Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (kcat/Km) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also observed.The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from  Candida antarctica, the half-life of the enzyme was increased by 4.5-fold at 50°C [Thermomyces lanuginosus increased approximately 10°C upon introduction of a disulfide bridge Q1C-Q24C [Pseudomonas fragi [A disulfide bridge is formed by the oxidation of two thiols each from two cysteines, thus linking the two cysteines and their respective main peptide chains, which can restrict the motion of the unfolded, random coil of protein or stabilize the folded state of protein ,2. One das fragi ,7.Aspergillus niger has been analyzed. The structure of this enzyme is based on an α/β hydrolase fold and consists of a major nine-stranded mixed β-sheet, two minor two-stranded β-sheet arrangements and seven helixes [Thermoanaerobacter tengcongensis, and type B Tx-Est1 from Thermobacillus xylanilyticus [AfFaeA from Aspergillus flavus and AnFaeA from A. niger [Feruloyl esterases  cleave the ester bond between polysaccharides and hydroxycinnamic acid in hemicellulose networks for the subsequent hydrolysis of hemicellulose by hemicellulose-digesting enzymes. Their potential for degradation and reutilization of the natural biomass is significant . Based o helixes ,11. More helixes . FAEs th helixes ,13. Unfoilyticus ,15. GeneA. niger ,17. AccoA. niger ,19.Trichoderma reesei endo-1,4-β-xylanase II [AufaeA encoding a mesophilic type A FAE (AuFaeA) from A. usamii was cloned and expressed in Pichia pastoris [The thermostability of AnFaeA was improved through multiple amino acid substitutions by error prone PCR technique . Some raanase II . These rpastoris . In the pastoris ,26 and dpastoris .Escherichia coli JM109 and plasmid pUCm-T  were used for gene cloning and DNA sequencing. A recombinant T-plasmid, pUCm-T-AufaeA, was constructed and preserved in our laboratory [E. coli DH5α and plasmid pPIC9K  were used for constructing the recombinant expression plasmids. E. coli JM109 and DH5α were cultured in the LB medium . P. pastoris GS115 and its transformants were cultured and methanol-induced in the YPD, MD, YPD with geneticin G418, BMGY and BMMY media, which were prepared as described in the manual of Multi-Copy Pichia Expression Kit (Invitrogen).boratory . E. colihttp://www.ncbi.nlm.nih.gov/) was performed using the BLAST server, while homology alignment of type A FAE primary structures was analyzed using the ClustalW2 program . Using the crystal structure of type A FAE from A. niger  as a template, three dimensional (3D) structures of A. usamii AuFaeA and its variants, sharing high primary-structure identities with A. niger AnFaeA, were homologically modeled and optimized using the MODELLER 9.9 program . The B-factor values of AnFaeA (1USW) were analyzed by B-FITTER software [http://pymol.org).Homology sequence search at the NCBI website (software . The 3D http://caps.ncbs.res.in/dsdbase/modip.html) and Disulfide by Design  were used to detect amino acid pairs, in the 3D structure of AuFaeA, where the disulfide bridges were likely to form. Based on the physicochemical properties of the located amino acids and their positions in the 3D structure of AuFaeA, several pairs of amino acids were selected for substitution with cysteine residues, creating a series of candidate variants of AuFaeA.Modeling of Disulfide Bonds in Proteins . The RMSD value was calculated using g_rms software from GROMACS 4.5 package, and statistical analysis was performed using Origin 9 software (http://www.originlab.com/).The root mean square deviation (RMSD) value is an important index for estimating the thermostability of a protein conformation. RMSD was defined as the C protein . To predA126C-N152C, a variant of AuFaeA, with the smallest RMSD value was selected. The variant-encoding gene, AufaeAA126C-N152C, was constructed by synchronously mutating Ala126- and Asn152-encoding codons of AufaeA into Cys126- and Cys152-encoding ones. Using pUCm-T-AufaeA as a template, AufaeAA126C-N152C was amplified using a QuikChange Mutagenesis Kit  with two pairs of forward-reverse PCR primers, A126C and N152C , was synthesized with a single-step method as reported previously [p-nitrophenol (pNP) released from pNPF as described [2HPO4–citric acid buffer (pH 5.5) containing 2.5% (v/v) Triton X-100, 1 volume of 10 mM pNPF in dimethyl sulfoxide and 1 volume of suitably diluted enzyme) was incubated at 40°C for 10 min. The released pNP was measured at 410 nm using a spectrophotometer. One unit (U) of FAE activity was defined as the amount of enzyme that released 1 μmol pNP per minute under the standard assay conditions as stated above.The substrate, eviously . FAE actescribed , with miSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method by Laemmli . The sepAufaeA and its mutant genes were excised from their recombinant T-plasmids by digestion with EcoRI and NotI, respectively. The isolated genes were inserted into pPIC9K, and then transformed into E. coli DH5α. The resultant recombinant expression plasmids, designated as pPIC9K-AufaeA,-AufaeAA126C-N152C,-AufaeAC29T,-AufaeAC91T and-AufaeAC234T, were separately linearized with SalI and electroporated into P. pastoris GS115. All P. pastoris transformants were respectively inoculated on YPD plates with increasing concentrations of G418 for the screening of multiple copies of integrated FAE genes. P. pastoris transformed with pPIC9K without AufaeA or any of the mutants was used as a control (P. pastoris GSC). Expression of respective FAE genes in P. pastoris was performed according to the instructions of Multi-Copy Pichia Expression Kit (Invitrogen) with minor modification [fication .P. pastoris was induced by 1% methanol for 72 h, and the expressed re-FAE in the supernatant was salted out by adding ammonium sulfate (NH4)2SO4 to 75% saturation. The collected precipitate was dissolved and dialyzed in 20 mM Na2HPO4–citric acid buffer (pH 5.5). The dialyzed solution was concentrated by ultrafiltration using a 10-kDa cut-off membrane  and was loaded on a Sephadex G-50 column , followed by elution with the same buffer at a flow rate of 0.4 mL/min. Aliquots of 2 mL eluent containing re-FAE were pooled and concentrated for further studies.The transformed A126C-N152C  [410 standard formula, which was established by testing the OD410 of cysteine concentration at a range from 0 to 0.4 mM. The number of disulfide bridges in the re-FAEs was deduced from the different value of the number of total cysteines in protein primary structure and free ones in 3D structure at the tested concentration.Guanidine hydrochloride  was added for 15 min to denature the purified re-AuFaeA or re-AuFaeA2 mg/mL) . Thiol t2 mg/mL) . The wilt1/2) was defined as the time when the residual activity of the re-FAE, determined under the standard conditions, was 50% of its original activity.Purified re-AuFaeA and its variants were functioned in the standard enzyme activity assay conditions except the changed temperatures to measure the temperature optima, and were incubated in the absence of substrate at different temperatures for 60 min or longer time to estimate their thermostability. The thermal inactivation half-life . Data were fitted to the Michaelis-Menten equation to generate Km and kcat values, using Graph-Pad Prism 5.0 software.The hydrolytic reaction rates (U/mg) of re-AuFaeA and re-AuFaeABacillus stearothermophilus, lipase from Rhizomucor miehei and acetylcholinesterase from Drosophila melanogaster [The primary structures of the AuFaeA and AnFaeA (PDB code: 1USW) were highly homologous (98.5%) in identity. Identification of three native disulfide bridges, C29-C258, C91-C94 and C227-C234, in AuFaeA , was an nogaster –37. In tTm) of 34°C, was more thermostable than its wild type, which was consistent with the evaluation of RMSD values [A24C-R66C, AuFaeAY80C-P200C, AuFaeAF176C-Y186C and AuFaeAY122C-Y125C . Purities of recombinant A. usamii xylanase and A. sulphureus β-mannanase expressed in P. pastoris GS115 and X-33 have been reported to be as high as 90 and 97%, respectively [P. pastoris was induced by 1.0% methanol for 72 h, the content of expressed re-AuFaeA or re-AuFaeAA126C-N152C was more than 88% in the cultured supernatant  of re-AnFaeA [A126C-N152C, towards pNPF, were 50.2 and 59.7 U/mg, respectively. SDS-PAGE assay displayed single protein bands of purified re-AuFaeA and re-AuFaeAA126C-N152C, with apparent molecular weights of approximately 36.0 kDa  under the denaturing conditions. Based on the cysteine standard formula y = 0.8727x + 1.3972 , the OD410 at the cysteine concentration of 0.056 mM was 1.446. The detected OD410 (1.452 and 1.437) of purified re-AuFaeA and re-AuFaeAA126C-N152C were almost unanimous with the standard value  of re-AuFaeA at 50, 55 and 60°C were 50, 15 and 4 min (t1/2) of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA  by approximately 1.6-fold compared to that of the re-AuFaeA, indicating a possible decrease in substrate affinity. However, the re-AuFaeAA126C-N152C also increased catalytic turnover frequency (kcat) by approximately 1.6-fold compared with that of re-AuFaeA. Consequently, the catalytic efficiency (kcat/Km) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Since