Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate β-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S-transferase NaGSTNu is the only enzyme needed to remove glutathione from both (R)- and (S)-GS-HPV in N. aromaticivorans. We solved the crystal structure of NaGSTNu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism  were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6  and Escherichia coli (which cannot break the β-aryl ether bond) can also cleave (R)- and (S)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S-transferases.As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the β-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the β-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-β-guaiacyl ether (GGE) and found that  As society looks to diversify its sources of fuels and chemicals, there are reasons to produce them from renewable resources, such as lignocellulosic plant biomass, the most abundant organic material on Earth. Lignin, which can compose ∼25% of lignocellulosic plant biomass , is a heSphingobium sp. SYK-6, Erythrobacter sp. SG61-1L, and Novosphingobium sp. MBES04, are known to contain this pathway  bond typically constitutes >50% of all the linkages between aromatic units in lignin . Thus, m bond typ pathway 6, identiSM 12444 , 8, Novosp. PP1Y , and SphC 107872 , for theC 107872  and find+-dependent dehydrogenases  to generate the α-ketone, β-(2-methoxyphenoxy)-γ-hydroxypropiovanillone (MPHPV) (–S-transferase (GST) LigG from Sphingobium sp. SYK-6 has been shown to remove the glutathione moiety from β(R)-GS-HPV in vitro (S)-stereoisomer. The Nu-class GST GST3 from Novosphingobium sp. MBES04 has been reported to react with both β(R)- and β(S)-GS-HPV in vitro (N. aromaticivorans (NaGSTNu) and Sphingobium sp. SYK-6 (SYK6GSTNu) can react with both β(R)- and β(S)-GS-HPV in vitro and that NaGSTNu is the sole enzyme required for these reactions in N. aromaticivorans. We also use kinetic and structural data to propose mechanisms for the glutathione lyase (deglutathionylation) reactions of NaGSTNu with both GS-HPV stereoisomers.Breaking the β-aryl ether bond of GGE via the β-etherase pathway involves three steps . First,  (MPHPV) . Next, s (MPHPV) –15. Finain vitro , but no in vitro; most, including EcYfcG and EcYghU from Escherichia coli, showed disulfide bond reductase activity toward small molecules, such as 2-hydroxyethyl disulfide - and β(S)-GS-HPV in vitro, although less efficiently than NaGSTNu. Furthermore, EcYghU complements growth of an N. aromaticivorans ΔNaGSTNu mutant, showing that it can perform these reactions in vivo. Our results thus suggest that glutathione lyase activity may be common throughout the Nu-class, and the lower catalytic efficiencies we find with EcYghU and EcYfcG toward GS-HPV may reflect the fact that this compound is not a natural substrate for E. coli.The most distinguishing characteristic of Nu-class (Main.2 ) GSTs is16–lfide 16–, 21, whieroxides . At leasne lyase . To inveN. aromaticivorans DSM 12444, Novosphingobium sp. PP1Y, and S. xenophagum NBRC 107872, we fed them erythro-GGE  gene has been deleted (12444Δ1879), so that we could make additional modifications to its genome using a sacB-containing cloning vector. When fed erythro-GGE alone, N. aromaticivorans 12444Δ1879 completely removed the compound from the medium (A and B) and Fig. S1) and incorporated ∼22% of its organic material ) into biomass (Table S1). The majority of the electrons from the erythro-GGE (∼53%) were probably combined with oxygen to support respiration during growth with the compound (Table S1). When fed erythro-GGE plus vanillate, 12444Δ1879 completely removed both substrates from the medium (G and H) and incorporated ∼41% of the COD from the vanillate  and ∼22% of the COD from the GGE into biomass (Table S1). The known β-etherase pathway intermediates threo-GGE, MPHPV, and HPV transiently appeared in the media of both erythro-GGE–fed 12444Δ1879 cultures (B and H) and Fig. S1), whereas the pathway intermediate guaiacol was only detected at a low level in the medium of the culture fed erythro-GGE plus vanillate (H). The predicted pathway intermediate GS-HPV was not detected in the medium of either culture. Wild-type N. aromaticivorans DSM 12444 behaved similarly under both of these growth conditions, justifying our use of 12444Δ1879 as a parent strain in this study.For these studies, we created a strain of e medium , G and Hanillate H. The prerythro-GGE, Novosphingobium sp. PP1Y slowly converted most of it into β-etherase pathway intermediates but did not completely remove these compounds from the medium and only achieved a low cell density . When fed erythro-GGE plus vanillate, Novosphingobium sp. PP1Y rapidly consumed the vanillate, but the maximum cell density and amount of COD incorporated into biomass in this culture were the same as when the strain was fed vanillate only , suggesting that the strain did not convert a significant amount of GGE into biomass in the presence of vanillate. S. xenophagum converted some of the erythro-GGE it was supplied into β-etherase pathway intermediates  but did not assimilate organic material from it into cellular biomass in any culture tested: when fed erythro-GGE alone, no cell density was detected, and when fed erythro-GGE plus glucose (we found this strain to be unable to metabolize vanillate), the maximum cell density and amount of COD incorporated into biomass were the same as when the strain was fed glucose only (C and G)Fig. S2 ( and Table S1). These results suggest that N. aromaticivorans is the best of the three organisms we analyzed for studying various aspects of the β-etherase pathway.When fed only N. aromaticivorans to GGE, we investigated expression levels of several of its genes predicted to code for enzymes of the β-etherase pathway, including Saro_2595, which encodes a Nu-class glutathione S-transferase (named here NaGSTNu). With the exception of ligL, transcript levels from these genes were higher (by >6-fold) in cells grown in the presence of GGE versus its absence , which can convert β(R)- and β(S)-GS-HPV into HPV in vitro (N. aromaticivorans lacks any homologues of LigG (the enzyme from Sphingobium sp. SYK-6 that cleaves the β(R)-stereoisomer of GS-HPV (Nu could cleave both β(R)- and β(S)-GS-HPV. We found that recombinant NaGSTNu cleaved both stereoisomers of GS-HPV in vitro , with slightly higher kcat and ∼5-fold higher mK with β(R)-GS-HPV than with β(S)-GS-HPV, resulting in a ∼4-fold higher kcat/mK with the β(S)-isomer (NaGSTin vitro  6). Bec. BecNu if GS-HPV  13)), w), wNu is)-isomer .in vivo role for NaGSTNu, we generated an N. aromaticivorans strain (12444Δ2595) lacking Saro_2595, the gene that encodes NaGSTNu. When 12444Δ2595 was provided only erythro-GGE, a small amount of MPHPV and threo-GGE appeared in the medium (D), but no cell density was detected (C), and no detectable COD was converted into biomass (Table S1). When 12444Δ2595 was fed both vanillate and erythro-GGE, all of the vanillate was consumed, and almost all of the GGE was converted into MPHPV (J). The maximum cell density and amount of COD incorporated into biomass were the same for this culture as for a culture fed vanillate only , suggesting that 12444Δ2595 cannot convert GGE into cell material. A small amount of guaiacol appeared in the medium of the 12444Δ2595 culture fed vanillate and erythro-GGE , showing that this strain cleaved some MPHPV. However, unlike for its parent strain (12444Δ1879), no extracellular HPV was detected in any erythro-GGE–fed 12444Δ2595 culture (D and J). These results show that NaGSTNu is necessary for complete GGE metabolism by N. aromaticivorans.To test for an e medium D, but nodetected C, and noto MPHPV J. The maNu, we incubated cell extracts of 12444Δ2595 and its parent strain (12444Δ1879) with racemic MPHPV and GSH. With the 12444Δ1879 extract, MPHPV was completely converted to roughly equimolar amounts of guaiacol and HPV, along with a small amount of GS-HPV (A). In contrast, the 12444Δ2595 extract incompletely cleaved the MPHPV, producing roughly equimolar amounts of guaiacol and GS-HPV . Thus, the 12444Δ2595 extract was defective in converting GS-HPV into HPV. When recombinant NaGSTNu was added to the 12444Δ2595 extract after 1 day of incubation with MPHPV , the initially accumulated GS-HPV rapidly disappeared, with a concomitant increase in HPV, showing that the defect in GS-HPV cleavage by the 12444Δ2595 extract was caused by the lack of NaGSTNu.To determine which step in the β-etherase pathway requires NaGSTf GS-HPV A. In conNu, we solved two structures of the enzyme with resolutions of 1.25 (PDB code 5UUO) and 1.45 (PDB code 5UUN) Å  domain ,135–Leu257), and N-terminal (Met1–Pro38) and C-terminal  extensions not present in most other characterized classes of GSTs (A).To gain insight into the mechanism of GS-HPV cleavage by NaGST 5UUN) Å . The str of GSTs A.e.g. PDB codes 3C8E and 4IKH) or a single GSSG molecule (e.g. PDB codes 4MZW and 3GX0) in each active site. Consistent with this, each NaGSTNu subunit contained electron density data that were best modeled as a mixed population of GSH1 and GSH2 thiols with an HS–SH distance of 2.4 Å (∼60% occupancy) and a GSSG disulfide with S–S distance of 2.0 Å (∼40% occupancy) , suggesting a heterogeneous population of protein molecules in the crystals in which each subunit contained either two GSHs or a single GSSG .Structures of Nu-class GSTs solved in the presence of GSH typically contain either two GSH molecules , and three residues make close contacts with GSH2  (B). These residues and contacts with the GSHs are conserved throughout much of the Nu class   B. These Fig. S3) , 20, altNu, a short channel connects the active site to the solvent . This channel is also present in Nu-class GSTs from E. coli . In most other structurally characterized Nu-class GSTs, the active site is more solvent-exposed, because these proteins lack N-terminal residues that contribute to one of the channel walls  and StrNu homodimer we solved (PDB codes 5UUN and 5UUO), the individual monomers differ in the positioning of residues Gln282–Phe288 (A–C). The C-terminal Phe288 resides near the active-site channel in one subunit  and ∼18 Å away from the channel entrance in the other , resulting in a difference between the monomers in the opening to the active-site pocket. The closed NaGSTNu configuration is stabilized by hydrogen bonds between Lys286 and both Lys262 and Arg220. The open configuration lacks these interactions, and its Arg220 side chain has two rotamer positions. In other structurally characterized Nu-class GST homodimers, the individual subunits are symmetric, with C termini that generally extend away from the active site ).In each NaGST2–Phe288 , A–C. ThR)- and β(S)-GS-HPV into the GSH2 active-site position (B) of the closed C-terminal configuration subunit of NaGSTNu and found that the HPV moieties extend into the active-site channel in different orientations without generating unfavorable steric clashes (A and B) and A and B)Fig. S7 . For both GS-HPV stereoisomers, there