The protective effect of GA was completely abrogated by the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the specific protein kinase B (Akt) inhibitor Akt VIII respectively, indicating that the protective mechanism of GA is mediated by the PI3K/Akt signaling pathway. The specific extracellular signal-regulated kinase (ERK1/2) inhibitor PD98059 could not block the neuroprotection of GA. GA attenuated the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by H2O2. Western blotting showed that GA promoted the phosphorylation of ERK1/2, Akt and endothelial nitric oxide synthase (eNOS), respectively, and effectively reversed the H2O2-inhibited phosphorylation of these three proteins. LY294002 completely inhibited the GA-activated phosphorylation of Akt, while only partially inhibiting eNOS. This evidence implies that eNOS may be activated directly by GA. PD98059 attenuated only partially the GA-induced phosphorylation of ERK1/2 with/without the presence of H2O2, indicating that GA may activate ERK1/2 directly. All these results put together confirm that GA protects RGC-5 cells from H2O2 insults via the activation of PI3K/Akt/eNOS signaling pathway. Whether the ERK1/2 signaling pathway is involved requires further investigations.Gardenamide A (GA) protects the rat retinal ganglion (RGC-5) cells against cell apoptosis induced by H RGC-5 c2 humidified atmosphere. Cultured media were replaced with fresh RPMI-1640 twice a week. Stock culture was routinely subcultured at 1:5 ratio weekly.RGC-5 cells were cultured in RPMI-1640 medium containing 10% FBS. RGC-5 cells were passaged by every 3–4 days using streptomycin (100 μg/mL) and penicillin (100 U/mL), and incubated at 37 °C with 5% COCell viability was estimated using an MTT assay. In short, after 24 h treatments, the culture medium was removed and replaced with 90 µL of fresh DMEM. Ten microliters of 5 mg/mL MTT in phosphate-buffered saline (PBS) was added to each well and the plates were incubated at 37 °C for another 3 h. Then, supernatants were discarded. Afterwards, DMSO (100 µL) was added to each well and the solutions were mixed thoroughly. Then, the plates were incubated at 37 °C for another 10 min. Each sample was mixed again and the resultant formazan was measured at 570 nm using a BIO-RAD680 plate reader . The experiments were repeated at least three times and compared with the control experiment.2O2. After washing with PBS, the cells were stained with Hoechst 33258 (5 μg/mL) for 10 min at 37 °C. Then, Hoechst 33258 was removed by washing cells with PBS and the rate of apoptosis was calculated by a high content screening system .The cells grown on slides were treated with GA and/or HCells from different experimental conditions were lysed with ice-cold RIPA lysis buffer as described previously . ProteinMitochondrial membrane potential was analyzed by using a fluorescent dye JC-1 . JC-1 penetrates live cells and healthy mitochondria. At low membrane potentials (apoptotic cells), JC-1 exists as a monomer which emits green fluorescence. JC-1 aggregates and emits red fluorescence at higher membrane potentials (non-apoptotic cells). Experiment was initiated by incubating RGC-5 cells with JC-1 (5 mg/L) for 20 min at 37 °C in the dark and the fluorescence of separated cells was captured by inverted fluorescence microscope , at the wavelengths of 490 nm excitation and 530 nm emission for green; and at 540 nm excitation and 590 nm emission for red. The ratios of red/green fluorescence were calculated.2O2 residues inside the cells.ROS level was evaluated using 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) . A membrane-permeable probe that is deesterified intracellularly. The non-fluorescent dye penetrates cells freely and then is hydrolysed to DCFH by intracellular esterases. The DCFH is then trapped inside the cells. Upon oxidation by ROS, DCFH yields the highly fluorescent product dichlorofluorescein (DCF). Treated cells were loaded with DCFH-DA  in RMPI-1640 media for 30 min in the dark. The cells were rinsed twice with 1× PBS solution and the fluorescence from the DCF was analyzed using a high content screening system  with the excitation wavelength set at 488 nm and the emission wavelength set at 525 nm. ROS level determined by this method included the H2O2 and left to grow to more than 90% confluence in 6-well plates. Cells were harvested and washed with PBS after 24 h. The MDA was measured using protocol described in the MDA detection kit from Beyotime Institute of Biotechnology, Nanjing, China.Malondialdehyde (MDA) reacts with thiobarbituric acid (TBA) to produce a fluorescent product. The level of MDA was measured in RGC-5 cells lysates with a microplate reader at a wavelength of 535 nm. RGC-5 cells were treated with GA for 2 h before exposed to H2O2 (100 µM), GA (10 µM) for 24 h, respectively; or treatment with GA (10 µM) for 2 h before treatment of H2O2 (100 µM) for another 24 h. The cells without the addition of either H2O2 or GA were set as control. The medium were removed and the adherent cells were washed with PBS for 1–2 times. Afterwards, the cells were digested by trypsin and passaged into an EP tube. PBS was added to wash the cells. The supernatants were discarded to remove trypsin by low-speed centrifugation. PBS (300 µL) were added to each EP tubes. The cells were disrupted by ultrasonic radiation  for 4 times at an interval of 30 s to 1 min. The temperature was maintained at 0–5 °C by ice-water bath during the whole ultrasonic process. NOS activities of the cells were assayed using the Typed Nitric Oxide Synthase (NOS) Detection Kit (A014) purchased from Institute of Nanjing Jiancheng Bioengineering according to the manufacturer’s instructions.RGC-5 cells were cultured in DMEM with 1% FBS for 24 h following the treatments of Hp values < 0.05 were considered statistically significant.All results are reported as means ± SEM for 3–5 experiments. Differences between groups were analyzed using ANOVA, followed by Dunnett’s multi-comparison test with PASW Software . 2O2. The protective effect of GA was completely abrogated by the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the specific protein kinase B (Akt) inhibitor Akt VIII, respectively, indicating that the protective mechanism of GA is mediated by the PI3K/Akt signaling pathway. The specific extracellular signal-regulated kinase (ERK1/2) inhibitor PD98059 could not block the neuroprotection of GA. GA attenuated the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by H2O2. Western blotting showed that GA promoted the phosphorylation of ERK1/2, Akt and endothelial nitric oxide synthase (eNOS), respectively, and effectively reversed the H2O2-inhibited phosphorylation of these three proteins. LY294002 completely inhibited the GA-activated phosphorylation of Akt, while only partially inhibiting eNOS. This evidence implies that eNOS may be activated directly by GA. PD98059 attenuated only partially the GA-induced phosphorylation of ERK1/2 with/without the presence of H2O2, indicating that GA may activate ERK1/2 directly. All these results put together confirm that GA protects RGC-5 cells from H2O2 insults via the activation of the PI3K/Akt/eNOS signaling pathway. Whether the ERK1/2 signaling pathway is involved requires further investigations.In short, gardenamide A (GA) protects the rat retinal ganglion (RGC-5) cells against cell apoptosis induced by H


The correlation between hnRNP A1 and RON tumoral expression suggests that these findings hold clinical relevance.The expression and role of RNA binding proteins (RBPs) controlling mRNA translation during tumor progression remains largely uncharacterized. Analysis by immunohistochemistry of the expression of hnRNP A1, hnRNPH, RBM9/FOX2, SRSF1/ASF/SF2, SRSF2/SC35, SRSF3/SRp20, SRSF7/9G8 in breast tumors shows that the expression of hnRNP A1, but not the other tested RBPs, is associated with metastatic relapse. Strikingly, hnRNP A1, a nuclear splicing regulator, is also present in the cytoplasm of tumor cells of a subset of patients displaying exceedingly worse prognosis. Expression of a cytoplasmic mutant of hnRNP A1 leads to increased translation of the mRNA encoding the tyrosine kinase receptor RON/MTS1R, known for its function in tumor dissemination, and increases cell migration  However, Y-box binding protein-1 (YB-1), a DNA/RNA binding protein known to shuttle between the nucleus and cytoplasm of cells, was among the genes found in the pro-invasion signature [The regulation of protein synthesis, established to be a crucial component of cancer cell survival and transformation , is emerignature . YB-1 waignature . Direct ignature  and for RON/MTS1R 5′UTR. RON encodes a tyrosine kinase receptor known for its function in tumor dissemination and a correlation exists between protein levels of hnRNP A1 and RON in breast tumors.To find out whether other RBPs regulate translation initiation of specific mRNAs during tumor progression, we first analysed by immunohistochemistry (IHC) the expression of several RBPs  in breast cancers. These RBPs were chosen based on their previously demonstrated translational activities –9. Here,p = 0.048) and a negative correlation between histological grade and expression of SRSF3 (p = 0.03). We found a positive correlation between lymph node metastasis and expression of hnRNP A1 (p < 0.01) or SRSF7 (p < 0.01) and a negative correlation between lymph node metastasis and the expression of hnRNP H (p = 0.03) or SRSF3 (p < 0.01). Associations were also found between estrogen receptor (ER) status and expression of SRSF3 (p < 0.01) and RBM9 (p < 0.01) as well as between HER2 status and expression of SRSF3 (p = 0.03) and SRSF7 (p = 0.017). No associations were observed between expression of hnRNP A1, SRSF1 or SRSF2 and histological grade, molecular subtype or the status of either hormonal receptors or HER2. Kaplan-Meier analysis showed that hnRNP A1 expression correlated with clinical outcome  in a collection of 277 breast cancer specimens . Due to  outcome . Indeed,p = 0.0260, vs 70.1%, p = 0.0005, Figure vs 70.4%, p = 0.0087).hnRNP A1, although predominantly nuclear and involved in the regulation of alternative pre-mRNA splicing, is able to shuttle between the nucleus and the cytoplasm  where itSince we are particularly interested in defining RBPs regulating the translation of mRNAs involved in metastatic process, we investigated hnRNP A1 translational targets. hnRNP A1 is known to regulate the translation of mRNA targets, either positively or negatively, by directly interacting with target 5′UTRs , 10–12. RON , which encodes macrophage stimulating 1 receptor (MST1R), a member of the Met family of receptor tyrosine kinases [RON 5′UTR using a UV crosslinking/immunoprecipitation assay. As expected, hnRNP A1 bound to one of its known targets, the human rhinovirus (HRV) IRES  IRES [RON mRNA levels  IRES , 10 but A levels . InteresA levels , in whicRON mRNA levels  Figure. The expA levels . ImportaA levels  reduced A levels . The obsA levels . These dRON mRNA, polysome gradients were prepared from cytoplasmic extracts of F1-transfected, hnRNP A1-transfected or control T47D cells  or the deleted mutant (Δ154), in which the two G4s are deleted) upstream of the luciferase Firefly (LucF) open reading frame (ORF). In vitro translation showed that deleting the G4 region in the Δ154 RNA led to an increase in translation  the fact that siRNA-mediated depletion of RON prevents the effect of F1 on cell migration. The increased cell migration observed in A1-overexpressing cells is however not dependent on RON. It is therefore possible that, in the nucleus, hnRNP A1 regulates alternative splicing of several genes involved in cell migration/invasion.In summary, among the 7 RBPs that were tested in this study, we found that hnRNP A1 was overexpressed in breast tumors of patients with a poor outcome. hnRNP A1 has been