Background: The P-gp drug-binding domain is linked to the two nucleotide-binding domains (NBDs) by NBD1 and NBD2 transmission interfaces.Results: Only mutations at the NBD2 transmission interface blocked P-gp assembly. Tariquidar repaired all 25 mutants.Conclusion: The mechanism of P-gp folding requires precise hydrophobic interactions at the NBD2 interface.Significance: We identified a linchpin for assembly and repair of ABC proteins. P-glycoprotein  is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp. The ATP-binding cassette (ABC)Human ABC proteins have been the subjects of intense research efforts since most are clinically important. Cystic fibrosis (CF), gout, intrahepatic cholestasis types 2 and 3 (liver bile diseases), Tangier disease (HDL deficiency), Dubin-Johnson syndrome (jaundice), hyperinsulimic hypoglycemia of infancy, pseudoxanthoma elasticum, multidrug resistance, secretory diarrheas, anemia, diabetes, and atherosclerosis are examples of potentially lethal or debilitating conditions caused by genetic mutations or altered activity of one of the 48 human ABC proteins (The most common genetic defect is expression of an ABC processing mutant that is defective in folding and trafficking. The classic example is the ΔF508-CFTR (ABCC7) mutant in CF . Other eHuman ABC proteins appear to be particularly sensitive to point mutations because they are multi-domain membrane proteins (core structure of two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs)) that require formation of specific domain-domain contacts to adopt a native structure . ABC pro6–The human P-glycoprotein (P-gp) drug pump has been a very useful model system for studying repair of ABC protein defective in processing/folding because processing mutations throughout the molecule can be repaired by expression in the presence of drug substrates  or by inThe interfaces between the domains play critical roles in the P-gp drug efflux mechanism. Drug substrates bind within a cavity located at the interface between the TMDs –15. Two –16–Coupling of ATP hydrolysis in the NBDs to drug efflux from the drug-binding sites in the TMDs is mediated by ball-and-socket joints  at the NContacts between the homologous halves (TMD1-NBD2 or TMD2-NBD1) appear to be particularly important for mammalian drug pumps. For example, the BCRP/ABCG2 drug pump lacks contacts equivalent to P-gp ICL1/ICL3 . In addiHypersensitivity to mutation at the IH2/NBD2 transmission interface compared with the IH1/NBD1 interface might explain the differences in the maturation of NBD2 deletion mutants of P-gp and CFTR . DeletioIn this study, we tested whether the NBD2 transmission interface was particularly critical for P-gp assembly and activity by comparing the effects of mutations at the NBD1-TMD and NBD2-TMD interfaces. We report that the interfaces are asymmetric since P-gp maturation was only sensitive to mutations at the NBD2 interface. The results suggest that the NBD2-TMD interface is a linchpin for assembly and repair of ABC proteins.N-linked glycosylation sites can be used to monitor maturation of human P-gp from an immature 150 kDa protein to a mature 170 kDa protein. P-gp cDNA was modified to contain a 10-histidine tag at the COOH-terminal end to facilitate purification of the expressed protein by nickel-chelate chromatography -2-aminoethanesulfonic acid, 280 mm NaCl and 1.5 mm Na2HPO4, pH 6.96). After 10 min at room temperature, 4 ml of HEK 293 cells  in Dulbecco's modified Eagle's medium (DMEM) with high glucose  bovine calf serum) was added and 1.5 ml of the mixtures were added to duplicate well of 6-well culture plates. After 5 h at 37 °C, the medium was replaced with fresh medium with or without 0.5 μm tariquidar. About 16 h later, the cells were harvested, washed with PBS, and cell pellets suspended in 150 μl of 2× SDS sample buffer  SDS, 4% (v/v) 2-mercaptoethanol containing 25 mm EDTA. Samples were applied to 6.5% SDS-PAGE gels . The gels were electroblotted onto a sheet of nitrocellulose and P-gp proteins detected using A52 monoclonal antibody, horseradish peroxidase conjugated anti-mouse secondary antibody, and enhanced chemiluminescence. The signals were imaged and levels of mature (170 kDa) P-gp relative to total P-gp (mature 170 kDa plus immature 150 kDa) determined using ChemidocTM XRS+ with Image LabTM software . An equivalent amount of the sample was loaded onto 10% (v/v) SDS-PAGE gels and subjected to immunoblot analysis with a monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GADPH) .The ICL mutant cDNAs were transiently expressed in HEK 293 cells by a calcium phosphate precipitation approach as described previously . Brieflym cyclosporin A. Cyclosporin A is a substrate of P-gp and acts as a potent pharmacological chaperone in promoting maturation and yield of P-gp . After 10 min at 4 °C, insoluble material was removed by centrifugation at 16 000 × g for 15 min at 4 °C. DNA in the supernatant was removed by passage through a DNA miniprep microfuge column . The flow-through material was then applied onto a nickel spin column  that had been pre-equilibrated with buffer A containing 50 mm NaPO4, pH 8.0, 500 mm NaCl, 50 mm imidazole and 20% (v/v) glycerol and 0.1% (w/v) n-dodecyl-β-d-maltoside. The column was then washed twice with 0.6 ml buffer B containing 10 mm Tris-HCl, pH 7.5, 500 mm NaCl, 80 mm imidazole, pH 7.0, 20% (v/v) glycerol and 0.1% (w/v) n-dodecyl-β-d-maltoside and twice with 0.6 ml buffer C (buffer B containing 50 mm imidazole). Histidine-tagged P-gp was then eluted with 0.2 ml of buffer B but containing 300 mm imidazole. Recovery of P-gp was monitored by immunoblot analysis with rabbit anti-P-gp polyclonal antibody (m verapamil.HEK 293 cells were plated onto fifty plates (10-cm diameter) and transfected with the cDNA of the histidine-tagged P-gp mutant when about 50% confluent. After 16 h at 37 °C, the medium was replaced with fresh medium containing 5 μantibody . A samplantibody  was measm copper phenanthroline (oxidant to promote disulfide bond formation). EDTA was then added to a final concentration of 2 mm. Immunoblot analysis and assay of ATPase activity were performed as described above.Cys-less P-gp or the ICL1/ICL4 double cysteine mutants I160C/F904C or F163C/R905C were transiently expressed in HEK 293 cells at reduced temperature (30 °C) to promote maturation. The P-gps were isolated by nickel-chelate chromatography. The isolated P-gps were incubated 20 °C for 10 min in the presence or absence of 0.5 mA and B). The IH located in the middle of the ICLs interacts with the NBDs to form ball-and-socket joints . The protein is initially synthesized as a 150 kDa core-glycosylated protein. If the protein correctly folds into a compact structure it can exit the endoplasmic reticulum for modification of the carbohydrate in the Golgi to yield a 170 kDa mature protein.A mutational approach was used to test if residues in the ICLs at the NBD2 transmission interface played more important roles in maturation of P-gp compared with residues in the ICLs at the NBD1 interface. Maturation of P-gp can readily be monitored in whole cell assays as the protein contains three N-glycosylation sites in the extracellular loop connecting TM segments 1 and 2 A. The prAccordingly, 84 mutants were constructed that contained point mutations to residues in IH4/ICL4 and residues in ICL1, ICL2, and ICL3 flanking IH1, IH2, and IH3, respectively (segments of about 30 residues). The 36 IH1, IH2, and IH3 mutants constructed in previous studies , 38 wereCaenorhabditis elegans . Mutants V907S (IH4) and Y790A (ICL3) represent examples of mutations that partially inhibited maturation. The V907S mutant yielded about equivalent levels of mature and immature P-gp. Mutation of Tyr-790 to alanine substantially reduced maturation as about 85% of the product was the 150 kDa immature protein. Mutant V908S (IH4) represents an example of a mutation that did not detectably reduce P-gp maturation. In general, the mutations did not markedly reduce the steady-state levels of P-gp expression 16 h after transfection. There was less than a 2-fold change in total P-gp compared with wild-type P-gp. For example, the total P-gp yields in mutants R789A, V907S, Y790A, and V908S (A) were about 60, 75, 75, and 98% of wild-type P-gp, respectively.Mutant R789A (ICL3) represents an example of a mutation that blocked maturation as only the 150 kDa immature form of P-gp was detected A. Mutantnd V908S A were abpanels B–E). Maturation of P-gp was quite insensitive to mutations at the NBD1 transmission interface. None of the 32 residues in ICL1 when mutated affected maturation of P-gp . Similarly, the mutations to residues in ICL4 (except for V907S) also did not affect maturation of P-gp. Mutant V907S yielded a slightly lower amount (about 55%) of mature P-gp.The effects of ICL mutations are shown in B and D). Fourteen of the twenty-eight mutations in ICL2  inhibited maturation of P-gp (<15% mature P-gp) while four other ICL2 mutations  partially reduced maturation of P-gp (about 55–60% mature P-gp). The results suggest that the NBD1 and NBD2 transmission interfaces make asymmetric contributions to maturation of P-gp. Maturation of P-gp was highly sensitive to mutations that could disrupt formation of the ICL2/ICL3 tetrahelix bindle at the NBD2 transmission interface.By contrast, the NBD2 transmission interface was highly sensitive to point mutations , B and Dpanels B–E). To test if maturation of the processing mutants could be restored, they were expressed in the presence of tariquidar. Tariquidar was selected because it is the most potent pharmacological chaperone for rescue of P-gp processing mutants  was expressed in the absence of drug substrate. Expression in the presence of tariquidar however, promoted maturation of the A250L mutant to yield mature 170 kDa P-gp as the major product. Accordingly, all 25 of the ICL processing mutants were expressed in HEK 293 cells in the absence or presence of tariquidar. A sample of whole cell SDS extracts was subjected to SDS-PAGE and immunoblot analysis and the amount of mature 170 kDa P-gp was quantified. It was found that all of the misprocessed mutants could be efficiently rescued with tariquidar to yield mature P-gp as the major product . The results show that the defects caused by the ICL3  or ICL4  point mutations at the NBD2 transmission interface could be overcome by binding of tariquidar to the TMDs. We previously showed that a P-gp truncation mutant lacking the NBDs could be efficiently rescued with tariquidar . The amount of mature P-gp in mutants L443A, L443S, and L443F P-gps were similar to wild-type while mutant L443R yielded about 35% mature P-gp. Mutants F1086A, F1086L, and F1086W yielded mature 170 kDa protein as the major product, whereas F1086R yielded little mature P-gp (<5%) .Residues equivalent to Phe-1086 and Tyr-1087 at the IH4-NBD1 site are Leu-443 and Tyr-444. To test if the IH4-NBD1 contact point was also hydrophobic we first compared the effects of changes to Leu-443 and Phe-1086 on maturation and activity. Accordingly, A52-tagged mutants L443X  were constructed. These mutants and A52-tagged mutants F1086X   were traFig. 