are predicted hydrogen bonds between the γ-hydroxyl and the hydroxyl of Tyr224 and between the HPV phenolic group and the carboxyl group of the C-terminal Phe288 (A and B). For β(R)-GS-HPV, the γ-hydroxyl is also predicted to hydrogen-bond with the α-ketone, which in turn is predicted to hydrogen-bond with the hydroxyl of Tyr166 (A). For β(S)-GS-HPV, the hydroxyl of Tyr166 is predicted to form a hydrogen bond with the HPV aromatic ring (B).We separately modeled the GS- moiety of β and the side-chain amide of Asn53 (3.2 Å) ). These residues are part of a Thr-Pro-Asn motif that is highly conserved throughout the Nu class, the Asn residue of which was found to be critical for the glutathione lyase activity of PcUre2pB1 . Extended hydrogen-bonding networks (“second-sphere interactions”) such as this are proposed to contribute to GSH thiolate stabilization in other GST classes (We propose that the thiol of a GSH molecule in the GSH1 active-site position of NaGSTcUre2pB1 . Interac GSTA3-3 ). In add classes .Nu, the GS1 thiolate attacks the thioether of GS-HPV to form a disulfide GS-SG. Because the C–S bond lyase reaction proceeds in the absence of a redox cofactor, such as flavin or pyridine nucleotide, we propose that a transient enolate (C and D) stores the 2e− reducing equivalents released by disulfide bond formation. Formation of this enolate is proposed to be facilitated by polarization of the GS-HPV α-ketone, which is stabilized by the predicted interactions between GS-HPV and Tyr166 and Tyr224 (C and D)). Due to active-site steric constraints, our modeling places the reactive portion (S-Cβ-(Cα=O)-aryl) of both β(R)- and β(S)-GS-HPV into roughly planar configurations in the NaGSTNu channel (A and B) and A and B)Fig. S7 , which should also promote enolate formation. Collapse of the proposed enolate intermediate proceeds with carbanion trapping of a solvent-derived proton, corresponding to reduction of the carbon atom originally containing the thioether bond (E and F).In our proposed reaction mechanism for NaGST enolate , C and Dher bond , E and FNu, in which the activated thiol of a glutathione molecule is used to break the thioether bond of GS-HPV, is different from that proposed for the Omega-class GST LigG, in which the activated thiol of a Cys residue in the enzyme breaks the thioether bond , whose thiol is >20 Å away from either GSH thiol, and so is unlikely to be involved in the lyase reaction.This proposed mechanism for NaGSTher bond . In contive site , 28. NaGNu predicted to be important for catalysis were separately mutated: Thr51 into Ala (T51A), Tyr166 into Phe (Y166F), and Tyr224 into Phe (Y224F). All three mutated enzymes had decreased reactivity with GS-HPV compared with wildtype NaGSTNu (B–D)Fig. S5 - and β(S)-stereoisomers of GS-HPV, respectively. These results suggest that the Tyr166 hydroxyl is critical for catalysis with both GS-HPV stereoisomers and is important for binding of β(S)-GS-HPV. Compared with wildtype NaGSTNu, the Y224F variant had kcat values that were ∼40- and ∼60-fold lower and mK values that were ∼7.5- and ∼6-fold higher for the β(R)- and β(S)-stereoisomers of GS-HPV, respectively. These results implicate the Tyr224 hydroxyl in both binding and catalysis, although it is less important to catalysis than that of Tyr166.To test the plausibility of our proposed reaction mechanism, three residues in NaGSTR)- and β(S)-isomers of a GS-conjugated syringyl phenylpropanoid into the NaGSTNu active site predicts that they should bind in essentially the same orientations as the corresponding GS-HPV isomers, because the active-site channel can accommodate each syringyl conjugate's additional methoxy group . Thus, Nu-class GSTs may be the only enzymes necessary for the glutathione lyase step in deconstructing natural lignin oligomers via the β-etherase pathway.Besides GS-HPV, depolymerization of natural lignin oligomers via the β-etherase pathway is expected to produce syringyl phenylpropanoids . ModelinSphingobium sp. SYK-6 can metabolize GGE (R)-GS-HPV (S)-GS-HPV has been identified in this organism (S)-GS-HPV, we tested a Nu-class GST from Sphingobium sp. SYK-6  encoded by SLG_04120 and named here SYK6GSTNu and found that recombinant SYK6GSTNu cleaved both β(S)- and β(R)-GS-HPV in vitro . SYK6GSTNu had higher kcat and lower mK with β(S)-GS-HPV than with β(R)-GS-HPV, leading to a ∼10-fold greater kcat/mK with the β(S)-isomer (Nu could cleave β(S)-GS-HPV and potentially contribute, along with LigG, to β(R)-GS-HPV cleavage in Sphingobium sp. SYK-6 -GS-HPV , no enzyorganism . To iden)-isomer . Thus, Sp. SYK-6 .E. coli (EcYghU and EcYfcG), an organism not known to metabolize lignin-derived glutathione conjugates. We found that recombinant EcYghU cleaves β(R)- and β(S)-GS-HPV in vitro ; whereas its mK values were comparable with those of NaGSTNu, its kcat values were much lower, resulting in kcat/mK values for EcYghU ∼100-fold lower than those of NaGSTNu (R)- and β(S)-GS-HPV into the active site of EcYghU (PDB code 3C8E). Whereas EcYghU is similar to NaGSTNu in sequence  and structure . The hydroxyls of Tyr167 and Tyr225 in EcYghU  are predicted to interact with the HPV moiety of both β(R)- and β(S)-GS-HPV (C and D). For β(R)-GS-HPV, Tyr225 is predicted to form hydrogen bonds with both the γ-hydroxyl and α-ketone, and Tyr167 is predicted to form hydrogen bonds with the α-ketone and the aromatic ring (C). For β(S)-GS-HPV, Tyr225 is predicted to form a hydrogen bond with the γ-hydroxyl, and Tyr167 is predicted to form a hydrogen bond with the aromatic ring and a long-range interaction with the γ-hydroxyl (D). These interactions could help stabilize an enolate intermediate in the reactions between EcYghU and GS-HPV, so EcYghU could follow a reaction mechanism similar to that proposed for NaGSTNu (β-(Cα=O)-aryl) of both β(R)- and β(S)-GS-HPV bound to EcYghU are predicted to be ∼45° out of alignment (C and D) and E and F)Fig. S7 , which could hinder formation of the enolate and lead to the lower kcat values for EcYghU than for NaGSTNu (for which these reactive atoms were in a roughly planar configuration (A and B) and A and B)Fig. S7 .To investigate the prevalence of glutathione lyase activity throughout the GST Nu-class, we tested two Nu-class GSTs from  NaGSTNu . To inve)-GS-HPV , C and Dtic ring C. For β in which Saro_2595 was replaced in the genome by the E. coli yghU gene completely removed the GGE from the media of both of its cultures and assimilated the GGE into biomass  and Table S1), whereas 12444Δ2595 (which lacks NaGSTNu) could not . 12444EcyghU metabolized GGE slower than 12444Δ1879 (which contains NaGSTNu) (Nu.Despite these lower NaGSTNu) , which wE. coli Nu-class GST we tested, EcYfcG, is less similar to NaGSTNu in sequence  and structure - and β(S)-GS-HPV in vitro , mK values were higher and kcat values were much lower than those of NaGSTNu, leading to kcat/mK values with GS-HPV ∼10,000-fold lower for EcYfcG than for NaGSTNu , which were found to be important for catalysis and substrate binding in NaGSTNu. Given the dramatically lower catalytic efficiencies of EcYfcG, it is unlikely that EcYfcG could substitute for NaGSTNu in N. aromaticivorans.The other  channel ). Wherea NaGSTNu . The lowS-transferase from N. aromaticivorans that acts as a glutathione lyase in the process (NaGSTNu).In developing bio-based systems to generate products from renewable materials such as lignin, optimized cellular and enzyme catalysts are needed. Toward this end, we tested sphingomonads for the ability to break the β-aryl ether bond commonly found in lignin  and idenN. aromaticivorans was the most effective species studied here at breaking the β-aryl ether bond of the dimeric aromatic compound GGE and assimilating GGE into cellular biomass. The rate of GGE β-aryl ether bond breaking by N. aromaticivorans  is comparable with rates for Erythrobacter sp. SG61-1L , and S. xenophagum  are slower and/or less efficient at breaking the β-aryl ether bond of GGE under laboratory conditions, although they each contain enzymes implicated in the β-etherase pathway. Understanding the bases for these differences can aid future efforts to develop microbial systems for converting lignocellulosic biomass into commodities.We found that threo-GGE, and HPV in N. aromaticivorans cultures fed erythro-GGE suggests that the bacterium excreted these metabolites and then subsequently transported them back into the cell  was unexpected. Cleavage of MPHPV into guaiacol and GS-HPV is catalyzed by LigF and LigE (B). We hypothesize that without NaGSTNu, GS-HPV accumulates in 12444Δ2595, and cells become limited for the free GSH that is needed to cleave MPHPV.Because NaGSTanillate J was uneand LigE , enzymesand LigE B. We hypB) resulted from activity of an unknown enzyme or spontaneous cleavage of GS-HPV. Even if there were another enzyme in N. aromaticivorans with low glutathione lyase activity, our results with the 12444Δ2595 strain and its extract show that NaGSTNu is sufficient for cleavage of GS-HPV and necessary for complete metabolism of GGE by N. aromaticivorans.It is unclear whether the trace amount of HPV formed in assays using 12444Δ2595 extract B resulteR)- and β(S)-isomers of lignin-derived GS-phenylpropanoids (both guaiacyl (HPV) and syringyl) in nature. This raises the question of why some sphingomonads (such as Sphingobium sp. SYK-6) contain both a Nu-class GST and LigG, an Omega-class GST that is reported to be specific for the β(R)-isomer -GS-HPV -GS-HPV (∼1700 mm−1 s−1 (Nu probably cleaves β(S)-GS-HPV in Sphingobium sp. SYK-6, LigG may play an important role in cleaving β(R)-GS-HPV in that organism.This work demonstrates that a Nu-class GST is probably the only enzyme necessary for metabolizing the β-isomer  13). In. InR)- amm−1 s−1 ). Thus, Novosphingobium sp. MBES04 contains not only a LigG homologue -GS-HPV (R)- and β(S)-GS-HPV in vitro  and so can probably react with β(R)- and β(S)-GS-HPV. Whereas the relative roles of these enzymes in Novosphingobium sp. MBES04 are unknown, given the effect of deleting NaGSTNu on N. aromaticivorans (its only known glutathione lyase) , perhaps they represent redundant enzymes or are specialized for different GS-conjugates.Nu, SYK6GSTNu, EcYghU, and EcYfcG (tested here), and GST3 (in vitro glutathione lyase (deglutathionylation) activity with the β(R)- and β(S)-stereoisomers of this substrate, although with a wide range of catalytic efficiencies. Phylogenetic analysis of Nu-class GSTs shows that these five enzymes lie in separate subclades , although the residues predicted to be involved in the binding of two GSH molecules (or one GSH molecule and the glutathione moiety of a GS-conjugate) (B) and in activating the thiol of the GSH1 molecule (Thr51 and Asn53) are conserved in many members of the class   not known or predicted to break the β-aryl ether bond of lignin. Whereas EcYghU and EcYfcG cleave β(R)- and β(S)-GS-HPV in vitro with lower catalytic efficiencies than NaGSTNu and SYK6GSTNu .All five of the Nu-class GSTs that have been tested for GS-HPV cleavage (NaGSTnjugate) B and in Fig. S3) , 19, 