  gambiae , howeverBNGR-A21 , D and E of cAMP , A–C, suA and B, BmCrz-activated BNGR-A21 triggered a significant increase in intracellular cAMP formation compared with the Gq-coupled human histamine H1 receptor (H1R), and cotreatment of BmCrz with forskolin induced a significant cAMP accumulation compared with treatment with only forskolin in both HEK293 and Sf21 cells. Moreover, BmCrz-induced luciferase activity was significantly inhibited by treatment with the Gq inhibitor YM-254890, and PKA inhibitor H89, PKC inhibitor Go6983, calcium chelators (EGTA and BAPTA-AM), and PLC inhibitor U73122 (C and D), indicating that both Gq and Gs are likely involved in BNGR-A21-mediated through PKA, PKC, Ca2+, and phospholipase C activation of CRE-driven reporter transcription.To confirm whether corazonin can bind to BNGR-A21 and trigger a second messenger cAMP signaling pathway, a direct cAMP assay by ELISA was used in this study. As shown in r U73122 , C and D2+ change in the A21-expressing cells using the calcium probe fura-2. As indicated in A and B, corazonin peptides elicited a rapid increase of Ca2+ in the FLAG-A21-expressing cells in a dose-dependent manner in both HEK293 and Sf21 cells. Preincubation with 1 μm Gq inhibitor YM-254890 led to a significant decrease in the intracellular Ca2+ mobilization (C). BNGR-A21-mediated intracellular Ca2+ mobilization was found to be sensitive to the PLC inhibitor U73122, calcium chelators EGTA and BAPTA-AM, and l-type calcium channel inhibitor nifedipine (D--F). Taken together, these results suggest that the BNGR-A21 receptor was specifically activated by the BmCrz peptide by primarily coupling to the Gq signaling pathway and also dually coupling to the Gs signaling cascade, leading to intracellular Ca2+ mobilization and cAMP accumulation. Therefore, we suggest designating BNGR-A21 as the Bombyx corazonin receptor (BmCrzR).We then examined the effects of corazonin peptides on the intracellular Calization C. BNGR-Afedipine , D--F. T4]BmCrz). Functional assays indicated that the Rho-[Lys4]BmCrz peptide could activate BmCrzR with an EC50 value of 86.1 nm (A). Using a displacement analysis method, Rho-[Lys4]BmCrz was found to compete with unlabeled BmCrz, with an IC50 value of 0.49 nm (B), suggesting that BmCrz directly binds and activates BmCrzR.To confirm the direct interaction of BmCrz peptides with BNGR-A21 receptors, we designed and synthesized a fluorescent BmCrz analog conjugated with rhodamine red. Although corazonin has been shown to be highly conserved in structure, sequence alignment demonstrates the existence of a few mutants including Q1E, Q4T, and R7H. Therefore, we synthesized the Q4K peptide and conjugated it with rhodamine red at the Lys residue  forms of these kinases  but not by the EGFR tyrosine kinase inhibitor AG1478, PI3K inhibitor wortmannin, and Src inhibitor PP2, confirming that BmCrz is the major agonist for BmCrzR.It has been well established that the activated GPCRs signal the ERK1/2 signaling pathway, which functions in transcriptional regulation that is involved in control of diverse processes ranging from proliferation and differentiation to apoptosis and can be used to assess the functional outcome of receptor stimulation , 36. We  kinases . As indir BmCrzR . In addir BmCrzR , E-G but2+ mobilization and CRE-driven luciferase activity (data not shown). Observation with confocal microscopy revealed that the fluorescence of BmCrzR-EGFP was primarily localized in the plasma membrane and was dramatically and rapidly internalized into the cytoplasm in response to the BmCrz peptide in HEK293 and BmN cells (A-D). The internalization of BmCrzR-EGFP was detectable 5 min after agonist stimulation and reached a maximum within 60 min. The internalized BmCrzRs were largely clustered in the perinuclear region at 30 min (B-D). The quantitative ELISA data are highly consistent with our observation by confocal microscopy. Moreover, we cloned kurtz, a novel nonvisual arrestin in insects, from B. mori and constructed vectors to express kurtz and arrestins fused with EGFP at the C terminus. HEK293 cells were cotransfected with BmCrzR and arrestin-EGFP or kurtz-EGFP and were examined by confocal microscopy after exposure to the agonist. As shown in E and F, in the presence of the BmCrz peptide, β-arrestin1-EGFP, β-arrestin2-EGFP, and kurtz-EGFP showed the same ability to be recruited to the plasma membrane, although we observed that β-arrestin2-EGFP and kurtz-EGFP, but not β-arrestin1-EGFP, remained associated with receptor throughout internalization (data not shown). Our data on receptor internalization confirm that BmCrz is the major agonist for BmCrzR.Agonist-induced internalization is a well characterized phenomenon for most GPCRs that is believed to contribute to the regulation of the strength and duration of receptor-mediated cell signaling . To visumN cells , A-D. Tht 30 min , B-D. ThA, in fifth instar larvae, BmCrzR expression was detectable in most of the tissues of which the silk gland was found as a major expression site, whereas other tissues, including brain, fat body, epidermis, midgut, Malpighian tubule, testis, and ovary, express BmCrzR at a lower level. This finding is in agreement with the observations made by Yamanaka et al. . These results suggest the possible role of BmCrZ and BmCrzR in the negative regulation of silkworm growth and silk production.To examine the tissue-specific expression of BmCrzR, RT-PCR was used to analyze the expression of BmCrzR in silkworm larvae. As shown in Fig. 8a et al. . To asseD. melanogaster 10 years ago, however, detailed information on the signaling and physiological roles for corazonin and the corazonin receptor remains largely unknown. Recently, the Bombyx neuropeptide GPCR A21 (BNGR-A21) was identified as a corazonin-like receptor in B. mori by genomic data mining and phylogenetic analysis .The insect neuropeptide corazonin was