reported to be involved in tumor dissemination , 24. HowRON mRNA described here, the RON gene is also regulated at the splicing level by hnRNP A1 [However, besides the hnRNP A1-mediated translation regulation of the hnRNP A1 . It therhnRNP A1 .Computational analysis revealed that G4-forming motifs are overrepresented in the 5′UTR of mRNAs , 27. G4-GGA TCC ACT AGA ATC GAT CCC GCG A-3′ (BamHI restriction site is underlined) and reverse 5′-CGG CCA TGG TAT CAT CGT GTT TTT CAA AGG A-3′ (NcoI restriction site is underlined). The hnRNP A1 cytoplasmic mutant F1 was amplified by PCR using the following primers: forward 5′-CGG CCA TGG ATG GGT TAC CCA TAC GAT GTT CCA GAT TAC GCT TCT AAG TCA GAG TCT CCT AAA-3′  and reverse-5′-CCG CTC GAG TTA AAA TCT TCT GCC GTC GCC ATA ATC GTC ATC GTC ATC GTC ACC GCC ATA GCC ACC TTG GTT TCG TGG-3′ . The trimolecular ligation was performed between the EMCV IRES PCR fragment digested by BamHI and NcoI, the F1 PCR fragment digested by NcoI and XhoI and the pcDNATM6.2-GW/EmGFP-LacZ plasmid from the BLOCK-iT™ Pol II miR RNAi ExpressionVector Kits (Invitrogen cat. K4935-00) digested by BamH I and XhoI. The lentiviral vector pLenti6/V5-EmGFP-IRES EMCV- HA F1 and the control pLenti6/V5- EmGFP- LacZ were then generated using the BLOCK-iT™ Pol II miR RNAi Expression system (Invitrogen cat. K4937-00) according to the manufacturer's protocol.EMCV IRES was amplified by PCR using the pCREL plasmid as a template  and the following primers : forward 5′-CGG The tumors selected for this study were retrieved from our institution's archives. Patient characteristics and clinicopathological features are described in i.e. date and type of relapse, treatment, date and cause of death) were recorded.Case selection for the TMA was based on the availability of archival paraffin blocks with sufficient tumor material for analysis. All tumor specimens included in this study were fixed in Bouin's solution. Elston-Ellis histological grade and nodal status. For the TMA construction, 600-μm diameter cores of histologically confirmed invasive breast carcinomas were extracted from the original (donor) paraffin blocks and re-embedded into a gridded recipient paraffin block using a Beecher Inc. tissue arrayer . For each case, 3 tumor cores and 1 normal breast core were taken from the original block. Control tissue cores from normal lymph node and placenta were also included in each recipient block. Medical reports of all cases were reviewed and clinical data (Immunohistochemistry (IHC) was performed on 4-μm-thick routinely processed paraffin TMA sections using a Techmate Horizon  slide processor. A prior antigen retrieval heating-based technique was used for all immunostainings except for hnRNP A1 and hnRNP C1/C2. The primary antibodies used were directed against Estrogen Receptor a (ER) , Progesterone Receptor (PR) , HER2 , Cytokeratins (CK) 5/6 , EGFR , hnRNP C1/C2 , hnRNP A1 , RBM9 , RON , SRSF1 , SRSF2 , SRSF3  and SRSF7 . After inhibition of endogenous peroxidase , slides were incubated with antibodies for 1h, followed by incubation with a dextran polymer enhancing system . Staining was visualized by using 3,3′-diaminobenzidine chromogen (Dako). Slides were counterstained with haematoxylin (Dako).® scoring system updated according to ASCO/CAP guidelines. CK5/6 and EGFR immunostains were interpreted using criteria described by Nielsen et al., in order to assess the molecular subtype (Nielsen CCR2004). For all RBPs and RON IHC, an immunoreactive score (IRS) was assessed, combining the percentage of positive tumor cells and staining intensity . Staining intensity was evaluated as 0 = negative, 1 = weak, 2 = moderate and 3 = strong. The percentage of labeled cells was categorized using a five-point scale i.e. 0 = 0%, 1 = 1 to 10%, 2 = 11 to 50%, 3 = 51 to 80%, 4 = 81 to 100%. The IRS (from 0 to 12) was obtained by multiplying intensity and percentage scores.The percentage of labeled tumor cells and staining intensity were reported for each immunostaining. A tumor was considered as ER or PR positive when at least 10% of tumor cells displayed a nuclear staining. The HER2 IHC score was given according to the HerceptestTissue cores that failed to adhere to the glass slide or that did not contain an invasive tumor component were excluded from the final analysis and reported as missing data.i.e. metastasis to any visceral or bone site) for metastasis free survival. For overall survival, patients alive at the last follow-up news were censored. For metastasis free survival, patients who never presented with metastatic disease were censored at the last follow-up news. Univariate analysis was performed using the log-rank test to identify associations with prognostic factors. All factors considered significant at the p < 0.05 level by this method were included in a Cox multivariate analysis to identify the major independent prognostic factors. All p values reported were two-sided. For all statistical tests, differences were considered significant at the 5% level. Multiple testing adjustment was done using Benjamini-Hochberg procedure . Sequences were read into R (v 3.0.2) using the Biostrings package  and scanned for the TAGGGW motif using the vmatchPattern function. Sequences containing that motif were then searched 5′ to the TAGGGW motif for a G4 sequence. G4 were defined as a series of at least 4 matches to the pattern 'GGG' separated by gaps with width between 1 and 7 bases  (n of QFP ). ResultExperiments were performed with T47D and MDA-MB-231 human breast cancer cell lines obtained from the American Type Culture Collection, and cultured in RPMI media or DMEM supplemented with 10% foetal bovine serum (FBS) (Invitrogen), respectively. Each cell line was first amplified to generate a cell master bank. All experiments were performed from this master bank. All cell lines were tested negative for mycoplasma contamination using VenorGeM advance PCR kit .in vitro transcribed luciferase-encoding capped and polyadenylated reporter mRNA  generated using mmessage mmachine kit (Ambion) according to the manufacturer's instructions.Cells were transfected, using lipofectamine2000 reagent (Invitrogen), with 250 ng of Cells were infected using lentivirus pLenti6/V5DEST expressing GFP, hnRNP A1 or the F1 mutated variant of hnRNP A1 (cytoplasmic variant)  using thSmall interfering RNA (siRNA) oligonucleotides against hnRNP A1 , HA , GFP , Ron  and actin . To validate the hnRNP A1 IHC technique, 3 breast tumors with different levels of expression were selected for cross-validation by Western blot.RON amplify a region spanning exons 14 and 15 that is present in the different alternatively spliced RNA isoforms of the RON gene.Total RNA was purified from cells using TRI Reagent solution (Applied Biosystems) according to the manufacturer's instructions. Reverse transcription was done with 1 μg of total RNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas) and random hexamers according to the manufacturer's instructions. Data were analysed by the threshold cycle (Ct) comparative method and normalized to the actin gene  or the Gn = 3 for each condition).Control and transfected cells in 500 μl of non-supplemented culture media were plated into the upper wells of 24-well transmigration chambers , and 850 μl of 10% FBS media was added into the lower wells as a chemoattractant. After a 24h-incubation at 37°C, the transmigration chambers were washed in PBS and the transmigration membranes were fixed with 10% TCA and stained with amidoschwarz. The number of migrating cells was counted on three distinct fields of each membrane (Cell proliferation assay was performed using the WST-1 reagent (Roche) according to the manufacturer's instructions.Cells were grown on sterilized glass slides (Dako) and were fixed in 3% paraformaldehyde for 15 min and permeabilized in 0.2% Triton for 5 min. Nonspecific binding sites were blocked with 0.1M PBS and 1% bovine serum albumin for 30 min at room temperature. Subsequently, cells were incubated for 1h with the anti-hnRNP A1 antibody , followed by 30 min incubation with an anti-mouse immunoglobulin G fluorescein isothyocyanate conjugated secondary antibody (Sigma-Aldrich).2 and 100 U/ml RNAsine). After 13 strokes of Dounce homogeneization, 400 μl of LSB containing 0.2% Triton X-100 and 0.25 M sucrose was added. Cellular debris was removed by centrifugation and the lysate was layered on a 11.3 ml continuous sucrose gradient (15-50% sucrose in LSB buffer). After 120 min of ultracentrifugation at 38,000 rpm in a SW41-Ti rotor at 4°C, fractions were collected with an ISCO density gradient fractionation system . The settings were as follows: pump speed, 0.75 ml/min; fraction time, 1.2 min/fraction; chart speed, 120 cm/h; sensitivity of the OD254 recorder, 2. The absorbance at 254 nm was measured continuously as a function of gradient depth; 16 fractions of 0.9 ml were collected. The fractions recovered from the gradient were either analyzed individually or divided into two groups, fractions containing actively translated mRNAs (polysomes (P)) and fractions containing untranslated mRNAs (non-polysomes (NP)). Equal amounts of RNA from the NP and P fractions were extracted by using Trizol LS (Invitrogen), analysed by agarose gel and subjected to qRT-PCR analysis to determine the polysomal mRNA distribution.Transduced T47D cells (30 millions) were treated with 0.1 mg/ml cycloheximide (CHX) for 15 min at 37°C, washed twice with ice-cold PBS/CHX, and scraped in PBS/CHX. After centrifugation for 5 min at 3000 rpm, the cell pellet was resuspended in 400 μl of LSB buffer  was annealed with 105 cpm of ATP-labeled primer (5′-ATGTTTTTGGCGTCTTCGGC-3′), followed by reverse transcription with 5 U of AMV reverse transcriptase (Promega).in vitro transcribed luciferase reporter mRNA (RON-LucF and RON Δ154-LucF) was incubated for 30 min at 90°C in 10 μl of RRL. When indicated, (i) the reaction was performed in presence of 50 ng GST or GST-A1 recombinant protein and 100X selex hnRNP A1 primer, (ii) the in vitro transcribed mRNA was pre-incubated 30 min at room temperature with increasing amount of G4 ligands Phen-DC(3) and Phen-DC(6).100 ng of each in vitro translation in RRL, 5 μl from each reaction was combined with Firefly luciferase-specific substrates and light emission was measured according to manufacturer's protocol (Promega). For the analysis of the luciferase activity in vivo, the T47D cells transfected with the different luciferase LucF and LucR mRNAs were harvested in 100 μl of Passive Lysis Buffer (Promega). 20 μl of this extract were analyzed.For the in vitro transcribed 32P-labeled RNAs  in buffer GS  for 10 min. Reaction mixtures were irradiated on ice with UV light (254 nm) in a Stratalinker (Stratagene) at 0.4 J/cm2 at a 10-cm distance. Five units of RNAse ONE (Promega) was then added and the reaction mixtures were incubated for 45 min at 37°C. SDS gel loading buffer was added and the samples were boiled 2 min before fractionation on a 10% SDS-polyacrylamide gel. For immunoprecipitation of UV cross-linked proteins, the RNAse ONE-treated samples were diluted in 150 ml of IP buffer , precleared and mixed with 1 μl of anti-hnRNP A1 mAb (4B10). The mixtures were allowed to rotate 1 h at 4°C. Then, 50 μl of protein A beads was added to the mixtures and incubation continued for an additional 1 h at 4°C. After extensive washing of the beads, bound proteins were eluted in SDS-loading buffer.UV cross-linking experiments and immunoprecipitation of cross-linked hnRNP A1 with the 4B10 monoclonal antibody (mAb) were performed as described previously . Protein