20.YK6GSTNu , this maN. aromaticivorans makes it an attractive organism for studying various aspects of the β-etherase pathway and may allow it to be developed into a biological system for converting lignin oligomers into useful compounds. Our finding that NaGSTNu is the only enzyme needed for the glutathione lyase step of the β-etherase pathway is notable, because the other pathway steps require multiple stereospecific enzymes (Nu and its variants define roles for residues conserved throughout the Nu class in GS-HPV cleavage. Our identification of SYK6GSTNu probably solves the enigma of how both β(R)- and β(S)-GS-HPV are cleaved in Sphingobium sp. SYK-6: based on kinetic analyses, we propose that SYK6GSTNu cleaves β(S)-GS-HPV, whereas LigG and SYK6GSTNu both contribute to cleaving β(R)-GS-HPV in that organism. Finally, the ability of EcYghU and EcYfcG to cleave GS-HPV shows that Nu-class GSTs from organisms lacking the β-etherase pathway can also act as glutathione lyases, offering new possibilities for the physiological roles of members of this large enzyme class.The rate of GGE metabolism by  enzymes . In addiTable S2. We deleted Saro_1879  from the N. aromaticivorans DSM 12444 genome to create a strain (12444Δ1879) amenable to markerless genomic modifications using a variant of pK18mobsacB (sacB and a kanamycin resistance gene. We used 12444Δ1879 as parent strain to generate strains in which Saro_2595 (SARO_RS13080 in the recently reannotated genome in NCBI) was deleted from the genome (12444Δ2595) and in which Saro_2595 was replaced in the genome by the E. coli DH5α yghU gene (12444EcyghU). Detailed methods for modifying the genome of N. aromaticivorans are described in the supporting information. All plasmids and primers used for genomic modifications are contained in Tables S2 and S3. Fig. S10 shows genotypes for 12444Δ2595 and 12444EcyghU.Strains used are listed in 8mobsacB , which cE. coli cultures were grown in lysogeny broth and shaken at ∼200 rpm at 37 °C. For routine storage and manipulation, sphingomonad cultures were grown in lysogeny broth or GluSis at 30 °C. GluSis is a modification of Sistrom's minimal medium  2SO4, and 20 ml of Hutner's vitamin-free concentrated base (adapted from Ref. supporting information) per liter, final pH 6.8. SMB was supplemented with carbon sources as described below. Where needed to select for plasmids, media were supplemented with 50 μg/ml kanamycin and/or 20 μg/ml chloramphenicol.Unless otherwise noted, l medium  in whichse (SMB)  used forN. aromaticivorans, 1 Klett unit (KU) is equal to ∼8 × 106 cfu/ml (Table S4). Experimental cultures of N. aromaticivorans and Novosphingobium sp. PP1Y were grown in SMB containing either vanillate or GGE alone  or a combination of vanillate and GGE . For S. xenophagum cultures, vanillate was replaced by glucose, because we found this strain to be unable to metabolize vanillate. N. aromaticivorans was also grown in SMB containing 165 μm GGE. Starter cultures were grown in SMB with 4 mm vanillate or glucose, and cells were pelleted and washed with PBS . Pellets were resuspended into culture medium and used to inoculate experimental cultures to initial cell densities of <5 KU.Cell densities were measured using a Klett–Summerson photoelectric colorimeter with a red filter. For e.g. Whatman Puradisc filters, GE Healthcare) before HPLC analysis of extracellular aromatics. Every culture was grown at least three times; data shown are from representative cultures.Cultures were grown aerobically at 30 °C, in 125-ml conical flasks containing 20–40 ml of medium and shaken at ∼200 rpm. Aliquots (400–600 μl) were removed at specified time points and filtered through 0.22-μm syringe tip filters  from Sphingobium sp. SYK-6 (codon-optimized for expression in E. coli) were individually cloned into plasmid pVP302K (8 tag connected to the N terminus via a tobacco etch virus (TEV) protease recognition site (construction of plasmids described in the supporting information). The plasmid containing Saro_2595 was modified via PCR to generate plasmids for expressing variants of NaGSTNu containing the single-residue mutations T51A, Y166F, and Y224F. Recombinant proteins were expressed in E. coli B834 (supporting information for modifications to the procedure). After removal of His8 tags using TEV protease, recombinant proteins retained a Ser-Ala-Ile-Ala-Gly- peptide on their N termini, derived from the linker between the protein and the TEV protease recognition site. Recombinant LigE and LigF1 from N. aromaticivorans were purified as described previously  and 25 mm NaCl. The β(R)- and β(S)-stereoisomers of GS-HPV were separately generated by incubating racemic β(S)- and β(R)-MPHPV (0.46 mm) in RB with 5 mm GSH and either 38 μg/ml LigF1 or 36 μg/ml LigE for several hours . This sample, containing a single GS-HPV stereoisomer, guaiacol, and the unreacted MPHPV stereoisomer (as well as LigE or F1), was diluted with RB to achieve the desired concentration of GS-HPV for the time course reaction . An additional 5 mm GSH (dissolved in RB) was added before initiation of each time course. At time 0, 100 μl of the indicated enzyme (resuspended in RB) was combined with 1800 μl of the diluted GS-HPV reaction mixture to achieve final concentrations of 8 nm NaGSTNu, 20 nm NaGSTNu (T51A), 100 nm NaGSTNu (Y166F), 10 nm NaGSTNu (Y224F), 195 nm EcYghU, 195 nm EcYfcG, or 47 or 18 nm SYK6GSTNu (for the β(R)- and β(S)-GS-HPV reactions, respectively). Reactions were performed at 25 °C. At specified time points, 300 μl of the reaction was removed and combined with 100 μl of 1 m HCl  to stop the reaction before HPLC analysis to quantify HPV formed.The reaction buffer (RB) consisted of 25 mN. aromaticivorans cells were grown in 500-ml conical flasks containing 267 ml of SMB medium with 4 mm vanillate and 1 mm GGE. When cell densities reached ∼1 × 109 cells/ml, cells were lysed by the sonication procedure used to generate E. coli lysates for protein purification (see supporting information). Samples were centrifuged at 7000 × g for 15 min, and the supernatants were used as crude cell extracts.m Tris-HCl (pH 8.0), 50 mm NaCl, 10 mm GSH, 0.407 mm racemic β(R)- and β(S)-MPHPV and cellular extract from 12444Δ1879 or 12444Δ2595  were performed at 30 °C. At defined time points, 300-μl aliquots were combined with 100 μl of 1 m HCl to stop the reaction before HPLC analysis. At the indicated time, recombinant NaGSTNu (30 μg of protein/ml) was added to the 12444Δ2595 cell extract reaction, along with an additional 10 mm GSH.Assays containing 50 msupporting information), routine analysis and quantification of aromatics were performed using an Ultra AQ C18 5-μm column (Restek) attached to a System Gold HPLC system (Beckman Coulter) with running buffers described in AFig. S11. The eluent was analyzed for light absorbance between 191 and 600 nm, and absorbances at 280 nm were used for quantification of aromatic metabolites by comparing peak areas with those of standards .After extracellular aromatics were identified using LC-MS with a PFP column ; at this point, ∼65% of the GGE initially present in the GGE-fed culture was converted into downstream intermediates. Because both cultures were actively metabolizing vanillate, and only one culture was actively metabolizing GGE, the differences in transcript levels between the cultures should largely be related to GGE metabolism.g. Cell pellets were resuspended into 2 ml of lysis solution  and then incubated at 65 °C for 5 min. RNA purification and cDNA synthesis were performed as described previously ). These mixtures were centrifuged at 4 °C for 12 min at 6000 × eviously , using S pathway .Table S5. Transcript levels were normalized to those of Saro_0141 .Real-time qPCR was performed on a 7500 real-time PCR system  using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich). Primers used to detect transcripts are contained in Initial COD values for cultures were obtained either from uninoculated medium or from inoculated medium that was immediately passed through a 0.22-μm filter. Final COD samples were collected when cultures reached their maximum cell densities, both from unfiltered culture (cells and medium) and filtered culture (medium). The difference in COD between the unfiltered and filtered final samples is defined as the COD of cellular biomass. Samples were diluted as needed and combined with High Range COD Digestion Solution . The mixtures were heated to 150 °C for 120 min to oxidize the materials before absorbances were measured at 600 nm. Standards with known COD values were analyzed in parallel.p-benzoquinone (DDQ) were purchased from Sigma-Aldrich. erythro-GGE was purchased from TCI America .Vanillate, guaiacol, GSH, and 2,3-dichloro-5,6-dicyano-erythro-GGE into ethyl acetate (Fisher) and then slowly adding 1.25 molar eq of DDQ and stirring for 30 min. The reaction was washed three times with saturated NaHCO3 to remove DDQH2 formed during the reaction. The MPHPV was purified via flash chromatography using hexane/ethyl acetate , as described previously ) with 0.2 μl of reservoir solution . This crystal was mounted directly from the growth solution by drawing it through a layer of fomblin oil, thinning the surrounding liquid with a paper wick, and plunging into liquid nitrogen. The best-diffracting ammonium sulfate–precipitated crystal was obtained at 293 K using 0.13 μl of protein solution and 85 nl of reservoir solution . This crystal was cryopreserved by adding 0.5 μl of a solution composed of 2 parts reservoir solution and 1 part undiluted glycerol to the droplet containing the crystal and equilibrating for 11 min before looping and plunging into liquid nitrogen.NaGST5UUN) and ammonium sulfate (PDB code 5UUO) crystal forms using 1.033 Å (for 1.45 Å resolution) or 0.7749 Å (for 1.25 Å resolution) X-rays, respectively. Diffraction data were reduced using XDS .Diffraction data were obtained at the GM/CA beamline at Argonne National Laboratory with an Eiger 16M detector . Data wesing XDS . Both crh Phaser  in the Ph Phaser , using a (EcYghU ) modifie - and β(S)-GS-HPV and their syringyl phenylpropanoid analogues into the active sites of NaGSTNu and EcYghU, the PyMOL Builder function (For modeling β(function  was usedfunction . For theW. S. K., D. R. N., and T. J. D. conceptualization; W. S. K., C. A. B., C. N. O., D. R. W., A. U., and L. M. Y. data curation; W. S. K., C. A. B., C. N. O., D. R. W., and B. G. F. formal analysis; W. S. K., C. N. O., D. R. W., and L. M. Y. investigation; W. S. K., C. N. O., A. U., D. L. G., and R. W. S. methodology; W. S. K., B. G. F., and T. J. D. writing-original draft; W. S. K., B. G. F., D. R. N., and T. J. D. writing-review and editing; B. G. F. and T. J. D. resources; B. G. F., D. R. N., and T. J. D. supervision; B. G. F., D. R. N., J. J. C., and T. J. D. funding acquisition; B. G. F. visualization; T. J. D. project administration; C. A. B. and R. W. S. crystallized NaGSTNu and solved its structure; A. U. and J. J. C. identified metabolites via LC-MS; D. L. G. purified LigE and LigF and synthesized MPHPV and HPV.