4Histidine-tagged versions of the L443X  and F1086X  mutants were expressed in HEK 293 cells in the presence of cyclosporine A to promote maturation of the mutants. Expression in the presence of cyclosporine A promoted maturation of all the mutants to wild-type levels (data not shown). The mutants were isolated and ATPase activity measured in the presence of verapamil. Verapamil was used because it is a substrate  that higB).It was found that P-gp was again less sensitive to changes at the Leu-443 position. While the F1086A and F1086R mutations blocked verapamil stimulated ATPase activity, the L443A mutant resembled wild-type activity, and the L443R mutant retained about 35% activity B.It was possible that the asymmetric effects of changes to positions 443 and 1086 were related to the fact that different amino acids were found at the two locations in the wild-type protein (Leu-443 at NBD1 and Phe-1086 in NBD2). Therefore, we next examined the effects of changes to the residues at the Tyr-444(NBD1) and Tyr-1087(NBD2) positions .A). By contrast, the amount of mature P-gp in Y444F and Y1087F was similar to that of wild-type P-gp . All the Y444X mutants except Y444E differed from their Tyr-1087 counterparts as they yielded detectable levels of mature 170 kDa P-gp. The Y444A and Y444L mutants yielded about 25% mature P-gp while the Y444R mutant yielded about 40% mature P-gp .A52-tagged mutants Y444X were constructed so that tyrosine was replaced with a small (Ala), hydrophobic  or charged  residue. For comparison, the previously constructed Y1087A, Y1087F, and Y1087L mutants were included  and addiFig. 4B shows that Y1087F retained about 30% of the activity of wild-type enzyme. The activity of wild-type P-gp was 2.2 + 0.2 μmol Pi/min/mg protein. The activity of Y1087 was almost completely inhibited when it was mutated to A, L, R, or E. In contrast, the Y444X mutations had less severe effects. With the exception of Y444E, the Tyr-444 mutations had less severe effects on P-gp activity. Both the Y444F and Y444R mutants showed over 75% of wild-type activity. Mutants Y444A and Y444L showed about 25% activity.We then tested whether the Y443X and Y1087X mutants had activity. Histidine-tagged mutants were expressed in HEK 293 cells in the presence of cyclosporine A to promote maturation of the mutants. The mutant P-gps were isolated, mixed with lipid and assayed for ATPase activity in the presence of saturating concentrations of verapamil. Fig. 4Results suggest that the IH4-NBD1 contact site is less sensitive to mutations and less hydrophobic than the equivalent IH2-NBD2 site. The difference is particularly evident when the aromatic residues were replaced with the positively charged arginine residue. Replacement of Phe-1086 or Tyr-1087 with arginines blocks P-gp maturation and activity. The L443R and Y444R mutations only showed modest reductions . The resClamping the IH segments in close proximity by cysteine cross-linking can activate or inhibit P-gp ATPase activity. For example, cross-linking of cysteines in ICL1 and ICL3 to hold IH1 and IH3 in close proximity activated P-gp ATPase activity over 10-fold in the absence of drug substrates . By contSince IH1 and IH4 at the NBD1 transmission interface were less sensitive to point mutations compared with IH2 and IH3 at the NBD2 transmission interface , it was A and B) were selected to test the effects of IH1/IH4 cross-linking as the mutants yielded mature P-gp that showed efficient cross-linking in the presence of oxidant (copper phenanthroline) (C). Cross-linking can readily be detected because cross-linking between different domains causes P-gp to migrate slower on SDS-PAGE gels (C). No cross-linked product was observed when the I160C, F904C, F163C, or R905C mutants were treated with oxidant (data not shown).Mutants I160C/F904C and F163/R905C , A and Bhroline) C. Cross-AGE gels . The croAGE gels C. No croD). Cross-linking of mutants I160C/F904C and F163C/R905C with oxidant however, inhibited more than 90% of the activity. Treatment with oxidant had little effect on the Cys-less parent. Activity of the cross-linked mutants was restored when the disulfide bond was reduced with dithiothreitol (D).To test for the effects of cross-linking on activity, isolated Cys-less P-gp and mutants I160C/F904C and F163/R905C were treated without or with copper phenanthroline for 10 min a 20 °C. The reaction was stopped by addition of EDTA. The samples were then assayed for verapamil-stimulated ATPase activity. Before treatment with oxidant, both mutants exhibited robust drug-stimulated ATPase activity. Mutants I160C/F904C and F163C/R905C exhibited over 70% of the activity of the Cys-less parent D. Cross-threitol D.The results show that IH1/IH4 and IH2/IH3 cross-linking had similar effects as they both severely inhibited the activity of P-gp. The results are in agreement with modeling studies that predict that the IH1/IH4 and IH2/IH3 segments undergo significant conformational changes during the reaction cycle that would alter the relative positions of residues at the IH1/IH4 and IH2/IH3 interfaces , 46. Altet al. (D).Pajeva et al.  predicteet al. D.A study of Q loop mutations Q475A(NBD1) and Q1118A(NBD2) suggested that the P-gp transport mechanism shows redundancy , 49.We constructed histidine-tagged R262A/R905A and T263A/T906A mutants (in wild-type background) to test if mutations in both IH2 and IH4 were needed to inactivate P-gp ATPase activity. It was found that both mutants retained about 70% of wild-type verapamil stimulated ATPase activity . A Q475AB and D).The major novel mechanistic findings in this study were that the transmission interfaces make asymmetric contributions to folding and activity of P-gp. The results show that the NBD2 transmission interface is particularly important for folding of P-gp and helps to explain why P-gp maturation is sensitive to deletion of NBD2 . The NBDBy contrast, maturation of P-gp was highly insensitive to changes at the NBD1 transmission as none of the ICL1 or ICL4 point mutations severely reduced maturation . ApparenThe NBD2 contact site required more precise and hydrophobic interactions for P-gp maturation and activity compared with NBD1. The F1086R or Y1087R mutations at the NBD2 socket blocked maturation and activity while P-gp showed substantial activity when the comparable mutations  were made to the NBD1 socket.B). Removal of the hydroxyl side chain may have detrimental effects on activity because the equivalent residue in the crystal structure of C. elegans P-gp (Tyr-1129) shows hydrogen bond interactions with Asp-846 in IH3  and Arg-286 in IH2   . In a moArg-262) . All othC. elegans P-gp structure showed that the equivalent residues at the NBD1 transmission interface made the same contacts  similar to the Cys-less parent. Their results showed that Asp-164 or Asp-805 were not critical for drug transport. In agreement with cysteine mutagenesis results, we found that the D164A mutation did not reduce verapamil-stimulated ATPase activity (A difference was that the D164A mutation did not reduce maturation of P-gp . The likOur results suggest that the NBD2 transmission interface is particularly sensitive to mutations relative to the NBD1 transmission interface. Studies on mutants of P-gp's structurally similar sister proteins, ABCB4 and CFTR, also suggest that the NBD2 transmission interface plays a key role in activity and protein assembly.Human ABCB4 is a phosphatidylcholine transporter that shows 78% amino acid identity to P-gp . An ABCBCFTR is a chloride channel predicted to be structurally similar to P-gp . The NBDNBD2 is more important for P-gp maturation than CFTR. A ΔNBD2 P-gp truncation mutant did not mature while ΔNBD2 CFTR showed robust maturation . The resAll of the ICL2/ICL3 processing mutations could be repaired with tariquidar. Binding of tariquidar to the TMDs  might st