A total of 45 healthy controls and 85 SLE patients were recruited. The C1245G polymorphism of the hOGG1 gene was determined by direct sequencing. The frequency of occurrence of the hOGG1 1245 GG genotype in SLE patients was 31.8% (27/85), which is lower than that of healthy controls of 53.3% (24/45). Thirty-three  SLE patients developed lupus nephritis. Significantly, SLE patients harboring the hOGG1 1245 GG genotype had a higher incidence to develop lupus nephritis than did those harboring the hOGG1 1245 CC or CG genotype . Divided into subgroups, SLE patients harboring the hOGG1 1245 GG genotype had the highest plasma levels of 8-OHdG among patients with all genotypes, with regard to the coexistence of lupus nephritis , including those with nephritis harboring the hOGG1 1245 CC or CG genotypes (p = 0.037), those without nephritis harboring the hOGG1 1245 GG genotype (p = 0.050), and those without nephritis harboring the hOGG1 1245 CC or CG genotype (p = 0.054). We conclude that the C1245G polymorphism of hOGG1 may be one of the factors that confer the susceptibility to lupus nephritis and modulate the plasma level of 8-OHdG in patients with SLE.We investigated whether the C1245G polymorphism of human 8-oxoguanine glycosylase 1 ( Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the generation of arrays of pathogenic auto-antibodies that cause cascades of organ damages . SimilarhOGG1) through the base excision repair mechanism [hOGG1 genotypes identified because of a C to G shifting at base-pair (bp) 1245 (C1245G) in exon 7 of the hOGG1 gene. Such a shifting causes a serine (Ser) to cysteine (Cys) substitution at codon 326 during translation. This phenomenon is referred to as hOGG1 C1245G polymorphism. Considering the DNA repair efficiency, homozygous hOGG1 1245 CC genotype has the highest DNA repair activity followed by the heterozygous hOGG1 1245 CG genotype and then the homozygous hOGG1 1245 GG genotype in order [hOGG1 repair activity implies that hOGG1 1245 GG genotype with lower efficiency in DNA repair might be related to a higher level of 8-OHdG in human tissues [hOGG1 C1245G polymorphism in the pathogenesis of SLE has remained obscure.In human cells, 8-OHdG is mainly repaired by human 8-oxoguanine glycosylase 1  [hOGG1 1245 GG genotype contributes to a higher rate of SLE development and higher plasma level of 8-OHdG; (2) whether the hOGG1 1245 GG genotype confers a higher incidence to the development of nephritis in SLE patients; and (3) whether the hOGG1 1245 GG genotype modulates the plasma level of 8-OHdG in SLE patients with or without nephritis.Lupus nephritis is a serious clinical manifestation of SLE. Studies revealed that excessive ROS production and oxidative stress may exacerbate inflammation, dampen antioxidant capacity and lead to tissue damage and the formation of lupus nephritis in SLE patients ,17. Furte (ESRD) ,19. In oe (ESRD) . In lighhOGG1 C1245G polymorphisms are illustrated in The genotypes of p = 0.017). For further comparisons, healthy controls harboring the CC or CG genotypes were designated as Group A, healthy controls harboring the GG genotype were designated as Group B, SLE patients harboring the CC or CG genotypes were designated as Group C and SLE patients harboring the GG genotype were designated as Group D, respectively.As shown in p < 0.001), regardless of their hOGG1 gene polymorphism of CC/CG genotypes  or GG genotype  . A mild elevation of plasma 8-OHdG levels was observed in SLE patients with GG genotype , although it was not obvious. Among all subjects, Group D (patients with the hOGG1 1245 GG genotypes) had the highest plasma levels of 8-OHdG  of the 45 healthy controls and 85 SLE patients were 0.157 ± 0.038 and 0.225 ± 0.082 ng/mL, respectively . SLE pat= 0.001) . Those hhOGG1 1245 GG genotype on SLE susceptibility or the elevation of 8-OHdG level in SLE patients deserves evaluation. In our study cohort, the percentage of hOGG1 1245 GG genotype in SLE patients was 31.8%, which was lower (p = 0.017) than that in healthy controls . On the contrary, the percentage of hOGG1 CC genotype in healthy control was 6.7% (3/45), which was lower than in SLE patients . As reported in the published data, hOGG1 1245 GG genotype frequency in SLE patients is variable, higher in normal Taiwanese (20.5%) population [hOGG1 1245 GG genotype in the normal Taiwanese population. Furthermore, the percentage of CC genotype in our healthy cohort was 6.7%, which was lower than those reported previously [hOGG1 1245 GG genotype does not predispose to SLE and is not the prominent contributor to a higher plasma 8-OHdG levels in SLE patients. However, if SLE patients harbor the hOGG1 1245 GG genotype (Group D), they might exhibit diminished capacity to ameliorate oxidative stress and resulted in higher plasma levels of 8-OHdG . There eviously ,19,21,22f 8-OHdG . Alternavs.18/58, 31.0%, p = 0.031). Thus, for further comparisons, the 85 SLE patients were divided into 4 groups according to the clinical presentations of lupus nephritis and hOGG1 C1245G polymorphism. These include SLE patients without nephritis and with the CC/CG genotypes (Group I), SLE patients without nephritis and with the GG genotype (Group II), SLE patients with nephritis and with the CC/CG genotypes (Group III), and SLE patients with nephritis and with the GG genotype (Group IV).As shown in vs. 0.201 ± 0.046 ng/mL, p = 0.165). In those who harbored the GG genotype (Group II and Group IV), Group IV had higher plasma levels of 8-OHdG than Group II . In the 33 SLE patients with nephritis (Group III and Group IV), Group IV had higher plasma levels of 8-OHdG than did Group III . However, such a difference was not observed between Group I and II in the 52 SLE patients without nephritis . Furthermore, SLE patients with nephritis who harbored the GG genotype (Group IV) had higher plasma levels of 8-OHdG than SLE patients without nephritis who harbored the CC or CG genotypes (Group I) . Group IV SLE patients with nephritis and with the GG genotype had higher levels of plasma 8-OHdG than did patients in the other groups  (In the patients who harbored the CC or CG genotypes (Group I and Group III), there was no significant difference in their plasma levels of 8-OHdG  .hOGG1 1245 GG genotype is related to the development of several human degenerative diseases, including type 2 diabetes mellitus [hOGG1 1245 GG genotype is associated with multiple vessel involvement in patients with coronary artery disease [hOGG1C1245G polymorphism in lupus nephritis remains unclear. Interestingly, we demonstrated that in our SLE cohort, the 27 patients harboring the GG genotype had a significantly higher rate  of nephritis than the other 58 patients harboring the CC or CG genotypes . Therefore, we suspect that the SLE patients harboring hOGG1 1245 GG genotype tend to have a higher risk of developing nephritis and accumulating more oxidative damage to DNA in affected tissues.It was reported that mellitus , Huntingmellitus , chronicmellitus  and Gravmellitus . Further disease , and wit disease . HoweverWith fulfillment of the American College of Rheumatology (ACR) criteria for the classification of SLE , we coll®, Greiner Bio-one, Monroe, NC, USA) that contained EDTA. After centrifugation at 3000× g for 10 min at 4 °C, the plasma and leukocyte-enriched buffy coat were collected separately. The plasma was then subject to the determination of 8-OHdG. After addition with the erythrocyte (RBC) lysis buffer , leukocyte DNA was extracted by standard phenol-chloroform procedures and kept at −20 °C until use [About 10 mL of venous blood was drawn in a specific tube  according to the manufacturer’s instruction ,27. EachhOGG1 C1245G polymorphisms were detected by direct genome sequencing. Briefly, 1 μL (10 ng/μL) of the leukocyte DNA was amplified in a 50 μL PCR reaction that containing 25 μL of RBC SensiZyme® Hotstart Taq Premix , 22 μL of PCR grade H2O, 1 μL of each primer (F: 5'-ACTGTCACTAGTCTCACCAG-3' and R: 5'-GGAAGGTGCTTGGGGAAT-3') to generate a 200-bp product [The  product . PCR con product ,21.t-test, Mann-Whitney U-test, ANOVA  or Kruskal–Wallis test when appropriate. A difference was considered significant when a p-value was less than 0.05.The plasma 8-OHdG concentrations are presented as the mean ± standard deviation (M ± SD). The continuous variables between two groups or among three or more groups were compared using the Student’s hOGG1 C1245G polymorphism might be one of the multiple factors that confer a higher susceptibility to lupus nephritis and modulate the plasma level of 8-OHdG in patients with SLE.In conclusion, we have provided evidence that


Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8) and S100A9 (MRP14) form bioactive antimicrobial heterodimers  that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model.Typhoid fever, caused by the Gram-negative bacterium -/-S100A9 mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but -/-S100A9 mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury.S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT) and S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response against S. Typhimurium in mice.S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of  Salmonella Typhi, the etiologic agent for typhoid fever, however harbors a number of factors, such as a polysaccharide capsule, which prevent the detection of these virulence factors, and thereby dampens the innate host response. Besides bacterial virulence factors, the host can detect endogenous danger molecules which are released upon tissue damage. S100A8/A9, an extracellular protein complex, is such a danger signal that is able to further amplify the systemic inflammatory response upon infection. In the present study we investigated the role of S100A8/A9 during invasive Salmonella infection and observed a marked increase of this protein in patients with typhoid fever, which correlates with disease stage and severity. Furthermore we found that S100A8/A9 directly inhibited the growth of Salmonella species in vitro thereby functioning as an antimicrobial. When mice were infected with Salmonella, the levels of S100A8/A9 were also elevated but mice lacking this protein did not have an altered host response to infection. The role and importance of the elevated levels of S100A8/A9 in human typhoid fever requires further study.Bacterial pathogens are recognized by the host upon infection through interactions between their virulence factors and host cell receptors leading to the activation and recruitment of innate immune cells.  