initially isolated from cockroaches in 1989 and the first CrzR was identified by functional expression from the fruit fly analysis –27. In t1 or AKH . We, theM. sexta, the corazonin receptor exhibits high sensitivity and selectivity for corazonin in Xenopus oocytes . BmCrzR-EGFP that was stably expressed in both HEK293 and Sf21 cells exhibited functional activities in intracellular cAMP accumulation and Calization . To conflization , from B.membrane . The arran GPCRs . The kurrrestin1 . No diffrrestin1 . Our dats-coupled AKHR system is well known to mobilize carbohydrates and lipids during energy-expensive activities such as long-distance flight (P. americana (Bombyx corazonin and its receptor in the negative regulation of growth and silk production. Therefore, further efforts should be focused on the molecular and functional dissection of the physiological roles played by Bombyx corazonin and its receptor.Phylogenetic studies have suggested that not only the AKH and corazonin neuropeptides but also the AKH and corazonin receptors are closely related, apparently originating from one ancestral receptor having one ancestral ligand , 28, 52.e flight , whereasmericana , inductimericana , reductimericana , initiatmericana , and regmericana . In thisBombyx orphan receptor BNGR-A21 as a receptor for the neuropeptide Bombyx corazonin. BmCrzR was activated by direct interaction with the corazonin peptide, leading to intracellular Ca2+ mobilization via a Gq-dependent signaling cascade, cAMP accumulation, and ERK1/2 phosphorylation through possibly Gs-and Gq-dependent signaling pathways. In response to the corazonin peptide, BmCrzR underwent a rapid internalization via an arrestin-dependent pathway (In summary, in the present study, we have identified the  pathway . Further


Streptomyces-E.coli shuttle vector pZRJ362 combining the xylose isomerase promoter and amylase terminator. A gene encoding the endoglucanase Cel6A in Thermobifida fusca was amplified by PCR, cloned into Streptomyces lividans host strain using the novel expression vector and Pichia pastoris GS115 host strain using the vector pPICZα-C, respectively. Afterwards, the expression pattern and the maximum expression level were comparatively studied in both expression systems. The maximum enzyme activity of Cel6A-(His)6 secreted in S. lividans supernatant after 84-h of cultivation amounted to 5.56 U/mL, which was dramatically higher than that secreted in P. pastoris about 1.4 U/mL after 96-h of cultivation. The maximum expression level of Cel6A-(His)6 in S. lividans supernatant reached up to 173 mg/L after 84-h of cultivation. The endoglucanase activity staining SDS-PAGE showed that there were some minor proteins in S. lividans supernatant which may be the Cel6A derivant by proteolytic degradation, while there was no proteolytic product detected in supernatant of P. pastoris.In this study, we constructed a novel  Escherichia coli [Pichia pastoris [Streptomyces lividans [P. pastoris and S. lividans was much higher than that achieved in E. coli, even expression of some actinomycetes enzymes in E. coli was unsuccessful. With respect to comparision of the expression yield in P. pastoris and S. lividans, the results obtained to date showed the expression yield of P. pastoris would seem to be superior to those of S. lividans [P. pastoris and actinomycetes. Theoretically, owing to similarities in their codon usage, with C or G preferentially as the third base of most codons, Streptomyces could be an ideal host for the expression of genes from actinomycetes, gram-positive bacteria with a high content of G + C nucleotides [Streptomyces was the inefficient expression vector or promoter. Thus, it is highly desirable to develop an efficient expression vector in Streptomyces to overproduct proteins of interest from actinomycetes, especially overexpress the thermostable enzyme from the thermophilic genera, such as Thermobifida and Thermoactinomyces because of their overall inherent stability.Microbial exploitation for the production of industrial enzymes like keratinase, pectinase, cellulase, xylanase, and lipase has attracted more and more attention. Actinomycetes constitute a formidable group of industrially important microorganisms that have been explored for the production of a variety of enzymes. A large number of enzymes have been found and isolated from actinomycetes, including phospholipase , phosphahia coli –16, Pichpastoris ,12,17 anlividans ,14,18. Clividans –21, despleotides . The mosThermobifida fusca is a major degrader of plant cell walls owing to its abundant extracellular cellulases and xylanases [cel6A (GenBank accession No. M73321) encoding an endoglucanase was selected to study the capacity of the novel expression vector we constructed in this paper. The reason for selecting endoglucanase Cel6A lies in the potential industrial application because of its thermostability, utility through a broad pH range and high activity [Among the thermophilic actinomycetes, ylanases . In thisactivity .T. fusca YX in Streptomyces has been reported recently, its maximum expression level was relatively low (about 64 mg/L) [Streptomyces-E. coli shuttle vector for overproduction of the endoglucanase Cel6A from T. fusca and to compare it with the production obtained in P. pastoris. In the course of these experiments, comparisons of the expression pattern, the maximum expression level in both expression systems were investigated.Although an expression vector for production of endoglucanase Cel6A from 64 mg/L) . TherefoE. coli and Streptomyces because of oriT. The vector was designed to enable selection with Amp and Thiostrepton antibiotic in E. coli and Streptomyces, respectively.As presented in cel6A and pZRJ362-cel6A-(His)6, containing approximately 1700 bp gene sequence which encoded endoglucanase Cel6A transcribed under the control of the Actinoplanes missouriensis xylose isomerase