the kinetics of both enzymes were done at 40°C showing equal activity, the mutant enzyme was expected to show higher activity at higher temperature.The kinetic parameters of re-AuFaeA and re-AuFaeAubstrate . The re-It has been reported that, after removal of the disulfide bridge constraints on the Ribonuclease T1 flexibility, the enzyme’s folding and functional abilities were maintained, but its stability was decreased . SimilarC29T, re-AuFaeAC91T and re-AuFaeAC234T in the cultured supernatants were up to 0.84, 0.42 and 0.45 U/mL, respectively, which were much lower than that (11.03 U/mL) of re-AuFaeA. The temperature optima of re-AuFaeAC29T, re-AuFaeAC91T and re-AuFaeAC234T were 25, 25 and 35°C, respectively  each eliminating a native disulfide bridge were expressed in P. pastoris GS115. Experimental results with the disulfide bridge mutants confirmed that the disulfide bridges contribute significantly to the thermostability of AuFaeA. The computational prediction combined with site-directed mutagenesis to enhance the thermostability and to explore the thermostable mechanism of AuFaeA also can be applied in other proteins or enzymes.Effects of disulfide bridges on the thermostability of AuFaeA were investigated by either introducing an extra disulfide bridge to, or by eliminating each native disulfide bridge from AuFaeA. Firstly, five pairs of amino acids  that can form disulfide bridges in AuFaeA with the highest possibility were located by both MODIP and DbD. Then, the variant AuFaeA


E (E denotes collision energy) data acquisition mode, which allowed the rapid separation and reliable determination of TCMP chemical constituents. By monitoring diagnostic ions in the high energy function of MSE, target peaks of analogous compounds in TCMPs could be rapidly screened and identified. “Re-Du-Ning” injection (RDN), a eutherapeutic traditional Chinese medicine injection (TCMI) that has been widely used to reduce fever caused by viral infections in clinical practice, was studied as an example. In total, 90 compounds, including five new iridoids and one new sesquiterpene, were identified or tentatively characterized by accurate mass measurements within 5 ppm error. This analysis was accompanied by MS fragmentation and reference standard comparison analyses. Furthermore, the herbal sources of these compounds were unambiguously confirmed by comparing the extracted ion chromatograms (EICs) of RDN and ingredient herbal extracts. Our work provides a certain foundation for further studies of RDN. Moreover, the analytical approach developed herein has proven to be generally applicable for profiling the chemical constituents in TCMPs and other complicated mixtures.The broad applications and mechanism explorations of traditional Chinese medicine prescriptions (TCMPs) require a clear understanding of TCMP chemical constituents. In the present study, we describe an efficient and universally applicable analytical approach based on ultra-performance liquid chromatography coupled to electrospray ionization tandem quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q/TOF-MS) with the MS Traditional Chinese medicine prescriptions (TCMPs), which are combinations of several medicinal herbs, have been widely employed for thousands of years in China and other Asian countries. In clinical practice, TCMPs often exhibit significant advantages of low therapeutic risk and remarkable effect for some chronic, multifactorial and systemic diseases –4. HowevE (E represents collision energy) technology provides an automated strategy to decrease analysis time and maximize duty cycles by using parallel alternating scans at low collision energy in the collision cell to obtain precursor ion information or at high collision energy to obtain accurate full-scan mass fragment, precursor ion and neutral loss information. Therefore, both precursor and fragmentation data in exact mass mode were collected in a single run; this method has provided excellent chromatographic and MS efficiencies for the rapid structural elucidation of multiple constituents in complex mixtures + by the successive losses of H2O and CO. Thus, compound 44 could be tentatively identified as L-phenylalaninosecologanin B . An obvious fragment ion [M+Na-Glc]+ at m/z 619.2036 was observed by the neutral loss of 162 Da. The additional loss of CH3OH (32 Da) produced [M+Na-Glc-CH3OH]+ at m/z 587.1668. The second predominant peak at m/z 549.1576 (C24H30O13Na) was formed through an retro-Diels-Alder (RDA) reaction with the neutral loss of C4H6O. The fragment ion at m/z 517.1330 was formed by successive loss of another CH3OH molecule from the ion at m/z 549.1576. A minor peak at m/z 387.1042 (C17H23O10) was formed by the cleavage of another C17H24O9 fragment. Further loss of CH3OH (32 Da) from 387.1042 (C17H23O10) produced m/z 355.0804 (C16H19O9). Thus, compound 37 was tentatively identified as (E)-aldosecologanin. Compounds 37 and 41 were further confirmed to be (E)-aldosecologanin and (Z)-aldosecologanin, respectively, based on comparing their retention times with the isolated compounds and fragmentation pathways observed in our MS/MS experiments.Compounds . In Fig  and 5b i+ ion of compound 35 was observed at m/z 747.2114, indicating an elemental composition of C33H40O18Na . The MS/MS spectrum of [M+Na]+ showed a base peak at m/z 377.0778 (C16H18O9Na) produced by cleavage of the C17H22O9 fragment. In addition, neutral loss of a glucose unit (162 Da) generated the [M+Na-Glc]+ at m/z 585.1559. Successive losses of CH3OH and H2O molecules formed [M+Na-Glc-CH3OH]+ at m/z 553.1147 and [M+Na-Glc-CH3OH-H2O]+ at m/z 535.1448. Loss of the C16H16O8 fragment produced [M+Na-C16H16O8]+ at m/z 411.1322 (C17H24O10Na), and successive loss of another H2O molecule from 411.1322 led to the formation of an obvious ion at m/z 393.0970. The fragment ion at m/z 231.1102 (C11H12O4Na) was produced by neutral loss of a glucose unit (162 Da). Other characteristic fragment ions were formed, such as m/z 215.0157 and 199.0407, by successive or simultaneous losses of an O atom and a CH3OH molecule from m/z 231.1102  were identified or tentatively characterized from RDN , hyperoside (68), luteolin-7-O-β-d-glucoside (70), luteolin (73) and quercetin (74) by comparison with standard constituents isolated from RDN. The other four flavonoids were tentatively identified as lonicerin (69), eriodictyol (71), artemetin (72) and eupatin (75) by matching their extract molecular weights with the chemical database and MS/MS fragmentation behavior.As shown in 87, 88 and 89, were unambiguously identified as scopoletin, 7-hydroxy-6,8-dimethoxyphenyl coumarin and coumarin, respectively, by comparison with the isolated reference standards. Among the four sesquiterpenes, compound 83 was tentatively assigned as 7,8,11-trihydroxyguai-4-en-3-one 8-O-β-d-glucopyranoside by matching its mass with the chemical database within 5 ppm. Compounds 84, 85 and 86 were confirmed by matching to the retention times of the isolated reference standards. Similarly, seven lignans  and one monoterpene (compound 90) were also identified.Another 15 obvious peaks in the extracted ion chromatogram of RDN were identified . Three cE data acquisition was developed to profile multiple chemical constituents in RDN. Diagnostic ions were used as invaluable markers for the screening of target compounds. A total of 53 compounds, including two new iridoids, were identified or tentatively characterized using this method. Due to the structural complexity of the chemical constituent types in TCMPs, the present analytical approach still has a limitation in the detection of low-abundance components. The remaining 37 compounds were identified according to their accurate mass measurements within 5 ppm error, tandem MS behaviors, database-matching and reference standards. The RDN herbal sources were unambiguously confirmed by comparing the extracted ion chromatograms (EICs) of RDN and ingredient herbal extracts. The results of our study not only provide a certain foundation for further studies of RDN but also demonstrate chemical profile analyses of TCMPs via UPLC-ESI-Q/TOF-MS and diagnostic ion screening using MSE.In this work, an approach involving UPLC-ESI-Q/TOF-MS coupled with MSS1 Fig(DOCX)Click here for additional data file.S2 FigE; (B) TIC of RDN in the high-energy function of MSE; (C) EICs of diagnostic ions 209.0814, 251.0532, 235.0582 and 215.0320 in the low-energy function of MSE; (D) TIC of RDN in the MSE low-energy function.(A) EICs of diagnostic ions 209.0814, 251.0532, 235.0582 and 215.0320 in the high-energy function of MS(DOCX)Click here for additional data file.S3 FigE; (B) TIC of RDN in the high-energy function of MSE; (C) EICs of diagnostic ions 285.0399 and 301.0348 in the low-energy function of MSE; (D) TIC of RDN in the MSE low-energy function.(A) EICs of diagnostic ions 285.0399 and 301.0348 in the high-energy function of MS(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 FigGardenia jasminoides Ellis, Lj = Lonicera japonica Thunb. and Aa = Artemisia annua L.Gj = (DOCX)Click here for additional data file.S6 FigGardenia jasminoides Ellis, Lj = Lonicera japonica Thunb. and Aa = Artemisia annua L.Gj = (DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.