Aeromonas veronii is a pathogenic gram-negative bacterium, which infects a variety of animals and results in mass mortality. The stalled-ribosome rescues are reported to ensure viability and virulence under stress conditions, of which primarily include trans-translation and alternative ribosome-rescue factor A (ArfA) in A. veronii. For identification of specific peptides that interact and inhibit the stalled-ribosome rescues, peptide aptamer library (pTRG-SN-peptides) was constructed using pTRG as vector and Staphylococcus aureus nuclease (SN) as scaffold protein, in which 16 random amino acids were introduced to form an exposed surface loop. In the meantime both Small Protein B (SmpB) which acts as one of the key components in trans-translation, and ArfA were inserted to pBT to constitute pBT-SmpB and pBT-ArfA, respectively. The peptide aptamer PA-2 was selected from pTRG-SN-peptides by bacterial two-hybrid system (B2H) employing pBT-SmpB or pBT-ArfA as baits. The conserved sites G133K134 and D138K139R140 of C-terminal SmpB were identified by interacting with N-terminal SN, and concurrently the residue K62 of ArfA was recognized by interacting with the surface loop of the specific peptide aptamer PA-2. The expression plasmids pN-SN or pN-PA-2, which combined the duplication origin of pRE112 with the neokanamycin promoter expressing SN or PA-2, were created and transformed into A. veronii C4, separately. The engineered A. veronii C4 which endowing SN or PA-2 expression impaired growth capabilities under stress conditions including temperatures, sucrose, glucose, potassium chloride (KCl) and antibiotics, and the stress-related genes rpoS and nhaP were down-regulated significantly by Quantitative Real-time PCR (qRT-PCR) when treating in 2.0% KCl. Thus, the engineered A. veronii C4 conferring PA-2 expression might be potentially attenuated vaccine, and also the peptide aptamer PA-2 could develop as anti-microbial drugs targeted to the ribosome rescued factors in A. veronii. Aeromonas veronii is a type of important pathogenic microbes to human and aquatic animals , aerolysin (AerA), lipase (Lip), flagellin (Fla), and out membrane protein (OmpA)  and the associated Small Protein B (SmpB) is predominant and essential for translation quality control to the vast majority of bacteria Keiler, . HoweverMycobacterium tuberculosis  as scaffold protein on which inserting 16 random amino acids to shape as a loop. In this study, the peptide aptamer PA-2 was isolated and demonstrated to interact specifically with both SmpB and ArfA by bacterial two-hybrid assay (B2H), resulting to dysfunction of both SmpB and ArfA, and subsequently intensifying the sensitivities under the conditions of temperatures, glucose, sucrose, potassium chloride, and antibiotics. Collectively, not only the peptide aptamer PA-2 could be as anti-microbial drugs targeted to the ribosome rescued factors, but also the transformant conferring PA-2 was potentially attenuated vaccine for the prevention of A. veronii.For this purpose, the peptide aptamers were selected to interact with SmpB and ArfA. The peptide aptamers are combinatorial proteins consisting of a stable scaffold protein and random amino acids, which typically embed as an exserted surface loop  to generate pTRG-SN, pTRG-SNΔC45, pTRG-SNΔN57C45, and pTRG-SNΔN57 , in which the residues E28I29K30 were substituted to alanines, PCR was performed using pBT-ArfA as template and F12/R12 as the primers to which the specific mutations were designed and introduced. In the reaction, 1 μl of DNA template (400 pg/μl), 1 μl primer either F12 or R12 (10 mM), 25 μl PrimeSTAR mix  were included in 50 μl volume for running 15 cycles, followed by a combination of both PCR products for an additional 15 cycles. Eventually the purified PCR was digested with Dpn I and transformed into E. coli. The mutated plasmids were extracted from positive colonies and verified by DNA sequencing . Similarly, a series of pBT-ArfA mutants including pBT (ArfA-D31N32), pBT (ArfA-A36L37), pBT (ArfA-L42F43), pBT (ArfA-K52G53K54), pBT (ArfA-G55S56Y57), pBT (ArfA-R59K60), and pBT (ArfA-K62) were constructed in which the residues were substituted to alanine except that K62 was substituted to proline  analysis  medium. All reagents were purchased from Stratagene and B2H assay was performed according to the instruction manual of BacterioMatch II Two-Hybrid System Vector Kit .Bacterial strains used in this study were listed in Table A. veronii C4 was isolated from Ctenopharyngodon idella  and 25 μg/ml chloramphenicol at 37°C respectively .The nuclease (SN) of smpB or arfA were cloned into the pBT bait to generate pBT-SmpB or pBT-ArfA, separately , 25 μg/ml chloramphenicol and 12.5 μg/ml tetracycline, and then continued to culture in dark location at room temperature for another 16 h  to determine 3D structure. Likewise, the structural model of either SmpB or ArfA was generated by submitting the amino acid sequence to the I-TASSER in comparison with SmpB (PDB code: 1k8h) or ArfA (PDB code: 2tmaA), accordingly. The output of I-TASSER was analyzed using Pymol version 1.4.1  server was applied to predict three-dimensional structure , glucose concentrations , sucrose , potassium chloride (KCl) concentrations , tetracycline concentrations , erythromycin concentrations  and kanamycin concentrations .The plasmid pRE112 and its derivatives were transformed into A. veronii C4 strains were cultured until to stationary phase at 30°C, 150 rpm in 5 ml LB complemented with 2.0% KCl. The amounts of RNA were isolated using the RNAiso Plus reagent according to the manufacturer's instructions . Residual genomic DNA was eliminated by treating with 4 μl of DNase I . Eventually the amount of RNA were precipitated by 75% ethanol and re-suspended in 50 μl of RNase free water . The qRT-PCR primer sequences were shown in Supplementary Table rpoS) and NhaP-type Na+/H+ antiporter (nhaP) were normalized utilizing 16S rRNA as internal standard. The relative expression level was calculated using the 2−ΔΔCt method, where ΔΔCt = (Ct target − Ct 16S rRNA)Treatment − (Ct target − Ct 16S rRNA) Control  with Statistical Package for the Social Sciences (SPSS) version 19.0 software  and GraphPad Prism 5 software . 63L64R65K66A67 of SN were replaced by 16 random amino acids exhibiting surface loop  and pTRG-SN, pBT-SmpB (G133K134) and pTRG-SN, or pBT-SmpB (D138K139R140) and pTRG-SN had growth defects on 5 mM 3-AT medium, indicating that the conserved sites G11S12, G133K134, and D138K139R140 of SmpB were important for the interaction with SN.B2H was employed to identify SN to interact with SmpB. The co-transformants endowing with pBT-SmpB and pTRG-SN appeared the abundance of colonies on 3-AT medium, whereas the colonies conferring pBT and pTRG-SN, pBT-SmpB and pTRG did not survive, revealing that only SN interacted with SmpB Figure . To detee Smith, , resultiA. veronii C4 were aligned with other pathogenic bacteria (Supplementary Table http://weblogo.berkeley.edu/logo.cgi)  and pTRG-PA-2 had growth defect on 5 mM 3-AT medium, demonstrating that the residue K62 of ArfA was vital for interacting with PA-2.The peptide aptamer library was constructed to insert a random and exposed surface loop on the scaffold protein SN Figure , and sev) Figure . A serie) Figure  were co-) Figure . The res138K139R140 and G133K134 interacted with the N-terminal region of SN  had slower growth as well as A. veronii C4 (pRE112), accompanied by severely impaired growth in A. veronii C4 (pN-PA-2)  and A. veronii C4 (pN-PA-2) grew defectively at different temperatures, signifying that expressions of SN and PA-2 could recognize and inhibit SmpB and ArfA in vivo individually.To analyze whether SN and PA-2 affected the functions by interacting with SmpB and ArfA in ) Figure . At 30°C) Figure , and theC Figure . Taken tA. veronii C4 derivatives were evaluated under different concentrations of glucose and sucrose. The growths of A. veronii C4 (pN-SN) and A. veronii C4 (pN-PA-2) showed extremely significant differences compared with those of wild type at 5, 10, 15% glucose and sucrose  and A. veronii C4 (pN-PA-2) at 5% glucose and sucrose, demonstrating that they had extremely significant differences (P < 0.01)  and A. veronii C4 (pN-PA-2) displayed extremely significant differences compared with those of wild type at 0, 0.5, 2% KCl, respectively (P < 0.01)  appeared slower than that of A. veronii C4 (pN-SN) (P < 0.01)  Figure . The gro) Figure . Besides) Figure . At 5 anA. veronii C4 strains were treated at tetracycline, kanamycin and erythromycin for measurements. The results showed that all engineered A. veronii C4 strains grew slower compared with wild type under various antibiotics  retarded the growth severely compared to wild type, while A. veronii C4 (pRE112) and A. veronii C4 (pN-SN) impaired moderately at 0−15 μg/ml kanamycin   and A. veronii C4 (pN-PA-2) at 2.0% KCl in contrast to those of wild type and A. veronii C4 (pRE112). Similarly, the transcriptions of rpoS and nhaP in smpB knock-out strain were significantly down-regulated compared to wild type (P < 0.01) Figure As the growths of A. veronii C4 (pN-SN) and A. veronii C4 (pN-PA-2) had significant slower in comparison with wild type and A. veronii C4 (pRE112) under 2.0% KCl culture  Figure , the totE. coli in the absence of trans-translation , because their sizes could not allow to penetrate the bacteria cell wall for interacting with SmpB and ArfA, which acted as cytoplasmic proteins in vivo , prompting us to construct the vectors pN-SN or pN-PA-2 for expression in vivo, in which utilizing neokanamycin promoter of pk18mobsacB for exogenous expression and replication origin of pRE112 for amplification  showed more observable retardation of growth in all engineered A. veronii C4 strains  and A. veronii C4 (pN-PA-2) were accordant by contrast with A. veronii C4 (pRE112)  Figure . At 42°Ce Figure . It probe Figure . Currente Figure .A. veronii C4 and its derivatives thereof were treated by parallel concentrations of sucrose or glucose, the strains always grew better in sucrose than those in glucoses  and A. veronii C4 (pN-PA-2), inferring the expressions of SN and PA-2 could interact and knockdown the functions of SmpB and ArfA, which resulted to growth defects under KCl stress. The gene nhaP encodes NhaP-type Na+/H+ or K+/H+ antiporter, which avoids excessive accumulation of potassium in the cytoplasm  and A. veronii C4 (pN-PA-2)  Figure . To furtP Figure .11S12, G133K134, and D138K139R140 of SmpB and K62 of ArfA for disturbing SmpB and ArfA actions, bringing about the interferes with their partner or substrates in A. veronii C4.When expressed SN and PA-2 in A. veronii C4, A. veronii C4 (pN-SN) and A. veronii C4 (pN-PA-2) exerted distinct growth defects at 25°C. Analogously, the growths of A. veronii C4 (pN-SN) and A. veronii C4 (pN-PA-2) showed significantly lower compared to those of wild type and A. veronii C4 (pRE112) by treating with glucose, sucrose, kanamycin and KCl. Further on, the down-regulated transcriptions of rpoS and nhaP genes eventually set off the growth defects, when employing PA-2 to disturb the functions of SmpB and ArfA. Collectively, the engineered A. veronii C4 (pN-PA-2) could be potentially attenuated vaccine for the prevention of A. veronii pathogen, and the peptide aptamer PA-2 could theoretically develop as anti-microbial drugs targeted to the ribosome rescued factors.In summary, peptide aptamer PA-2, in which scaffold protein SN interacted with SmpB and the surface loop interacted with the ArfA, was screened from the peptide aptamer library. It binds primarily to GYZ and ZL designed the experiments. PL, YC, DW, YT, HS, and HT performed and analyzed for the work. PL, QS, YZ, and ZL interpreted the data. ZL, PL, and DW drafted the work. All the authors agreed to submit for Frontiers in Microbiology Journal.