promoter, were conjugated in S. lividans and the plasmid vector pZRJ362 was used as a negative control. The resultant S. lividans transformants were cultured in TSB medium for 96 h. As a result, a large amount of T. fusca Cel6A or Cel6A-(His)6 was secreted in S. lividans supernatants using its signal sequence, and the enzyme activity in supernatants amounted to 5.06 and 5.20 U/mL, respectively. SDS-PAGE staining with Coomassie Blue  and S. lividans/pZRJ362-cel6A-(His)6  transformants as the major protein but not in culture of S. lividans transformed with pZRJ362. Analysis by SDS-PAGE revealed that the target proteins were exceptionally highly expressed and secreted in S. lividans using its own signal sequence, constituting up to 80.6% of all soluble protein in supernatants ).The expression plasmids, pZRJ362-sie Blue  confirme6 (6.26 U/mg) in the supernatant after 96-h of cultivation showed the hexahistidine tag at the C-terminal has no adverse on the activity of Cel6A. Therefore, we merely selected S. lividans/pZRJ362-cel6A-(His)6 for purification and further study. Concurrent with the growth of S. lividans/pZRJ362-cel6A-(His)6 transformant, the measurement for endoglucanase activity and total protein content in the supernatants was taken. The maximum endoglucanase activity was observed at 84-h of cultivation (5.56 U/mL) and the production of Cel6A-(His)6 remained basically constant at longer incubation time  and Cel6A-(His)ion time . The accanalysis . Unlike analysis  and it wred 96-h , we chos6-tag affinity column purification (6 (32.3 U/mg), the maximum expression level of Cel6A-(His)6 reached to 173 mg/L after 84-h of cultivation of S. lividans harboring pZRJ362-cel6A-(His)6. With the combination of xylose isomerase promoter and amylase terminator, the protein concentration of the secreted Cel6A-(His)6 was approximately 3-fold higher than that with the combination of Streptoverticillium cinnamoneum phospholipase D promoter and terminator [The SDS-PAGE of the purified enzyme exhibited a single band after (His)fication . Estimatrminator .cel6A-(His)6 or pPICZα-cel6A carrying the mature cel6A gene was regulated by a promoter of alcohol oxidase gene and the production of Cel6A was induced with 0.5% methanol. Using pPICZα-cel6A or pPICZα-cel6A-(His)6, the maximum Cel6A activity in the BMMY medium reached approximately 1.6 U/mL and 1.4 U/mL after 96-h of cultivation, respectively. P. pastoris transformants. Several protein bands corresponding to a molecular mass about 55 kDa were observed, probably because of the formation of different numbers of glycosylated side chains. The result is consistent with the previous studies which indicated the generation of several glycosylated proteins in the supernatants of P. pastoris transformants [6 and Cel6A accounted for 59.6% and 54.7% of all soluble protein in the supernatants (determined by Bandscan 5.0 software), respectively. As seen in SDS-PAGE gels, the expression level of Cel6A in P. pastoris was quite low in comparison with that in S. lividans. Therefore, for T. fusca endoglucanase Cel6A, the use of S. lividans/pZRJ362 as an expression system represents a dramatic improvement in comparision with the use of P. pastoris/pPICZα-C.The plasmid pPICZα-formants . AnalyseS. lividans 1326, the expression pattern of Cel6A-(His)6 in S. lividans 1326 was different from that in P. pastoris. As shown in 6 by proteolytic degradation. This result was consistent with previous observations, which also indicated the generation of proteolytic products in the supernatants of S. lividans transformants [6 also exhibited CMCase activity (P. pastoris carrying pPICZα-cel6A-(His)6 .It was perhaps because of the endogenous protease of formants . These dA-(His)6 . As showE. coli DH5α  were used, restriction endonucleases and T4 DNA Ligase were purchased from New England Biolabs . For expression, P. pastoris GS115 and pPICZα-C vector were purchased from Invitrogen . Genomic DNA from T. fusca YX was purchased from ATCC . S. lividans 1326 and pZRJ362 constructed in this paper were used for heterologous protein production. Methylation-deficient E. coli ET12567/pUZ8002 was used to transfer DNA from E. coli into S. lividans by conjugation. S. lividans 1326 and transformants were grown at 30 °C on mannitol soya flour agar for sporulation. For the production of Cel6A, S. lividans transformants carrying expression plasmid were inoculated in tryticase soy broth (TSB) medium  with continuous shaking (200 rpm). When applicable, antibiotics were used to select recombinant strains including 100 μg/mL Ampicillin, 50 μg/mL Kanamycin, 25 μg/mL Chloramphenicol and 5 μg/mL Thiostrepton which were purchased from Sigma . For production of Cel6A in P. pastoris, yeast extract and peptone were obtained from OXOID , yeast nitrogen base (YNB) without amino acids was purchased from BD , biotin was obtained from Amersco , and Zeocin was purchased from Invitrogen .For cloning, pMD19-T vector  and S. lividans as a host was constructed as follows. The plasmid pSP72  was digested with XbaI and EcoRI to obtain the fragment carrying ori (E. coli) and Ampicillin (Amp) resistance gene bla. The plasmid pHZ1358, which was kindly provided by K. Bao and Z. Deng , was also digested with XbaI and EcoRI to obtain the fragment containing oriT of RP4, tsr gene for Thiostrepton resistance and the replication (rep) ORF of pIJ101 for autonomous replication of the plasmid in Streptomyces. The two fragments were mutually ligated to generate pZRJ. Afterwards, the synthetic fragment of XbaI-Xi promoter-Shine-Dalgarno sequence-MCS-amyA2 terminator-SacI was subcloned into the XbaI-SacI gap of the plasmid pZRJ, yielding the expression vector pZRJ362. The gene fragment of Xi promoter sequence encoded the promoter of xylose isomerase of Actinoplanes missouriensis and the amyA2 fragment encoded the terminator region of amylase from Streptomyces avermitilis MA-4680. Diverse