Recent studies suggest that H2S modulates many cellular processes and it has been recognized to play a central role in inflammation, in the cardiovascular and nervous systems. By infecting monocytes/macrophages with Mycoplasma fermentans (M.F.), a well-known pro-inflammatory agent, we evaluated the effects of H2S.Hydrogen sulfide (H2S donors (NaHS and GYY4137) on MF-infected cells was determined by treating infected cells with H2S and then testing the culture supernatants for MCP-1 and on MMP-12 production by ELISA assay. In order to identify the pathway/s mediating H2S- anti-inflammatory activity, cells were also treated with specific pharmaceutical inhibitors. Cytoplasmic and nuclear accumulation of NF-κB heterodimers was analyzed.M.F.-infected cells were analyzed by ELISA and real time RT-PCR to detect the M.F. effects on MCP-1 and on MMP-12 expression. The role of two different H2S was able to reduce the production of pro-inflammatory cytokine MCP-1, that was induced in monocytes/macrophages during M.F. infection. Moreover, MCP-1 was induced by M.F. through Toll-like receptor (TLR)-mediated nuclear factor-κB (NF-κB) activation, as demonstrated by the fact that TLR inhibitors TIRAP and MyD88 and NF-κB inhibitor IKK were able to block the cytokine production. In contrast H2S treatment of M.F. infected macrophages reduced nuclear accumulation of NF-κB heterodimer p65/p52.We show that H2S is effective in reducing Mycoplasma-induced inflammation by targeting the NF-κB pathway. This supports further studies for possible clinical applications.Our data demonstrate that under the present conditions H After washing, cells were incubated for 1 hour at 37° in 4 μM Calcein-AM solution . Finally, fluorescence was measured at an excitation wavelength of 494 nm and an emission wavelength of 530 nm, in a plate reader. The percentage of viable cells was calculated using the following formula:% Live Cells = , suggesting that early treatments with H2S, and in particular with GYY4137, markedly reduce inflammation caused by M.F. , suggesting that at this time point the protective effect of HDifferent signal pathways including NF-κB , and MAP2S plays a role in NF-κB signaling, we analyzed cytoplasmic and nuclear NF-κB subunits  during M.F. infection, with or without H2S [Figure In order to determine if H2S had a protective effect, particularly with the slow releasing donor GYY4137. Indeed, treatment of infected cells with GYY4137 caused a 10% reduction in the nuclear p50 subunit (p = 0.019), while NaHS had no effects. H2S had a more potent effect in reducing the amount of nuclear p65 subunit in infected cells: while NaHS caused 11% of reduction in nuclear p65 (p = 0.05), GYY4137 completely abrogated the increase in nuclear p65 caused by M.F. infection (45.6 ± 1.41 versus 51.7 in the control) (p = 0.009).M.F. infection caused an increase of p65 (74.1 ± 12.13% in M.F.-infected cells versus 45.5% in the control) (p = 0.01) and p50 subunits (57.9 ± 2.24% in M.F.-infected cells versus 51.7% in the control) (p = 0.02) in the NF-κB nuclear fraction, while no change was observed for the p52 subunit (p = 0.004). Treatment of the M.F.-infected cultures with H2S interfere with the activation and nuclear translocation of p52 and p65 subunits, thus preventing NF-κB dimer binding to the promoters of a variety of pro-inflammatory genes.These results suggest that H2S as a gaseous signaling molecule with a variety of effects on multiple systems. The protective effects of H2S, especially in the cardiovascular and in the nervous systems, are being increasingly explored. Several recent reports provide evidence suggesting a role for H2S in inflammation [There is increasing evidence showing Hammation .2S during bacterial infection of macrophages with M.F.Our work highlights the anti-inflammatory effects of HM.F. is associated with several chronic inflammatory diseases, in particular with arthritis  and it hIn our experiments, primary macrophage cultures and U937 monocytoid cell line were infected with M.F., inducing the production of pro-inflammatory cytokines; in particular MCP-1 was induced both at RNA and protein levels. The amount of MCP-1 induced in U937 cell lines (325.4 ng/ml) was constantly higher compared to the amount of MCP-1 induced in monocytes/macrophages (50.7 ng/ml) and was comparable to the MCP-1 plasma levels detected in human sepsis (2.00-128.00 ng/ml). It is known in fact that MCP-l plasma levels are detectable and increased in human sepsis and septic shock with respect to healthy patients: both surviving and non-surviving patients with sepsis or septic shock showed increased MCP-1 plasma levels compared with those of controls (<2.00 ng/ml) .M.F. also induced MMP-12 that, in addition to be a pro-inflammatory molecule, is required for processing MCP-1 in its active form, suggesting that M.F.-induced MCP-1 in our systems was functionally active.2S donors NaHS and GYY4137 were used to test the anti-inflammatory effects of H2S in M.F. infected cells. Both H2S donors showed anti-inflammatory activity by reducing MCP-1 production in M.F. infected cells. However while GYY4137 anti-inflammatory effect was more potent in U937 cells, NaHS was more effective in macrophages. H2S thus may represent a novel therapeutic molecule capable of limiting M.F.-induced inflammation, since it influences the levels of key chemokines like MCP-1 involved in the inflammatory process. Furthermore our results show that pre-treatment with H2S could more efficiently impact the resolution of inflammatory process.Two different HTreatment of M.F.-infected macrophages with pharmacological inhibitors demonstrated that M.F. induces MCP-1 expression through TLR-NF-κB pathway. In particular TIRAP and MyD88 inhibitor peptides were able to completely block MCP-1 production in M.F.-infected macrophages. This pathway is essential for the activation of most pro-inflammatory genes and the constitutive activation of NF-κB pathway is associated with inflammatory diseases such as rheumatoid arthritis, bowel disease, multiple sclerosis and asthma .2S, particularly GYY4137, completely blocked the nuclear translocation of NF-κB heterodimer p65/p50. However NaHS only had a slight effect on NF-κB inhibition suggesting that this H2S fast donor might use a different mechanism to inhibit bacterial inflammation.NF-κB subunits exist in the cytoplasm in inactive forms, as a result of their association with the IkB proteins . In resp2S inhibits NF-κB nuclear accumulation indicate that H2S may target directly TLR or any of the TLR adaptors proteins, as well as signaling proteins involved in NF-κB activation. Further studies are needed to see whether a specific component of the TLR-NF-κB pathway is directly inhibited by H2S. Besides it is important to establish the mechanism of H2S inhibition of TLR-NF-κB pathway. A recent study showed that S-sulfhydration of NF-κB by H2S is responsible for its anti-apoptotic actions [2S-mediated protein S-sulfhydration affect the TLR-NF-κB pathway.Our findings that H actions , therefo2S inhibits the activation and the nuclear translocation of the NF-κB, reducing the transcription of pro-inflammatory genes, including MCP-1 gene.Our data show that H2S-releasing compounds are currently being developed as candidate drugs [2S-mediated mechanism of action during inflammatory response to infection.Hte drugs  and it i2S: Hydrogen sulfide; NaHS: Sodium hydrosulfide; GYY4137: Morpholin-4-ium-4-methoxyphenyl-(morpholino)-phosphinodithioate; NF-kB: nuclear factor-kB; M.F: Mycoplasma fermentans; MCP-1 (CCL-2): Monocyte Chemoattractant Protein-1; MMP-12: Matrix metalloproteinase-12; TLR: Toll like receptor; MyD88: Myeloid differentiation primary response gene (88); IKK: IκB kinase; TIRAP: Toll-interleukin 1 receptor domain containing adaptor protein.HThe authors declare that they have no competing interests.FB designed and performed experiments, analyzed data, and wrote the manuscript; SD designed and performed experiments; SK performed experiments and contributed to the writing of the manuscript; RCG provided logistic and budget support and contributed to the writing of the manuscript; GS contributed to the writing of the manuscript; DZ provided logistic and budget support and contributed to the writing of the manuscript; SC designed and performed experiments, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.