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective. In this study, we investigated whether RAAS gene single nucleotide polymorphisms (SNPs) and their interactions were associated with end-stage renal stage (ESRD). Methodology and Results. This was a case-control study for 647 ESRD cases and 644 controls. AGT (M235T (rs699) and T174M (rs4762)), AGTR1 (A1166C (rs5186) and C573T (rs5182)), ACE (I/D (rs1799752) and G2350A (rs4343)), and CYP11B2 C-344T (rs1799998) were genotyped and compared between cases and controls to identify SNPs associated with ESRD susceptibility. Multifactor dimensionality reduction (MDR) was used to identify gene-gene interactions. Several RAAS genes were associated with ESRD: AGT M235T, ACE I/D, ACE G2350A, and CYP11B2 C-344T. By MDR analysis, a three-locus model (ACE ID/ACE G2350A/CYP11B2 C-344T) of gene-gene interaction was the best for predicting ESRD risk, and its maximum testing accuracy was 56.08% and maximum cross-validation consistency was 9/10. ESRD risk was higher with the simultaneous occurrence of ACE I/D DD-ACE G2350A AA. AGT, ACE, and CYP11B2 gene polymorphisms are associated with ESRD. Conclusions. The gene-gene interaction effects of ACE I/D, ACE G2350A, and CYP11B2 C-344T polymorphisms are more important than individual factors for ESRD development among Han Chinese. Taiwan has the third highest incidence and the highest prevalence of end-stage renal disease (ESRD) in the world. This not only burdens healthcare resources but also has a major impact on patients and their families . ESRD isThe RAAS is a key blood pressure, renal hemodynamics, and volume homeostasis regulator . Thus, gSeveral studies recently identified RAAS gene mutations and polymorphisms that affected host susceptibility to several diseases, including hypertension , type 2 In this study, we genotyped seven different loci in four RAAS genes in ESRD patients and healthy controls and examined gene-gene interactions using multifactor dimensionality reduction (MDR) and a logistic regression model (LRM).n = 256; 39.6%), chronic glomerulonephritis , hypertensive nephropathy , systemic nephropathy , and unknown .This case-control study included 647 ESRD patients  receiving hemodialysis at Cardinal Tien Hospital and five other hemodialysis centers in Taipei, Taiwan. These patients were stable  and had undergone hemodialysis for >6 months. Autoimmune disease, malignancy, and any acute or chronic infection were exclusion criteria. ESRD causes included diabetes mellitus  with an estimated glomerular filtration rate (eGFR) of ≥60 mL/min/1.73 mThis study was approved by the ethics committee of Cardinal Tien Hospital (CTH-100-3-5-025). After thoroughly explaining this study, written informed consent was obtained from all participants.http://genepipe.ngc.sinica.edu.tw/primerz/beginDesigndo).AGT (M235T (rs699) and T174 M (rs4762)), AGTR1 (A1166C (rs5186) and C573T (rs5182)], ACE [I/D (rs1799752) and G2350A (rs4343)), and CYP11B2 C-344T (rs1799998) polymorphisms previously shown to be significantly associated with kidney diseases in genetic polymorphism studies of Chinese Han populations , 16 werePCR amplification was performed as follows. Cycling conditions were an initial denaturation at 95°C for 5 min, followed by 35 denaturation cycles at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. PCR products were digested with the respective restriction endonucleases , and the resulting fragments were separated in 3.0% agarose gel containing 0.5 g/mL of ethidium bromide by electrophoresis at 100 V and visualized under UV light. Genotyping was performed after blinding for case or control status. Two independent investigators interpreted the images for each gel, and all ambiguous samples were analyzed again. To validate genotyping results, at least 10% of samples were randomly selected for repeated genotyping.t-test for continuous variables and expressed as mean ± standard deviation (SD). Hardy-Weinberg equilibrium was assessed by a goodness-of-fit χ2 test and performed to examine possible genotyping errors for each SNP among controls. Genotype and allele frequencies were compared between ESRD patients and healthy controls using a χ2 test or Fisher's exact test as appropriate. LRM was used to estimate crude and adjusted  odds ratios (ORs) and 95% confidence intervals (CIs) as a measure of association with ESRD risk. Statistical analysis was performed using SPSS for Windows version 20.0 . Two-tailed P values of <0.05 were considered significant.The demographics were evaluated by Student's P < 0.05. Stepwise logistic regression based on backward selection was used to confirm the results of interaction analyses.Gene-gene interactions among the loci were evaluated using multiple dimensionality reduction (MDR) and MDR-permutation testing software (version 1.0 beta). MDR reduces the dimensionality of multilocus information that has reasonable power to identify interactions among two or more loci in relatively small samples and improves the identification of polymorphism combinations associated with disease risk. Average prediction errors were calculated using permutation tests considered significant at P < 0.05).P > 0.05). AGT (M235T and T174M), AGTR1 (A1166C and C573T), ACE (I/D and G2350A), and CYP11B2 (C-344T) genotype distributions were all compatible with Hardy-Weinberg equilibrium for the controls (P = 0.005). The genotype and allele distributions of ACE I/D and G2350A were significantly different between groups.There was a significant association between the AGT M235T polymorphism and ESRD risk, with a mutation carrier having a lower risk . For ACE G2350A, when the GG genotype was used as a reference, GA and AA genotypes appeared to be associated with a higher ESRD risk .For ACE I/D, when genotype II was used as a reference, ID and DD genotypes were apparently associated with a higher ESRD risk .Significant associations were found in the CYP11B2 C-344T polymorphism between groups. For CYP11B2 C-344T, the TC genotype compared with the TT genotype was a protective factor for ESRD  but not for diabetic nephropathy, hypertensive nephropathy, systemic nephropathy, or nephropathy for unknown reasons.There were significant differences in the genotypes of ACE I/D  and G2350A  between patients with diabetic nephropathy and controls. The genotypes of ACE I/D  and G2350A  were also significantly different for patients with hypertensive nephropathy compared with controls. The CYP11B2 genotype was significantly different between patients with systemic nephropathy and controls . No associations were found between patients with different underlying causes of their renal disease and AGT T174M .P = 0.001; We presented statistical evidence for significant interactions among the ACE I/D, ACE G2350A, and CYP11B2 C-344T genes and the ESRD risk. These results were corroborated by permutation testing. Four genetic polymorphisms in AGT, ACE, and CYP11B2 were found to be significantly associated with the ESRD risk; however, these single SNP analyses did not remain significant after correction for multiple testing (data not shown). Our data also indicated that underlying ESRD etiology differed based on RAAS genes.Several studies investigated relationships between RAAS gene polymorphisms and ESRD. Many of these focused on the associations of single polymorphisms among RAAS genes with ESRD. The AGT M235T gene polymorphism, which is correlated with circulating and cellular AGT concentrations, has been implicated in ESRD etiology and investigated in epidemiologic studies , 19. TheAn increasing number of studies have focused on interactions, which can be partly attributed due to new statistical theories –28. MDR Recently, many research articles have focused on RAAS gene polymorphisms associated with risk of different ESRD causes. Reis et al. showed that the AGT M235T TT genotype may be linked to a DN risk in the Turkish population . ACE I/DOur results suggest that AGT, ACE, and CYP11B2 gene polymorphisms are associated with ESRD and that an interaction effect of ACE I/D, ACE G2350A, and CYP11B2 C-344T polymorphisms may play a more important role than individual factors for ESRD development. A higher ESRD risk was found for the simultaneous occurrence of ACE DD-ACE AA. This investigation was done with Han Chinese patients. The applicability of our results to other ethnic groups is uncertain and warrants further study.