Background. Recent studies have revealed that inflammatory processes are involved in the pathogenesis of Parkinson's disease (PD). Multiple lines of evidence have suggested that chemokines and their receptors are involved in several neurodegenerative disorders. We have examined whether genetic polymorphisms at the genes encoding chemokines IL-8 (-251A>T), MCP-1 (-2518A/G), and RANTES (-28C>G) and chemokine receptors CCR2 (V64I) and CCR5 (-Δ32) were associated with sporadic PD risk in Isparta, Turkey. Method. The pilot case-control association study included 30 PD patients and 60 control subjects, who were all genotyped with PCR-RFLP for the five polymorphisms. Their genotype and haplotype frequencies were compared statistically. Results. One SNP (-28C>G) in RANTES revealed a significant association with PD  < 0.0001, p-trend = 0.0007). The risk allele (G) in the homozygous and dominant models  suggests additional PD risk. The haplotype TGCAN from the IL-8 (-251A>T), MCP-1 (-2518A>G), RANTES (-28C>G), CCR-2 (V64I), and CCR-5 (-Δ32) has protective effect . Conclusions. Our data are the first indication of the role of RANTES (-28C>G) in PD risk. Parkinson's disease (PD) affects about 2% of the elderly population and is the second most common neurodegenerative disease after Alzheimer's disease (AD) –3. CliniChemokines are a large family of structurally homologues cytokines that have a role in the mediation and regulation of immune and inflammatory reactions. They are small polypeptides with a molecular weight of 7–15 kDa. Chemokines are classified by the number and location of N-terminal cysteine residues. The two major groups are CC chemokines (beta-chemokines), in which the cysteine residues are adjacent, and CXC chemokines , in which cysteine residues are separated by one amino acid. The genes encoding CXC chemokines are clustered mainly in the 4q13 chromosome locus, whereas the members of CC chemokines are encoded by genes that are located mainly in the 17q112–12 locus , 13. IL-8/CXCL8 (-251A/T), MCP-1/CCL2 (-2518G/A), RANTES/CCL5 (-28C/G), CCR2 (V64I), and CCR5 (-delta32) have been studied in different populations and ethnic groups with PD and late onset AD [Chemokines, in particular inflammatory cytokines, can exert toxic effects via a direct mechanism, through binding to dopaminergic neurons, or by an indirect mechanism, through glial cell activation and the expression of inflammatory factors . Chemoki), CCR2 V4I, and Conset AD . IL-8, MCP-1, RANTES, CCR2, and CCR5.Genetic predisposition has been suggested as an important factor in the etiopathogenesis of PD. In this study, our aim was to assess whether there were genetically driven differences between the immune responses of healthy people and those with PD, specifically in terms of polymorphisms in chemokines and their receptor genesThirty patients with clinically definite idiopathic PD, according to the PD Society Brain Bank criteria, were enrolled for the study. All patients were recruited from the outpatient clinic of the Neurology Department, Faculty of Medicine, Isparta Süleyman Demirel University (SDU). Isparta is in southwest Turkey, has a low influence of migration from other countries and interregional movement, and has a population of 230,000 that only 5 percent of them are resettlers.The average age of disease onset was 62.87 years (range 37–77) and the mean duration of disease was 2.37 years (with a range of 1–4) for PD patients . A total of 60 healthy control subjects  were recruited from relatives or friends of patients in general medical clinics. Their mean age was 60.52 years (range 39–80). Healthy control volunteers and patients were excluded from the study if there were any indications of dementia, stroke, secondary parkinsonism, hypertension, neoplastic or hematological disorders, alcoholism, diabetes, recent infection, hepatic or renal insufficiency, or systemic inflammatory disease.The study was approved by the Research Ethics Committee of Süleyman Demirel University Hospital (07.15.2015/166) and a written consent statement was signed by all subjects. IL-8 (-251A>T), MCP-1 (-2518A>G), RANTES (-28C>G), CCR-2 (V64I), and CCR-5 (-Δ32) polymorphisms were administered, as shown in A 2 ml blood sample was obtained from patients and control subjects. All DNA samples were obtained using a DNA isolation kit (Thermo Scientific). Genotyping was carried out by the polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) method. The PCR-RFLP methods of RANTES -28C>G polymorphisms, the relevant area was amplified with PCR and cut with MnlI restriction enzymes. Following the application of the PCR-RFLP method for -28C>G polymorphism, 114 + 27 + 20 and 13 bp band patterns were observed in normal individuals (CC), 134 + 27 and 13 bp patterns in homozygous individuals (GG), and 134 + 114 + 27 + 20 and 13 bp patterns in heterozygous individuals (CG), respectively (To determine theectively .http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) for allele and genotype frequencies (Pearson χ2 statistics), odds ratios (OR), and p values as well as genetic models such as the homozygous comparison, dominant, recessive, and allele models. When some of the analyzed frequencies were zero, OR was adjusted by Haldane's modification, which adds 0.5 to all cells to accommodate possible zero counts [http://bioinfo.iconcologia.net/SNPstats). All statistical tests were conducted at the 0.05 significance level.Genotypes were identified based on electrophoresis banding patterns. Allele and genotype frequencies were calculated by simple allele counting. Genotypes and alleles were expressed as numbers and percentages. Each SNP was analyzed with the FINETTI program (o counts . A Hardyo counts . The hap IL-8 (-251A>T), MCP-1 (-2518A>G), CCR-2 (V64I), or CCR-5 (-Δ32) polymorphisms. However, there were significant differences for the RANTES -28C>G polymorphism  polymorphism was identified in 63.3% of PD patients and 86.6% of control subjects. Nine patients (30%) were heterozygous for the T allele and 10 (33.3%) were homozygous. In the control group, 22 (36.6%) were homozygous and 30 (50%) were heterozygous. There was no statistically significant difference between the patient and control groups in terms of genotype and allele frequency (p = 0.107).The MCP-1 (-2518A>G) polymorphism was identified in 30% of PD patients and 45% of control subjects. Four PD patients (13.3%) were heterozygous for the G allele and 5 (16.6%) were homozygous, while in the control group, 22 (36.67%) were heterozygous and 5 (8.33%) were homozygous. Still, there was no statistically significant difference between the groups (p = 0.664).The CCR2 (V641) polymorphism was identified in 23.3% of PD patients and 36.6% of control subjects. Seven (23.3%) PD patients and 22 (36.6%) control subjects were heterozygous for the I allele while no homozygous I allele was detected in either group. The two groups were similar in terms of CCR2 (V641) polymorphism (p = 0.201). Neither group included the CCR5 (-Δ32) polymorphism.The RANTES -28C>G polymorphism, 85% of PD patients expressed C alleles and the remaining G alleles. In the control group, all subjects had C alleles. This allelic difference was found to increase PD risk in our study group at a statistically significant level . Genotype analysis showed the RANTES -28C>G polymorphism was present in 12.5% of PD patients but it was absent in the control group. Of the PD patients, 3 (10%) were heterozygous and 3 (10%) were homozygous for the G allele. Analysis of possible relationship of genotype frequencies of the RANTES -28C>G polymorphism revealed significant differences in the GG (homozygous) model compared to CC , in the CG+GG (dominant) model compared to CC , and in the GG (recessive) model compared to CC+GG . The common odds ratio was 4.580 and the p value was p = 0.0007 in Armitage's trend test. According to the statistical data, the C allele of RANTES -28C>G polymorphism has a protective effect on the disease and the G variant was significantly associated with PD at both allelic and genotypic levels.In terms of the A, A, C, G, and N alleles of the IL-8 (-251A>T), MCP-1 (-2518A>G), RANTES (-28C>G), CCR-2 (V64I), and CCR-5 (−Δ32) variants. Four haplotypes had a frequency of greater than 5%. The most common haplotype (A-A-C-G-N) was used as a reference haplotype in our analysis ( (T-G-C-A-N) was found to be significantly protective for PD . In addition, the global association of haplotype was also found to be significant (p < 0.0001) between PD patients and control subjects. None of the other haplotypes were found to be significantly associated with PD  polymorphism, we found no significant difference between PD patients and control subjects. Ross et al. (2004) investigated the relationship of proinflammatory cytokines and Parkinson's disease in an Irish community and found a statistically significant relationship between increased IL-8 levels with IL-8-251A>T polymorphism and the disease [ TT genotype frequency decreased and AT genotype frequency increased significantly in PD patients. However, the difference in genotype frequencies in the PD group did not reach a statistically significant level compared to the control group (p = 0.1068). They suggested that the decrease in TT genotype, which reduces IL-8 production, might play a protective role against PD. In contrast to Ross et al.'s results, TT and TA allele frequencies were about the same in PD patients and control subjects in our study  were homozygous and 80 (47%) were heterozygous for the G allele. It was concluded that -2518A>G polymorphism increases the expression of MCP-1 and causes higher microglia activation, which might be effective during the early appearance of PD symptoms [ MCP-1 polymorphism [ MCP-1 when compared to control subjects [ MCP-1 -2518A>G polymorphism in the control group outnumbered those in the patient group (The monocyte chemoattractant protein-1 (MCP-1) is a member of the C-C chemokine family. It is potent chemotactic factor for monocytes. MCP-1 is produced by many cells including astrocytic, monocytic, and microglial cells. Nishimura et al. (2003) reported a relationship between thesymptoms . In anotmorphism . Howeversubjects , 23. In nt group . CCR2 and CCR5 genotypes . The frequency of haplotype T-G-C-A-N in PD patients (2.28%) was lower than in control subjects (19.2%), suggesting that haplotype T-G-C-A-N was statistically related to PD as a protective haplotype.Haplotype analyses further confirmed the role of chemokine receptors in PD development. In our study, between-group differences in haplotype frequencies had a global  IL-8, MCP-1, RANTES, CCR2, and CCR5 polymorphisms in a Turkish population with PD. Our results suggest that the -28C>G polymorphism in the promoter region of RANTES might increase the protein expression and consequently contribute to the mechanisms underlying inflammation in neurodegeneration.This is the first study to evaluate