Salmonella (S.) enterica serovar Typhi, which is the Gram-negative intracellular bacterium, results in a bacteremic disease characterized by protracted and high-grade fever. Mortality usually results from intestinal perforation and peritonitis or from severe toxic encephalopathy associated with myocarditis and hemodynamic shock [Typhoid fever constitutes a global public health problem with more than 21 million cases worldwide each year resulting in over 217,000 deaths predominantly in Asia . Infectiic shock ,4.Salmonella spp. are recognized by pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) [Salmonella spp. are the type 3 secretion system (T3SS)-1 and 2, Vi antigen, flagella, lipopolysaccharide (LPS), bacterial DNA and exotoxin [S. Typhi can mask Salmonella specific outer surface PAMPs (LPS and flagella) during specific stages of disease [S. Paratyphi A, and most other Salmonella serovars. It is likely that there are a number of other of mechanisms for down regulating and dampening the host immune responses during enteric fever. We hypothesize that besides PAMPs the host response during typhoid fever is also driven by danger-associated molecular patterns  that can contribute to PRR-mediated tissue damage in systemic inflammation [s (TLRs)  and Nod-s (TLRs) ,7 via cos (TLRs) . Importaexotoxin ,9. The p disease ,11. The  disease . The Vi ammation ,14.The S100 proteins S100A8  and S100A9 (MRP14) are DAMPs, which are cytoplasmic phagocyte-specific proteins that represent danger signals and share some functional features of cytokines . S100A8 Salmonella enterica serovar Typhimurium in colonizing the inflamed gut when S100A8/A9 is overexpressed [Escherichia (E.) coli [Previous reports have demonstrated a dual role for S100A8/A9 in severe infection depending on the pathogen it encounters and the stage of disease. Overwhelming release of S100A8/A9 could be detrimental for the host during sepsis contributing to ongoing inflammation, organ damage and/or exhaustion of the immune system . Endogenxpressed . A recenE.) coli .S. Typhi has not been studied before. We have determined the levels of S100A8/A9 in patients with acute typhoid fever and during convalescence. We have also measured the levels of S100A8/A9 in a murine Salmonella model, using wild type and S100A9-/- mice, which due to instability of S100A8 in the absence of its binding partner S100A9 are considered deficient for S100A8/A9 at protein level [S. Typhimurium and S. Typhi replication in vitro.The expression and function of S100A8/A9 in typhoid fever caused by in level ,29, and The Indonesian patient study protocol was approved by the Indonesian National Institute of Health Research and Development (Litbangkes) and provincial authorities. The Bangladesh patient study protocol was approved by the National Research Ethics Committee of Bangladesh (BMRC/NREC/2010-2013/1543) and the Oxford Tropical Research Ethics committee (OXTREC reference 25–11). Patients and parents of all children recruited to the study gave witnessed, informed and written consent before study enrolment. Animal procedures were performed in accordance with the Dutch Experiment on Animals Act and approved by the Animal Care and Use Committee of the University of Amsterdam (DIX102331).S. Typhi and S. Paratyphi A and malaria blood smear. There were 28 individuals with blood-culture or PCR confirmed typhoid fever caused by S. Typhi. There were no cases with S. Paratyphi A detected. Healthy local controls (n = 38) were recruited during the same period. The controls had no documented fever or signs of illness at the time the blood was drawn. In these patients complicated typhoid fever was defined as the presence of one or more of the following: gastrointestinal bleeding (e.g. visible blood in stool); intestinal perforation; encephalopathy ; hemodynamic shock; renal impairment (creatinine >2.0 mg/dL); hepatitis (defined as AST or ALT ≥1000 U/L); pneumonia  or pleural effusion; presence of focal septic complications; severe anemia (hematocrit ≤20%); need for blood transfusion or in-hospital death [S. Typhi and 60 healthy local controls were included during a community-based case-control study in Jatinegara, the eastern district of Jakarta, from 2001–2003 [Febrile adults n = 144) admitted to Chittagong Medical College Hospital, Chittagong, Bangladesh during 2012 were investigated by blood culture, blood real-time PCR for 4 admitteal death ,31. In I001–2003 .Blood samples were obtained at the time of admission, and day 10 post fever onset or at hospital discharge (Bangladesh study) and at household visits 3 weeks post diagnosis (Indonesian study). Plasma was prepared from EDTA or heparin tubes (BD vacutainer) and stored immediately at −20°C pending analysis. For hospitalized patients, stool samples were taken within 48 hours after hospital admission, and stored at −70°C.S100A9-/- mice, backcrossed >10 times to a C57BL/6 background, were generated as described [S100A9-/- mice, however due to a lack of S100A9 as a binding partner these mice do not release S100A8/A9 complexes extracellularly and are therefore considered functional knockout (KO) for S100A8/A9 [S. Typhimurium strain 14028 (ATCC) was grown to mid-logarithmic phase in Lysogeny Broth (LB). Mice were starved for 12 hours followed by oral inoculation with 100 μl (105 or 106 colony forming units (CFU)/100 μl HBSS) bacterial suspension as described [Pathogen-free C57Bl/6 male wildtype (WT) mice were purchased from Charles River Laboratories. escribed , and bre100A8/A9 ,29. Age-escribed –36. At 2Liver, spleen, mesenteric lymph nodes (MLN), terminal ileum and cecum were harvested after sacrifice, fixed in 10% formalin and embedded in paraffin. Sections of 4 μm were stained with hematoxylin–eosin (HE), and read by a pathologist who was blinded to groups as described . In shorS. Typhimurium 14028 was heat-inactivated (HI) at 70°C for 30 minutes. Whole blood and PMs (after multiple washings) were incubated with 1x105, 1x106, or 1x107 HI S. Typhimurium or 100 ng/ml LPS  diluted in RPMI to a final volume of 200 μl for 4,5 or 20 hours before measurement of cytokine or S100A8/A9 release. Phagocytosis was assayed as described [S. Typhimurium was labeled with carboxyfluorescein succinimidyl ester . 50 μl heparinized whole blood was incubated with 50 μl bacteria in PBS (end concentration 1x106 CFU/ml) at 4°C or 37°C. After 0, 15 and 60 minutes, samples were put on ice to stop phagocytosis. Afterwards, red blood cells were lysed using isotonic NH4Cl solution [Peritoneal macrophages (PMs) were harvested as described ,38. PMs escribed ,38,39. Isolution . NeutropS. Typhimurium (14028) and the TY21a vaccine strain of S. Typhi  were grown to log phase and diluted to approximately 10,000 CFU/ml in RPMI 1640 medium. 100 μl of this bacterial suspension was added to 100 μl of recombinant murine S100A8/A9 heterodimer in HBSS (end concentration 50 μg/ml unless indicated otherwise) without Ca2+ and Mg2+ [For the growth inhibition assays, recombinant mouse S100A8/A9 heterodimers were generated as described . S. Typhand Mg2+ . BacteriHuman plasma S100A8/A9 was measured by in-house ELISA and confirmed with Western blots as described . S100A8/U or unpaired t test where appropriate. We log-transformed plasma and feces S100A8/A9 concentrations to correct for heteroscedasticity. For linear regression analysis, P value and Spearman rho are reported. Analyses were performed in GraphPad Prism version 6.0 for Mac OS X (GraphPad Software) and data are expressed as mean and SD unless stated otherwise.Differences between human and controls or murine groups were analyzed by the Mann-Whitney S. Typhi in Bangladesh and 60 out-patients with culture confirmed S. Typhi in Indonesia. The baseline characteristics of both patient cohorts are shown in We studied 28 hospitalized patients with culture or PCR confirmed There were no differences in S100A8/A9 complex levels between uncomplicated and complicated typhoid fever patients . HoweverS. Typhimurium (105 and 106 CFU) showed increased levels of S100A8/A9 in different compartments corresponding with stage and severity of disease . In uninfected WT mice positive immunostaining for S100A8 , nor at distant organ sites , nor at the systemic compartment (blood), between WT and S100A9-/- mice (6 CFU). This inoculum rapidly led to death within a week for virtually all mice; however, again, no differences were seen between groups (5 CFU). However, also with this inoculum size no effect of S100A9 deficiency was seen on bacterial clearance: equal bacterial counts were observed between WT and S100A9 deficient mice in all organs  and chemokines (MCP-1/CCL-2) in liver homogenates and plasma harvested from S100A9-/- and WT mice after oral infection. In contrast to data derived from a murine endotoxemia model using the same mouse strain [S. Typhimurium infection  mice 5 days after infection , and tissue damage (indicated by elevated plasma LDH), were observed 5 days after infection. However, consistent with the pathology data, these effects were not influenced by S100A9 deficiency  [S100A9-/- mice showed decreased hepatocellular inflammation compared to WT mice in an E. coli induced sepsis model [Salmonella model we did not find any significant evidence for increased hepatocellular injury in WT mice compared to S100A9-/- mice.The S100A8/A9 complexes are cytoplasmic proteins of phagocytes that elicit a variety of host defense mechanisms. Specifically, S100A8/A9 inhibits the growth of numerous microorganisms by binding of divalent cations ,23,25. W E. coli  but alsohimurium  have a Z100A8/A9 . Furthernfection . Howevers (NETs) . S100A9is model ,49. Bactis model . In the S. Typhi antigen stimulation, and determining their associations with S100A8/A9 levels. Some of the complications observed in the typhoid patients in this study, such as hepatic abscess and septic arthritis, are uncommon [S. Typhi was the causative pathogen. Based on the clinical picture, we assumed that they are related. The existence of pneumonia due to typhoid is debated and it is possible that pneumonia in our patients was a secondary phenomena caused by another pathogens. Furthermore, care should be taken in translating results obtained from mouse data towards human pathology, especially since S. Typhi is an exclusive human pathogen. Although S. Typhimurium is widely used in murine studies as a surrogate for S. Typhi [S. Typhimurium, Salmonella spp. have the ability to produce differing clinical manifestations depending on the host infected. For example, increased leukocyte levels and elevated systemic cytokine levels are observed in mice following S. Typhimurium infection, but not in typhoid fever patients [Salmonella mouse model, negative results from the murine model do not exclude a functional role of S100A8/A9 in human typhoid fever.Our study has several limitations. The comparison of acute with convalescence levels in the hospitalized group is limited in that the samples obtained at discharge or day 10 may be too early a timepoint to be a true "convalescent" sample. Therefore, as a comparison, we added the Indonesian study were we look at the convalescence phase in outpatients were blood was drawn during household visits 3 weeks after infection. We have published the details of this Indonesian comparator cohort earlier . Furtheruncommon . BecauseS. Typhi ,36,51 anS. Typhi have markedly increased S100A8/A9 plasma levels which correlate with liver damage and fever duration. Persistent increase in plasma and feces S100A8/A9 levels are observed in acute disease as well as during convalescence. Furthermore S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro. In a murine Salmonella infection model the levels of S100A8/A9 were also elevated but S100A8/A9 deficiency in mice did not influence bacterial growth and dissemination, organ damage or mortality. The role and importance of the elevated levels of S100A8/A9 in human typhoid fever requires further study.In conclusion, we here document that patients with S1 FigS. Typhimurium (106) a marked increase of S100A8 in both liver (B) and spleen (D) is seen corresponding with increased inflammatory cell influx. Magnification 10 × and 40 ×.Positive immunostaining for S100A8 in liver parenchyma (A) and the red pulp of the spleen (C) tissue in uninfected control animals. Five days after infection with (TIF)Click here for additional data file.S2 FigS. Typhimurium (106) S100A8 (E) or S100A9 (F) is not markedly increased compared to the control IgG (D) in the intestines. Magnification 10 ×.Immunostaining for rat IgG , S100A8 (B) and S100A9 (C) of the colon (and feces (*)) in uninfected control animals. Five days after infection with (TIF)Click here for additional data file.S3 FigS. Typhimurium (106). Positive S100A9 staining corresponds with HE staining suggesting that the presence of S100A9 increases with inflammatory cell influx. Magnification 4 × and 20 ×.Representative slides of liver and spleen for hematoxylin and-eosin  and S100A9 staining  in wildtype (WT) mice five days after infection with (TIF)Click here for additional data file.S4 FigS100A9-/- mice (grey bars) with 1x106S. Typhimurium or Escherichia (E.) coli LPS. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation . * P<0.05, determined using a non-parametric t tests.Tumor necrosis factor (TNF)-α levels after 5 and 20 hour stimulation of peritoneal macrophages  and whole blood  obtained from individual wildtype  and (TIFF)Click here for additional data file.