IL27 p28 gene  on the outcome of HCV infected patients.Interleukin 27 (IL-27) has pleiotropic properties that can either limit or enhance immune responses. Recent studies revealed that single nucleotide polymorphisms (SNPs) of the IL-27 promoter region modulate the development of infectious diseases and individual's susceptibility to therapeutic response. Little is known about the relationship between IL-27 single nucleotide polymorphisms and therapy response in patients infected by hepatitis C virus (HCV). In this study we have investigated the potential role of SNPs in the promoter region of rs153109, corresponding to position c.-964A>G of the IL-27 locus, was amplified from genomic DNA extracted from 15 patients with chronic hepatitis C stratified by sustained viral response (SVR), relapser and non-responder, after treatment with peginterferon-α (PegIFN- α) combined with ribavirin (RBV). Amplification products were studied by direct sequencing.rs153109 SNPs of IL-27p28. The genotype distribution of the c.-964 A>G polymorphism was more present in patients who did not achieve a SVR. By contrast, the genotype G/G was absent in non-responder and relapser patients. Moreover, the analysis of allelic distribution of rs153109 highlighted a predominance of allele A in all genotypes in spite of allele G.This method has been applied in a preliminary study on patients with chronic hepatitis C to provide information for a standardized assay useful to genotyping of rs153109, and suggests its utility as a candidate approach to evaluate IL-27 p28 polymorphisms as additional clinical predictors of response to therapies in HCV infected patients.Our work provides preliminary information for a standardized method potentially useful for genotyping  In agreement to the broad role that cytokines play in shaping many aspects of innate and adaptive immunity, there was a concerted effort toward identifying single nucleotide polymorphisms (SNPs) of cytokine associated with human diseases .rs153109) and 2905T/G (rs 181206) polymorphisms, corresponding to promoter region of IL-27p28 gene, were identified to be associated with individual susceptibility of asthma, inflammatory bowel diseases [et al., [rs 181206). Moreover, Robinson et al. [Mycobacterium tuberculosis infection while Peng et al., [Interleukin 27 (IL-27) is a novel Interleukin 12 (IL-12) family member, formed by the dimerization of two subunits, Epstein-Barr virus-induced gene 3 (EBI3) and IL-27p28, which engages a receptor composed of gp130 and IL-27Rα activating Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and mitogen activated protein kinase (MAPK) signaling. The human IL-27 gene is located on chromosome 16p11 and consists of five exons. Recently, both c.-964A/G  rs13109 and Furthermore, recent reports have shown that IL-27 significantly induces interferon (IFN)-inducible antiviral genes, suggesting that IL-27 inhibits human immunodeficiency virus (HIV), influenza virus and hepatitis C virus (HCV) replication by eliciting an IFN-like response . The mecHowever, this approach is considered limitative because the evolution of liver disease and the response to treatment appear to be the result of a dynamic process in which genetic factors of host are mainly involved.IL-28B are strongly correlated with HCV containment, spontaneous clearance, host response to antiviral treatment and disease progression [Indeed, information about genetic variants, either mutant or polymorphic, represents the basis for the development of new clinical approach for the management of chronic HCV infected patients . Severalgression -19.All these findings prompted us to hypothesize that interleukin-27 could influence the HCV infection susceptibility and response to therapy.IL27 p28 gene ( rs153109) and report preliminary data on HCV RNA-positive patients undergoing treatment with peginterferon-α (PegIFN- α) combined with ribavirin (RBV).In this report, we show the development of an assay useful to detect SNP of the promoter region of The study was designed as a retrospective analysis. The sample of individuals studied was composed of 15 patients with chronic hepatitis C. All subjects were treated with Peg-IFN-α-2a at a fixed dose of 180 μg/week or with Peg-IFN-α-2b at 1.5 μgr/kg/week and ribavirin 800-1.200 mg/day . Patients with HCV genotype 1 were treated for 24-48 weeks and 24 weeks in patients with genotype non-1.10 after 24 weeks of therapy). Baseline characteristics of 15 patients with hepatitis C are summarized in Table The sample was stratified according to therapeutic response in: sustained viral response , relapser (reappearance of HCVRNA in serum after therapy is discontinued) and non-responder , according to the manufacturer's directions. DNA quality was assessed by calculating the absorbance ratio OD260nm/280nm using NanoDrop (Thermo scientific 1000 spectrophotometer) and by electrophoresis on 1% agarose gel.rs 153109; length of the amplification product was 306bp. The primers used in this study were the following: rs153109_F 5'-gTg TACAGCTgAACTCACAg-3' and rs153109_R 5'-gCCTCTACAgAgCAgAAAC-3'  and 200 μM dNTP. PCR was carried out under the following thermal cycler conditions: initial denaturation at 95°C for 3 minutes, followed by 38 cycles consisting of denaturation at 95°C for 30 seconds, annealing at 52° C for 30 seconds, extension at 72°C for 40 seconds; a final extension at 72°C for 5 minutes was performed.Polymerase Chain Reaction (PCR) was performed in a thermocycler VERTI 96well (Applied Biosystems) in a total volume of 50 μl containing 150 ng genomic DNA, 25 μM primers, 1,25 U Taq DNA Polymerase, 50 m M KCl, 30 m M Tris- Tris-HCl, 1,5 mM MgPCR Illustra MicroSpin S-300 Colonne HR (Gelifesciences UK), amplification products were used as template DNA for sequencing analysis . PCR products were studied by direct sequencing .SNP rs153109 alleles of the IL27p28 gene in patients with chronic hepatitis C using a direct sequencing approach. In particular, we analyzed the genotype of this SNP on c. -964A>G. Our data show a different distribution of genotype and allele of SNP in our patients. Indeed, the A/G genotype is present in non-responders and relapser compared to patients with a sustained viral response (SVR). In addition, the G/G genotype was absent in patients who negatively respond to antiviral therapy. Moreover, the analysis of allele distributions of the SNPs highlights a predominance of allele A in all genotypes in spite of allele G , Interleukin 28B (IL28B), Interleukin 29 (IL 29), interferon-γ (IFN- γ), mannan binding lectin (MBL), Interleukin 10 (IL−10), Interleukin 18 (IL-18), human cytotoxic T-lymphocyte antigen 4 (CTLA 4), Tumor necrosis factor-related apoptosis inducing ligand receptor 1 (TRAIL), Transforming growth factor (TGF-β), Osteopontin, low molecular mass polypeptides (LMP 7), oligoadenylate synthetase (OAS1) genes, insulin resistance, obesity and ethnicity, have been found to modulate treatment response . MoreoveIL-27 is one of the critical cytokines that functions as a mediator between the innate and adaptive immune system. Moreover, IL-27 synergizes with IL-12 to potentiate Interferon-γ (IFN-γ) production by activated naïve T- and Natural Killer (NK) cell populations . Recent rs153109 and might suggest an association between treatment response and genetic variability of IL-27p28 SNPs.Our work provides information for a standardized method useful to genotyping of IL-27p28 SNPs as novel genetic markers which may correlate with therapy response and, possibly, with liver disease progression in HCV infected patients.Given the small number of patients studied, the statistical association demonstrated in this study may have occurred by chance. However, this approach could represent an interesting starting point for future investigations. Therefore, a larger sample size should confirm our results. Moreover, prospective studies should support the causal relationship between the presence of A/G genotype in non-responder and relapser patients, as well as the major presence of A allele. So, could allow us to identify SNP: Single Nucleotide Polymorphism; IL-27: Interleukin 27; IL-12: Interleukin 12; EBI3: Epstein-Barr Virus-Induced Gene 3; JAK: Janus Kinase; STAT: Signal Transducer and Activator of Transcription; MAPK: Mitogen Activated Protein Kinase; IFN: Interferon; HCV: Hepatitis C Virus; GWAS: Genome Wide Association Studies; PegIFN- α-2a: Peginterferon-α-2a; PegIFN- α-2b: Peginterferon-α-2b; RBV: Ribavirin; SVR: Sustained Viral Response; EtBr: Ethidium Bromide; PCR: Polymerase Chain Reaction; IL28A: Interleukin 28A; IL28B: Interleukin 28B; IL29: Interleukin 29; IFN- γ: Interferon-γ; MBL: Mannan Binding Lectin; IL-10: Interleukin 10; IL-18: Interleukin 18; CTLA4: Human Cytotoxic T-Lymphocyte Antigen 4; TRAIL: Tumor Necrosis Factor-Related Apoptosis Inducing Ligand Receptor 1; TGF-β: Transforming Growth Factor; LMP7: Low Molecular Mass Polypeptides; OAS1: Oligoadenylate Synthetase; NK: Natural Killer.The authors declare that they have no competing interests.E.Z., F.T., N.P., C.T., M.C.M., and A.F. designed research; E.Z., N.M., S.N., F.F. performed research; N.M., A.S., and C.T. collected clinical samples; E.Z., A.Q., N.M. F.T., N.P., A.S., C.T., M.C.M., and A.F. analyzed data; E.Z., F.T., N.P., C.T., M.C.M, and A.F wrote the paper.


The aim of this study was to investigate the role of K101Q, Y181C and H221Y emerging in HIV-1 reverse transcriptase with different mutations patterns in phenotypic susceptibility to currently available NNRTIs  and NRTIs  in China.50) compared with the reference, while P ≤ 0.01 was considered to be statistically significant considering multiple comparisons.Phenotype testing of currently available NNRTIs  and NRTIs  was performed on TZM-b1 cells using recombined virus strains. P ≤ 0.05 was defined significant considering the change of 50% inhibitory drug concentration (ICTriple-mutation K101Q/Y181C/H221Y and double-mutation K101Q/Y181C resulted in significant increase in NVP resistance (1253.9-fold and 986.4-fold), while only K101Q/Y181C/H221Y brought a 5.00-fold significant increase in EFV resistance. Remarkably, K101Q/H221Y was hypersusceptible to EFV (FC = 0.04), but was significantly resistant to the three NRTIs. Then, the interaction analysis suggested the interaction was not significant to NVP  but significant to EFV and other three NRTIs.Copresence of mutations reported to be associated with NNRTIs confers significant increase to NVP resistance. Interestingly, some may increase the susceptibility to EFV. Certainly, the double mutation (K101Q/H221Y) also changes the susceptibility of viruses to NRTIs. Interaction between two different sites makes resistance more complex. They were infected HIV-1 subtype B by blood donation. Patients complied with treatment regimens well. We had followed up with interval for approximately six months (ten times) since the very start of therapy. Every time, we collected 10 ml anticoagulated whole blood samples, separated them by centrifugation to obtain blood plasma and peripheral blood mononuclear cells (PBMCs) and then stored them at -80°C.Clonal sequencing approach was adopted in this study. RNA andpol to construct the first clone as previously described[th hour. Then, transfection supernatant infected MT-2 cells and viral cultures were grown in 4 to 6 days. Supernatants were stored at -80°C and sequenced to confirm the presence of the desired mutations. Although we constructed the single mutation (H221Y) virus as mentioned above to clarify the contribution