In cancer, upregulated Ras promotes cellular transformation and proliferation in part through activation of oncogenic Ras-MAPK signaling. While directly inhibiting Ras has proven challenging, new insights into Ras regulation through protein-protein interactions may offer unique opportunities for therapeutic intervention. Here we report the identification and validation of Aurora kinase A (Aurora A) as a novel Ras binding protein. We demonstrate that the kinase domain of Aurora A mediates the interaction with the N-terminal domain of H-Ras. Further more, the interaction of Aurora A and H-Ras exists in a protein complex with Raf-1. We show that binding of H-Ras to Raf-1 and subsequent MAPK signaling is enhanced by Aurora A, and requires active H-Ras. Thus, the functional linkage between Aurora A and the H-Ras/Raf-1 protein complex may provide a mechanism for Aurora A's oncogenic activity through direct activation of the Ras/MAPK pathway. The Ras family of proteins  are well characterized oncogenic drivers in a variety of cancer types , 2. Ras Activated Ras recruits distinct effector proteins to initiate cellular signaling cascades and physiological processes –15. SpecRas-MAPK signaling can upregulate transcription of the mitotic kinase, Aurora kinase A (Aurora A) . Aurora Interestingly, literature provides evidence of cooperation between Ras-MAPK signaling and Aurora A beyond transcriptional regulation. Aurora A amplification co-occurs in several cancer types with deregulated Ras signaling –35. AlsoHere we report that the protein-protein interaction of Aurora A and H-Ras is a mechanism by which Aurora A functions upstream of H-Ras to promote MAPK signaling. The Aurora A and H-Ras interaction validated in this study provides a novel link and potential positive feedback loop between two oncogenic proteins known to drive proliferation and survival in cancer. Blocking this interaction may have promising therapeutic potential to inhibit Ras-MAPK activity in cancer.To gain insight into Aurora A signaling pathways and oncogenic activities, we tested whether Aurora A directly interacted with a variety of signaling proteins, including Ras. We first used the homogeneous, solution-based time-resolved Föster resonance energy transfer (TR-FRET) assay to detect binding . The assTo confirm the Aurora A/H-Ras interaction detected by TR-FRET, a GST pull-down was performed as a secondary affinity-based binding assay. GST pull-downs were conducted with lysates from HEK 293T cells co-expressed with GST H-Ras with Venus-Flag Aurora A. Aurora A was found to pull down with GST H-Ras complex, but not in control lanes with GST Figure , demonstin vitro cell lysate-based assays, thus, we further validated the interaction of Aurora A with H-Ras in vivo by utilizing a fluorescence (Venus)-based protein-fragment complementation assay (PCA). In this assay, N-Venus or C-Venus fragments are fused to two interacting proteins. The association of these proteins leads to functional reconstitution of Venus and allows the detection of green fluorescence signal using imaging. For this purpose, Aurora A and H-Ras were fused with N-Venus and C-Venus, respectively, and co-expressed in HEK 293T cells. The percentage of cells with positive protein-protein interactions (reconstituted Venus) was revealed by fluorescence imaging. Co-expression with N-Venus or C-Venus established background  function as oncogenic drivers in many cancer types by transmitting pro-growth and proliferative signals through the Ras-MAPK pathway. In this study, we identify a novel protein-protein interaction between Aurora A and Ras that provides a mechanism by which Aurora A acts as a positive regulator of Ras-MAPK signaling –35.Using complementary protein-protein interaction assays, we demonstrated that Aurora A interacts with H-Ras and Raf-1, functioning upstream of Ras in the MAPK pathway to potentiate Ras-mediated MAPK signaling. Cooperation between Aurora A and Ras-MAPK signaling pathway is implicated in various cancer models. For example, Aurora A overexpression and Ras alterations co-occur in pancreatic, colon, and bladder cancers –35. AddiIn characterizing the structural domains that mediate the Aurora A/H-Ras interaction, we identified that a region within the kinase domain of Aurora A (amino acids 130 – 383) interacts within the N-terminal domain of H-Ras (amino acids 1-66). Since Aurora A binding is maintained with the deletion of the switch I domain of H-Ras, these data suggest that the required Aurora A binding site on Ras may be narrowed to amino acids 36-66, near the switch II domain of H-Ras.Although most of our characterization was conducted with H-Ras, the demonstrated interaction of Aurora A with the three isoforms of Ras suggests a broad role of Aurora A in regulating the Ras-MAPK signaling mediated by isoforms of Ras in various cellular environment Figure . ConsideOur finding that Aurora A interacts with Ras isoforms adds to previous reports of binding between Aurora family proteins and other GTPases and Ras-binding proteins. Aurora A interacts with RalA , Aurora Raf-1 was identified to associate with Aurora A and other cell cycle machinery during mitosis , 58. ThiBeyond the interactions we discovered, how Aurora A leads to enhanced Ras-MAPK signaling remains to be established. It is possible that the Aurora A/H-Ras interaction may increase GEF activity, prevent GAP activity, or induce an active conformation of H-Ras. Ras G12V is a mutant of Ras that binds GAP but is unable to hydrolyze GTP. Because we were able to demonstrate that co-expression of Aurora A and H-Ras G12V also enhances ERK activation, the mechanism of action of Aurora A may be Ras-GAP independent despite reports that both Aurora A and Aurora B both associate with Ras GAP , 59.Attempts to directly target Ras proteins for cancer treatment have been largely unsuccessful in the clinic , 60. AnoHEK 293T and MCF7 cells were utilized in the described experiments . HEK 293T and MCF7 cells were cultured in DMEM  with 10% FBS  and 1% pen/strep at 5000 I.U/ml penicillin and 5000 μg/ml streptomycin . Between passages, cells were trypsinized with 0.25% Trypsin with 2.21 mM EDTA . All cells were maintained at 37°C in a humidified atmosphere of 5% CO2.Primary antibodies used for western blotting include Flag M2 at 1:3000 , Flag-HRP at 1:1000 , GST Z-5 at 1:3000 , rabbit GST-HRP at 1:1000 , Aurora kinase A at 1:500 , rabbit pERK and ERK , pMEK and MEK , pRaf-1  and Raf-1  at 1:1000, and Ras  at 1:1000. Secondary antibodies include goat anti-rabbit IgG  and goat anti-mouse IgG  and were used at either 1:2500 or 1:5000 dilutions. Antibodies used for immunoprecipitation include Aurora A , Ras , and IgG  at 1:50 dilution.Sorafenib p-Toluenesulfonate Salt (S-8502) and U0126 (U-6770) inhibitors were obtained from LC Laboratories . Compounds were dissolved in dimethyl sulfoxide (DMSO) as 10 mM stock and stored at −20°C. Cells were treated for 24 hours with 10 μM of compounds diluted in DMSO.5 cells per well and cultured in 600 μl of complete medium . Cells were then transfected 24 hours after plating. Complete media was replaced with DMEM media without FBS supplementation (serum free media) 24 hours after transfection. Samples were collected for the 0 minutes-post serum stimulation time point following 24 hours in serum-free media. Then, serum (10% FBS) was added to all remaining wells. Remaining cells were collected at time points 5, 10, 15, 45, and 90 minutes after serum stimulation. Cells were collected directly into 1X SDS loading buffer, boiled for 5 minutes, and subjected to SDS-PAGE and western blotting.MCF7 cells were plated in a 24-well plate at 1×10For experiments with ectopically expressed proteins, HEK 293Ts were transfected using X-tremeGENE . MCF7 cells were transfected with FugeneHD . Plated cells were transfected at a density of 60-80% confluency and performed with a ratio of 3 μl transfection reagent to 1 μg DNA to 100 μl of serum-free media. DNA was mixed at appropriate concentrations prior to the addition of serum-free DMEM. Transfection reagent was then added and incubated at room temperature for 15 and 20 minutes . Transfection complexes were then added drop-wise to plated cells.All plasmids of full length and truncated proteins were constructed using Gateway® technology  per the manufacturer's protocols. For GST-tagged and Venus-Flag tagged plasmids used for Time Resolved-Föster Resonance Energy Transfer (TR-FRET) and glutathione-S-transferase (GST) pull-downs, pDEST27 and pFUW vectors were used as destination cloning vectors, respectively. Amino (N-Venus) and carboxy (C-Venus) plasmids used for Venus protein-fragment complementation assay (PCA) were generated previously in the lab. Aurora A or H-Ras cDNA was PCR amplified and inserted into the pDONR201 (Invitrogen) vector using a BP reaction to generate entry cloning vectors. A LR reaction was used to clone the desired DNA into the appropriate destination vectors. Constructs were verified by restriction digest using BSRGI  or FastDigest Bsp1407I , both cutting at the T^GTACA attB1 and attB2 (entry clone) or attR1 and attR2 (destination vector) recombination sites, and DNA sequencing. Clones in pDEST-27 (GST) vectors were sequenced with forward primer 5′-AAGCCACGTTTGGTGGTG-3′ and the standard T7 reverse primer. Clones in pFUW (Venus-Flag) vectors were sequenced with forward primer 5′-CGATCACATGGTCCTGCTG-3′ and the standard SP6 reverse primer.Site-directed mutagenesis was performed on the GST H-Ras vector to create the catalytically-inactive mutant (S17N) using the QuikChange™ Site-Directed Mutagenesis Kit according to the manufacturer's protocol . The H-Ras S17N mutant was generated using the oligonucleotide forward primer 5′-GGCGGTGTGGGCAAGAATGCGCTGACCATC-3′ and reverse primer 5′-GATGGTCAGCGCATTCTTGCCCACACCGCC-3′. Successful mutagenesis was confirmed by DNA sequencing as described previously.4, where F486 and F520 are fluorescence counts at 520 nm and 486 nm for Venus and terbium emission signal, respectively. Data were presented as mean with standard deviation calculated from duplicate samples.TR-FRET was performed in 384-well black solid bottom plates  in a total volume of 30 μL in each well. Briefly, HEK 293T cells were transfected as described above. Cells were lysed using 0.5% NP-40 lysis buffer  and Protease Inhibitor Cocktail ). Lysates were collected and centrifuged at 13,500 g for 10 minutes at 4°C to remove cellular debris. Cleared cell lysates were serially diluted in FRET buffer  in a 384-well plate, bringing the final volume of diluted cell lysate to 15 μL per well. Then, 15 μL of diluted anti-GST-Terbium antibody  was added to all wells at a final dilution of 1:1000. The TR-FRET signals were detected with an EnVision Multilabel plate reader (PerkinElmer) with laser excitation at 337 nm, emission1 at 486 nm and emission2 at 520 nm. TR-FRET signal is expressed as ratio and calculated by the following equation: TR-FRET signal = F520/F486 × 10Cells were seeded in to a 6-well plate and allowed to reach 60-80% confluency. Cells were then harvested by adding 200 μL of 0.5% NP-40 lysis buffer to each well, collected by scraping, transferred to an eppendorf tube, and incubated at 4°C for 30 minutes. Lysis buffer components consisted of 0.5% NP-40, 150 mM NaCl, 10 mM HEPES lysis buffer, and Phosphatase Inhibitor Cocktail  and Protease Inhibitor Cocktail  at 1:1000. After incubation, lysates were centrifuged to remove cellular debris. After removing 20 μl of the lysate for an input control and the debris pellet, 20 μl of a 50% glutathione-conjugated sepharose bead slurry  was added to the remaining lysate and incubated by slowly rotating for 3-4 hours at 4°C. Beads were then washed three times in 0.5% NP-40 lysis buffer by inverting 8 times with 200 μl of fresh lysis buffer added each time. GST-bound protein complexes were then eluted by the addition of 20 μl of 2x SDS loading buffer, boiled for 5 minutes, resolved by SDS-PAGE subjected to western blotting along with input controls.Micro automated imaging high-content imaging system (Molecular Devices) with 20X objective. The standard filter set for FITC (excitation 482/35 nm and emission 536/40 nm) and DAPI (excitation 337/50 nm and emission 447/60 nm) was used for Venus and Hoechst 33342 imaging, respectively. The number of green (Venus) and total cells (Hoechst 33342) from the images were calculated using the Metamorph Analysis Cell Scoring module and presented as percent of Venus positive cells compared to the total number of cells.Cells were seeded into 24-well plates and transfected at 60% confluency with N-Venus or C-Venus constructs. After 24 hours, cell nuclei were stained with the addition of Hoechst 33342  at 5 μg/ml. Images were then acquired using the ImageXpressCells were seeded in 15 cm dishes and grown to confluency. Cells were then washed with PBS and lysed in 0.5% NP-40 lysis buffer containing protease inhibitors (as previously mentioned), then incubated on ice for 30 minutes. Lysates were then centrifuged for clarification at 4°C. To preclear, protein A/G beads  were added and rotated with lysates for 30 minutes at 4°C. Cleared lysates were then transferred to a new tube for immunoprecipitation where they were then incubated with antibodies against Aurora A, Ras, or IgG at 1:50 dilution for 16 hours at 4°C, followed by adding 25 μl of protein A/G beads and rotating for an additional 8 hours. Aurora A or Ras immunocomplexes on beads were washed three times with 0.5% NP-40 lysis buffer containing protease inhibitors and eluted into 2X SDS loading buffer prior to SDS-PAGE and western blot analysis.Cell lysates were subjected to western blot analysis following protein separation by SDS-PAGE (10% acrylamide gels) and subsequent transfer to PVDF membranes at 100V for 1.5 hours. Membranes were blocked in TBST  containing 5% dry milk for 30 minutes – 1 hour at ambient temperature, then incubated at 4°C or ambient temperatures with primary antibodies diluted in 5% milk in TBST. After primary antibody incubation, membranes were washed three times with TBST for 5 minutes each, then incubated with secondary antibody for 1 hour at ambient temperatures. For HRP conjugated antibodies, membranes were blocked with milk, then washed three times with TBST for 10 minutes each, then incubated with GST-HRP or Flag-HRP for 1 hour. Membranes were then washed three times with TBST for 10 minutes each and chemiluminescent signal  or ECL  was added for 5 minutes prior to developing by autoradiography. Proteins with the Venus-Flag epitope tag were detected by blotting with anti-Flag antibody.