EOLA1, GenBank number AY074889) as a lipopolysaccharide (LPS) responsive gene in ECV304 cells. The previous studies have further demonstrated the association of EOLA1 with metallothionein 2A (MT2A), while the role of EOLA1 during LPS-induced inflammatory response in ECV304 cells is unknown. In this report, we determined the subcellular localization of EOLA1 and the regulatory capacity of EOLA1 on vascular cell adhesion molecule-1 (VCAM-1) in response to LPS in ECV304 cells. Our results show that EOLA1 is broadly diffuse in the cells, and EOLA1 expression is dramatically induced by LPS. EOLA1 knockdown results in significant enhancement of LPS-induced VCAM-1 production. Consistent with this, overexpression of EOLA1 leads to the reduction of LPS-induced VCAM-1 production. Furthermore, MT2A knockdown reduces LPS-induced VCAM-1 production. Collectively, our results demonstrate a negative regulatory role of EOLA1 on LPS-induced VCAM-1 expression involving its association with MT2A in ECV304 cells.Our research group firstly discovered endothelial-overexpressed lipopolysaccharide-associated factor 1 ( EOLA1, GenBank number 074889, also named as CXorf40A) has been identified by our research group from the genes with differential expression after stimulation of ECV304 cells with lipopolysaccharide (LPS) in 2004 [ EOLA1 is located at human chromosome Xq28 and contains 6,248 bases in genomics and 5 exons separated by 4 introns. The EOLA1 gene is highly conserved in chimpanzee, rhesus monkey, dog, mouse, rat, chicken, zebrafish, and frog. EOLA1 protein comprises 158 amino acids and has several glycosylation and phosphorylation sites and the helix-turn-helix motif without signal peptide and membrane spanning domain. These structural features indicate that EOLA1 is an intracellular protein and may interact with other unknown proteins in the cytoplasm and nucleus to regulate cell function. The bioinformatics analysis reviewed that EOLA1 gene belongs to the activating signal cointegrator-1 (ASC-1) homology (ASCH) superfamily, which is frequently observed in many organisms [Endothelial-overexpressed lipopolysaccharide-associated factor 1  has been confirmed by yeast double-hybridization and coimmunoprecipitation assay . MT2A isyclin D1 . Based oyclin D1 . These rLPS is the major outer surface membrane component present in almost all Gram-negative bacteria and acts as ligand in triggering innate immune response via specific receptors in diverse eukaryotic species ranging from insects to humans , 11. TheTo further understand the biological function of EOLA1 in regulating inflammatory immune response, we focused on determining the subcellular distribution of EOLA1 and its efficacy in the regulation of LPS-induced inflammatory gene response including VCAM-1 in ECV304 cells. E. coli serotype 0111: the B4 (Sigma) was dissolved in phosphate-buffered saline (PBS) to the dilution of 1 mg/mL and stored at −20°C. For experiments, the LPS stock solution was further diluted into supplemented tissue culture media to the working concentration of 100 ng/mL. ECV304 cells (ATCC) were cultured in Medium 199 supplemented with 10% fetal bovine serum, 3 mg/mL HEPES, 100 U/mL penicillin G, 100 mg/mL streptomycin, and 6 U/L insulin on gelatin coated dishes in a humidified atmosphere of 5% CO2. Confluent cultures were detached with EDTA-trypsin solution and split into 1 : 3 for further use.Purified native LPS phenol extracted from EcoR I and BamH I endonuclease sites were introduced into the terminals of forward and reverse primers, respectively. ECV304 cells were stimulated with LPS (100 ng/mL) for 6 h, and then the total RNA was extracted for RT-PCR (TaKaRa), and finally the ORF sequence of EOLA1 was amplified. The PCR product and pEGFP-N2 plasmid (Clontech) were cut with endonucleases EcoR I and BamH I, then the enzyme cutting products were recovered by electrophoresis and linked with DNA ligase (TaKaRa), and finally the target ORF sequence was inserted into pEGFP-N2 plasmid and single colonies were selected for sequencing (TaKaRa) after transformation of competent Escherichia coli DH5α.A pair of primers was synthesized according to the open reading frame (ORF) sequence of EOLA1 ;5/well and continually cultured. Then, the fusion protein expression plasmid pEGFP-EOLA1 and the control plasmid pEGFP-N2 were transfected into cells using the liposome Lipofectamine 2000 (Invitrogen) for 48 h. The transfection efficiency was determined by GFP positive versus negative cells.For cell transfection, ECV304 cells were split onto 6-well culture plate (precovered with a sterile coverslip) by 1.5 × 10After cell transfection for 48 h, the culture medium was removed, and then the cells were rinsed twice with PBS and fixed with 4% PFA. A part of cells was used to examine the subcellular location; the cells were incubated with rat anti-GFP antibody (primary antibody) and goat anti-rat antibody (secondary antibody), stained with DAPI, and photographed under LSM510 META laser confocal microscope (Zeiss). For observation under immunoelectron microscope, the cells were fixed with 4% PFA and then oxidized with 3% hydrogen dioxide and incubated with serum. The incubation was performed with the previously prepared rabbit anti-EOLA1 polyclonal antibody as primary antibody and HRP-labeled goat anti-rabbit IgG antibody as secondary antibody. Then the cells were stained with HRP-labeled streptoantibiotin and diaminobenzidine (DAB), refixed with 2.5% glutaral and fixed with 0.1% osmic acid, dehydrated with acetone at a gradient, and then preserved overnight in the saturated uranyl acetate prepared with 70% acetone. The cells were further rinsed with pure acetone, soaked with the mixture of acetone and epoxy resin (v : v = 1 : 1), treated with pure embedding medium (without epoxy resin curing promoter DMP-30) and embedding medium (with DMP-30) at 40°C, and embedded with epoxy resin 618. Finally the cells were made into ultrathin sections, stained with lead, and observed and photographed under TECNA110 transmission electron microscope (Philips).μg/mL in a final volume of 500 μL. The cells were washed after 4 h incubation and resuspended in complete growth medium with serum for further experiments.Knockdown of EOLA1 was accomplished using RNA interference (RNAi). The oligonucleotide sequences of EOLA1 shRNA  were as follows: sense: 5′-AGGAGGCAAGGATGTATTCTTCAAGAGAGAATACATCCTTGTTTTTTT-3′ and antisense: 5′-AATTAAAAAAAGGAGGCAAGGATGTATTCTCTCTTGAAGAATACATCCTTGCCTCCTGGCC-3′. For knockdown of MT2A, the oligonucleotide sequences of MT2A-shRNA were as follows: sense: 5′-GATCCGCCGTG ACCGTTTGCTATATTTCAAGAGAATATAGCAAACGGTCACGGTTTTTTGGAAA-3′ and antisense: 5′-AGCTTTCCAAAAAACCGTGACCGTTTGCTATATTCTCTTGAAATATAGCAAACGGTCACGGCG-3′. The shRNA expression cassette driven by H1 promoter was engineered by inserting those oligonucleotides into the plasmid pSilencer 3.1/H1 hygro (Ambion). An unengineered plasmid, pH1, was used as a blank control. The cells were transfected with pH1, pH1-EOLA1 shRNA, or pH1-MT2A shRNA with Lipofectamine 2000 in serum-free OPTI-MEMI (Invitrogen) according to the instruction manual. The concentration of DNA vectors for transfection was 8 β-actin: upstream: 5′-CGGGAAATCGTGCGTGACATT-3′, downstream: 5′-CTAGAAGCATTTGCGGTGGAC-3′). For VCAM-1 assay, the cells were homogenized in PBS  containing 1% protease inhibitors and then centrifuged at 12,000 ×g for 20 min at 4°C. The anti-EOLA1 polyclonal antibody was produced from New Zealand white rabbits immunization and identified by Western blot. IgGs against EOLA1 were purified on a protein A Sepharose column. The efficiency of polyclonal antibody was measured by ELISA analysis. The lysates of ECV304 cells were analyzed by Western blotting with antibodies to EOLA1. The supernatants were analyzed using ELISA kit (Sigma) according to the manufacturer's instructions.The total RNA was isolated from the cells using TRIzol reagent following the manufacturer's instructions (Invitrogen). To measure the gene mRNA expression level, RT-PCR was performed using one-step reagent kit (TaKaRa). The primers are synthesized from commensal  were loaded on SDS-9% polyacrylamide gels and then transferred to Protan nitrocellulose membranes in an electroblotting apparatus, using standard procedures. Immunodetection was performed as described previously [β-actin as loading control. The anti-EOLA1 polyclonal antibody was produced from New Zealand white rabbits immunization and identified by Western blot. IgG against EOLA1 was purified on a protein A Sepharose column. The efficiency of polyclonal antibody was measured by ELISA analysis.Transfected cells were lysed with RIPA buffer supplemented with Complete Protease Inhibitor Tablets (Roche). Lysate protein concentration was