of H221Y to resistance, the virus was low virus titer. So we did not obtain a reliable result of single H221Y.Amplification of viral genome was performed using a nested PCR procedure, and patient-derived HIV-1 RT fragment carrying K101Q/Y181C/H221 replaced the partner sequence  in pNL4-3 escribed. There w4cells/well) in 96-well plates growing in 100ul Dulbecco’s minimal essential medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin[50. Forty-eight hours after the TZM-b1 got infected, with the condition of 37°C and 5% CO2, relative luminescence units (RLU)/well were measured by a luminometer  according to Bright-gloTM Luciferase assay system (Promage E2650) instructions. All the experiments were performed at least in duplicate on three different days. The IC50 was calculated using the GraphPad Prism program.The study tested the susceptibility of viruses to currently available RT inhibitors  in China with recombined viruses. They were performed in TZM-b1 cells as previously described. In brieptomycin. Immedia50 values between any two viruses, and Kruskal-Wallis method was used to correct the testing. P ≤ 0.05 was defined significant considering the change of 50% inhibitory drug concentration (IC50) compared with the reference, while P ≤ 0.01 was considered to be statistically significant considering multiple comparisons.Multiple comparisons statistical method was used to assess the significance of differences in ICThe study was approved by the Ethical Board of the Beijing Institute of Microbiology and Epidemiology in January 2009. All the patients were compliance with the first antiviral therapy program in China and were selected with informed consent. Data were managed anonymously.th month of antiviral therapy with the frequency more than 30% in quasispecies. From the 22nd month on, the frequency of H221Y/Y181C in quasispecies was 100% in plasma. However, not until the 28th month did the frequency of H221Y/Y181C in quasispecies become 100% in PBMCs. Remarkably, K101Q was observed along with the double mutations at the early time of antiviral therapy.H221Y, a novel NNRTI-resistance mutation relevant to NVP, emerged in all the six patients. There were 204 sequences of Patient 1 blood plasma and 160 of PBMCs. Combination of H221Y and Y181C was detected in the 10Apart from the above, we also observed more mutations in plasma than in PBMCs at each interview time. In other words, some mutations which emerged in plasma were not found in PBMCs. With the antiviral therapy going, mutations in PBMCs and plasma would be consilient. However, certain mutations were momentary and were absent in the last mutations patterns. All the mutations present in the last patterns were nearly 100% in both PBMCs and plasma.WT and 4 recombined HIV-1 viruses separately harboring K101Q/Y181C/H221Y, K101Q/Y181C, K101Q/H221Y and K101Q. By analyzing sequences, we confirmed that the desired mutations did exist. Then, we calculated IC50 of 5 drugs  by making dose-effect relationship using the GraphPad Prism program. Moreover, the fold changes (FC) of IC50 were calculated in Table We obtained one reference virus pNL4-350 data of NVP and EFV in Table 50 respectively  in the presence of NVP, but the interaction was statistically significant with EFV . Comparing K101Q/Y181C/H221Y with K101Q in the presence of EFV, we found the contribution of Y181C plus H221Y to resistance was 13.22-fold which was higher than the sum of the folds of K101Q/Y181C versus K101Q and K101Q/H221Y versus K101Q. We guessed the interaction between Y181C and H221Y was synergetic. We also analyzed the interaction between sites 181 and 221 in NRTIs at the background of K101Q. Results showed the interaction was significant in AZT, 3TC and d4T , but only a 4.0-fold increase in NVP resistance. How these mutations cause NNRTI resistance is not clear. It is conceivable that more mutations or associated mutations than currently known are involved in the development of drug resistance and lead to therapeutic failure[Long NNRTIs exposure may trigger the accumulation of additional mutations, leading to even higher levels of drug resistance. Here, we evaluated the contribution of K101Q plus Y181C plus H221Y to resistance of NNRTIs . The result showed K101Q/Y181C/H221Y viruses led to a 1253.9-fold great increase in NVP resistance and a 5.00-fold significant increase in EFV resistance. The copresence of K101Q and Y181C in viruses resulted in a little increase in EFV resistance, but a 986.4-fold increase in NVP resistance. However, K101Q was interpreted as a relatively non-polymorphic mutation that occurred slightly more commonly among patients receiving NNRTIs. It was reported that single K101Q induced a 3.2-fold NVP resistance and a 5.6-fold EFV resistance and that Y181C conferred 100-fold NVP resistance and 1.1-fold EFV resistance at the pNL4-3 background. Moreoveparallel,33. K101We also investigated whether these mutations conferred NRTI resistance. K101Q/H221Y double mutation showed significantly increase in AZT, 3TC, d4T resistance. It is worth noting that triple-mutation K101Q/Y181C/H221Y only shows a less extensive increase. The situation is made complicated by the fact that resistance mutations do not accumulate independently within each other. Instead, they disappear and occur in time order along the pathway of resistance evolution, leading to distinct mutational complexes or clusters. The morWe evaluated the interaction between Y181C and H221Y considering K101Q as the background. Result of statistical analyses showed the interaction between sites 181 and 221 in NVP was not statistically significant . In EFV, H221Y increased the susceptibility while Y181C increased resistance. However, Y181C significantly reversed the K101Q/H221Y phenotypic susceptibility to EFV. Then, as for NRTIs, Y181C significantly decreased the H221Y resistance. Mutational pathways may interpret the complexity at a certain extent. Regrettably, we have not observed which one first emerges during mutational pathways, Y181C or H221Y. So far, some researchers have reported the molecular mechanism between the Y181C and other mutations, but theIn summary, some copresence of the mutations reported to be associated with NNRTIs in our study confer significant increase of NVP resistance. Interestingly, some may increase the susceptibility of EFV. Certainly, the double mutation (K101Q/H221Y) also changes the susceptibility of viruses to NRTIs. Interaction between different sites makes resistance more complex.Our data in this study are based on the recombined subtype B’ viruses and pNL4-3 wild-type. Viral evolution pathways toward drug resistance may proceed through distinct steps and at different rates among different HIV-1 subtypes. To asse50: 50% inhibitory concentration; RT: HIV-1 reverse transcriptase; NVP: Nevirapine; EFV: Efavirenz; AZT: Zidovudine; 3TC: Lamivudine; d4T: Stavudine.HIV-1: Human immunodeficiency virus type 1; HAART: Highly active antiretroviral therapy; NRTIs: Nucleoside reverse transcriptase inhibitors; NNRTIs: Non-nucleoside reverse transcriptase inhibitors; ICThe authors declare that they have no competing interests.Conceived and designed the experiments: JL, WG, HL, DZ. Performed the experiments: WG, DZ, SL, LJ. Analyzed the data: WG, LJ, TG. Wrote the paper: WG, LL, YL. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/237/prepub


Bifidobacterium longum 105-A shows high transformation efficiency and allows for the generation of gene knockout mutants through homologous recombination. Here, we report the complete genome sequence of strain 105-A. Genes encoding at least four putative restriction-modification systems were found in this genome, which might contribute to its transformation efficiency. Bifidobacterium strains, in part due to their restriction-modification (R-M) systems (2–Bifidobacterium longum 105-A (4 to 106 transformants/μg DNA) with several plasmid DNAs , 56 tRNA genes, 1 transfer-messenger RNA (tmRNA) gene, and 4 rRNA operons were predicted by g-MiGAP , BL105A_1441 (a specificity subunit), and BL105A_1439 (an endonuclease); one type II system comprising BL105A_1060 (an endonuclease) and BL105A_1059 (a methyltransferase); another type II system comprising BL105A_0366 (an endonuclease/methyltransferase); and a type IV system comprising BL105A_0073 (an endonuclease). Further functional analysis of the R-M system genes will clarify their contributions to transformation efficiency. The complete genome sequence of B. longum 105-A will largely contribute to postgenomic or functional genomics studies of bifidobacteria.The circular chromosome of  g-MiGAP . The ave NCC2705  revealedB. longum 105-A has been deposited in the DDBJ/EMBL/GenBank database under accession no. AP014658.The complete genome sequence of


N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD). We expressed CatD of mouse AMCase as a recombinant fusion protein with Protein A and V5-His in Escherichia coli (Protein A-CatD-V5-His), evaluated its functional properties and compared them to the full-length AMCase (Protein A-AMCase-V5-His). Under our experimental conditions, the chitinolytic activity of both proteins against 4-nitrophenyl N,N'-diacetyl-β-d-chitobioside was equivalent with regard to their specific enzymatic activities, optimal pH and temperature as well as to the pH and temperature stability. CatD bound to chitin beads and cleaved the N-acetylglucosamine hexamer, colloidal and crystalline chitin as well as the shrimp shell, and released primarily N,N'-diacetylchitobiose fragments at pH 2.0. These results indicate that the primary structure of CatD is sufficient to form a proper tertiary structure required for chitinolytic activity, recognize chitin substrates and degrade them in the absence of a CBD. Our recombinant proteins can be used for further studies evaluating pathophysiological roles of AMCase in different diseases.Mouse acidic mammalian chitinase (AMCase) plays important physiological roles in defense and nutrition. AMCase is composed of an  N-acetyl-d-glucosamine (GlcNAc). Chitin is an integral component of the exoskeletons of crustaceans and insects, and the microfilarial sheaths of parasitic nematodes and fungal cell walls [Chitinases are enzymes that digest chitin, a polymer of β-1,4)-linked ,4-linkedll walls . Chitinall walls ,3 and arDespite the absence of endogenous chitin, humans and mice express two active chitinases, designated as chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Both enzymes show sequence homology to bacterial chitinases and belong to the family 18 of glycoside hydrolases ,5,6. ChiAMCase has attracted substantial attention due to its increased expression under certain pathological conditions. Significant increase of the AMCase mRNA and protein levels was detected in an induced asthma mouse model . MoreoveEscherichia coli expression system that allows for the periplasmic production of active AMCase as a recombinant protein fused with Protein A, V5 epitope and (His)6 tag (V5-His) (Protein A-AMCase-V5-His) [E. coli-produced AMCase showed a robust peak of activity at pH 2.0, cleaved colloidal chitin and primarily released N,N'-diacetylchitobiose fragments, displaying properties similar to those of the protein expressed in CHO cell-expressed [Recently, we reported that AMCase mRNA is synthesized in mouse stomach at extraordinarily high levels that are comparable to the mRNA level of pepsinogen C (progastricsin), a major component of the gastric mucosa ,23. To b-V5-His) . The E. xpressed .N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD) [N,N'-diacetylchitobiose fragments at pH 2.0. Thus, the CatD of mouse AMCase is sufficient for chitin substrates recognition and degradation.Mouse AMCase is composed of an in (CBD) . It has in (CBD) . HoweverE. coli, mature AMCase-V5-His cDNA or its CatD region was cloned into pEZZ18 vector, containing the signal sequence of Staphylococcus aureus Protein A to express precursor forms of recombinant proteins of pre-Protein A-AMCase-V5-His and pre-Protein A-CatD-V5-His  was purified by IgG Sepharose chromatography, followed by Ni Sepharose chromatography. The purified fraction was subjected to SDS-PAGE and then visualized with Coomassie Brilliant Blue R-250 staining (CBB). As shown in N,N′-diacetyl-β-d-chitobioside chromogenic substrate  and pH 7.0 (McIlvaine buffer). It showed a strong chitinolytic activity as measured using 4-nitrophenyl ubstrate C, which ubstrate C.E. coli-expressed CatD of AMCase, we first evaluated the optimal pH for Protein A-CatD-V5-His activity against the chromogenic substrate at 37 °C for 30 min from pH 1.0 to 10.0. As shown in To further characterize N,N′-diacetyl-β-d-chitobioside for 15 min. The rate of the recombinant CatD-catalyzed reaction increased with the rising temperature, reached the maximum level at 54 °C, and then abruptly declined  followed by analysis of the enzymatic activity at 37 °C and pH 2.0. The recombinant CatD was stable in both acidic and basic conditions (between 1.0 and 10.0) during the pre-incubation on ice C.We next assessed the thermal stability of the Protein A-CatD-V5-His by measuring the chitinolytic activity at pH 2.0  or pH 7.0 . Samples were pre-incubated at the indicated pH for 20 min from 30 to 58 °C. After pre-incubation, we measured the residual activity against the chromogenic substrate at pH 2.0. Recombinant CatD was heat-stable up to 54 °C, at both pH 2.0 and 7.0 D while iN,N′-diacetyl-β-d-chitobioside were basically identical with those of the full-length AMCase with regard to the pH and temperature optima as well as pH and thermal stabilities [Thus, the enzymatic properties of the recombinant CatD against 4-nitrophenyl bilities .The full-length AMCase contains a CBD at the C-terminus of AMCase, whereas it is deleted in CatD A,B. To dE. coli-expressed recombinant proteins, we incubated GlcNAc hexamer and colloidal chitin with Protein A-AMCase-V5-His and Protein A-CatD-V5-His. Mono- and oligosaccharides produced in the reaction were covalently labeled at the reducing end groups with a fluorophore. The resulting fluorescent derivatives were then separated by high-resolution PAGE, as described previously [N,N'-diacetylchitobiose fragments at pH 2.0 at a similar rate . This method can only be used if the fusion product is stable under these conditions. The CatD showed acid stability at pH 1 to 3 C. Thus, E. coli cells. We have reported that E. coli-expressed AMCase showed properties similar to those of the native enzyme from mice [N,N′-diacetyl-β-d-chitobioside was equivalent and comparable to the E. coli-produced full-length AMCase as for the specific activity, pH and temperature optima as well as pH and thermal stabilities. Furthermore, the CatD degraded colloidal chitin and produced primarily N,N'-diacetylchitobiose. These results indicate that the E. coli-expressed Protein A-AMCase-V5-His and Protein A-CatD-V5-His are equivalent under our experimental conditions. Our results clearly indicate that the primary structure of CatD is sufficient to form a proper tertiary structure for chitinolytic activity in the absence of CBD.The objective of the studies described here was to compare the enzymatic properties of the full-length AMCase and its CatD expressed in rom mice  or CHO-erom mice . Here, wThe recombinant CatD of AMCase bound to chitin beads and cleaved the GlcNAc hexamer, colloidal and crystalline chitin and shrimp shell at pH 2.0. These results indicate that the CatD of mouse AMCase can recognize high molecular weight chitin substrates and degrade them in the absence of CBD. A high-resolution analysis by X-ray crystallography shows that a long cleft, which is lined with aromatic residues, is the site of chitin binding . These aSerratia marcescens 2170 produced chitinase C1 and C2 [et al. reported that chitinase C1 is the full-length chitinase consisting of CatD and CBD, whereas chitinase C2 contains CatD and is generated by proteolytic removal of CBD from chitinase C1 [The CatD showed reduced hydrolytic activity towards crystalline chitin and shrimp shell, when compared with the full-length AMCase. 1 and C2 . Suzuki inase C1 . These aWe recently reported that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach ,23,35. TAMCase is potentially an important downstream effector of interleukin-13 stimulation in Th2 helper cell-mediated immune responses to pathogens, parasites and ovalbumin. Elevated AMCase levels in lungs or eye may lead to diseases such asthma, allergic inflammation, ocular allergy, and dry eye syndrome (reviewed in ). InhibiCATGGAATTCGTACAATCTGATATGCTATTTCACC-3’) for CatD contains a 6 bases-long EcoRI recognition sequence (underlined) and a 25 bases-long AMCase sequence corresponding to nucleotides 80~103 of the AMCase cDNA (GenBank accession number AK160173.1). We designed the reverse primer, which is in frame with the C-terminal region of V5-His. The reverse primer (5'-GTGACCTCGAGCACTCCCACTTCCTGGAGGAGTAG-3') contains the XhoI recognition sequence (underlined) and is complementary to nucleotides 1230~1253 of the AMCase cDNA. Both primers contain the 4~5 bases-long extra nucleotides (boldfaced) to efficiently cleave close to the end of the amplified cDNAs by a restriction enzyme. The amplified DNA contains one EcoRI and one XhoI sites anchored with the PCR primers. The PCR product was purified using the Wizard SV Gel and PCR Clean-Up System  and then digested with EcoRI and XhoI. The DNA fragment was purified using 1.5% agarose gel electrophoresis and the Clean-Up System and was finally subcloned into a similarly digested pEZZ18 expression vector. The entire nucleotide sequence of the resulting plasmid DNA (pEZZ18/CatD/V5-His) was confirmed by sequencing .To express the CatD of mouse AMCase as a recombinant fusion protein with Protein A and V5-His, the CatD region was amplified from the full-length mouse AMCase expressing plasmid DNA (the pEZZ18/AMCase-V5-His) using KOD Plus DNA polymerase  and oligonucleotide primers  anchored with the restriction sites EcoRI and XhoI as described previously . The forE. coli BL21 (DE3) strains were grown in 1 L LB medium containing 100 µg/mL ampicillin at 37 °C for 18 h. Cells were harvested by centrifugation at 5000× g for 20 min at 4 °C. The recombinant CatD was prepared from the periplasmic fraction of the E. coli and purified by IgG Sepharose chromatography, followed by Ni Sepharose chromatography as described previously [Transformed eviously . The proWe also produced Protein A-AMCase-V5-His and Protein A-V5-His as described previously .The obtained protein fractions were analyzed using standard SDS-polyacrylamide gel electrophoresis (PAGE), followed by Western blotting using anti-V5-HRP monoclonal antibody . We used All Blue molecular weight marker .N,N′-diacetyl-β-d-chitobioside (Sigma–Aldrich), at a concentration of 200 μM. Each reaction was performed in triplicate. All enzymatic reactions for the determination of optimum pH and temperature were conducted in a volume of 50 μL containing E. coli-expressed protein in 0.1 M Gly-HCl buffer or McIlvaine’s buffer. Reactions performed for the optimum pH and kinetic assays were conducted for 30 min at 37 °C. The reactions were halted with the addition of 20 μL of 1 M sodium carbonate to the reaction mixture. The absorbance of the liberated 4-nitrophenolate ion was measured at 405 nm.Chitinolytic activity was determined using the synthetic chromogenic substrate, 4-nitrophenyl Protein A-AMCase-V5-His, Protein A-CatD-V5-His and Protein A-V5-His (each 20 µg) were mixed after adjusting the binding buffers’ pH values to pH 2.0 or 7.6 in 0.5 M NaCl. The mixed solutions were loaded onto a chitin bead  column equilibrated with the binding buffers (0.5 M NaCl in 100 mM Gly-HCl (pH 2.0) or 0.5 M NaCl in 20 mM Tris-HCl (pH 7.6)) and recirculated ten times to bind the recombinant proteins to the chitin beads. The flow-through fractions were pooled as an unbound fraction. The columns were washed with 10-column volumes of the same buffer and then washed with 5-column volumes of 2 M NaCl in 100 mM Gly-HCl (pH 2.0) or 2 M NaCl in 20 mM Tris–HCl (pH 7.6), respectively. Bound proteins were eluted with 8 M Urea and 20 mM Tris–HCl (pH 7.6). The bound and unbound fractions were analyzed by Western blot using an anti-V5-HRP monoclonal antibody.E. coli-expressed proteins in 0.1 M Gly-HCl buffer (pH 2.0). The reactions were incubated for 10 min, 1 h or 16 h at 37 °C. The chitin fragments generated from these reactions were labeled covalently at their reducing end groups with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid , and the resulting fluorescent derivatives were separated by high-resolution PAGE, as described by Jackson [N-acetyl chitooligosaccharides  were used as standards.Colloidal chitin was prepared from shrimp shell chitin (Sigma-Aldrich), as described previously, and used as a substrate to determine the chitinase activity . Shrimp  Jackson . N-acetyE. coli-expressed Protein A-AMCase-V5-His and Protein A-CatD-V5-His are equivalent under our experimental conditions and that the primary structure of CatD is sufficient to form a proper tertiary structure for chitinolytic activity in the absence of CBD. Thus, the CatD of mouse AMCase can recognize chitin substrates and degrade them in the absence of CBD. Our results suggest that the development of potentially druggable AMCase-specific inhibitors should focus on compounds binding to CatD enabling a more potent chitinolytic activity inhibition of AMCase.Our results indicate that the