In this study, we tested whether polymorphisms in human leukocyte antigen G (HLA-G) were associated with event-free survival (EFS) in pediatric Hodgkin's lymphoma (HL). We evaluated the association of HLA-G 3′-UTR polymorphisms with EFS in 113 pediatric HL patients treated using the AIEOP LH-2004 protocol. Patients with the +3027-C/A genotype  showed lower EFS than those with the +3027-C/C genotype . Female patients and systemic B symptomatic patients with the HLA-G +3027 polymorphism showed lower EFS. Multivariate analysis showed that the +3027-A polymorphism  was an independent prognostic factor. Immunohistochemical analysis showed that HL cells from patients with the +3027-C/A genotype did not express HLA-G. Moreover, HLA-G +3027 polymorphism improved EFS prediction when added to the algorithm for therapeutic group classification of pediatric HL patients. Our findings suggest HLA-G +3027 polymorphism is a prognostic marker in pediatric HL patients undergoing treatment according to LH-2004 protocol. Hodgkin lymphoma (HL) originates mostly from transformed B-lymphocytes and represents 6% of the childhood tumors . MononucClass I histocompatibility leukocyte antigen (HLA) molecules play an important role in the ability of tumor cells to evade immune surveillance . MoreoveHLA-G is a non-classical HLA class I molecule that is expressed in physiologic conditions in immune privileged tissues  . The altPatient and disease characteristics are listed in Table As shown in HLA-G encompassing the +3027-A variant [We observed that the +3010-C, +3027-A and +3142-G alleles were part of the unique 3′-UTR-7 haplotype of  variant . TherefoWe found that 27 (23.9%) of the 113 patients that received AIEOP LH-2004 treatment experienced a tumor-event in 1.2±1.048 y (range 0.4-6 y) after diagnosis. Kaplan Meier survival analysis demonstrated that the patients carrying the heterozygous +3027-C/A genotype showed lower EFS time than patients carrying the C/C genotype  demonstrated that +3027-A polymorphism (UTR-7 haplotype) was an independent prognostic factor for poor EFS  based on increased radiation dosage and aggressive chemotherapy (both length and intensity) to reduce long-term toxicities in younger patients [In the present study, we included the HLA-G +3027 risk factor in the algorithm with the AIEOP LH-2004 GR score to predict the EFS risk Figure . We perfCox regression analysis showed that patients in the GR123-C/A and GR3-C/C groups were associated with decreased overall survival and lower EFS score according to the HLA-G +3027 algorithm Figure . Nine ofImmunohistochemical analysis of HLA-G expression in tumor cells from 25 lymph node biopsies that were obtained at diagnosis showed that patients carrying a +3027 C/A genotype were all negative for HLA-G and only 5 cases having the wild type +3027 C/C genotype showed HLA-G expression  HLA-G polymorphism was associated with a low EFS in pediatric HL patients treated according to the AIEOP LH-2004 protocol, more evident in female gender and patients with systemic B symptoms. Multivariate analysis demonstrated that +3027-C/A genotype was an independent prognostic factor associated with poor EFS. Moreover, incorporating the HLA-G +3027 polymorphism into the AIEOP LH-2004 therapeutic stratification improved risk evaluation. Patients belonging to the GR123-C/A and the GR3 groups showed lower 5 year-overall survival than patients belonging to GR1 and GR2 groups (9.7% vs. 0%). The effect of HLA-G expression on HL patient survival needs further investigation.et al. showed that HLA-G expression was a positive prognostic factor in patients with advanced-stage classical HL [et al. and Caocci et al. (about 55%). However, all of our HLA-G-positive cases demonstrated positive prognosis as reported earlier. Patients that carried the +3027 C/A variant were associated with poor prognosis and were all HLA-G-negative.Many studies have reported association of polymorphisms in the HLA region with HL , 40. Howsical HL . In the HLA-G expression can exert beneficial or detrimental effects of immune recognition, which depend on the pathological context or situation , 43. HLAOf note, the impact of chemotherapy and radiotherapy on HLA-G expression in patients carrying the HLA-G +3027-C/C and the HLA-G +3027-C/A genotypes is not known. Previous reports have shown that gamma radiation and/or chemotherapy modulates surface HLA-G expression in B-cell lines (M8 and K562), melanoma cell lines (OCM-1A and JEG-3) and basal cell carcinoma of the skin , 48. ThuHowever, it is also possible that +3027-C/A polymorphism could be in strong linkage disequilibrium with another critical gene, which causes the disease progression and poor EFS.Overall these issues needs to be investigated in detail.In conclusion, we demonstrated that +3027-A HLA-G polymorphism was associated with poor EFS in pediatric HL patients treated with the AIEOP LH-2004 protocol. In the future, comprehensive prospective studies are necessary to confirm that HLA-G polymorphisms are prognostic indicators in pediatric HL therapy.Peripheral blood samples were collected from 113 pediatric HL patients (range 3-18 years) at the AIEOP Centers in the laboratory of Padua University from December 2004 to August 2014. Table The GR1 patients (low risk) were administered 3 cycles of ABVD  without radiotherapy (RT) were administrated. In case of unsatisfactory response after 2 cycles of ABVD or residual disease after 3 cycles, GR1 patients received chemotherapy/RT or RT. The GR2 (intermediate risk) and GR3 (high risk) patients were administered 4 or 6 alternating cycles of COPP-ABV chemotherapy  to ABVD treatment. Radiotherapy was administered with Involved Field Technique (IFRT) at cumulative dose of 14.4 or 25.2 Gy depending on the remission status  at the end of chemotherapy. Patients in complete remission at the end of chemotherapy were not administered radiotherapy. Complete response (CR) was defined as absence of clinical, radiological  and FDG-PET evidence of disease. We enrolled 259 local blood donors (BD) as a control group. Study was approved by the ethics committee of all participating institutions. Written consent forms were obtained from parents or legal guardians of all patients.Genomic DNA was extracted from peripheral blood with the Qiagen DNAeasy Kit . HLA-G genotyping was performed by first PCR amplifying using the 3′UTR specific primers   followedParaffin-embedded primary HL lymph node tissue was retrieved. HLA-G expression was assessed in 25 paraffin-embedded tissues by Immunohistochemical staining with primary 4H84 mouse monoclonal antibody , which recognizes all the HLA-G isoforms. If intense membrane HLA-G staining was found in more than 10% of Hodgkin or Reed-Sternberg cells, then the sample was scored as positive.http://faculty.vassar.edu/lowry/VassarStats.html and the SKDM software (http://sourceforge.net/projects/skdm). The HLA-G 3′UTR haplotypes were defined according to Castelli [The frequencies of HLA allele and genotype were compared between patients and controls chi-square test (VassarStats Castelli . HLA hapCastelli . FrequenCastelli  currentl


Hepatitis B virus (HBV) infection, which is a major health concern worldwide, can lead to liver cirrhosis and hepatocellular carcinoma. Although current nucleos(t)ide analogs efficiently inhibit viral reverse transcription and viral DNA load clinically, episomal viral covalently closed circular DNA (cccDNA) minichromosomes and transcripts from cccDNA continue to be expressed over the long term. We hypothesized that, under these conditions, viral transcripts may have biological functions involved in pathogenesis. Here, we show that the host protein DExH-box helicase 9 (DXH9) is associated with viral RNAs. We also show that viral-derived circular RNA is produced during HBV replication, and the amount is increased by knockdown of the DHX9 protein, which, in turn, results in decreased viral protein levels but does not affect the levels of HBV DNA. These phenomena were observed in the HBV-producing cell culture model and HBV mini-circle model mimicking HBV cccDNA, as well as in human primary hepatocytes infected with HBV. Based on these results, we conclude that, in HBV infection, the RNA binding factor DHX9 is a novel regulator of viral circular RNA and viral protein levels. Despite the availability of effective vaccines, hepatitis B virus (HBV) infection remains a major global health problem. Approximately 2 billion people are infected with HBV, and over 350 million patients are chronic HBV carriers . ChronicHBV is a DNA virus with a 3.2-kb partially double-stranded relaxed circular DNA (rcDNA) as its genome. Covalently closed circular HBV DNA (cccDNA) is formed by conversion from rcDNA following HBV infection. HBV cccDNA exists persistently in the hepatocyte nucleus in an episomal state, where it acts as a viral transcription template. Host RNA polymerase II transcribes HBV genes to produce five viral RNAs: a transcript that encodes precore, a transcript that encodes core and pregenomic RNA (pgRNA), a preS1 transcript that encodes the large S surface protein, a transcript that encodes preS2 and S protein , and the X gene mRNA –6.It has been recently revealed that RNAs, especially non-coding RNAs, have diverse biological functions . AlthougIn this study, to discover unknown biological functions of HBV RNAs, host proteins that can associate with viral RNAs were comprehensively identified using RNA immunoprecipitation followed by proteomic approaches. We found that circular RNA is produced from HBV RNA and that the host RNA-binding protein DExH-box helicase 9 (DXH9) is involved in its production. Knockdown of DXH9 leads to increased production of this viral circular RNA but to decreased viral surface protein levels. These results represent novel findings regarding HBV RNAs and the regulation of viral protein levels.To comprehensively identify host proteins that interact with HBV-derived RNAs, we performed an RNA precipitation assay after mixing cell lysates with biotinylated HBV-RNAs including the S, X, and core regions. After precipitation, proteins were dissolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and possible interacting protein bands were excised for identification by mass spectrometry . Among tTo determine whether knockdown of the identified proteins affected HBV replication or HBV-DNA levels, we performed southern blotting to detect HBV-derived DNA levels including open circular DNA, double-stranded linear DNA, and cccDNA levels in HepAD38 cells with knockdown of each protein. Southern blotting revealed that HBV-derived DNA levels were not significantly changed by knockdown of any of the identified proteins . Thus, wTo further confirm the existence of circular RNA derived from HBV-RNA, we next examined RNAs derived from cells transfected with HBV pgRNA-expressing plasmids. Whereas RNase R treatment clearly digested host linear Mcl1 mRNA, primer set #2 could amplify the amplicon derived from HBV RNA even after RNase R treatment Figure . These rAmong the proteins identified as interacting with HBV-RNAs, DHX9 was recently reported to be involved in the production of circular RNAs. In particular, the loss of DHX9 leads to an increase in circular RNAs . We deciAs noted above, DHX9 was identified as an HBV-RNA-interacting protein. To examine whether DHX9 also interacted with HBV circular RNA, we performed an RNA-immunoprecipitation (RIP) assay by pull-down of the DHX9 protein in HepAD38 cells with or without HBV expression. Precipitation of a similar amount of DHX9 protein was confirmed under both conditions Figure . BecauseTo determine if the HBV circular RNAs interacted with the host factors involved in translation, we next performed an RIP assay using an antibody