restriction sites were available within the MCS to facilitate gene cloning, including NheI, KpnI, AflII, AfeI, MluI and BamHI. All the constructs were sequenced by Invitrogen .The plasmid pZRJ362 for protein expression using cel6A gene including the native signal peptide was PCR amplified from genomic DNA of T. fusca YX with the primers cel6A-f (5′-GCTAGCTCCCCCAGACCTCTTCGCGCTCTTC-3′) and cel6A-r (5′-GGATCCTCAGCTGGCGGCGCAGGTAAGGGTCG-3′), which included NheI and BamHI restriction sites (underlined), respectively. To simplify the purification process, we introduced (His)6-tag into the C-terminus of recombinant Cel6A. To obtain His-tagged Cel6A, the reverse primer cel6A-His (5′-GGATCCTCAGTGGTGGTGGTGGTGGTGGCTGGCGGCGCAGGTAAGGGTCG-3′) was used. These two PCR products were cloned in plasmid pMD19-T. After the digestion of NheI and BamHI, 1.6 kb fragments were ligated into the NheI-BamHI gap of pZRJ362 to generate the expression vectors pZRJ362-cel6A and pZRJ362-cel6A-(His)6, respectively. From the latter expression vector, the corresponding protein was produced with a C-terminal 6× His tag.The T. fusca YX genome and obtain His-tagged cel6A gene, three primers  were designed. The mature cel6A gene was amplified by PCR with cel6A-F (5′-ATCGATGAATGATTCTCCGTTCTACGTC-3′) as forward primer and cel6A-R (5′-TCTAGATTATCAGCTGGCGGCGCAGGTAA-3′) as reverse primer with ClaI and XbaI restriction sites denoted as the underline, respectively. To obtain His-tagged cel6A, the reverse primer cel6A-His-R (5′-TCTAGATTATCAATGATGATGATGATGATGGCTGGCGGCGCAGGTAA-3′) was used with XbaI restriction site and (His)6-tag denoted as the underline too. The PCR products were cloned into pMD19-T, yielding plasmids pMD19-cel6A and pMD19-cel6A-(His)6, respectively.To achieve the target gene encoding the mature region of endoglucanase Cel6A from P. pastoris-E. coli shuttle vector pPICZα-C and recombinant plasmid pMD19-cel6A/pMD19-cel6A-(His)6 were both digested with ClaI and XbaI and mutually ligated. Afterwards transformants were selected on the low salt LB agar plates  containing 25 μg/mL Zeocin. Then recombinant plasmids designated as pPICZα-cel6A and pPICZα-cel6A-(His)6 were achieved, respectively. After being digested with PmeI, the linearized pPICZα-cel6A and pPICZα-cel6A-(His)6 were transformed into P. pastoris GS115 by electroporation using MicroPulser  under the PIC setting. The P. pastoris transformants were selected at 30 °C on the YPDS agar plates  containing 100 μg/mL Zeocin for 3–4 days. Then, the multi-copy transformants were screened by using YPDS agar plates containing higher Zeocin concentration (2000 μg/mL) and incubated at 30 °C for 2 days. Finally, the multi-copy transformants were checked for the integration by genomic PCR.To achieve secretive expression, the 6, one spore of each S. lividans 1326 carrying the corresponding expression vector was inoculated into a test tube containing 5 mL of TSB medium supplemented with 5 μg/mL Thiostrepton, followed by cultivation at 30 °C for 3 days. Then 1 mL of the preculture medium was seeded into a 250 mL baffled flask containing 100 mL TSB medium with 5 μg/mL Thiostrepton, and incubated at 30 °C for 4–7 days.For the production of heterologous protein Cel6A or Cel6A-(His)6 from P. pastoris, a single colony was inoculated into 25 mL BMGY medium , 13.4 g/L YNB, 4 × 10−4 g/L biotin, 1% (v/v) glycerol) in a 250 mL baffled flask and shaken (220 rpm) at 29 °C for 18 h. Then the cells were harvested by centrifuging at 3000× g for 5 min and resuspended in 100 mL BMMY medium , 13.4 g/L YNB, 4 × 10−4 g/L biotin, 0.5% (v/v) methanol) in a 500 mL baffled flask, shaken (220 rpm) for 4 days. To maintain induction, 100% methanol was added to a final concentration of 0.5% during the induction phase.For the production of heterologous Cel6A or Cel6A-(His)To purify the His-tagged interest protein, the mycelium was separated from the supernatant by filtrating with 0.22 μm Millex-GP Filter Unit . Afterwards, the His-tagged recombinant protein was purified from the supernatant using a Ni Sepharose™ 6 Fast Flow  column according to the standard procedures of the manufacturer.2SO3) was added and vortexed. Subsequently, samples were boiled for 10 min, and the absorbance of 200 μL sample at 540 nm was measured on iMark Microplate Reader . One enzyme unit (U) is defined as an average of 1 μmol of glucose equivalent released per min in the assay reaction. All the enzyme activity values presented were averages obtained from triplicate measurements.The cells were harvested at a selected time point, afterwards the cell extracts were centrifuged and the activities of culture supernatants were measured. The dinitrosalicyclic acid (DNS) method  was usedSDS-PAGE analyses were performed to detect the extracellular proteins. Activity staining was used for visualization of carboxymethyl-cellulase (CMCase) activity bands on the gel with slightly modifications . The proStreptomyces-E. coli shuttle vector pZRJ362. T. fusca YX endoglucanase Cel6A was highly expressed as secretory proteins in S. lividans 1326 using this novel expression vector. A most remarkable experimental finding was the higher capacity of S. lividans/pZRJ362 to produce Thermobifida heterologous protein compared to P. pastoris. This newly constructed vector may be applicable to efficient production of other actinomycetes enzymes.In this study, combining the xylose isomerase promoter and amylase terminator, we have constructed a