The same cloned DENV-4 was found to yield a single E-Glu327Gly (E-E327G) mutation after passage in FRhL cells and cause the loss of immunogenicity in rhesus monkeys. Here, we used site-directed mutagenesis to generate the E-E345K and E-E327G mutants from DENV-4 and DENV-4Δ30 infectious clones and propagated in Vero or MRC-5 cells. The E-E345K mutations were consistently presented in viruses recovered from MRC-5 cells, but not Vero cells. Recombinant E-DIII proteins of E345K and E327G increased heparin binding correlated with the reduced infectivity by heparin treatment in cell cultures. Different from the E-E327G mutant viruses to lose the immunogencity in rhesus monkeys, the E-E345K mutant viruses were able to induce neutralizing antibodies in rhesus monkeys with an almost a 10-fold lower level of viremia as compared to the wild type virus. Monkeys immunized with the E-E345K mutant virus were completely protected with no detectable viremia after live virus challenges with the wild type DENV-4. These results suggest that the E-E345K mutant virus propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development.Knowledge of virulence and immunogenicity is important for development of live-attenuated dengue vaccines. We previously reported that an infectious clone-derived dengue type 4 virus (DENV-4) passaged in MRC-5 cells acquired a Glu Aedes aegypti and Aedes albopictus mosquitoes in tropical and sub-tropical areas. Disease severity ranges from asymptomatic infections to a febrile fever, or potentially life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). According to the World Health Organization (WHO), two-fifths of the world's population is at risk of DENV infection. 50–100 million cases occur each year, resulting in hundreds of thousands of incidents of DHF and DSS Dengue virus (DENV) is a vector-borne virus that is transmitted to humans by 345Lys (E-E345K) mutation was detected in 50% of DENV-4 virus propagated in MRC-5 cells 327Gly (E-E327G) mutation with enhanced heparin bindings, also resulting in a loss of infectivity and immunogenicity in rhesus monkeys Passaging of DENVs or their derived chimeras in Vero cells has been shown to generate mutations that are specific for host cell adaptation, virus attenuation, or other properties yet to be characterized 345K and E-E327G mutant viruses were obtained from DENV-4 and DENV-4Δ30 infectious clones and passaged in Vero and MRC-5 cells, respectively. The genetic stability and replication kinetics in Vero and MRC-5 cells were characterized. Enhanced heparin bindings and reduced mouse neurovirulence were observed for both E-E345K and E-E327G mutants compared to DENV-4 wild type. Since DENV-4 E-E327G mutant failed to induce viremia and neutralizing antibodies in monkeys 345K mutant induced potent neutralizing antibodies with low viremia in rhesus monkeys and protective immunity after live virus challenges. Our findings may have important implications regarding the development of live-attenuated DENV vaccines.In this study, we conducted site-directed mutagenesis on the infectious clones of DENV-4 and DENV-4Δ30 2) following ISCIII IACUC guidelines to ameliorate suffering. All experimental procedures involving monkeys were approved by the Animal Experiment Committee of Beijing Institute of Microbiology and Epidemiology, China. The animals were individually housed in cages and the room was cleaned and disinfected every 10 days. Fresh fruits and vegetables were provided twice a week. All procedures were performed under sodium pentobarbital anesthesia by trained technicians from the animal center and all efforts were made to ameliorate the welfare and to minimize animal suffering in accordance with the “Weatherall report for the use of non-human primates” recommendations.The mouse studies were conducted in accordance with guidelines established by the Laboratory Animal Center of National Tsing Hua University (NTHU). Animal use protocols were reviewed and approved by the NTHU Institutional Animal Care and Use Committee . Mice survived from experimental studies were sacrificed using carbon dioxide  assay. For FFU assay, the infected cells at 6 days post-incubation were fixed in 4% formaldehyde, permeabilized with 0.05% Tween 20, reacted with HB-114 monoclonal antibody, anti-mouse HRP secondary antibody, and stained with Liquid DAB-Plus Substrate Kit (Invitrogen).Vero, Vero E6, and MRC-5 cells were obtained from the Bioresource Collection and Research Center of the Food Industrial Research and Development Institute, Hsinchu, Taiwan. Cells were grown in Minimum Essential Media (MEM) (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/ml of penicillin G sodium-streptomycin (Invitrogen). Stock viruses were prepared from the supernatants of infected C6/36 cells grown in Hank's MEM medium (Gibco-BRL) plus 10% FBS for 6 days at 28°C. To prepare high DENV titers, virus supernatant was concentrated with a Centriplus device (10-kDa cutoff)  and centrifuged at 2,800 rpm for 30 min before each assay. All virus stocks were stored at −80°C until used for analysis. Virus titers were measured using 10-fold serial dilutions of culture supernatant in duplicate infections of Vero-E6 cell monolayers in 6-well plates. After incubation at 37°C for 1 h, 4 ml of medium containing 1×MEM, 1.1% methylcellulose, and 100 U/ml of penicillin G sodium-streptomycin was added to each well (4 ml/well). After 6 days post-incubation, virus plaques were stained with 1% crystal violet dye and Infectivity titers were measured in plaque forming units per ml (PFU/ml). To improve the visualization of E-E345K, DENV-4 E-E327G, and DENV-4Δ30 E-E345K. To construct DENV-4 E-E345K we used the primer pairs NsiI-f and E345K-r and E345K-f and StuI-r. In the second pair, E345K-f changed the E gene amino acid no. 345 from Glu to Lys  into a pJET1.2/blunt cloning vector (Fermentas Life Sciences) for amplification. To introduce changes at E protein residues 345 and 327, a 2 kb region flanked by NsiI and StuI restriction enzyme sites in the DENV-4 or DENV-4Δ30 infectious clone was replaced with NsiI-StuI fragments derived from confirmed clones containing E-E345K or E-E327G mutations. All the nucleotide sequences were confirmed by Mission Biotech Inc., Taipei, Taiwan.The infectious clones of DENV-4 and its 3′ NCR deletion mutant DENV-4Δ30 contained the full-length DENV cDNA corresponding to the vaccine candidate strain 814669 KpnI restriction enzyme, added to a transcription reaction mixture, and transcribed using SP6 RNA polymerase and the RiboMAX large-scale RNA production system (Promega). Full-length RNA transcripts were further capped with m7G(5′)ppp(5′)G at the RNA 5′ end using a ScriptCap Capping enzyme (EPICENTRE). After incubation at 37°C for 1 h, RNA product was purified with TRIzol LS reagent (Invitrogen) according to the manufacturer's instructions. Prior to RNA transfection, subconfluent Vero and MRC-5 cells (in 6-well plates) were rinsed with serum-free medium. The transfection mixture was prepared by adding 4 µl of DMRIE-C reagent (Invitrogen) to 1 ml of serum-free medium, followed by mixing with 10 µg of the RNA product. The transfection mixture was added directly to cell monolayers. After incubation for 18 h at 37°C, DMEM +10% FBS was added to each well. Culture supernatants were collected at 8 days post-transfection. All virus stocks were stored at −80°C until used.Infectious clone plasmids were linearized by cleavage with the PfxDNA polymerase (Invitrogen). Four overlapping fragments that span the entire DENV-4 genome were produced using 4 sets of forward and reverse primers: (i) W01/W02R, (ii) W03/W04R, (iii) W05/W06R, and (iii) W07/W08R reported previously(9). The nucleotide sequences of each of the four fragments were determined by Mission Biotech Inc., Taipei, Taiwan. The cloned DNA segments that span the entire DENV-4 genome were mapped using the software Lasergene version 6.00  to determine the extent of overlapping sequences.Viral RNAs were extracted from culture supernatants using TRIzol reagent (Invitrogen), and the cDNA synthesized by reverse transcription using Superscript II RTase (Invitrogen). The double-stranded DNA was then generated by PCR using Platinum 345K and E-E327G mutants were incubated with the concentrations of 10 ug/ml of heparin (Sigma), heparan sulfate (Sigma), chondroitin 4-sulfate  (Sigma), β-Heparin  (Sigma), chondroitin 6-sulfate  (Sigma), hyaluronic acid sodium salt (Sigma), heparin disaccharide IV-A, I-H, III-S or I-S sodium salt (Sigma) for 1 hour at 37°C in a CO2 incubator. Confluent monolayers of Vero-E6 cells in 6-well plates were rinsed with PBS and incubated with the mixture at 37°C for 1 hour. Next, 4 ml of medium containing 1× EMEM, 1.1% methylcellulose, and 100 U/ml of penicillin G sodium-streptomycin were added to each well. Virus titers were determined by plaque or focus-forming assays of Vero-E6 cells. The relative infectivity was determined as the percentage of GAGs-treated infectious titers to protein A sepharose bead –treated infectious titers.100 PFU of DENV-4 or clone-derived DENV-4 E-E5 FFU of DENV-4 wild type, E-E345K and E-E327G mutants was diluted in the tube before mixing with or without Sepharose beads at 4°C for 6 hr. All viruses were diluted in HBSS-BSA plus with or without Sepharose beads. Virus-bead mixtures were centrifuged for 5 min at 6,000×g at 4 °C to pellet the Sepharose beads. Infectious titers in supernatants were determined by plaque or focus-forming assays of Vero-E6 cells. The relative infectivity was determined as the percentage of beads-treated infectious titers to beads-untreated infectious titers.50 µM heparin-Sepharose, hyaluronic acid-EAH Sepharose and and Control protein A-Sepharose beads  were respectively suspended in phosphate-buffered saline (PBS) and equilibrated before use by pelleting and washing three times in Hank's balanced salt solution (Invitrogen) containing 0.2% bovine serum albumin (Gibco) (HBSS-BSA) plus 10 mM HEPES (pH 8.0). Next, 10345K and E-E327G viruses were cloned into pET22b (+) expression vector (Novagen) with an additional C-terminal 6x histidine gene to facilitate purification. The vectors were transformed into E.coli BL21 (DE3) cells (Invitrogen) and stimulated with 1 mM IPTG. DIII proteins obtained from inclusion bodies were purified using nickel-chelated resin (TOSOH) affinity chromatography under denaturing condition after cells were homogenized at 15K psi and re-suspended in 8M urea. Purified DIII proteins were eluted on 30–40% buffer B , concentrated by 30,000 centrifugal filters (MILLPORE), and analyzed by SDS-PAGE gel stained with Coomassie blue. For heparin binding ELISA assay, 5 mg heparin was added to 5 mg N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (Sigma) in 500 µl of ethanol and incubated at room temperature for 1 h, then 10 mg of BSA was added and incubated at 4°C overnight. Unconjugated heparin was removed by centrifugation at 3000 g using a spin concentrator (50-kDa cutoff) . ELISA plates were coated with 50 µl BSA–heparin (5 µg per ml) per well in a carbonate buffer  pH 9.6 at 4°C overnight. Plates were blocked with 200 µl blocking buffers (PBS with 1% BSA) for 1 h and then recombinant DIII proteins added for 1 h at room temperature. Plates were washed with PBST (PBS with 0.05% Tween-20) and incubated with mouse anti-His antibody for 1 h at 37°C. Plates were washed with PBST and then probed with anti-mouse–HRP conjugate antibody. Plates were again washed and TMB substrate buffers (BioLegend) was added for 15 min. Color reaction was arrested with 2N H2SO4 and plates were read at 450 nm. reader.The envelope protein domain III (DIII) genes of wild type, E-E4 FFU of the wild type and mutant DENV-4 and DENV-4Δ30 as described previously All mice were housed at the National Tsing Hua University barrier facility. All experiments were conducted in accordance with guidelines established by the Laboratory Animal Center of National Tsing Hua University (NTHU). Animal use protocols were reviewed and approved by the NTHU Institutional Animal Care and Use Committee (permit number: 09734). Inoculum were prepared by diluting virus stocks in HBSS containing 0.4% bovine serum albumin fraction V (Gibco) (HBSS-0.4% BSA fraction V) immediately prior to inoculation. For measuring neurovirulence, litters of newborn (<24 h) outbred white ICR mice  were inoculated intracranially with 30 µl of Mock diluent or diluent containing 10345K mutant containing 105 FFU. Blood was collected daily for 10 days to detect viremia. Blood samples for neutralizing-antibody tests were taken before immunization and then on days 0, 14 and 28 post immunization. On day 157 post immunization, the E-E345K mutant immunized monkeys were challenged by s.c. inoculation of 0.5 ml containing 105 PFU of DENV-4 wild type. Blood was collected daily for 10 days to detect viremia after live virus challenges. Blood samples for neutralizing-antibody were measured by neutralization assay on days 15 and 30 post-challenge. The levels of viremia in immunized monkeys were measured by quantitative real-time reverse transcriptase PCR. Briefly, viral genomic RNA was extracted from 200 µl of serum or an equal volume of sample serum by using the PureLink RNA minikit  according to the manufacturer's instructions. RNA was eluted in 50 µl of RNase-free water, aliquoted, and stored at −80°C until use. The target region for the assays was based on the C protein region of the genome of DENV-4. The number of PFU in serum detected was calculated by generating a standard curve from 10-fold dilutions of RNA isolated from a serum sample containing a known number of PFU, the titer of which was determined by plaque assay. Neutralizing antibody titers in sera were measured using a serum dilution-plaque reduction neutralization test (PRNT). We incubated 100 PFUs of the wild type DENV-4 with equal volumes of serial two-fold dilutions of heat-inactivated serum at 37°C for 1 h. Six-well plates of Vero-E6 cells were inoculated with the serum-virus mixture and incubated at 37°C 1 h. Plates were treated as described for the plaque titration protocol. Neutralizing antibody titers were identified as the highest serum dilution reducing the number of virus plaques by 50%. The 50% neutralization inhibition dose  was obtained using using GraphPad Prism version 5.01 Six monkeys, weighing 3.6 to 4.8 kg, were prescreened for negativity for antibodies against DENV by IFA. Animals were randomly divided into two groups and immunized subcutaneously (s.c.) in the deltoid region with 0.5 ml of cloned DENV-4 or DENV-4 E-E345K and E-E327G mutations  was based on the DENV-4 E-DIII structural model as determined by nuclear magnetic resonance (NMR) spectroscopy (Protein Data Bank code 2H0P). Surface mapping of the DENV-4 E-DIII electrostatic field was displayed using PyMOL software . Blue and red colors in Molecular modeling of E-Et test. Differences with a P value of less than 0.05 (*) were considered statistically significant.Statistical analyses were carried using GraphPad Prism . Statistical significance of differences between groups was assessed using two-tailed Student's 345K mutant viruses. RNA transcripts were obtained by incubating the cDNAs with SP6 RNA polymerase and rNTPs for 2 h, and then capping the transcribed RNA in vitro with GTP and a 5′-Cap capping enzyme for 1 h at 37°C. The in vitro RNA transcripts were analyzed by RNA gel electrophoresis to confirm high quality (data not shown). RNA transcripts were transfected into Vero or MRC-5 cells (i.e. P0) and propagated via four consecutive passages  at MOI  = 0.01 to avoid the influence by accumulation of defective interfering particles and enhance the possibility for cell adaptation. Small plaque formation with reduced cytopathogenicity was observed in Vero-E6 cells for E-E345K mutants derived from both DENV-4 and DENV-4Δ30 infectious clones.We conducted site-directed mutagenesis of these mutations on the two infectious cDNA clones, DENV-4 and DENV-4Δ30, to generate E-Ein vitro RNA transcripts (P0) to rule out the in vitro transcription errors. Results indicate that the E-E345K mutations of the DENV-4 and DENV-4Δ30 clones were consistent during all passages in MRC-5 cells . However, sequencing the virus stocks from Vero cells revealed the reversion of E345K to E345 WT sequence. , including heparan sulfate, heparin, chondroitin sulfate A, B, C, and hyaluronic acid. 100 FFU of DENV-4 wild type, E-E345K or E-E327G mutants were pre-treated with various GAGs at the concentration of 10 µg/ml. Compared to the untreated controls (no GAGs), the FFU percentages of DENV-4 wild type, E-E345K and E-E327G mutant were reduced by the treatment with heparin, but not with heparan sulfate, hyaluronic acid, chondroitin sulfate A, B, or C  with 105 FFU dose. The results demonstrated that two out of three monkeys inoculated with DENV-4 E-E345K mutant had peak viremia during two to five days post inoculation, and the mean peak virus titer of these three monkeys was 17.7 FFU/ml with 2.3 days duration  (Since DENV-4 E-Eduration . All of duration . Therefoto 1∶634 . By day (1∶1675) .345K immunization, the three immunized monkeys were challenged with 105 DENV-4 wild type on 157 day post immunization. The results indicated that DENV-4 E-E345K did not elicit viremia in any of these three immunized monkeys  substitution in DIII of the E protein. We used site-directed mutagenesis to construct the E-E345K mutant viruses from DENV-4 and DENV-4Δ30 infectious clones and passaged in Vero or MRC-5 cells to demonstrate that genetic stability and replication kinetics were consistently presented in MRC-5 cells. The E-E345K mutant viruses showed greater attenuation in mouse neurovirulence, and were able to induce neutralizing antibodies in rhesus monkeys with an almost 10-fold lower titer of viremia as compared to the wild type virus. Monkeys immunized with the E-E345K mutant viruses were completely protected with no detectable viremia after live virus challenges with the wild type DENV-4.The present study was based on Liu et al.'s (2008) report 345K mutant viruses were stably maintained in MRC-5 cells but not in Vero cells. We also generated the E-E327G mutant viruses from both DENV-4 and DENV-4Δ30 infectious clones, propagated in MRC-5 cells, but appeared as a mix of G and A nucleotides at P4 sequences (data not shown). However, the E-E327G mutation was consistent in P0 and P4 Vero cells (data not shown). Additionally, we constructed the mutant viruses with other positively charged amino acid substitutions, E-Gly345Arg (E-E345R) and E-Gly345His (E-E345H), and the sequencing results indicated that only E-E345R but not E-E345H mutants were stably maintained in MRC-5 cells (data not shown). These findings suggest that a gain of more positive charges by lysine  and arginine , but not histidine , may account for DENV adaptation in MRC-5 cells.According to sequencing analyses, E-E291 and E-K295345K 327G 345K and E-E327G mutant viruses may occur via electrostatic and hydrogen-bond interactions with the cationic amino acid side chains, we conducted a molecular structure modeling and surface mapping on E-DIII for E-E345K and E-E327G mutations according to the structural models determined by NMR spectroscopy (Protein Data Bank code 2H0P) 345K and E-E327G mutations indicated that E-E345K and E-E327G mutations increase the net positive charge in adjacent areas  DENV-4 E-E327G mutant.Effects of heparin and different form of heparin disaccharide on the inhibition of virus infectivity in Vero-E6 cells as determined by plaque percentages for (A) DENV-4 wild type, (B) DENV-4 E-E Bars represent means with standard deviation from two independent sets of experiments. .(TIF)Click here for additional data file.Figure S2Molecular modeling and surface mapping of DENV-4 E protein electrostatic fields. Blue and red denote positive and negative charges, respectively. White arrows: amino acid positions 345 and 327. Parental and variant structures were modeled into the DIII nuclear magnetic resonance-derived solution structure of DENV-4 E. Molecular modeling structure based on Protein Data Bank code 2H0P. Sum of partial charges analyses were carried using the PyMOL Molecular Graphics System software Version 1.1veal (Delano Scientific LLC).(TIF)Click here for additional data file.Checklist S1ARRIVE Checklist.(PDF)Click here for additional data file.