against the ribosomal P0/P1/P2 protein. Because the ribosomal proteins P0, P1, and P2 exist as a pentameric complex, P0(P1)2(P2)2, on the large subunits of eukaryotic ribosomes, this complex plays a crucial role in the recruitment of translation factors to the ribosome . The RIPin vitro HBV replication model using primary human hepatocytes. On day 1, primary hepatocytes were infected with HBV genotype C with or without shDHX9-expressing lentiviruses. The media was changed the following day as well as 2 days after infection. After that, the media was changed every 5 days; at 17 days after infection, cells were harvested for further analyses  [in vitro. All cells were incubated at 37° C, 20% O2, and 5% CO2.HepAD38 cells, which express HBV pgRNA under the control of the inducible tetracycline promoter, were kindly provided by Prof. C. Seeger . The celThe TL7 plasmid, which expresses HBV pgRNA , was kindly provided by Prof. Loeb . MinicirEscherichia coli transformed with pre-mcHBV-GLuc, which expresses inducible ƟC31 integrase and I-SceI endonuclease to eliminate the bacterial plasmid DNA backbone. No contamination of genomic and parental DNA was confirmed by agarose gel electrophoresis. The Gluc signal comes from pgRNA, providing a surrogate of cccDNA activity, and it secretes from the cells into the media. Gluc activity in the aliquots of the media was measured using the reagent for measurement of renilla luciferase activity in the Dual Reporter Assay system  because Gaussia and Renilla luciferases catalyze the light reaction using the same substrate.Minicircle mcHBV-GLuc cccDNA was produced using the Minicircle DNA Vector Technology MC-Easy System  from ZYCY10P3S2T HepG2 cells were transfected with mcHBV-GLuc- or HBV pgRNA-expressing plasmids using Effectene transfection reagent  according to the manufacturer's instructions.in vitro transcription reaction using MEGAScript  according to the manufacturer's instruction. Briefly, PCR amplification of the corresponding regions using the TL7 plasmid as a template was performed with T7 promoter sequences in the forward primers. The primers used were: S region, Fw, 5′-TAATACGACTCACTATAGGGAGAATGGGGCAGAATCTTTCCACC-3′ and Rv, 5′-TTAAATGTATACCCAAAGACAAAAG-3′, X region, Fw, 5′-TAATACGACTCACTATAGGGAGAATGGCTGCTAGGCTGTGCTG-3′ and Rv, 5′-TTAGGCAGAGGTGAAAAAGTTG-3′, and Core region, Fw, 5′-TAATACGACTCACTATAGGGAGAATGGACATCGACCCTTATAAAGA-3′ and Rv, 5′-CTAACATTGAGATTCCCGAGATT-3′. PCR products were purified by gel extraction using the DNA MinElute kit (Qiagen), followed by in vitro transcription. In vitro transcribed RNAs were treated with DNase, purified using MEGAclear Transcription Cleanup Kit (Ambion), and subjected to ethanol precipitation. Aliquots were run in a denatured gel to confirm the integrity of the obtained RNA. Biotinylated cytidine bisphosphate was attached to the 3′ end of the 50 pmol RNAs by T4 RNA ligase using Pierce RNA 3′ End Biotinylation Kit .To perform RNA pull-down, we produced HBV-RNAs containing S, X, or core regions by an 7 cells). Streptavidin beads and biotinylated RNAs containing HBV S, X, and core regions were mixed, followed by the addition of 60 μl of cell lysate to the each mixture for 60 min at 4° C. After five washes, 25 μl of elution buffer was added and subjected to SDS-PAGE. After gel electrophoresis, proteins were stained with the Silver Staining Kit for Mass Spectrometry . Possible specific bands, determined by comparing the results from the negative control (sample without RNA), were excised and the proteins were identified by liquid chromatography tandem mass spectrometry . The resulting tryptic peptides were separated and analyzed using reversed phase capillary high-performance liquid chromatography directly coupled to a Finnigan LCQ ion trap mass spectrometer (LC-MS/MS) with a slight modification. The individual MS/MS spectra were processed using TurboSEQUEST software . The generated peak list files were used to query either the MSDB database or NCBI using the MASCOT program (http://www.matrixscience.com).To screen proteins interacting with RNAs, the Pierce Magnetic RNA-Protein Pull-Down Kit was used (Thermo Fisher) according to the manufacturer's instructions. HepAD38 cell lysates without HBV expression were prepared by adding M-PER Mammalian Protein Extraction Reagent , and semi-quantitative RT-PCR was performed. The primers used were as follows: S, Fw, 5′-ACCTCTATGTATCCCTCCTG-3′ and Rv, 5′-GACTCAAGATGCTGTACAGAC-3′; X, Fw, 5′-ACTCTCTCGTCCCCTTCTCCG-3′ and Rv, 5′-AGGCAGAGGTGAAAAAAGTTGC-3′; and core, Fw, 5′-ACAGTTATAGAGTATTTGGTG-3′ and Rv, 5′-GAGATCTTCTGCGACGCGGCG-3′. The antibodies used were: snRNP200 (A303-454A) and LRPPRC (A304-731A) from Bethyl Laboratories , DNA-PK (#ab70250) from Abcam, and DHX9 (RN063PW) from MBL.To examine the endogenous binding of candidate proteins to HBV RNAs, the RIP assay kit  was used according to the manufacturer's instructions. Briefly, 6.0 × 107 HepAD38 cells were harvested and subjected to the RIP assay according to the manufacturer's protocol. The antibodies used were anti-DHX9 (RN063PW) and anti-ribosomal protein P0/P2/P3 antibodies , which were RIP-certified. After immunoprecipitation, samples were divided and subjected to western blotting for confirmation of immunoprecipitation or subjected to RNA extraction. Three micrograms of RNA were either reverse transcribed or treated with RNase R, purified, and reverse transcribed. PCR was performed using the indicated primers.For the RIP assay, RNase R treatment, and RT-PCR, 1 × 10shRNA-expressing lentiviruses against each protein were purchased from Santa Cruz Biotechnology : shDNA-PK (sc-35200-V), shsnRNP200 (sc-75243-V), shILF3 (sc-106301-V), shDHX9 (sc-45706-V), and shLRPPRC (sc-44734-V). HepAD38 cells were transduced with these viruses using polybrene, followed by puromycin selection to establish stably shRNA-expressing cells. In the case of primary hepatocytes, coinfection with HBV was done without selection.HepAD38 cells were harvested, and HBV DNA, including cccDNA, was extracted with the Hirt method  for SoutSouthern blotting was performed using digoxigenin (DIG)-labeled probes  according to the manufacturer's instructions. Briefly, DNA samples were separated on an agarose gel. After depurinating the DNA in 250 mM HCl and denaturing in 0.5 M NaOH/1.5 M NaCl, DNA was transferred to a Hybond N+ membrane  with the capillary transfer method in 20× SSC. After UV crosslinking, the membrane was incubated with a DIG-labeled DNA probe at 42° C overnight. After hybridization, membranes were stringently washed at room temperature in 2× SSC containing 0.1% SDS and at 65° C in 0.5× SSC containing 0.1% SDS, twice each, and the bound probe was visualized with a DIG wash and block buffer set (Roche Diagnostics), according to the manufacturer's instructions. The DNA marker was purchased from Roche Diagnostics (DIG-labeled DNA Molecular Weight Marker VII).in vitro transcription and a DIG RNA labeling kit (Roche Diagnostics). The template was a PCR product with T7 primer. The primers used were: Fw, 5′-TAATACGACTCACTATAGGGGGTGCGCAGACCAATTTATGC-3′ and Rv, 5′-GCACCATGCAACTTTTTCAC-3′.To synthesize an RNA probe for Southern blotting, HBV full-length RNA was synthesized by Reverse transcription was performed using SuperScript III First-Strand Synthesis Supermix (Invitrogen). Oligo d(T) or random primers were used for priming as described in the figure legends. In the case of RNase R treatment, RNA was incubated with RNase R , which specifically digests linear RNAs, at 10 U/μg for 15 min [PCR was performed using the primers described in in vitro transcription using MEGA Script (Ambion) following PCR amplification of the region with T7 promoter-containing primers. The primers were as follows: Fw, 5′-ATGGACATCGACCCTTATAAA-3′ and Rv, 5′-TAATACGACTCACTATAGGGTAAGGTTTAATACCCTTATCC-3′.Northern blotting was performed as described previously , with slHuman primary hepatocytes were infected with five equivalent copies of HBV (genotype C) (PhoenixBio) with 40% polyethylene glycol 8,000 , as described previously . dHCGM mFor TaqMan RT-PCR, 1.0 μl of template was subjected to quantitative real-time PCR using SYBR universal mix 2  with the StepOnePlus real-time PCR system. The threshold cycle number (Ct), at which the amount of amplified target reached a fixed threshold, was measured. Real-time PCR experiments were performed in triplicate for each sample.ddPCR was performed using the QX100 Droplet Digital PCR system . Briefly, 1.0 μl of template cDNA reverse transcribed from 3 μg of RNA treated with DNase and RNase R with 20× primer and a TaqMan probe set was partitioned into approximately 20,000 droplets by the QX100 Droplet Generator (Bio-Rad Laboratories). Cycling conditions were as follows: 95° C for 10 min, followed by 45 cycles of 94° C for 30 s and 56° C for 1.5 min, and a final 10 min incubation at 98° C. The droplets were subsequently read automatically by the QX10 droplet reader. Assays were performed in duplicate for each sample. The data were analyzed with QuantaSoft analysis software 1.3.2.0 (Bio-Rad Laboratories). Duplicate data were combined into a single metawell before Poisson analysis. The probe set was designed to amplify between positions 1,561 and 1,883 of the HBV genome , and the detection probe was FAM-5′-AGGCTGTAGGCATAAATTGGTCT-3′-BHQ, which covers the HBV-DNA gap, as described previously .t-test when the variances were equal. When the variances were unequal, Welch's t-test was used instead. P values less than 0.05 were considered to indicate statistical significance.Statistically significant differences between groups were identified using Student's


Methanol oxidation catalysts comprising an outer Pt-shell with an inner Ni-core supported on carbon, (Pt-Ni/C), were prepared with either crystalline or amorphous Ni core structures. Structural comparisons of the two forms of catalyst were made using transmission electron microscopy (TEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), and methanol oxidation activity compared using CV and chronoamperometry (CA). While both the amorphous Ni core and crystalline Ni core structures were covered by similar Pt shell thickness and structure, the Pt-Ni(amorphous)/C catalyst had higher methanol oxidation activity. The amorphous Ni core thus offers improved Pt usage efficiency in direct methanol fuel cells. Increasing Methanol oxidation in direct methanol fuel cells affords an important alternative fuel source for future energy requirements . In the Pt shell thickness in the Pt shell-core structure has been found to effect methanol oxidation activity in other catalysts ,18. The A/C and Pt-NiC/C respectively) through modification using a two-step method. Pt-NiA/C was found to be more active toward methanol oxidation catalyst than Pt-NiC/C.In the present study, catalysts containing an outer Pt shell with an inner Ni core supported on carbon were prepared with either amorphous or crystalline Ni cores , (200), (220) and (311) indices [morphous . For the, (200), 0 and  and Ni/C, indicating that no alloy phase was formed.After depositing Pt on Ni nanoparticles via a reduction reaction, both Pt-NiC/C and Pt-NiA/C are shown in 7/2 and 4f5/2 is ca. 3.3 eV, which also adheres to the reference value [C/C, that of Pt-NiA/C shifts to the lower binding energy, which is likely contributed from lattice strain on the Pt shell and charge transfer between Ni atoms and Pt atoms [C/C and Pt-NiA/C had different electronic structure.X-ray photoelectron spectroscopy (XPS) technique is a useful tool to study changes in the electronic structure of different surface preparations and compositions . The regce value . ComparePt atoms . The aboC/C and Pt-NiA/C, which could be deconvoluted into three pairs of doublets. In the 7/2 and 4f5/2 lines respectively. The second and weaker doublet (at 72.4 and 75.7 eV), with binding energy at 1.1 eV higher than Pt(0), could be assigned to the Pt(II) oxidation state species in PtO and Pt(OH)2 [C/C, the peak positions of the Pt 4f correspondent to the different valence were listed in A/C was predominately metallic (53.0%). The Pt 4f7/2 spectrum , Pt(II) and Pt(IV), and the Pt on the surface of Pt-NiC/C catalyst was also predominately metallic.In order to evaluate the composition of Pt species, the spectra were fitted as two pairs of overlapping Lorentzian curves.  