Glioblastoma is the most common type of primary brain tumors. Cisplatin is a commonly used chemotherapeutic agent for Glioblastoma patients. Despite a consistent rate of initial responses, cisplatin treatment often develops chemoresistance, leading to therapeutic failure. Cellular resistance to cisplatin is of great concern and understanding the molecular mechanisms is an utter need.Glioblastoma cell line U251 cells were exposed to increasing doses of cisplatin for 6 months to establish cisplatin-resistant cell line U251R. The differential miRNA expression profiles in U251 and U251R cell lines were identified by microarray analysis and confirmed by Q-PCR. MiRNA mimics were transfected into U251R cells, and cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis.U251R cells showed 3.1-fold increase in cisplatin resistance compared to its parental U251 cells. Microarray analysis identified Let-7b and other miRNAs significantly down-regulated in U251R cells compared to U251 cells. Transfection of Let-7b mimics greatly re-sensitized U251R cells to cisplatin, while transfection of other miRNAs has no effect or slightly effect. Cyclin D1 is predicted as a target of Let-7b through bioinformatics analysis. Over-expression of Let-7b mimics suppressed cyclin D1 protein expression and inhibited cyclin D1-3’-UTR luciferase activity. Knockdown of cyclin D1 expression significantly increased cisplatin-induced G1 arrest and apoptosis.Collectively, our results indicated that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression. Let-7b may serve as a marker of cisplatin resistance, and can enhance the therapeutic benefit of cisplatin in glioblastoma cells. Glioma is the first commonly diagnosed types of intracranial tumors, accounting for more than 50% among all primary brain tumors . GliomasCurrent options for treatment of glioblastoma include surgical resection of the primary tumor to reduce the tumor size, followed by radiotherapy and adjuvant chemotherapy with temozolomide (TMZ) . HoweverMicroRNAs (miRNAs), a class of 22-nucleotide small non-coding RNAs, can regulate gene expression at post-transcriptional level. MiRNAs are evolutionarily conserved and negatively regulate gene expression. They are transcribed by RNA polymerase II, spliced, and then poly-adenylated to generate primitive miRNAs (pri-miRNAs) . The steCisplatin has dramatically been used as the first line treatment for several types of solid tumors, such as breast, head and neck, ovarian, and lung cancers . CisplatTo study chemoresistance in glioma, we established a cisplatin-resistant glioblastoma cell line U251R, which is 3.1 fold resistant to cisplatin compared to its parental cell U251. MiRNA expression signature analyzed by microarray identified 16 miRNAs as down-regulated in U251R. Let-7b is one of the most significantly suppressed miRNA. Furthermore, over-expression of Let-7b significantly re-sensitized U251R cells to cisplatin through inhibition of cyclin D1 expression. Cyclin D1 knockdown dramatically increased cisplatin-induced apoptosis and G1 arrest. Taken together, our results suggested that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression, resulting in resistance to cisplatin. Therefore, Let-7b may be considered as a marker for early diagnosis of cisplatin resistance, and restoration of Let-7 in glioblastoma could be a new strategy for cisplatin-resistant cancer treatment in the future.Fetal bovine serum for cell culture and Lipofectamine 2000 were purchased from Invitrogen . Anti-β-actin antibody was from Santa Cruz Biotechnology . Anti-Bcl-2, Bax, and ppRb antibodies were from Cell Signaling Technology . Anti-cyclin D1 antibody was from Abcam . Let-7b mimics expression vector was purchased from Wuhan Genesil Biotechnology .Human neuronal glioblastoma cell line U251 was a gift from Dr. Zhongping Chen . U251 cell line was maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum (Invitrogen), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen), in a 5% CO2 humidified atmosphere at 37°C.To generate a cisplatin-resistant cell line, U251 cells were exposed to increasing concentrations of cisplatin. Cisplatin concentrations were increased stepwise from 0.1 μg/mL to 0.5 μg/mL when the cells resumed growth kinetics similar to the untreated parental cells. Cells with the ability to grow in 0.5 μg/mL of cisplatin were obtained 4 months after the initial drug exposure, named as U251R.3 cells/100 μL medium per well. After adherence, cells were treated with various concentrations of cisplatin for 48 h, with DMSO as negative controls. At the end of treatment, the tetrazolium compound, 3--2,5-diphenyl tetrazolium bromide  was added and then incubated for additional 4 h at 37°C in the dark. The formazan crystals were dissolved by DMSO, and the absorbance was recorded using an ELISA plate reader.Cell lines were seeded into 96-well plates at a density of 5 × 10Cyclin D1 shRNA (cyclin-sh) and negative scramble shRNA (SCR) were inserted into pGPHI vector. The primers were as follows: For cyclin-sh, forward primer 5-CACCGATCGTCGCCACCTGGATGTTCAAGAGACATCCAGGTGGCGACGATCTTTTTTG-3, and reverse primer 5-GATCCAAAAAAGATCGTCGCCACCTGGATGTCTCTTGAACATCCAGGTGGCGACGATC-3; for SCR, forward primer 5-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTG-3, and reverse primer 5-GATCCAAAAAA TTCTCCGAACGTGTCACGTAATCTCTTGACGTGACACGTTCGGAGAAC-3. Cyclin D1 3’-UTR sequence was cloned into pGL3-Luc vector. The primers were as follows: forward primer 5-GCTCTAGAGCTGACTCCAAATCTCAATGAAGCCA-3, and reverse primer 5-GCTCTAGAGCTAACCAGAAATGCACAGACCCAG-3.Total RNA was extracted from each cell line using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The RNA samples were submitted to KangChen Bio-tech , then labeled with Hy3™ fluorescent dye for hybridization on a miRCURY™ LNA microRNA array . Expression levels of selected miRNAs differed by at least 2-fold between cisplatin-resistant U251R cell line and parental U251 cell line.Cell lysates were loaded onto 10% SDS–polyacrylamide gels, electrophoresed and transferred to PVDF membranes . Membranes were blocked in TBS-Tween-20 containing 5% non-fat milk at room temperature for 1 h and then incubated with primary antibodies at 4°C overnight. On the second day, the blots were incubated with HRP-linked secondary antibodies at room temperature for 1 h. After three times’ wash in TBST buffer, the