Meanwhile, the expression levels of p-IκB and NF-κB were downregulated, while IκB expression was upregulated. H-PHC also exerted long-term cardioprotection in a rat model of I/R injury by decreasing infarct size and improving cardiac function. These results suggest that PHC can efficiently protect the rats against I/R-induced myocardial injury.To investigate the impacts and related mechanisms of penehyclidine hydrochloride (PHC) on ischemia/reperfusion (I/R)-induced myocardial injury. A rat model of myocardial I/R injury was established by the ligation of left anterior descending coronary artery for 30 min followed by 3 h perfusion. Before I/R, the rats were pretreated with or without PHC. Cardiac function was measured by echocardiography. The activities/levels of myocardial enzymes, oxidants and antioxidant enzymes were detected. Evans blue/TTC double staining was performed to assess infarct size. Cardiomyocyte apoptosis was evaluated by TUNEL assay. The release of inflammatory cytokines and inflammatory mediators was detected by ELISA. Western blot was performed to analyze the expression of COX-2, IκB, p-IκB and NF-κB. Meanwhile, the rats were given a single injection of H-PHC before I/R. The effects of PHC on myocardial infarct and cardiac function were investigated after 7 days post-reperfusion. We found that PHC remarkably improved cardiac function, alleviated myocardial injury by decreasing myocardial enzyme levels and attenuated oxidative stress in a dose-dependent manner. Additionally, PHC preconditioning significantly reduced infarct size and the apoptotic rate of cardiomyocytes. Administration of PHC significantly decreased serum TNF-α, IL-1β, IL-6 and PGE Acute myocardial infarction (AMI) is a common disease with high morbidity and mortality . More thPenehyclidine hydrochloride (PHC) is an anti-cholinergic drug synthesized by Academy of Military Medical Sciences in China , which pIn the present study, we used a rat model of I/R injury and then the rats were pretreated with three different dose regimens of PHC to explore the dose-effect relationship and probably mechanisms in therapeutics. Next, we further assessed the possible effects of H-PHC on myocardial infarct and cardiac function at day 7 post-I/R.ad libitum. The rats were housed in a constant temperature (22°C) condition with 12 h light/12 h dark cycles. The animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Capital Medical University, and approved by the Ethics Committee of Capital Medical University.Male Wistar rats (8 weeks of age) were purchased from Charles River Laboratories  and were fed a standard chow diet and water We performed coronary artery ligation (CAL) to induce I/R injury in Wistar rats. Before the surgery, the rats were anesthetized with 10% chloral hydrate (300 mg/kg bodyweight) by intraperitoneal injection and ventilated with a HX-300S animal respirator  . The heart was exposed via a left thoracotomy and left anterior descending (LAD) coronary artery was ligated for 30 min followed by 3 h reperfusion. The signs, including muscle tone and tail/pedal withdrawal response, were used to monitor the adequacy of the anesthesia. At the end of the experiment, the rats were sacrificed by an administration of pentobarbital sodium (200 mg/kg) by intraperitoneal injection and phlebotomy.The rats were randomly divided into 6 groups (n = 18 in each group): (i) Sham group, the rats underwent sham surgery; (ii) Sham+H-PHC group, the rats were injected intravenously with high dose of penehyclidine hydrochloride  30 min before sham surgery; (iii) I/R group, the rats were subjected to a 30 min LAD coronary artery ligation followed by 3 h reperfusion; (iv) I/R+L-PHC group, the rats were injected with low dose of PHC  30 min before I/R; (v) I/R+M-PHC group, the rats were injected with moderate dose of PHC  30 min before I/R; (vi) I/R+H-PHC group, the rats were injected with H-PHC (1 mg/kg bodyweight) 30 min before I/R.After 3 h reperfusion, Philips IE33 ultrasound system  was used to measure ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic pressure (LVEDP) and left ventricular systolic pressure (LVSP).After 3 h reperfusion, the levels of serum creatine kinase (CK), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in serum were measured using commercial kits  according to the manufacturer’s instructions.After 3 h reperfusion, superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in serum were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute) following the manufacturer’s instructions.After 3 h reperfusion, the homogenated myocardial tissues were centrifuged at 12000 rpm for 10 min and the supernatant was collected. Total reduced glutathione (GSH) level was determined using GSH assay kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions. Total level of nitric oxide (NO) was determined using Total NO Assay Kit (Beyotime Institute of Biotechnology). The activities of glutathione peroxidase (GSH-Px) and catalase (CAT) in myocardial tissues were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute).After 3 h reperfusion, myocardial tissues were collected, minced, homogenated in mitochondria isolation reagent A (Beyotime Institute of Biotechnology) containing PMSF and centrifuged at 600 g for 5 min (4°C). The supernatants were then transferred to a new tube and centrifuged at 11000 g for 10 min. The supernatants were aspirated and the pellets were lysed with mitochondria lysis buffer (Beyotime Institute of Biotechnology). The concentrations of mitochondrial proteins were determined using a bicinchoninic acid (BCA) kit (Beyotime Institute of Biotechnology). The proteins were incubated with DCFH-DA solution (1 mM) and incubated for 30 min at 37°C and then analyzed by a microplate reader ELX-800 .After 3 h reperfusion, LAD coronary artery was retied. Evans blue dye  was injected through the jugular vein. Ischemic region and area at risk (AAR) were defined. Subsequently, the hearts were removed, frozen at -20°C for 30–60 min and sliced into slices. Then, the slices were incubated with 1% TTC solution in PBS  at 37°C in the dark and images were photographed. Myocardial infarct size was expressed as percentage of infarct area (IA) over AAR (IA/AAR×100%).2O2 to quench endogenous peroxidase activity. Then, the sections were incubated with DAB (Solarbio) for 1 h at 37°C and dyed with hematoxylin. TUNEL-positive cells were counted under a microscope with 400× magnification . The apoptotic rate was calculated as the ratio of TUNEL-positive cells to the total number of cardiomyocytes.After 3 h reperfusion, myocardial tissues were obtained, embedded in paraffin and cut into 5-mm thick slices. After deparaffinization with xylene and rehydration through graded alcohols, the sections were permeabilized in 0.1% Triton X-100 for 8 min and then blocked with H2 (PGE2) levels were determinated using ELISA kits  according to the manufacturer’s instructions.After 3 h reperfusion, blood samples were collected and serum was obtained by centrifugation. Then, serum (TNF)-α, interleukin (IL)-1β, IL-6 and prostaglandin ERIPA lysis buffer containing 1% PMSF was added into myocardial tissue and the supernatant was harvested by centrifugation . Protein concentration was determined using a BCA Protein Assay Kit . Protein lysates (40 μg) were separated on SDS-PAGE and transferred to PVDF membranes . Subsequently, the membranes were blocked with 5% non-fat milk/1% BSA and then incubated overnight at 4°C with cyclooxygenase (COX)-2 antibody (1:1000), IκB antibody (1:1000), NF-κB antibody  and p-IκB antibody . Then, the membranes were incubated for 45 min at 37°C with HRP-conjugated anti-rabbit IgG antibody (Beyotime Institute of Biotechnology). Protein bands were detected using ECL agent (Wanleibio) and the band intensities were quantified using Gel-Pro-Analyzer Software .The rats were separated into three groups: The rats in the Sham group (i) and I/R group (ii) underwent the same operation as previously described. The I/R+H-PHC group (iii) rats were given a single injection of H-PHC (1 mg/kg bodyweight) 30 min before LAD ligature, and then the rats were subjected to I/R. After 7 days, the rats were anesthetized. Cardiac function was measured by echocardiography. Infarct size was determined using Evans blue/TTC staining.P<0.05 was considered statistically significant.Results were expressed as the mean±SD. Data were analyzed and images were processed using GraphPad Prism 5.0 software . Statistical analyses were performed with one-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests. A value of P<0.01). However, pretreatment with H-PHC significantly elevated EF , FS  and LVSP  when compared with the I/R group. Both M-PHC and H-PHC downregulated LVEDP .We investigated the impacts of PHC on cardiac function. There were no significant differences in EF , FS Fig, LVEDP  and LVSPP<0.01). However, pretreatment with PHC showed a significant reduction in the activities of all serum myocardial enzymes in a dose-dependent manner when compared with the I/R group .To determine the effects of PHC on myocardial enzymes, we detected the activities of CK, AST and LDH in serum. Higher activities of CK , AST Fi and LDH P<0.01). Compared with the Sham group rats, total GSH level (a non-enzymatic antioxidant) (P<0.01), SOD (P<0.01), CAT (P<0.05) and GSH-Px activities (P<0.01) were markedly decreased in the I/R group rats. PHC pretreatment restored the all these markers in a dose-dependent manner in comparison with the I/R group . However, there were no significant differences in the activity of CAT and total GSH level between the treatment groups and the I/R group.To determine the effects of PHC on oxidative stress, we measured several oxidative stress markers in serum and myocardial tissues. I/R injury significantly increased total NO level , ROS prooxidant) , P<0.01,01), SOD , P<0.01,01), CAT , P<0.05 tivities , P<0.01 P<0.01] when compared with the Sham group. Myocardial infarct sizes were significantly reduced in M-PHC-  and H-PHC-treated I/R rats  compared with the I/R group rats. However, L-PHC pretreatment did not influence infarct size .As shown in P<0.01]. The apoptotic rates in the I/R+M-PHC group  and I/R+H-PHC group  were significantly reduced compared with the I/R group.To analyze the effects of PHC on cardiomyocyte apoptosis in a rat model of I/R, we performed a TUNEL assay. The apoptotic rate was significantly increased in the I/R group compared with the Sham group .The levels of serum TNF-α , IL-1β , IL-6 F, PGE2 (F2  and myoc2  in the IP<0.01). However, pretreatment with PHC diminished phosphorylation of IκB and significantly decreased NF-κB expression and p-IκB/IκB ratio, while IκB expression was markedly increased .We analyzed the impacts of PHC on p-IκB, IκB and NF-κB protein expression by western blot. The results showed that the levels of p-IκB and NF-κB and p-IκB/IκB ratio in the I/R group were higher and IκB expression level was lower than the Sham group  (P<0.01)P<0.01). However, infarct size was significantly reduced at 7 d after H-PHC pretreatment (P<0.01). We then evaluated the long-term effects of PHC on hemodynamic parameters (P<0.01). However, H-PHC pretreatment resulted in a significant elevation of EF (P<0.01), FS (P<0.05) and LVSP (P<0.05), and a downregulation of LVEDP.As expected, I/R injury greatly increased myocardial infarct size , P<0.01.rameters . Comparein vivo.Myocardial ischemia followed by reperfusion is always applied to the therapies of acute coronary syndrome, open heart surgery and heart transplantation. Although reperfusion is essential for the survival of ischemic tissue, reperfusion itself may cause additional injury, which is called I/R injury . PenehycCardiac function depends on the ventricular systolic and diastolic functions . Recent −) [The levels of serum myocardial enzymes  are used to assess the degree of myocardial injury . Their e−) . It also−) . CAT, SO−) ,26. GSH −) . MDA is −) . Our res−) .Myocardial I/R-induced injury involves necrosis and apoptosis . Accumul2 is an inflammatory mediator regulated by COX-2 [2 and COX-2 in vitro [The inflammatory responses play a major role in the pathogenesis of acute myocardial infarction (AMI) ,35. In aby COX-2 . In presin vitro , suggestNF-κB is an important transcriptional regulatory factor and plays an important role in myocardial I/R injury . I/R injIn the present study, we further evaluated the long-term effect of H-PHC (at 7 d after single-injection of H-PHC) on myocardial infarct and cardiac function. We found that a single injection of H-PHC significantly decreased infarct size and improved cardiac function.Our studies demonstrated that PHC protected the myocardium from I/R injury by attenuating oxidative stress, inflammatory response and apoptosis through the inhibition of NF-κB and the activation of IκB. Moreover, H-PHC also exerted long-term cardioprotection in a rat model of I/R injury. However, the definite mechanisms underlying the effects of PHC require further study. The above results suggest that PHC can be used as a novel strategy for the therapy of myocardial I/R injury.