measured by Lowry assay. The protein samples  (ECV304 cells were grown to 80% confluence and then stimulated with LPS (100 ng/mL) for a time-course as indicated. The cells showed that EOLA1 expression was increased in a time-dependent manner at both gene transcription  and tran < 0.05) . All theTo further characterize the functional effects of EOLA1 on VCAM-1 induction in ECV304 cells, the cells were cotransfected with pOPRSVI-EOLA1-EGFP and pCMV-LacI plasmids to stably overexpress EOLA1 after induction by IPTG . The celTo further investigate the efficacy of EOLA1 on LPS-induced VCAM-1 production, the ECV304 cells were transfected with EOLA1 shRNA to silence EOLA1 gene. A significant downregulation of EOLA1 expression was confirmed in EOLA1 shRNA transfected cells by Western blot. Knockdown EOLA1 also showed the reduction of MT2A expression . KnockdoEOLA1 has an association with MT2A in ECV304 cells, and EOLA1 regulates the expression of MT2A, suggesting EOLA1 mediates VCAM-1 production through association with MT2A. We further investigated the role of MT2A in LPS-induced immune response. MT2A knockdown cells were treated with LPS, and the cell supernatant of VCAM-1 was measured by ELISA assay in 6 h after LPS treatment. The results show that knockdown MT2A downregulates LPS-induced VCAM-1 protein secretion in ECV304 cells .EOLA1 is a novel gene firstly identified by our research group from the screening of LPS-induced gene expression in ECV304 cells; however, little is known about its biological function in inflammation. To understand the subcellular distribution of EOLA1, we directionally cloned the ORF sequence of EOLA1 into pEGFP-N2 carrier and thus successfully constructed the eukaryotic expression plasmid of EGFP-EOLA1 fusion protein to transfect ECV304 cells. Furthermore, we observed the transfected cells under laser confocal microscope after transient expression. The observations showed strong green fluorescence signals in both the cytoplasm and nucleus. The DAPI staining confirmed that the fusion protein was expressed in the cytoplasm and aggregated in the nucleus. Besides, we further observed the subcellular localization of EOLA1 at a level of cell ultrastructure by the use of immunoelectron microscopic enzyme labeling technique, and the results confirm that the expression of EOLA1 is mostly localized in the cytoplasm. The above observations indicate that EOLA1 is an intracellular protein and has a capacity to translocate into the nucleus from the cytoplasm, although such phenomenon still needs to be further confirmed in the future.κB, which ultimately leads to the transcription and release of VCAM-1 in endothelial cells to attract mononuclear macrophage accumulation for inflammatory response [ECV304 cells belong to human umbilical vein endothelial cells, which can be activated by LPS stimulation. LPS has been shown to initiate TLR4 mediated intracellular signaling events, including the activation of NF-response , 16. Recresponse , 18. OurκB, through upregulating IκB-α. Our results show that EOLA1 regulates the expression of MT2A and affects the production of VCAM-1 by association with MT2A; therefore the excessive activation of ECV304 cells could be avoided, and the fine regulation and timely control of inflammatory reaction could be thereby achieved. This will avoid the damage of excessive inflammatory reaction to tissues and cells. The detailed mechanism of EOLA1 association with MT2A and its functional effects on the NF-κB signaling pathway needs to be further characterized.MT2A is a low-molecular-weight protein in the cytoplasm, plays multiple roles in cell metabolism, and has the effects on anti-inflammation and antiendotoxin and inhibiting tumor growth. Recent report indicates that MT2A reduces cell inflammatory response by inhibiting the activation of NF-In conclusion, EOLA1 plays a negative regulatory role in LPS-induced VCAM-1 expression under the association with MT2A in ECV304 cells.


Familial Mediterranean fever (FMF)(MIM 249100) is a hereditary autoinflammatory disorder characterized by episodes of inflammation in the absence of high-titer autoantibodies or antigen-specific T cells. The Mediterranean fever (MEFV) gene(OMIM 608107) located on chromosome 16p13.3, which encodes the 781-amino-acid protein pyrin, is the causative gene for this monogenic Mendelian disease. This study presents the molecular analysis of an MEFV gene mutation screen of 268 patients from Azerbaijan Republic, with clinical diagnoses of FMF.Genomic DNA was obtained from peripheral blood. 4 exons of MEFV gene were analysed by PCR and direct DNA sequencing method. Furthermore the obtained nucleotid sequence compaired with reference sequence published in NCBI (NM_000243.2).%56 of 268 patients were found to have mutations. Allele frequency of common five major mutation of MEFV gene E148Q,M680I, M694V, V726A and R761H were 8.2%;1.3%;,3.1%, 9.8%; and %4.2 respectively. it is revealed one novel MEFV mutation E148D in 3 patients wich updated to INFEVERS. The mutation carrier frequency of majör common alleles was 15% in healthy individuals from Azerbaijan population. In addition, the frequency of R202Q polymorphism in patients group was 45%. and 32% in control group in this population.The mutation carrier frequency and disease causative common major MEFV gene mutations in Azerbaijan population are similar of certain ethnic groups. Moreover the mutation of R761H more frequently, compared to other ethnic groups, may be a founder effect.


Sensitive molecular techniques are needed for rapid detection of the R292K oseltamivir-resistant mutant of influenza A(H7/N9) virus strain to monitor its transmission and guide antiviral treatment. We developed a real-time reverse transcription PCR and single nucleotide polymorphism probes to differentiate this mutant strain in mixed virus populations in human specimens. An unusually high proportion of severe cases and a high case-fatality rate have been observed for patients infected with this virus (An outbreak of human infections with a novel reassortant avian-origin influenza A(H7N9) virus occurred in several provinces of China during March 2013  gene (R292K) and its association with severe clinical outcome in infected persons (The NA R292K assay has 2 reactions with 1 pair of primers. One reaction contained a FAM-labeled SNP probe specific for the 292K mutant strain and a second reaction contained a VIC-labeled probe specific for the R292 wild-type strain .1–1011 copies) in sterile water and used to test the assay. The linear range of sensitivity was 102–108 copies. The lower limit of detection was 100 copies/reaction (3/3 reactions detected) for wild-type and mutant virus  virus and confirmed by using Sanger sequencing . The 2 pnt virus . HoweverOf 35 respiratory samples tested, 6 were infected with influenza A(H3N2), 2 with influenza A(H1N1) virus, 6 with influenza A(H1N1)pdm09 virus, 4 with parainfluenza virus, 4 with human rhinovirus, 4 with human coronavirus, 5 with influenza B virus, and 4 with respiratory syncytial virus. In addition, 6 other respiratory samples were virus negative. Cross-amplification was not observed during sample testing. Thus, the assay is highly specific for detecting the mutant NA gene of influenza A(H7N9) virus.4 copies/reaction were prepared at the following ratios of mutant virus to wild-type virus: 2:98, 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, and 98:2. The ΔCtK – R of the mixture a ratio of 50:50 was used as the assay-specific normalization value in determination of the percentage of 292K mutant in mixed population as described by Liu et al.  virus who had various disease outcomes . The timThe 7 samples that contained the 292K mutant were obtained from 4 patients: 2 patients who died (patients 2 and 3) and 2 patients who recovered (patients 8 and 10). In our previous study, sequencing of the NA gene was not successful for the first throat swab specimens from patients 8 and 10 because of low viral load  binding preference, which favor greater replication in the lower respiratory tract. A similar phenomenon was observed in patients infected with the HA D222G mutant of influenza A(H1N1)pdm09 virus; this virus showed preferential replication in the lower respiratory tract and this infection was correlated with severe outcomes or deaths  virus. The sensitivity of the assay is lower than that of an HA7-specific real-time RT-PCR  virus were not detected by this RT-PCR). However, as a screening tool, this assay is sensitive, specific, fast, and inexpensive.This assay had a detection threshold of 10% for a mutant strain in a mixed viral population, which is more sensitive than Sanger sequencing  (PCR for detection of oseltamivir resistance in influenza A(H7N9) virus.