blots were visualized by ECL Reagent  as previously described .This assay was performed as previously described . BrieflyTotal RNA was isolated from tissues using Trizol (Invitrogen) following the manufacturer’s instructions. RNA was converted to cDNA with Reverse Transcription System (Promega) according to the manufacturer’s instructions. Q-PCR was performed using the miRNA SYBR Real-time PCR kit  on the ABI 7300 Real-Time PCR system . To calculate relative expression, the (ΔΔCT) method was used in comparing miRNA expression in U251R cells to U251 parental cancer cells according to ABI’s protocol.This assay was performed according to the manufacturer’s instructions . Briefly, after treatment, cells were collected, washed with PBS and pelleted. Cell pellets were resuspended in 100 μL of Annexin V-FITC labeling solution and incubated at room temperature in dark for 30 minutes. After incubation, reaction was stopped by adding 300 μL ice-cold PBS and measured on FACSCalibur flow cytometer .Caspase-3 activity was measured by Caspase-Glo3/7 assay kit (Promega) according to the manufacturer’s instructions.This assay was performed as previously described . Brieflyp < 0.05 was considered as indicating a significant difference.The Student′s t-test was used to compare the difference between two tested groups. A value of 50 values for cisplatin over 48 hours treatment: 1.4±0.1 μg/mL and 4.4±0.9 μg/mL, respectively . To compare the sensitivity of the parental U251 and U251R cells to cisplatin, cells were treated with different concentrations of cisplatin for 72 hours and dose–response curves were plotted as shown in Figure Dysregulation of miRNA expression has been reported to be associated with chemoresistance of human cancers. Herein, we performed microRNA microarray containing 3100 probes to analyze differential miRNA expression profiles in U251 and U251R cell lines. As shown in Figure The microarray results were then validated by real-time PCR. Consistent with microarray data, miR-182 and miR-224 were up-regulated in U251R cells; Let-7b, miR-125b, miR-107 and miR-203 were significantly suppressed in U251R cells  . Notably, the apoptosis in U251R transfected with Let-7b is comparable to that in U251 parental cells  . In fact, the activity of caspase-3 in U251R-Let-7b cells is similar to U251 parental cells  (Figure The caspase-3 activity was determined. After 0.625 μg/mL cisplatin treatment for 48 hours, caspase-3 activity was significantly increased in U251 cells, but less increased in U251R cells. Interestingly, compared with scramble transfection, cisplatin-induced caspase-3 activity in U251R cells was partially enhanced by transfection of Let-7b mimics (3.92±0.08 vs. 6.23±0.30, To clarify the mechanism of Let-7b-induced changes in chemosensitivity, we first used miRBase and TargetScan to predicted Let-7b target genes, and potential Let-7b binding site is found in 3'-UTR of cyclin D1 Figure A.To validate if cyclin D1 is a real target of Let-7b, Let-7b mimics was transfected into U251 and U251R cells. As shown in Figure Amplification, mutation, and high expression of cyclin D1 are reported to be associated with resistance to chemotherapy and poor prognosis in breast tumors, brain tumors and testicular germ cell tumors. To test if cyclin D1 plays an important role in cisplatin resistance in U251R cells, cyclin D1 expression was knockdown by shRNA Figure A. CisplaCurrent anti-cancer chemotherapeutic agents for glioblastoma have not significantly improved the survival of glioblastoma patients during the past ten years . Those pRecently, miRNA has been proved as one of the critical regulators during glioma progression. Both up-regulation and down-regulation of miRNAs are involved in the development of glioblastomas and chemoresistance. Shi et al. showed that over-expression of miR-21 could attenuate TMZ-induced apoptosis in U87MG cells through up-regulation of Bax, reduction of Bax/Bcl-2 ratio and caspase-3 activity, demonstrated that miR-21 over-expression is associated with resistance to chemotherapeutic drug TMZ . FurtherIt is known that Let-7 modulates chemosensitivity in various types of cancer. Let-7 inhibited gemcitabine chemoresistance in pancreatic cancer , and couCyclin D1 belongs to a family of protein kinases that involved in cell cycle regulation. Cyclin D1 has been proved to be associated with chemoresistance to cisplatin-based therapy. Noel et al. demonstrated that cyclin D1 expression was significantly higher in chemoresistant testicular germ tumor cell lines comparing with the parental cells. Furthermore, cyclin D1 knockdown in combination with cisplatin treatment inhibited tumor cell growth more effectively than single treatments . In pancTo sum up, we generated a cisplatin-resistant glioblastoma cell line U251R, and analyzed miRNA expression profiles in U251R compared with its parental cell line U251. Microarray data indicated that Let-7b was dramatically down-regulated in U251R cells compared with U251 cells. Furthermore, ectopic expression of Let-7b remarkably inhibited U251R cell chemoresistance to cisplatin through cyclin D1 expression blockade. Cyclin D1 knockdown significantly promoted cisplatin-induced apoptosis and G1 arrest. In conclusion, Let-7b could be considered as a novel marker of cisplatin resistance during early diagnosis, and more importantly, restoration of Let-7 in tumor cells could offer a novel therapeutic approach for cisplatin-resistant glioblastoma treatment.The authors declare no competing financial interests.YG, KY, and QQ were involved in the design of the study, performance of the experiments, data analysis, and manuscript writing. JF and MZ participated in the experimental design and data analysis. FC conceived of the study, and was involved in financial support, the design of the study, data analysis, and final approval of the manuscript. All the authors read and approved the manuscript.