Aedes aegypti mosquito. Transcriptomic and bioinformatics analyses revealed that ~7500 transcripts are differentially expressed in four sequential waves during the 72-h reproductive period. A combination of RNA-interference gene-silencing and in-vitro organ culture identified the major regulators for each of these waves. Amino acids (AAs) regulate the first wave of gene activation between 3 h and 12 h post-blood meal (PBM). During the second wave, between 12 h and 36 h, most genes are highly upregulated by a synergistic action of AAs, 20-hydroxyecdysone (20E) and the Ecdysone-Receptor (EcR). Between 36 h and 48 h, the third wave of gene activation—regulated mainly by HR3—occurs. Juvenile Hormone (JH) and its receptor Methoprene-Tolerant (Met) are major regulators for the final wave between 48 h and 72 h. Each of these key regulators also has repressive effects on one or more gene sets. Our study provides a better understanding of the complexity of the regulatory mechanisms related to temporal coordination of gene expression during reproduction. We have detected the novel function of 20E/EcR responsible for transcriptional repression. This study also reveals the previously unidentified large-scale effects of HR3 and JH/Met on transcriptional regulation during the termination of vitellogenesis and remodeling of the fat body.In multicellular organisms, development, growth and reproduction require coordinated expression of numerous functional and regulatory genes. Insects, in addition to being the most speciose animal group with enormous biological and economical significance, represent outstanding model organisms for studying regulation of synchronized gene expression due to their rapid development and reproduction. Disease-transmitting female mosquitoes have adapted uniquely for ingestion and utilization of the huge blood meal required for swift reproductive events to complete egg development within a 72-h period. We investigated the network of regulatory factors mediating sequential gene expression in the fat body, a multifunctional organ analogous to the vertebrate liver and adipose tissue, of the female  In addition to being vectors of devastating human diseases, mosquitoes represent outstanding model organisms for studying regulatory mechanisms of differential gene expression due to their rapid reproductive cycles. About 7500 transcripts are differentially expressed in four sequential waves during the 72-h reproductive period in the fat body, a critical reproductive organ. The major regulators for these waves of gene expression are the two very important insect hormones, 20-hydroxyecdysone (20E) and Juvenile hormone (JH), their respective receptors Ecdysone Receptor (EcR) and Methoprene-Tolerant (Met), amino acids and the orphan nuclear receptor HR3. These key regulators are responsible for activation and repression of co-regulated gene sets, at different time points, within the 72-h reproductive period. Importantly, this study, apart from providing an insight into the regulatory complexity involved in the temporal coordination of gene expression, also reveals the previously unidentified roles of 20E/EcR, JH/Met and HR3 during the 72-h period post blood meal. Anopheles gambiae and the Dengue virus vector mosquito Aedes aegypti . At the end of the gonadotrophic cycle, it undergoes programmed autophagy and transforms itself back to reserve storage . Hence,  factors .A. aegypti mosquito.Here, we investigated the network of regulatory factors responsible for sequential gene expression in the PBM fat body. We show that systemic factors—JH, 20E and nutritional amino acids (AAs)—differentially regulate this gene-expression program. Moreover, our study has revealed that JH and 20E signaling in the PBM fat body is mediated by Met, EcR and HR3. Importantly, we report the previously unidentified role of JH in controlling gene expression during the PBM period. Furthermore, we have demonstrated the repressive function of 20E, which downregulates large cohorts of PBM genes in this mosquito tissue. Finally, we have shown that EcR mediates this repressive function. Taken together, our study provides new insights into the complexity of regulatory mechanisms responsible for temporal coordination of gene expression during reproduction in the female A. aegypti genes  in a chronological time order . 20E and JH titers (dashed lines), labeled on the right y-axis, are from Hagedorn et al. (Number of transcripts significantly upregulated and downregulated (left y-axis) in the microarray experiment [fold change ≥1.75  and a false-discovery rate Click here for additional data file.S2 Fig(A-C) Comparison of microarray (blue) and qRT-PCR (red) expression data for representatives of the Early Genes (EGs) set that were used for further analysis. (D-F) Comparison of microarray (blue) and qRT-PCR (red) expression data for the three Yolk Protein Precursor genes, that were used as representatives of the Early-Mid Genes (EMGs) set for further analysis. (G-I) Comparison of microarray (blue) and qRT-PCR (red) expression data for representatives of the Late-Mid Genes (LMGs) set that were used for further analysis. (J-L) Comparison of microarray (blue) and qRT-PCR (red) expression data for representatives of the Late Genes (LGs) set that were used for further analysis. The X-axis depicts the log2-fold change when the expression is compared to that at 72h PE and the Y-axis represents the time-points for tissue collection.(TIFF)Click here for additional data file.S3 Fig(A-B) Relative expression of genes—AAEL002488, Dead box atp dependent RNA helicase and AAEL004345, Cysteinyl t-RNA synthetase, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (C-D) Relative expression of the same genes detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (E-F) Relative expression in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (G-H) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments (C-D and G-H). All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001.(TIFF)Click here for additional data file.S4 Fig(A) AAEL009600—EcR, (B) AAEL001746—Met and (C) AAEL009588—HR3 genes detected by qRT-PCR, in fat body tissues collected from female mosquitoes post (A) EcR, (B) Met and (C) HR3 knock-downs, respectively. Injecting double stranded RNA for the Luciferase gene (iluc) served as the control. All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001.Relative expression of (TIFF)Click here for additional data file.S5 FigA-B) Relative expression of genes—AAEL007585, Cathepsin b and AAEL010434, Vitellogenin, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E). (C-D) Relative expression of the same genes detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (E-F) Relative expression in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (G-H) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (I-J) Relative expression of the same genes in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus Insulin (AA+INS), Insulin and 20E (INS+20E), amino acids plus 20E (AA+20E), and amino acids plus 20E and Insulin (AA+20E+INS). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments (C-D and G-H). All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001.((TIF)Click here for additional data file.S6 Fig(A-B) Relative expression of genes—AAEL004398, g protein-coupled receptor and AAEL014671, protease S51 alpha-aspartyl dipeptidase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (C-D) Relative expression of the same genes detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (E-F) Relative expression in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (G-H) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments (C-D and G-H). All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001.(TIF)Click here for additional data file.S7 Fig(A-B) Relative expression of genes—AAEL010075, oxidoreductase and AAEL002638, Cytochrome 450 detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (C-D) Relative expression of the same genes detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (E-F) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (G-H) Relative expression in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (I-J) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments . All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001.(TIF)Click here for additional data file.S8 Fig(A-B) Relative expression of genes—AAEL015143 –Glycine rich ribosome binding protein and AALE003352 –Ribosomal Protein l7ae_E2, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (C-D) Relative expression of the same genes in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (E-F) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments. All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SEM, * P < 0.05; ** P < 0.01; *** P < 0.001.(TIF)Click here for additional data file.S9 Fig(A-B) Relative expression of genes—AAEL002675, arginase and AAEL001623, proteasome subunit, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (C-D) Relative expression of the same genes in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (E-F) Relative expression of the same genes detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments. (G-H) Expression profiles of the same genes post first blood meal. (I-J) Expression profiles of the genes after the completion of the first reproductive cycle (egg laying) and post second blood meal. All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001.(TIFF)Click here for additional data file.S10 Fig(A-B) Relative expression of the genes—AAEL003347, CRAL/TRIO domain containing protein and AAEL000705, Steroid dehydrogenase, detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet); injecting double stranded RNA for the Luciferase gene (iluc) served as the control. (C-D) Relative expression of the same genes detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E). (E-F) Relative expression of the same genes detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR) injecting double stranded RNA for the Luciferase gene (iluc) served as the control.(TIFF)Click here for additional data file.S11 Fig(A-C) Relative expression of the early genes—AAEL002269, Purine nucleoside phosphorylase (A), AAEL002488, Dead box atp dependent RNA helicase (B) and AAEL004345, Cysteinyl t-RNA synthetase (C) detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA), with amino acid plus low concentration of 20E (AA+20E LO) and with amino acid plus high concentration of 20E (AA+20E HI). (D-F) Relative expression of the late-mid genes—AAEL002781, Galactokinase (D), AAEL003347, CRAL/TRIO domain containing protein (E) and AAEL000705, Steroid dehydrogenase (F) detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA), with amino acid plus low concentration of 20E (AA+20E LO) and with amino acid plus high concentration of 20E (AA+20E HI).(TIFF)Click here for additional data file.S12 Fig−8 M and 10−6 M) of 20E, along with AAs (AA+20E) for 8 h (4 h + 4 h respectively), in IVBFC, than, when incubated for 8 h with the higher concentration of 20E plus AAs (AA+20E NC). The YPP genes–(A) Vitellogenin, (B) Cathepsin beta and (C) Carboxypeptidase were used for this test. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001.Genes respond significantly better when incubated with two different concentrations (5x 10(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S1 Reference(DOCX)Click here for additional data file.


Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.During  Agrobacterium genus are phytopathogenic bacteria with the unique ability to transfer and integrate a segment of their own DNA (T-DNA) into the genome of their plant hosts1e.g., crown galls in the case of Agrobacterium tumefaciens) and production of opines, small molecules providing a source of carbon and nitrogen for the bacteriaAgrobacterium T-DNA relies on a set of proteins encoded by bacterial vir (virulence) genes located on a specialized Ti (tumor inducing)-plasmid, and on interaction of these Vir proteins with host cell factors1vir genes absolutely required for transformation, several non-essential vir genes are conserved in Ti-plasmids of different Agrobacterium strains. They likely represent host range factors required for efficient transformation of specific hosts, or provide a competitive advantage in the complex rhizosphere environment where several strains may be competing for the same host. virF, one of these non-essential genes, was originally described as responsible for difference of virulence between two main A. tumefaciens strains: octopine and nopaline (named after the nature of the opines produced in tumors) on several specific hosts. Indeed, a nopaline-specific Agrobacterium strain (C58) was only weakly virulent on Nicotiana glauca, as opposed to strains  harboring the octopine-type Ti-plasmid; the difference in virulence was found to depend on the virF locus of the octopine-specific Agrobacterium strainsvirF resulted in efficient transformation of N. glauca by the C58 strain, whether virF was expressed in bacteriavirF locus at all or its virF was not functional. Subsequently, it was found that octopine-type VirF is translocated into the host cell789Members of the  strain C8 was onls e.g., A harborinAgrobacterium strains does not make biological sense. Indeed, the Ti-plasmid from C58 Agrobacterium strain contains in its vir region a gene—Atu6154, which we term here C58virF—whose protein product C58VirF shares homology with the octopine-type VirF. A virF locus was also found in several Ti-plasmids from Agrobacterium vitis, suggesting that the presence of virF homologs is widespread in different Agrobacterium species and strainsAgrobacterium on different hosts depends, at least in part, on specificity of their VirF F-box proteins.That such an important virulence function as an F-box protein is not conserved between major virF gene is located in the vir region of the Ti-plasmid of the Agrobacterium C58-C1 strain, between virH and the region containing the virA-E loci. By comparison with octopine-type VirF from the A6 strain (A6VirF), the C58virF-encoded protein, C58VirF, is noticeably longer, i.e., 312 amino acid residues versus 202 residues, respectively . A study of another nopaline-specific Agrobacterium strain, SAKURA, which is almost identical to C58 in its vir region sequence, showed that expression of SAKURAvirF is induced by acetosyringonevirF also represents a true vir-type gene, albeit belonging to a group different from that of the classical VirF protein, A6VirF.A phylogenetic tree constructed with VirF protein sequences from the two major t groups : one conAgrobacterium to plant via the T4SShttp://psort.hgc.jp) detected no known specific subcellular localization signals in C58VirF. However, C58VirF is a small protein, and its molecular mass of ca. 34.5 kDa is within the 40–60 kDa size exclusion limit of the nuclear porePrevious studies suggested that C58VirF is transferred from Agrobacterium.Surprisingly, although octopine VirF-VIP1 complexes are known to accumulate in the cell nucleusArabidopsis Skp1-like, ASK, proteins using a yeast-two-hybrid assay, in which protein interaction is indicated by histidine prototrophyArabidopsis has been reported to contain 21 ASK genes in its genomeAgrobacterium proteins known to be translocated to host plant cell617252728Although previous studies indicated that C58VirF lacks apparent biological function4Within the SCF complex, interaction between the F-box protein and its Skp1/ASK partner is mediated by the F-box domainin planta, using bimolecular fluorescence complementation (BiFC). For these verification studies, we chose ASK1 as a representative ASK family member that is recognized by C58VirF  at 22 °C.A. tumefaciens C58-C1 as template—was inserted into the EcoRI-SalI sites of the LexA plasmid pSTT91 [TRP1+ +, Clontech). LexA fusion of VirE2 in pSTT91, and Gal4AD fusions of VirD2, VirE3, ASK1, VIP1 and VIP2 in pGAD424 were described previously12252829Arabidopsis Col0 cDNA library as template, and inserted into EcoRI-SalI  or BglII-PstI sites  of pGAD424.For the yeast-two-hybrid experiments, the C58VirF coding sequence—amplified using the primer pair 5′CCGGAATTCATGGAGCCCAGCCAACGAAGC3′/5′CCGCTCGAGTTATCGCGATAGTCCAGAGCGAC3′ and purified Ti plasmid of the wild-type 91 TRP1+ , and GalFor generation of C58VirFmut, coding sequences of the two overlapping N- and C-terminal segments of C58VirF were first amplified with the primer pairs 5′CCGGAATTCATGGAGCCCAGCCAACGAAGC3′/5′GCCGCAAGCTCGGGAGCCGCATCCC3′ and 5′GATGCGGCTCCCGAGCTTGCGGCTAAG3′/5′CCGCTCGAGTTATCGCGATAGTCCAGAGCGAC3′, respectively, introducing the following three mutations: M28A, P29A, and L33A. Then, using these two PCR products as templates, the full coding sequence of C58VirFmut was amplified and cloned into EcoRI-SalI sites of pSTT91 as described above for C58VirF.Agrobacterium VirD2 proteinFor transient expression of the GFP-GUS-C58VirF fusion, the coding sequence of C58virF was amplified using the primer pair 5′CCGGAATTCATGGAGCCCAGCCAACGAAGC3′/5′CCGCTCGAGTTATCGCGATAGTCCAGAGCGAC3′ and digested with EcoRI and SalI, and coding sequence of GUS was amplified using the primer pair 5′GGAAGATCTATGTTACGTCCTGTAGAAACCCC3′/5′CCGGAATTCTTGTTTGCCTCCCTGCTGC3′ and digested with BglII and EcoRI. Both fragments were then inserted by triple ligation into the BglII-SalI sites of pSAT5-MCSCauliflower mosaic virus (CaMV)For BiFC experiments, the coding sequence of ASK1 was amplified with the primer pair 5′AAAGGATTCATGTCTGCGAAGAAGATTGTGTTGAAG3′/5′AAACTGCAGTCATTCAAAAGCCCATTGGTTCTCTC3′ and inserted into the BglII-PstI sites of pSAT4-cCFP-CThe assay was performed using the yeast strain L40N. benthamiana leaf epidermis at a pressure of 140–160 psi using a portable Helios gene gun system , essentially as describedFor biolistic gene delivery, DNA preparations of tested constructs (20 μg of each plasmid) was absorbed onto 10 mg of 1-μm gold particles (Bio-Rad) and microbombarded into How to cite this article: Lacroix, B. and Citovsky, V. Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein. Sci. Rep.5, 16610; doi: 10.1038/srep16610 (2015).


TET2 rs3733609 C/T genotype had a higher incidence  in JAK2V617F-positive sporadic MPN patients than that in normal controls (n = 236) , which was predisposing to MPN (odds ratio(OR) = 2.361; P = 0.01). MPN patients with rs3733609 C/T genotype had increased leukocyte and platelets counts, elevated hemoglobin concentration in comparison with T/T genotype. Thrombotic events were more common in MPN patients with rs3733609 C/T than those with T/T genotype (P < 0.01). We confirmed that rs3733609 C/T genotype downregulated TET2 mRNA transcription, and the mechanism may be involved in a disruption of the interaction between CCAAT/enhancer binding protein alpha (C/EBPA) and TET2 rs3733609 C/T locus.TET2 rs3733609 C/T genotype stimulated the erythroid hematopoiesis in MPN patients. Altogether, we found a novel hereditary susceptible factor-TET2 rs3733609 C/T variant for the development of MPN, suggesting the variant may be partially responsible for the pathogenesis and accumulation of MPN.Common germline single-nucleotide polymorphisms (SNPs) at JAK2 locus have been associated with Myeloproliferative neoplasms (MPN). And, the germline sequence variant rs2736100 C in TERT is related to risk of MPN, suggesting a complex association between SNPs and the pathogenesis of MPN. Our previous study (unpublished data) showed that there was a high frequency distribution in rs3733609 C/T genotype at Ten-Eleven Translocation 2 (TET2) locus in one Chinese familial primary myelofibrosis. In the present study, we evaluate the role and clinical significance of rs3733609 C/T genotype in JAK2V617F-positive sporadic MPN ( The most common BCR-ABL negative MPN includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) . In the Loss-of-function mutations in TET2 are found in 5%–17% of MPN and have been shown to decrease 5-hmc levels –21. TET2http://www.ncbi.nlm.nih.gov/snp/?term = rs3733609) was much higher in one familial PMF than sporadic PMF (unpublished data), suggesting rs3733609 may be a MPN predisposition allele. In the present study, we attempted to determine whether the C allele of the rs3733609 is associated with the predisposition to sporadic MPN harboring JAK2V617F mutation and especially with the clinical phenotypes and the complications of MPN. Furthermore, we also evaluated the effects of the rs3733609 on myeloid lineage proliferation and explored in part the possible mechanism of rs3733609 C/T genotype in developing MPN.Previously, we noted that the frequency of rs3733609 C/T genotype (located in TET2 exon 9) = 2.361; 95% Confidence Interval (CI) = 1.206–4.624), suggesting that TET2 rs3733609 C/T genotype was associated with a predisposition to sporadic MPNs.To determine whether rs3733609 C/T genotype was associated with the predisposition to sporadic MPN, we analyzed the distribution of TET2 rs3733609 C/T genotype using PCR-direct sequencing in 181 MPN patients with JAK2V617F-positive and 236 healthy controls. A typical DNA sequencing of TET2 rs3733609 genotype was shown in Figure P < 0.001, P = 0.042 and P = 0.019, respectively). Compared to T/T genotype, the MPN patients with C/T genotype had higher proliferative activity, which was defined as the hyperplasia degree of bone marrow (P = 0.005). Meanwhile, thrombotic events and hepatosplenomegaly were more common in C/T genotype (P < 0.01 for both). However, no significant differences were observed in age, sex and cardiovascular risk factors such as tobacco use, hypertension, or diabetes mellitus between the two genotypes.Our