Bactrocera caudata is a pest of pumpkin flower. Specimens of B. caudata from the northern hemisphere (mainland Asia) and southern hemisphere (Indonesia) were analysed using the partial DNA sequences of the nuclear 28S rRNA and internal transcribed spacer region 2 (ITS-2) genes, and the mitochondrial cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit II (COII) and 16S rRNA genes. The COI, COII, 16S rDNA and concatenated COI+COII+16S and COI+COII+16S+28S+ITS-2 nucleotide sequences revealed that B. caudata from the northern hemisphere  was distinctly different from the southern hemisphere , without common haplotype between them. Phylogenetic analysis revealed two distinct clades (northern and southern hemispheres), indicating distinct genetic lineage. The uncorrected ‘p’ distance for the concatenated COI+COII+16S nucleotide sequences between the taxa from the northern and southern hemispheres (‘p’ = 4.46-4.94%) was several folds higher than the ‘p’ distance for the taxa in the northern hemisphere (‘p’ = 0.00-0.77%) and the southern hemisphere (‘p’ = 0.00%). This distinct difference was also reflected by concatenated COI+COII+16S+28S+ITS-2 nucleotide sequences with an uncorrected 'p' distance of 2.34-2.69% between the taxa of northern and southern hemispheres. In accordance with the type locality the Indonesian taxa belong to the nominal species. Thus the taxa from the northern hemisphere, if they were to constitute a cryptic species of the B. caudata species complex based on molecular data, need to be formally described as a new species. The Thailand and Malaysian B. caudata populations in the northern hemisphere showed distinct genetic structure and phylogeographic pattern. Bactrocera (Zeugodacus) caudata (Fabricius 1805) is a pest of pumpkin infesting the host at the flowering stage  as descAGGTT -3'. The COIB. caudata from the GenBank . The first 200 trees from each run were discarded as burn-in , and the remaining trees were used for the construction of a 50% majority-rule consensus tree. The effective sample size for COI+COII+16S was 1374.35 and for COI+COII+16S+28S+ITS-2 was 9268.35.Kakusan v.3  was usedhttp://www.fluxus-engineering.com). PAUP* 4.0b10 software [The median joining (MJ) network  was usedsoftware  was usedThe number of haplotypes based on the median-joining (MJ) network of the individual and concatenated aligned nucleotide sequences (outgroup was not included) is shown in Of the four COI haplotypes, C1 was the most common among the populations of Peninsular Malaysia and East Malaysia. Haplotype C2 occurred only in Indonesia, C3 in Pulau Tioman  and C4 in Thailand. The largest difference of 37 bp was found between haplotypes C2 (Indonesia) and C4 (Thailand). Of the eight COII haplotypes, D1 was the most common. The Indonesian taxa were characterized by haplotypes D3 and D8. The largest difference of 28 bp was between haplotypes D3 (Indonesia) and D5 (Thailand). Three 16S haplotypes were present, with haplotype R2 present only in Indonesia. The largest difference of 13 bp was between haplotypes R2 (Indonesia) and R3 .The concatenated COI+COII+16S rDNA nucleotide sequences yielded eight haplotypes, with haplotypes M3 and M8 present only in Indonesia. The largest difference of 79 bp was between haplotypes M3 (Indonesia) and M8 (Thailand). A great diversity of haplotypes (12) was evident in the concatenated COI+COII+16S rDNA+28S rDNA+ITS-2 nucleotide sequences. The Indonesian taxa were characterized by haplotypes N4 and N12, with N4 having 82-bp difference from N11 (Thailand).The total length of the aligned sequences of 28S rRNA, ITS-2, COI, COII, 16S rRNA, 28S+ITS-2+COI+COII+16S rRNA, and COI+COII+16S rRNA genes as well as the selected models used for ML and BI analyses are summarized in B. caudata varied from one marker to the others. The nucleotide sequences of the slower-evolving nuclear ITS-2 and 28S rRNA genes could not differentiate unequivocally the taxa of B. caudata from northern  and southern (Indonesia) hemispheres  taxon differed from the other northern hemisphere  taxa with uncorrected 'p' distances of 0.56–0.77%; the 'p' distance for the Malaysian taxa was 0.00–0.21%. The uncorrected 'p' distance between the southern hemisphere (Indonesia) and Malaysian taxa was 4.46–4.53%, and 4.94% between Indonesian and Thailand taxa.For concatenated COI+COII+16S+28S+ITS-2 nucleotide sequences, the uncorrected 'p' distance between Indonesian and Malaysian taxa was 2.34–2.50% and between Indonesian and Thailand taxa 2.69%. The uncorrected 'p' distance for Malaysian taxa was 0.00–0.19%, and between Malaysian and Thailand taxa 0.34–0.41%.B. caudata from the northern  and southern (Indonesia) hemispheres form clearly two distinct genetic lineages  [B. caudata species complex based on molecular data, need to be formally described as a new species.The occurrence of two distinct genetic lineages of the taxa attributed to he wing) ,2 indicaB. caudata in the northern hemisphere had an uncorrected 'p' distance of 0.56–0.77%, compared to 'p' = 0.00–0.21% for the Malaysian taxa. The distinct difference is also reflected by the concatenated COI+COII+16S+28S+ITS-2 nucleotide sequences. It indicates the occurrence of distinct genetic structure and phylogeographic pattern of the B. caudata populations in the northern hemisphere.In the present study, based on concatenated COI+COII+16S rDNA nucleotide sequences the Thailand and Malaysian taxa of S1 FileBactrocera species from various localities. Table B in S1 File. Variation sites in DNA sequences for different haplotype of mitochondrial COII of Bactrocera species from various localities. Table C in S1 File. Variation sites in DNA sequences of Bactrocera species for mitochondrial 16S rDNA from various localities. . Table D in S1 File. Variation sites in DNA sequences of Bactrocera species for 28S rDNA from various localities. Table E is S1 File. Variation sites in DNA sequences of Bactrocera species for ITS-2 from various localities. Table F is S1 File. Percentage of uncorrected “p” distance matrix of Bactocera caudata from various geographical locations in northern and southern hemispheres based on COI. Table G in S1 File. Percentage of uncorrected “p” distance matrix of Bactocera caudata from various geographical locations in northern and southern hemispheres based on COII. Table H in S1 File. Percentage of uncorrected “p” distance matrix of Bactocera caudata from various geographical locations in northern and southern hemispheres based on 16SrDNA. Table I in S1 File. Percentage of uncorrected “p” distance matrix of Bactocera caudata from various geographical locations in northern and southern hemispheres based on 28S rDNA. Table J in S1 File. Percentage of uncorrected “p” distance matrix of Bactocera caudata from various geographical locations in northern and southern hemispheres based on ITS-2.Table A in S1 File. Variation sites in DNA sequences for different haplotype of mitochondrial COI of (DOCX)Click here for additional data file.


Several reader domain proteins that specifically recognize methyllysine-containing histones contain the negatively-charged aspartate or glutamate residues as part of the aromatic cage. Herein, we report thermodynamic analyses for the recognition of histone H3K4me3 and H3K4me2 by the tandem tudor domain of Sgf29 and its recognition site variants. Small uncharged and large aromatic substitutions on the Asp266 site resulted in a significant decrease in binding affinities for both H3K4me3 and H3K4me2, demonstrating the role of the negative charge of Asp266 in the readout process by Sgf29. This study emphasizes the essential contribution of electrostatic interactions to the overall binding affinity, and reveals that the underlying mechanisms for the recognition of Kme2/3 depend on the composition and arrangement of the aromatic cage. In order to structure the vast amount of genetic material stored within the cell, several mechanisms of DNA condensation have evolved in eukaryotic organisms. The first level of condensation is achieved by wrapping DNA around a protein assembly of eight histone proteins to form the nucleosome. The hisOne of the most widespread histone PTMs is lysine methylation, which can be found in three different methylation states: monomethylated (Kme1), dimethylated (Kme2) and trimethylated (Kme3). The process of lysine methylation is controlled by three types of proteins, namely histone lysine methyltransferases (writers), histone lysine demethylases (erasers), and histone lysine reader domain proteins , that are capable of specifically recognizing posttranslationally-modified histones,6. A comSgf29 (SAGA-associated Factor 29) is a subunit of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex, a chromatin modifying complex, which is capable of acetylating and deubiquitinating histone proteins.. CrystalThis study examines the molecular basis for the involvement of the negatively-charged aspartate/glutamate in the biomolecular recognition of Kme2/3-containing histone peptides using Sgf29 as a reader domain protein. We chose the tandem tudor domain of Sgf29 for this study for the following reasons: 1) the Kme2/3 recognition site contains three aromatic residues and one negatively-charged Asp266; 2) X-ray crystal or NMR solution structures for Sgf29, Sgf29-H3K4me2 and Sgf29-H3K4me3 complexes have been solved ; 3) bindf-C18 preparative HPLC and the lyophilized peptides were used for binding studies  on position 4 using solid-phase peptide synthesis. The two peptides were purified by R studies  Figs. Wipurified . In ordepurified . Multiplpurified . Collectof Sgf29 . Upon abAdditionally, differential scanning fluorimetry (DSF) was employed as a secondary method to determine the structural stability of the Sgf29 variants , Table 1Kd, free energy of binding ΔG°, enthalpy of binding ΔH°, and entropy of binding -TΔS°) for the association of reader-histone complexes , whereas the entropy of binding is slightly unfavorable  1173.69, found [M+H](TIF)Click here for additional data file.S2 Fig+ 1187.6.Calculated [M] 1187.71, found [M+H](TIF)Click here for additional data file.S3 FigAmino acid substitutions are displayed in single letter code. From left to right: Sgf29 wild-type, D266A, D266E, D266F, D266N, D266W, D266Y, Y238F and Y245F, respectively.(TIF)Click here for additional data file.S1 TableMutations are underscored and codons are indicated in grey.(DOCX)Click here for additional data file.


Agropyron cristatum (L.) Gaertn. , a wild relative of common wheat, possesses many potentially valuable traits that can be transferred to common wheat through breeding programs. The wheat-A. cristatum disomic addition and translocation lines can be used as bridge materials to introduce alien chromosomal segments to wheat. Wheat-A. cristatum 2P disomic addition line II-9-3 was highly resistant to powdery mildew and leaf rust, which was reported in our previous study. However, some translocation lines induced from II-9-3 have not been reported. In this study, some translocation lines were induced from II-9-3 by 60Co-γ irradiation and gametocidal chromosome 2C and then identified by cytological methods. Forty-nine wheat-A. cristatum translocation lines were obtained and various translcoation types were identified by GISH (genomic in situ hybridization), such as whole-arm, segmental and intercalary translocations. Dual-color FISH (fluorescent in situ hybridization) was applied to identify the wheat chromosomes involved in the translocations, and the results showed that A. cristatum 2P chromosome segments were translocated to the different wheat chromosomes, including 1A, 2A, 3A, 4A, 5A, 6A, 7A, 3B, 5B, 7B, 1D, 4D and 6D. Many different types of wheat-A. cristatum alien translocation lines would be valuable for not only identifying and cloning A. cristatum 2P-related genes and understanding the genetics and breeding effects of the translocation between A. cristatum chromosome 2P and wheat chromosomes, but also providing new germplasm resources for the wheat genetic improvement. Triticum aestivum L.), widely planted in different parts of the world, is the third most important cereal behind maize and rice. However, modern breeding was challenged by the narrow genetic variation [Agropyron cristatum chromosome 6P with the genes controlling large numbers of florets and kernels per spike and multiple fertile tiller numbers per plant [Dasypyrum villosum chromosome 1V including the seed storage protein genes [Thinopyrum ponticum containing stem rust resistance gene Sr43 on chromosome 7E [Psathyrostachys huashanica chromosome 3Ns with the gene(s) for resistance to stripe rust [Wheat . Therefore, the production of wheat-A. cristatum 2P translocation lines may transfer genes conferring resistance to powdery mildew and leaf rust from chromosome 2P into wheat.It was observed that -A. cristatum 2P translocation lines induced by 60Co-γ irradiation and gametocidal chromosomes 2C, and identify and characterize these translocation lines by GISH/FISH. These newly developed translocation lines will not only lay a solid groundwork for taking advantage of desirable genes on the chromosome 2P for wheat improvement, but also provide novel germplasms and valuable materials for studying gene expression, balance and interaction between