Actinobacillus pleuropneumoniae genome serotype 5b strain L20 and serotype 3 strain JL03. The initial identification was based on a bioinformatic analysis of the amino acid similarity between these putative heptosyltrasferases with others of known function from enteric bacteria and Aeromonas. The putative functions of all the Actinobacillus pleuropneumoniae heptosyltrasferases were determined by using surrogate LPS acceptor molecules from well-defined A. hydrophyla AH-3 and A. salmonicida A450 mutants. Our results show that heptosyltransferases APL_0981 and APJL_1001 are responsible for the transfer of the terminal outer core D-glycero-D-manno-heptose  residue although they are not currently included in the CAZY glycosyltransferase 9 family. The WahF heptosyltransferase group signature sequence [S(T/S)(GA)XXH] differs from the heptosyltransferases consensus signature sequence [D(TS)(GA)XXH], because of the substitution of D261 for S261, being unique.We experimentally identified the activities of six predicted heptosyltransferases in  Actinobacillus pleuropneumoniae is a non-motile Gram-negative bacterium causing porcine pleuropneumonia, a highly contagious respiratory disease transmitted through aerosols or close contact with infected animals including asymptomatic carriers To date, two biovars have been described on the basis of NAD growth dependence; and fifteen serotypes based on capsular antigens A. pleuropneumoniae virulence factors showed that, as in other pathogenic bacteria, there is an array of these factors. However, the most relevant one appears to be the Apx toxins Studies aimed at the identification of A. pleuropneumoniae infection, including adhesions to lower respiratory tract, induction of lesions, and persistence in the upper respiratory tract reviewed in The LPS appears to play a role in virulence in different stages of the A. pleuropneumoniae consist of three domains: an endotoxic glycolipid (lipid A), an O-polysaccharide chain (O-PS or O-antigen), and an intervening core oligosaccharide (core-OS). The structures the O-PS present in fourteen out of the fifteen capsular serotypes have been determined A. pleuropneumoniae i.e., serotypes 1 (strain 4074), 2 (strain 4226), 5a (strain K17), and 5b (strain L20) glycero-D-manno-heptose  and one D-glycero-D-manno heptose  residues. Relative to serotype 1, an additional D,D-HepV residue was found in serotypes 2, 5a, and 5b  A. pleuropneumoniae strains. The cladogram obtained is shown in The core LPS structures of residues  requirinhttp://www.cazy.org) five glycosyltransferases  belonging to family GT9, that includes known heptosyltransferases, are reported for the genome strain L20 (serotype 5b) A. pleuropneumoniae genome strains JL03 (serotype 3) In the Carbohydrate-Active EnZymes database (CAZy)  and as expected their core LPS oligosaccharides only contain phosphorylated Kdo Pasteurella multocida contains both versions of heptosyltransferase I and analysis of the core LPS of strains Pm70 and VP161has shown that their core LPS contains two major oligosaccharides only differing in their Kdo phosphorylation A. pleuropneumoniae strains studied OpsX and WaaC transfer the first L,D-Hep residue to phosphorylated and unphosphorylated Kdo, respectively Aeromonas hydrophyla and salmonicida transferring L,D-Hep II (WaaF) and L,D-Hep III (WahF) cluster with putative heptosyltransferases from A. pleuropneumoniae L20 (APL_1403 and APL_0421), and Pasteurella multocida Pm70  and A. salmonicida WasD (ASA_0098) with A. pleuropneumoniae APL_0979 and P. multocida PM1144. WahD and WasD transfer a D,D-Hep and L,D-Hep residue attached by an α 1,6 linkage to the β-D-Glc residue in A. hydrophila and A. salmonicida, respectively A. pleuropneumoniae  and P. multocida, respectively, suggest that APL_0970 and PM1144 are homologues of WahD and WasD.A fourth cluster encompasses A. hydrophila and APL_0981, APLJ_1001, and APP7_1037 from A. pleuropneumoniae genomic strains L20 (serotype 5b), J03 (serotype 3) and AP76 (serotype 7). WahC has been shown to be the heptosyltransferase responsible for the transfer of the terminal D,D-Hep residue of the A. hydrophila AH-3 core LPS A. pleuropneumoniae are actually included in a non-classified group of glycosyltransferases  the present analysis could suggest a common function for all these proteins.Two proximal clusters includes WahC from A. hydrophila and salmonicida involved in the transfer of a L,D-Hep residue by an α 1,6 linkage to L,D-Hep I A. pleuropneumoniae or P. multocida fall in this cluster.As a control for this analysis we used the WahE proteins from A. hydrophila AH-3 strain mutants as a source of surrogate LPS acceptor molecules. These mutants were previously constructed by either KmR insertion or internal in-frame deletion in such a way to avoid polar effects on downstream genes In order to unambiguously identify the functions of the above putative heptosyltransferases we decided to use LPS from several ARA promoter (pBAD33-Gm-APL_0186). The core LPS from A. hydrophila AH-3ΔopsX mutant only contains a phosphorylated Kdo residue and is devoid of O-antigen A. hydrophila AH-3ΔopsX mutant and LPS from this strain was analyzed by SDS-PAGE after growth in inducing conditions. The APL_0186 expression plasmid fully complemented the opsX mutation leading to the production of full length LPS including O-antigen (A. pleuropneumoniae strain JL03 (serotype 3)  , lane 4.A. hydrophila AH-3ΔwaaF is devoid of O-antigen and contains a trisaccharide core  A. pleuropneumoniae waaF mutant  and it was expected that the putative WaaF enzymes APL_1403 and APJL_1428 would be able to recognized the A. hydrophila AH-3ΔwaaF