results have shown that TET2 rs3733609 C/T genotype is a risk factor for the development of MPN. Whether MPN cases with rs3733609 C/T genotype exhibited a different clinical features and thrombotic complication from those of MPN with T/T genotype is also the concerned problems. We compared the hematological parameters and clinical characteristics at newly diagnosed 181 MPN patients with JAK2V617F-positive. According to the rs3733609 genotype stratification, there were evident difference on clinical phenotypes and hematological parameters between two groups (Table P < 0.001), rs3733609 C/T genotype (P < 0.001), WBC count ≥ 10 × 109, (P < 0.001) and cardiovascular risk factors (P < 0.001) were correlated with thrombotic complications; while the sex, hemoglobin levels and platelet numbers were not risk factors for thrombotic events (P > 0.05) (Table P = 0.022); rs3733609 C/T genotype ; increased WBC count  and cardiovascular risk factors  were independent risk factors of thrombosis respectively  had thromboembolic events. Among them, 31 cases were arterial embolism (17.13%) and 12 cases were vein thrombosis (6.63%). Since the occurrence of thrombotic complication was higher in MPN patients with rs3733609 C/T genotype than those with T/T genotype, we analyzed the risk factors of thrombosis using univariate analysis and then definited the thrombotic risk factors from a shortlist of seven candidates comprising rs3733609 C/T genotype, sex, age, WBC count, hemoglobin levels, platelet numbers and cardiovascular risk factors with reference to related literatures . The uni.75% had n = 18) and T/T genotype (n = 35). The results demonstrated that TET2 mRNA levels were decreased by 71.2% in rs3733609 C/T group in comparison to T/T group . Compared with the T/T genotype vector, the luciferase activity of C/T genotype vector of TET2 gene was reduced (P = 0.009) on transfected HEL cells, suggesting that the rs3733609 T locus in TET2 exon 9 was a critical positive regulatory element, and the T to C variant at this position impeded its transcriptional activity.To investigate the effect of the TET2 gene with rs3733609 C/T genotype on transcript activity, we cloned the exon 9 fragment of TET2 gene with both rs3733609 T/T or C/T genotype sequences  into pGL3-promoter-vector Figure  and condin vitro, we performed burst-forming unit-erythroid (BFU-E) assays on BMMNCs from 39 cases of JAK2V617F positive MPN patients with (n = 16) or without rs3733609 C/T genotype (n = 23). The results showed that the percentages of BFU-E formation in C/T genotype was higher than that in T/T genotypes (P = 0.005)  (Table P = 0.001) (Table To clarify the effect of rs3733609 C/T genotype on the erythroid hematopoiesis  P = 0.00 (Table 51) Table . AltogetP = 0.01), suggesting that rs3733609 C is a MPN predisposition allele.In MPN, TET2 mutations have been associated with an increased risk of leukemic transformation and short survival . InteresGiven these findings, we then wished to determine whether the MPN cases with rs3733609 C/T genotype display different characteristics of clinical and laboratory from those with T/T genotype. To do so, we compared and analysed some variables by univariate and multivariate analysis. Our results showed that compared with T/T genotype, MPN cases with rs3733609 C/T genotype had elevated leukocyte and platelet counts, increased hemoglobin levels; and an higher incidences of hepatosplenomegaly and thrombotic events, indicating C/T genotype contributes to myeloid hematopoiesis of MPN Table . In addiP = 0.006). To further confirm this change, we performed luciferase activity assay, and obtained a consistent results with RT-PCR in that the transcriptional activities produced by pGL3-pro-TET2-exon9-Mut96 were lower than the wild-type construct (P = 0.009), indicating the rs3733609 T/T locus was involved in the basal activation of the transcription of TET2.We also attempted to reveal the mechanisms of rs3733609 C/T genotype on the pathogenesis and the susceptibility of MPN. We analyzed BMMNCs samples from MPN patients with or without rs3733609 C/T genotype using a real-time quantitative-PCR assay, which showed that the expression of TET2 mRNA in rs3733609 C/T genotype decreased by 71.2% than that in T/T genotypes  is a key factor in driving the development of myeloid cells through interacting with a variety of factors, and mutations of C/EBPA have been observed in about 10% of patients with acute myeloid leukemia (AML) , 28. As In conclusion, we report a novel germline variant in TET2 that associates with JAK2V617F-positive MPN. The risk regulated by rs3733609 C/T variant is higher (OR = 2.361) than the reported JAK2 germline signal rs1034072 A (OR = 1.85). Moreover the variant also exerts effect on myeloid hematopoiesis by promoting proliferation of myeloid progenitor cells, which is related to the decreased transcript activity of TET2. and, this study characterizes the mechanism by which rs3733609 C/T variant escapes from the positive regulation of C/EBPA on TET2 transcription.One hundred and eighty-one consecutive MPN patients , diagnosed between 2005 and 2015, were enrolled into the study after carefully revising all of diagnosis according to the criteria of World Health Organization classification of MPN . The conThrombotic events were defined as arterial or venous events occurring prior to diagnosis or at any time post diagnosis of MPNs. For arterial events, cardiovascular events included myocardial infarction and unstable angina, which were confirmed by an elevated troponin I level or with electrocardiogram changes. Cerebrovascular events included radiologically verified cerebrovascular accidents or transient ischaemic attacks with consistent history. Peripheral vascular events included new onset of intermittent claudication or acute limb ischaemia. Venous thromboembolic events included radiologically or ultrasonically confirmed portal vein, abdominal vein and deep venous thrombosis with elevated D-D levels.Genomic DNA was obtained from peripheral blood or bone marrow using a commercially available BloodGen Kit . The JAK2 V617F mutational status was analyzed in all MPN patients by a allele specific PCR method, as previously described . A 475 b6 cells/mL was extracted by Trizol reagents (Invitrogen). Random primers and M-MLV reverse transcriptase (TaKaRa) were used to generate cDNA. Real-time quantitative PCR was carried out on a LightCycler 96  using SYBR® Premix Ex Taq TM II Kit (TaKaRa). Primers sequences were as follows: Human TET2-specific primers: forward primer: 5′-CTATTGCTAAGTGGGTGGTTCG-3′; reverse primer: 5′-GCCGTATTTCCTCAGCGTCTC -3′. Human β-actin amplifation was designed as internal control. The PCR reactive system comprised cDNA template 2 uL, 2xSYBR mix 10 uL, forward or reverse primers 10 uM.To assess whether TET2 mRNA expression was influenced by rs3733609 SNP, real-time quantitative PCR was performed for evaluating the expressions of TET2 mRNA. 35 of MPN with rs3733609 T/T genotype and 18 of MPN with rs3733609 C/T genetype were compared for TET2 mRNA expressive abundance. In brief, bone marrow (5 mL) mononuclear cells (BMMNCs) were isolated by Ficoll centrifugation; total cellular RNA from 1 × 10http://www.gene-regulation.com/cgi-bin/pub/programs/patch/bin/patch.cgi), and the predicted results indicated that rs3733609 (T/T genotype) loci could bind to C/EBPA protein domain . Briefly, BMMNCs (1 × 107/ml) from 16 of MPN patients with rs3733609 C/T genotype and 20 of MPN cases with rs3733609 T/T genotype were crosslinked respectively for 10 minutes in 1% formaldehyde solution. Then, crosslinked chromatins were prepared and sonicated to an average size of 500 bp. Chromatin-protein complexes were immunoprecipitated with antibody specific to C/EBPA  or normal mouse IgG at 4°C for overnight. Input DNA was precipitated without antibody. After reversal of cross-linking, the region of interested was amplified from precipitated samples by real-time quantitative PCR. For the amplification of TET2 gene fraction, the primers sequences were as follows: forward primer: 5′-CCGTCTGGGTCTGAAGGAAG-3′; reverse primer: 5′-CTGTCCTCAGCCCAACTTACC-3′. Each sample was assayed in triplicate, and the amount of precipitated DNA was calculated as the percentage of input sample.The prediction of transcriptional factor binding site for TET2 rs3733609 SNP was performed with Transfac-Patch 7.0 Software  containing 5 ug of the pGL3 promoter construct and 0.5 ug pRL-TK plasmid using T016 electroporation program [To further testify whether rs3733609 C/T genotype is able to affect the transcriptional activity of the TET2 gene, a corresponding reporter gene plasmid was constructed as follows: 1). pGL3-promoter vector , containing the SV40 promoter of driving the firefly luciferase gene; 2). pGL3-pro-TET2-exon9-wt, containing the luciferase reporter gene and carrying 138 bp of TET2 exon 9 (rs3733609 T/T genotype); 3). pGL3-pro-TET2-exon9-Mut96 (rs3733609 C/T genotype), containing the TET2 exon 9 c.138 + 96T > C variation. The exon 9 and its variant form (+96 T > C variation) were respectively inserted into the junction region between Kpn I and MLuI restriction locus Figure . The con program . At 48 h5 BMMCs from MPN patients with rs3733609 C/T variant were plated in 1 ml of RPMI-1640 methylcellulose culture without any cytokines. Meanwhile, the BMMCs from MPN patients with T/T genotype were used as the control. The culture medium consist of 0.8% methylcellulose , 1% deionised bovine serum albumin (BSA), 30% FCS and 2-β-mercaptoethanol (10−4 M). Culture dishes were incubated at 37°C for 14 d in a highly humidified atmosphere with 5% CO2. The colonies were identified by benzidine staining  was performed. In briefly, 1 × 10g Figure  and enumt-test (two-tailed) or the Mann-Whitney U-test. Logistic regression analysis was used for multivariate analysis. SPSS Version 19.0  was used for all statistical tests. In all tests, P < 0.05 was defined as statistically significant.The frequencies of the genotypes between MPN cases and controls were compared by Pearson's Chi-square test or Fisher's exact test when required. The data was expressed as the means ± SD. Univariate analysis was treated using Student's