different 2P chromosome segments of A. cristatum and common wheat.The aim of this study was to produce various types of wheatA. cristatum 2P disomic addition line II-9-3 (2n = 44) was obtained by hybridization between A. cristatum accession Z559  and Triticum aestivum cv. ‘Fukuhokomugi’  and was inherited stably through several generations of backcrosses. This line was obtained and provided by Dr. Lihui Li of Chinese Academy of Agricultural Sciences. T. aestivum cv. ‘Chinese Spring’-Aegilops cylindrica Host 2C addition lines (CS-G2C) (2n = 44) were provided by Professor Jilin Li of Harbin Normal University, China.Wheat-A. cristatum 2P disomic addition line II-9-3 was overwintered in the field and transplanted into pots before jointing. The plants at the booting stage were irradiated with 60Co gamma rays at a dose of 20 Gray (Gy) and a dose rate of 0.5 Gy/min at the cobalt source chamber of Beijing University [1BC1 population. Pollen collected from ‘Fukuhokomugi’ was used to pollinate the untreated spikes of wheat-A. cristatum 2P disomic addition line II-9-3 as a control.Wheat-iversity . Fresh pA. cristatum 2P disomic addition line II-9-3 as the male parent. Mature hybrid seeds were harvested and selfed to produce a F2 population.Crosses were carried out using the CS-G2C addition line as the female parent and the wheat-All the materials surveyed in this study were planted at the Chinese Academy of Agricultural Science Experiment Station in Beijing  during the 2013–2014 growing season.The exposed seeds were dipped into water in Petri dishes with double moistened filter papers at room temperature for one day, and then the water was absorbed by dry filter papers. The seeds were transferred into a refrigerator at 4°C for 48 h and germinated at 23°C in an incubator. Roots were sampled when the length of the roots was approximately 1.5–2.0 cm; these samples were pretreated in ice water for 24–48 h, fixed in a solution of absolute ethanol-acetic acid  at 4°C for 48 h, and then kept in 70% ethanol solution at -20°C. The chromosome slides were treated with 45% glacial acetic acid . CytologA. cristatum and ‘Fukuhokomugi’ genomic DNA was isolated by a modified CTAB (hexadecyl trimethyl ammonium bromide) method [P-genomic DNA and ‘Fukuhokomugi’ genomic DNA were used as probe and block, at a ratio of 1:40, respectively, to identify the A. cristatum chromosomal fragments. The total genomic DNA of A. cristatum was labelled with digoxigenin-11-dUTP and used as a probe for GISH. The GISH procedure followed that described by Liu et al. [GISH was used to analyse the mitotic metaphase cells of the materials used in this study. ) method . The puru et al. . The GISA. cristatum as probes. The clones pAs1, pHvG39 and pSc119.2 were labelled with digoxigenin-11-dUTP, biotin-16-dUTP and biotin-16-dUTP, respectively, and were used as probes for dual-color FISH. Chromosomes were counterstained with DAPI . The hybridization signals were examined using a Nikon Eclipse E600 (Japan) fluorescence microscope, and the FISH images were captured using a CCD camera. After FISH, the hybridization signals were washed with PBS . The total genomic DNA of A. cristatum was labelled with digoxigenin-11-dUTP as a probe for subsequent GISH. Finally, according to the two hybridization results for the same cell, the homoeologous groups of the translocated wheat chromosomes were analysed by reference to the standard idiogram of the chromosomes of Chinese Spring wheat showing the locations of FISH signals [FISH was used to characterize the translocated wheat chromosome, and dual-color FISH/GISH was employed by using repetitive DNA clones, i.e., pAs1, pHvG39, pSc119.2 and the total genomic DNA of  signals ,35.1BC1 progenies from 60Co-γ irradiation showed that 39 plants had translocations between chromosome 2P and the wheat chromosomes, 204 plants had a whole chromosome 2P and 20 plants had no chromosome 2P. The translocation frequency induced by irradiation was 14.83%.The GISH results were shown in 2 hybrid progenies of the CS-G2C and wheat-A. cristatum 2P addition lines, 51 plants had P chromosomal segments, accounting for 34.46% of the examined plants. Among them, there were 10 translocated plants (6.76%), 4 double P fragments addition plants (2.70%) and 37 single P fragment addition plants (25.00%). At the same time, neither translocations nor any other types of chromosomal structural changes were detected in any of the wheat-A. cristatum 2P disomic addition line/‘Fukuhokomugi’ F1 plants (control). This indicated that the translocation lines in this experiment totally resulted from the irradiation and gametocidal chromosome effect. Moreover, the frequency of alien translocation induced by irradiation was significantly higher than that induced by gametocidal chromosome 2C.Among the 148 F1BC1 and F2 progenies, 49 translocation plants were verified and various translocation types were obtained. The translocation types and frequencies between wheat and A. cristatum 2P chromosomes were shown in P.Among the 411 MP, including one plant (2.04%) with both small and large alien segmental translocation, as well as 2 plants (4.08%) with small alien segmental translocation (P: 2 plants (4.08%) having large alien segmental and whole-arm translocation , 25 small alien segmental translocations (SASTs), 8 intercalary translocations (ITs) and 17 whole arm translocations (WATs) . This re60Co-γ irradiation and gametocidal chromosome 2C had very high efficiency in inducing interstitial breakage. Sixty-six translocations  involved one-breakage fusion events, while only 8 ITs involved two-breakage fusion events. This indicated that the major effect of irradiation and gametocidal chromosome 2C was to induce one-breakage fusion events.The 74 translocated chromosomes involved 82 breakage-fusion events, including 65 in interstitial regions (79.27%) and 17 in centric regions (20.73%) ; Fig 2, A. cristatum chromosome 2P, dual-color FISH/GISH was performed in wheat root-tip cells. The FISH results were shown in A. cristatum alien chromosomal translocation lines. A. cristatum 2P chromosome fragments were translocated with wheat chromosomes 1A, 2A, 3A, 4A, 5A, 6A, 7A, 3B, 5B, 7B, 1D, 4D and 6D.To identify the wheat translocated chromosomes with P genomic DNA and the clone pAs1 labelled with digoxigenin-11-dUTP, the clones pHvG39 and pSc119.2 labelled with biotin-16-dUTP. Plant 2P-23 has 2 translocated chromosomes; the small alien segmental translocation of 2P-23 showed an obvious green fluorescent signal near the centromeric region of the short arm and a faint green signal in the middle of the long arm; the arm ratio is large. Accordingly, it was a 5BS.L-2P translocation with a small 2P fragment. Further, the large alien segmental translocation of 2P-23 exhibited a faint green signal at the end of the arm; it was a 3A-2P translocation with a large 2P fragment  have sufficient pollen. Furthermore, the treatment of booting stage plants in the pots was more convenient than that on spikes. Additionally, chromosomal structural variants can be directly identified in the M1BC1 generation by GISH.Ionizing irradiation has been proven as an effective method to induce chromosomal translocations . Dry seeA. cristatum disomic addition lines [A. cristatum has been developed [A. cristatum disomic addition lines and to produce wheat-A. cristatum alien translocation lines with different breakpoints and P segments of different sizes. The translocation lines can be further used to construct a deletion bin map of each P chromosome.To date, many translocation lines have been screened and identified involving three wheat-on lines –31. A neeveloped . This stA. cristatum has many potentially valuable traits that can be used in wheat improvement, so it is important to produce wheat-A. cristatum 2P small-segment translocations to transfer useful genes to wheat for broadening wheat genetic diversity. Huang et al. [A. cristatum 6P small intercalary translocation lines. These lines did not carry undesirable genes and had a good compensation effect of A. cristatum chromatin, which further confirmed that the wheat-A. cristatum small alien segments translocations were desirable. Twenty-five small fragment alien translocations were obtained in the present study by ionizing irradiation and gametocidal chromosome 2C, which would make it possible to utilize desirable genes from A. cristatum 2P chromosome.The transfer of desirable genes from the tertiary gene pool could be an efficient way to increase genetic diversity and improve cultivated wheat ,48. Trang et al.  identifiAgropyron are cross-pollinating plants. The tetraploid A. cristatum derived from the hybridizations between diploid A. cristatum and A. mongolicum. Although the diploid A. cristatum and A. mongolicum contained the same basic P genome, their P genomes exhibited rearrangements and variation [P genomes by identifying four different types of wheat-A. cristatum 6P disomic addition lines and speculated that genomic rearrangements may occur in the wheat-A. cristatum addition line. Accordingly, we speculated that A. cristatum chromosome 2P may have genetic arrangement to some extent. Although the A. cristatum chromosome 2P fragment was translocated to wheat homoeologous groups  in this study, some wheat-A. cristatum 2P translocation lines were not agronomically poor in the field environment. Thus, we could not exclude the possibility that the wheat-A. cristatum 2P translocation lines may have a subordinate complementary relationship with wheat homoeologous groups  and that the offspring may have a partial compensation effect because of genomic rearrangement. The present study showed that A. cristatum chromosome 2P was translocated to all three wheat genomes and that the wheat A genome had the highest recombination frequency. The results were inconsistent with previous studies, which showed that the wheat B genome possessed the highest number of chromosome arrangements [P chromosome segment could be transferred to different wheat chromosomes/genomes. Different 2P chromosome segments could also be transferred to the same wheat chromosome/homoeologous group. These lines would be useful in studies to better understand recombination, interaction and genetic balance between wheat chromosomes and the A. cristatum 2P chromosome. In addition, they could provide a theoretical basis for the utilization of desirable genes of the chromosome 2P in wheat breeding.Many wheat-alien chromosomal translocation lines carrying desirable genes have been reported in recent years ,54,55, bariation ,59. Han ariation  detectedngements . The traBlumeria graminis f. sp. tritici (Bgt) and Puccinia recondita f. sp. tritici, respectively, are two devastating diseases that cause severe yield losses in most of the wheat production areas [A. cristatum 2P addition line possessed high resistance to powdery mildew and leaf rust compared with ‘Fukuhokomugi’ in our laboratory (Submitted). By preliminary phenotypic evaluation, newly developed 2P alien translocation lines 2P-173 and 2P-205 were highly resistant to powdery mildew Click here for additional data file.S2 FigA. cristatum alien 2P disomic addition line II-9-3. 2: Wheat-A. cristatum 2P alien translocation line 2P-205. 3: Fukuhokomugi.1: Wheat-(TIF)Click here for additional data file.


Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease characterized by pancreatic beta-cell destruction, hyperglycemia and progressive insulin deficiency, affecting mainly children and adolescents genetically predisposed. About 10% (2.4-16.4%) of T1D individuals develop celiac disease (CD), an immune-mediated enteropathy triggered by gluten exposure. Both diseases have a common autoimmune origin and share a similar genetic Background, the major histocompatibility complex class II antigen (HLA-DQ). Some genetic association studies have also identified susceptibility polymorphisms non-HLA associated to both diseases, located in different genes: RGS1 (rs2816316), IL2-IL21 (rs6822844), BACH2 (rs11755527) and IL18RAP (rs917997).The aim of the present study was to determine the allelic and genotypic frequencies of polymorphisms in RGS1, IL2-IL21, BACH2 and IL18RAP genes in a sample of 317 T1D individuals and to compare the frequency of the risk alleles in patients with negative (N=264) or positive (N=53) serology for CD.Saliva or blood sample was collected and DNA extraction and genotyping performed by PCR Real-Time. All polymorphisms were in Hardy-Weinberg equilibrium. The comparison between the allele and genotype frequencies was calculate by Chi-square and Fisher's exact test.The frequency of the allele risk for the genes RGS1 ; IL2-IL21 ; BACH2  e IL18RAP  in T1D individuals and negative serology for CD was respectively: 95.5%, 97.4%, 79.8% and 46.6%. In T1D with seropositive, the frequency of the same alleles were: 98.1%(p=0.703), 100.0% (p=0.604), 86.0% (p=0.433) and 46.2%(p=1.000). Both genotype and allele frequencies were not significantly between T1D with negative or positive serology for CD.Our data did not evidence differences between the polymorphisms non-HLA analyzed in T1D with or without seropositive for CD, being not possible to assure that the presence of these polymorphism increase or decrease the predisposition to celiac disease.