core. As expected, expression of APL_1403 (pBAD33-Gm-APL_1403) or APJL_1428 (pBAD33-Gm-APJL_1428) into A. hydrophila AH-3ΔwaaF resulted in the production of full length A. hydrophila AH-3 core including O-antigen - On the other hand, both APL_0421 or APJL_0446 were able to restore wild type AH-3 core when expressed into A. A. hydrophila AH-3 is involved in the transfer of the D,D-Hep residue linked to Glc by an α 1,6 linkage while WasD from A. salmonicida transfer the L,D-Hep to Glc using the same linkage  consistent with the loss of the outer-core trisaccharide fragment α-D,D-Hep-(1→6)-[β-D-Gal-(1→4)]-α-D,D-Hep from the full core LPS  . Taken together, these data allow to attribute a heptosyltransferase function involved in the transfer of the first D,D-Hep residue of the outer-core in A. pleuropneumoniae to APL-0979, and APJL_0999.To further confirm the function of APL_0979 and APJL_0999, LPS from core LPS . By cont1857.617  in agreeep6Kdo1)  [25]. Th core OS . SimilarA. hydrophila AH-3 WahC is responsible for the transfer of the terminal D,D-Hep residue of the AH-3 core LPS A. pleuropneumoniae APL_0981 and APJL_1001. No homologous protein from A. salmonicida was detected  motif. On the basis of the presence of a terminal D,D-Hep in the A. pleuropneumoniae core OS of serotype 2, 5a, and 5b (genome strain L20) The detected  in agreemonicida . A majorA. hydrophila AH-3ΔwahC. As previously shown, the LPS of the AH-3 mutant still contains O-antigen , other signals were attributed to Hex1HexN1Hep6Kdo1 (m/z 1695.573) and Hex1HexN1Hep5Kdo1 (m/z 1503.495)  . SimilarwahC harboring pBAD33-Gm-APL_0981 (data not shown). These results, strongly suggests that A. pleuropneumoniae APL_0981 and APJL_1001 are functional homologues of A. hydrophila WahD and are responsible for the transfer of the terminal outer-core D,D-Hep residue (AH-3Δ residue .E. coli heptosyltranferase I (WaaC) 13, K192, E222, D261, and H266, the last two (D261and H266) present in the proposed heptosylytansferases signature sequence D261(T/S)(A/G)XXH266. These conserved residues were shown to interact with ADP-2-deoxy-2-fluoro-heptose 13 and H266. The K192 was found to interact with the O5 and O6 atoms of the heptose, and it was present in L,D- heptosyltransferases WaaC, OpsX, WaaF, and WaaQ. It has been suggested that in D,D-heptosyltransferases the K192 could be substituted by other residues as by R in Helicobacter pylori192 is substituted by R or N or R, respectively. We also found K192 in all the analyzed L,D-heptosyltransferases except WahF with N or T substituting K192  can be grouped on the basis of their amino acid similarity , while ting K192 . The E. ted by S . The WahThis analysis stresses the importance of experimental identification of the function of hypothetical heptosyltransferases. Two approaches are possible; one is based on the construction of individual mutations and analysis of the core LPS defects A. pleuropneumoniae heptosyltransferases from strains L20 and JL03. The same functions are expected from the homologous proteins from strain AP76  or tryptic soy agar (TSA) at 30°C unless stated otherwise. A. pleuropneumoniae strains were grown on Brain Herat Infusion (BHI) at 37°C and E. coli strains were grown on Luria-Bertani (LB) Miller broth and LB Miller agar at 37°C Bacterial strains, and plasmids used in this study are listed in E. coli DNA polymerase (Klenow fragment), and alkaline phosphatase were used as recommended by the suppliers. Deduced amino acid sequences used from different Enterobacteriaceae and Aeromonadaceae were obtained from the GenBank, EMBL, and SwissProt databases. ClustalW General DNA manipulations were done essentially as described A. pleuropneumoniae genes  were PCR-amplified by using specific primer pairs  D-glucose, and induction was obtained by adding l-arabinose to a final concentration of 0.2% (w/v). The cultures were grown for 18 h at 30°C in TSB medium supplemented with gentamycin and 0.2% glucose, diluted 1∶100 in fresh medium (without glucose) and grown until they reached A600 nm of about 0.2. Then l-arabinose was added, and the cultures were grown for another 2 h. Repressed controls were maintained in glucose-containing medium.For complementation studies, the er pairs  and liga2 pH 7.63 (10 mL g -1) were treated at 37°C with RNAse, DNAse , and then with Proteinase K . The suspension was dialyzed and lyophilized. The phenol/chloroform/light petroleum ether method For screening purposes LPS was obtained after proteinase K digestion of whole cells g, 20 min) and the supernatant fractionated on a column (56×2.6 cm) of Sephadex G-50 (S) in 0.05 M pyridinium acetate buffer pH 4.5 with monitoring using a differential refractometer .A portion of the LPS (∼50 mg) from each strain was heated with aqueous 2% HOAc (6 mL) at 100 °C for 45 min. The precipitate was removed by centrifugation  were dissolved in a 1∶1 (v/v) water-acetonitrile mixture and sprayed at a flow rate of 3 µL min-1. Capillary entrance voltage was set to 4.5 kV and exit voltage to −150 V; drying gas temperature was 180°C. The spectra showing several charge states for each component were charge deconvoluted, and mass numbers given refer to monoisotopic molecular masses.Electrospray ionization mass spectra were run in the negative ion mode using a micrOTOF II instrument . Mass spectra were acquired using standard experimental sequences as provided by the manufacturer. Samples (∼50 ng µLAeromonas or Pasteurella multocida with those of A. pleuropneumoniae strains L20 (serotype 5b) The analysis was performed by running the phylogenetic inference software Protpars from the PHYLIP (Phylogeny Inference Package) package version 3.5c