Pt(OH)2 ,28. The  Pt(OH)2 . For Pt-spectrum a of Pt-NC/C and Pt-NiA/C respectively. It can be seen that the Pt-NiC and Pt-NiA nanoparticles were highly dispersed on the carbon support with narrow size distribution. By counting one hundred particles, the average particle size of Pt-NiC/C and Pt-NiA/C was approximately 3.2 nm and 3.4 nm respectively. The selected-area electron-diffraction (SAED) pattern  interplanar distance of pure Pt (0.23 nm), suggesting a Pt shell formed on the Ni nanoparticles. At the same time, the reduction in the interplanar distance of Pt in Pt-NiC/C and Pt-NiA/C, compared to pure Pt, most likely results from the strain effect on the Pt shell from the Ni core. From the EDS spectrum, C/C was 1:1.92, similar to that of Pt-NiA/C (1:1.93), the metal loading for the two catalysts is ca. 35%, which was also confirmed by ICP analysis.A/C and Pt-NiC/C after stabilization (20 cycles) in a N2-saturated 0.5 mol L−1 H2SO4 solution at a scan rate of 50 mV s−1. There are three characteristic potential regions of polycrystalline Pt on both Pt-NiA/C and Pt-NiC/C: the hydrogen adsorption and desorption region (−0.2 to 0.1 V vs. Ag/AgCl), double layer plateau region (0.1~0.5 V vs. Ag/AgCl), and the formation and reduction of surface metals oxide (0.5~1.0 V vs. Ag/AgCl). The coulombic charge for hydrogen adsorption (HQ) is used to evaluate the electrochemical surface area (ESAS) of the electrocatalysts using Equation (1) [m represents the platinum loading (g) on the electrode, HQ is the charge for hydrogen desorption electro-oxidation in microcoulomb (C), and C is the charge required to oxidize a monolayer of H2 on the catalyst, its value is 0.21 mC cm−2.ESAS of the Pt-NiA/C and Pt-NiC/C were 106.1 and 89.5 m2 gPt−1 respectively. The larger ESAS of the Pt-NiA/C was slightly larger than that of the Pt-NiC/C catalyst.tion (1) , in whic−1 H2SO4 + 0.5 mol L−1 CH3OH solution  chloride hexahydrate (NiCl2·6H2O) dissolved in a solution containing 25 mL ultrapure water and 25 mL ethanol, to which 413.4 mg of sodium citrate was then added with stirring. The solution was adjusted to pH 14 by addition of 5 wt % KOH/EG solution. Pretreated carbon black Vulcan XC-72R (300 mg) was added to the mixture under stirring, followed by addition of 10 mL of hydrazine hydrate. The mixture was transferred to a Teflon-lined autoclave container and heated at 120 °C for 4 h. The final product was collected by filtration, washed with ultrapure water (until the filtrate wash water reached pH 7), and finally dried under vacuum at 323 K for 12 h.NiA/C nanoparticles were prepared as reported previously [2·6H2O) was added to the above solution and stirred for 20 min to completely dissolve the mixture. The pH value of solution was adjusted to 9~10 by addition of a 5 wt % KOH/EG solution with stirring. Pretreated carbon black Vulcan XC-72R (300 mg) was added and the solution was mixed in a flask using ultrasonic stirring, followed by heating at 433 K for 20 h. The final product was cooled, collected by filtration and washed with ultrapure water until the wash water read pH 7. The NiA/C nanoparticles were finally dried at 323 K for 12 h under vacuum.Nieviously . Here, 4−1 chloroplatinic acid (H2PtCl6·6H2O) aqueous solution and 30 mL ethylene glycol (EG) were added to a flask, and the pH value was adjusted to 9~10 using 5 wt % KOH/EG solution. Then, 100 mg of either NiC/C or NiA/C powder was added to the flask and the mixture was stirred for 6 h at 433 K. The final products were cooled and collected by filtration, washed with ultrapure water, and dried under vacuum at 323 K for 12 h, the obtained products were denoted as Pt-NiC/C and Pt-NiA/C, respectively.Pt shell deposition onto the surface of Ni/C nanoparticles was carried out as follows: 3.32 mL of20 mg mLhν =1486.6 eV) was used. The chemical composition of the samples was determined by an IRIS advantage inductively coupled plasma atomic emission spectroscopy (ICP-AES) system  and energy dispersive X-ray analysis (EDX) coupled to TEM.X-ray diffraction (XRD) patterns of the catalysts were recorded on a Shimadzu XD-3A (Japan), using filtered Cu-Kα radiation. Transmission electron microscopy (TEM) measurements were carried out on a JEM-2010 Electron Microscope (Japan) with the acceleration voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) (Phi-5702 America) with a monochromatic Al Kα X-ray source (−1 KCl) electrode, respectively. The working electrode i.e., the catalyst-film electrode, was prepared as follows: 5 mg of catalyst was dispersed ultrasonically in 1.00 mL Nafion/ethanol (0.25% Nafion) for 15 min. 8 μL of the dispersion was transferred onto the glassy carbon disk (5 mm in diameter) using a pipette and then dried in the air. Then, the working electrode was prepared. The catalysts were characterized by cyclic voltammetry (CV) and chronoamperometry (CA) tests at room-temperature. Before each measurement, the solution was purged with high purity N2 for at least 10 min to ensure O2 free measurements. All current densities reported in this article were normalized to the electrode surface area (EASS).Electrochemical measurements were carried out on a CHI650d electrochemical work station. A common three-electrode electrochemical cell was used, in which the counter and reference electrodes were a platinum wire and an Ag/AgCl (3 mol LA/C has greater methanol oxidation activity than Pt-NiC/C, mainly because of the decrease in adsorption energy toward CO species owing to the stronger Pt-Ni interaction in the Pt-NiA/C catalyst. Thus, controlling the crystallinity of the core metal affords a promising approach to improve the catalytic activity of core-shell-structure catalysts.Carbon-supported Pt-Ni/C catalysts with either amorphous or crystalline Ni cores were synthesized and characterized. The XRD results showed that neither amorphous nor crystalline Ni structure as a seed crystal affected the structure of the Pt shell. However, the Pt-Ni interaction was different, as shown by the shift of Pt 4f XPS. Electrochemical results indicate that Pt-Ni


Hepatitis B virus (HBV) genotypes have distinct geographical distributions and are associated with different clinical courses. HBV genotype G (HBV/G) is extremely rare among Human immunodeficiency virus (HIV) infected populations in Japan. Genetic analysis and clinical course of recombinant forms with HBV/G infection are seldom reported in the literature.A 36-year old homosexual man with HIV infection was referred to a general hospital for assessment of chronic HBV infection. We cloned full-length HBV isolates and determined the complete genome sequences of 2 obtained clones, although mixture of multiple variant with different length is detected by HBV-DNA genotyping. The Bootscaning analysis using a full-length HBV genome revealed the clones represented as the HBV/A2 and the HBV/G/A2 recombinant strain. The HBV-DNA decreased from >9.1 to 2.5 log copies/mL after 24 months of antiretroviral therapy.This patient was co-infected with HBV/A2 and HBV/G/A2 recombinant strain. This recombinant strain was not identical to HBV/G/A2 strains previously reported from Japan. Recombination with other genotypes could alter the clinical manifestations of chronic hepatitis B in people living with HIV. Hepatitis B virus (HBV) infection is a major public health issues among human immunodeficiency virus (HIV) -infected people. HIV-HBV co-infection can accelerate the progression of chronic hepatitis resulting in fibrosis and hepatocelullar carcinoma  is the most frequently detected, followed far behind by genotype C and genotype B which are common in the entire chronic hepatitis B population  at a local clinic during follow-up for flu-like symptoms. He was referred to the hepatology division, in a general hospital for further assessment of chronic hepatitis B. The liver biopsy showed septal fibrosis and infiltrating lymphocyte in his liver tissue associated with degeneration of limiting plate, and histologic activity index—METAVIR score , 93,297 IU/mL; antibodies to the HBsAg (anti-HBsAg), negative; total hepatitis B core antibodies, 11.9 S/CO; HBeAg, 42.9 S/CO; antibodies to the HBeAg (anti-HBeAg), negative; HBV-DNA, >9.1 log copies/mL; hyaluronic acid, 124 ng/mL; Type IV Collagen 7 s, 7.3 ng/mL. HBV drug resistance mutations related to lamivudine, adefovir, entecavir or tenofovir were not detected. He received antiretroviral therapy (ART) consisting of tenofovir/emtricitabine (TDF/FTC) and raltegravir to treat HIV-HBV/G/A2 recombinant infection. Three months after the initiation of ART, while the HBV-DNA decreased steadily, the levels of liver enzyme increased progressively with the rapid decline of HBeAg and HBsAg. The CD4 T-cell count increased to 487 cells/μL. This presentation was consistent with HIV-HBV immune reconstitution inflammatory syndrome (IRIS). The levels of liver enzymes improved without specific treatments. The HBV-DNA decreased to 2.5 log copies/mL after 24 months of ART.The HIV-RNA and CD4 T cell count were 4300 copies/mL and 226 cells/μL, respectively. The HIV subtype was CRF-01 AE and no primary mutations associated with drug resistance mutations were detected. Laboratory findings were as follows: platelet count, 10.6 × 10HIV infection accelerates chronic hepatitis B related liver diseases, and precise assessment of HBV infection is imperative in the management of HIV-HBV co-infected patients. Different HBV genotypes induce different clinical features in HBV-infected patients. Recently, acute HBV infection in metropolitan areas of Japan has been increasingly attributed to the HBV/A2, that is transmitted through sexual contacts (Tamada et al. There was a discrepancy between results of the HBV-DNA genotyping and the serological genotyping test by EIA in this case. The reason was unclear, but recombination in the preS2-region might affect the results of the serological test by EIA that used monoclonal antibodies of a combination of epitopes on preS2-region products. HBV/G may be overlooked if a tool designed to detect recombinant forms with other genotypes is not used. It is assumed that detection technique could affect the epidemiologic study of HBV genotype. Full-genome sequence was occasionally difficult to be determined because of the presence of mixture of multiple variant with different length.Two studies suggest that HBV/G may accelerate liver fibrosis progression and associated with HBeAg positivity and high HBV-DNA levels (Lacombe et al. Clinical data regarding treatment for HBV/G is limited. As a rule, HIV-HBV co-infected patients regardless of HBV genotype receive TDF/FTC-containing ART. A study reported three of six patients who experienced HBV-DNA rebound with correct TDF serum concentration were infected with HIV-HBV/G or HIV-HBV/G/A2 mixture (Karine Lacombe et al. This patient was infected with HBV/A2 and HBV/G/A2 recombinant which is extremely rare in Japan. Although these HBV strains had no mutations related to anti-HBV drugs, detectable HBV-DNA levels have lasted for 2 years under TDF/FTC-containing ART. Clinicians should keep the HBV genotyping in mind using tools designed to detect recombinant forms in the management of HIV-HBV co-infected patients.