The exact mechanisms of interplay between host and viral factors leading to severe dengue are yet to be fully understood. Even though previous studies have implicated specific genetic differences of Dengue virus (DENV) in clinical severity and virus attenuation, similar studies with large-scale, whole genome screening of monophyletic virus populations are limited. Therefore, in the present study, we compared 89 whole genomes of DENV-2 cosmopolitan clade III isolates obtained from patients diagnosed with dengue fever , dengue hemorrhagic fever  and dengue shock syndrome  in Singapore between July 2010 and January 2013, in order to determine the correlation of observed viral genetic differences with clinical outcomes. Our findings showed no significant difference between the number of primary and secondary infections that progressed to DHF and DSS (p>0.05) in our study cohort. Despite being highly homogenous, study isolates possessed 39 amino acid substitutions of which 10 substitutions were fixed in three main groups of virus isolates. None of those substitutions were specifically associated with DHF and DSS. Notably, two evolutionarily unique virus groups possessing C-P43T+NS1-S103T+NS2A-V83I+NS3-R337K+ NS3-I600T+ NS5-P136S and NS2A-T119N mutations were exclusively found in patients with DF, the benign form of DENV infections. Those mutants were significantly associated with mild disease outcome. These observations indicated that disease progression into DHF and DSS within our patient population was more likely to be due to host than virus factors. We hypothesize that selection for potentially less virulent groups of DENV-2 in our study cohort may be an evolutionary adaptation of viral strains to extend their survival in the human-mosquito transmission cycle. Dengue fever is the most widespread arbovirus disease at present with an annual estimate of 50 million infections worldwide . The disDENV is a single stranded positive sense RNA virus with a 11.8 kb genome flanked by two untranslated regions 5’ and 3’ UTRs) and a single coding region for three structural: capsid (C), the precursor of membrane (prM) and envelope (E); and seven non-structural (NS) proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 ’ UTRs an. Even thOne of the classical examples of genotype-based severity of dengue is the transition from mild to severe dengue outbreaks subsequent to emergence of a DENV-2 genotype of Asian origin in Latin and central America . AccordiThis study was part of the Early Dengue Infection and Outcome (EDEN) and Prospective Adult Dengue (PADS) studies under the STOP Dengue Translational Clinical Research Flagship Program. The National Healthcare Group Domain Specific Review Board approved the study .The study included 89 patients infected with DENV-2 between July 2010 and January 2013 in Singapore during which DENV-2 was the dominant serotype . In overDENV infections were classified as DF, DHF and DSS based on the World Health Organization (WHO) 1997 guidelines . PatientViral RNA was extracted from patient sera/plasma by using the QIAamp Viral RNA Mini Kit  according to manufacturer’s instructions. Detection and serotyping of DENV was achieved by amplifying viral RNA using a quantitative real-time polymerase chain reaction (PCR) protocol described previously . CrossinAe. albopictus clone C6/36 mosquito cell line (ATCC CRL-1660). Briefly, the monolayer of C6/36 was inoculated with serum in Leibowitch L-15 medium  supplemented with 3% FCS at 33°C to allow for virus adsorption and replication. The infected fluid was harvested after 4–8 days. The presence of DENV in cell supernatants was confirmed by an immunofluorescent assay (IFA) using DENV group-specific and serotype-specific monoclonal antibodies derived from hybridoma cultures . The fluorescein isothiocyanate-conjugated goat anti-mouse antibody was used as the detector. A maximum of two passages was performed for each sample based on the original virus load.DENV was isolated from sera/plasma using the All virus strains isolated from patient sera were subjected to whole genome sequencing. RNA was reverse transcribed using the Superscript III First-strand Synthesis System . Each DENV-2 whole genome was amplified in 6 fragments, together with 5’ and 3’ untranslated regions (UTRs) using the Phusion Flash High-Fidelity PCR Master Mix  as described elsewhere . AmplifiContiguous sequences were assembled using the Lasergene package version 8.0 . The resulting sequences were aligned in the BioEdit Sequence Alignment Editor version 7.0.9.0 .The phylogeny of study sequences was inferred by the maximum-likelihood (ML) method based on the General Time Reversible (GTR) substitution model and gamma distributed rates with invariant sites in MEGA 6.06 . The robGenome-wide dN/dS ratios were computed using Hyphy package in the Datamonkey web server . The sinhttp://mfold.rna.albany.edu/?q=mfold under standard conditions (37°C) [The secondary structures of the variable region of 3’ UTR and complete 5’ UTR of wild type (NS2A-V83) and mutant (NS2A-V83I) variants of DENV-2 cosmopolitan clade III were predicted using the mfold web server at s (37°C) .The statistical significance of the association of primary and secondary infections within the DHF and DSS categories was calculated using the exact binomial test. Student’s t-test was used to calculate the significance of age, number of days of fever at presentation and crossing point values (virus titres) between DF and DF/DSS categories. Student’s t-test was also used to determine any statistically significant differences of crossing point values  between mutants and non-mutants.In order to determine relationships among the three variables; substitutions, clinical outcome (DF and DHF/DSS) and primary/secondary infection status, we first used Chi-squared and Fisher’s exact tests under the null hypothesis that each variable is independent from one another. The data were organized into 2-way tables to estimate the association between any two variables as compared to the third variable. Next, in order to understand the association between individual mutants and clinical outcome in both primary and secondary infections, we calculated relative odds of the occurrence of severe clinical outcome (DHF/DSS) upon exposure to a specific mutant compared to non-mutants using the Cochran-Mantel-Haenszel test. In parallel, the confounding effect of primary/secondary infection in clinical outcome was also addressed through the Breslow and Day test. These two tests are widely used to test the strength of association after controlling for the observed confounders. The data was stratified into multiple 2×2×2 tables by the defined confounders. Odds ratio was calculated to estimate the increased/decrease odds of the pre-defined outcome given the condition of any mutant compared to non-mutants. All the statistical analyses were carried out in R software package version 2.15 . ProbabiOf 89 patients recruited, 58 patients developed DF and the remaining developed either DHF (n = 30) or DSS (n = 1). Of them, 45 patients had secondary dengue infections and 27 patients had primary infections . The priE) gene-based phylogeny revealed that 89 virus strains isolated from patient sera belonged to a monophyletic group (clade III) of cosmopolitan genotype (Envelope (genotype . At compgenotype . Twenty genotype . The remgenotype .The largest group (n = 20) possessed NS3-R337K substitution. There were six additional substitutions fixed in the NS3-R337K group; C-P43T (n = 19), NS1-S103T (n = 18), NS2A-V83I (n = 15), NS3-I600T (n = 4), NS5-P136S (n = 4) and NS5-N645D (n = 3). It was noteworthy that all substitutions (C-P43T+NS1-S103T+NS2A-V83I+NS3-R337K+ NS3-I600T+ NS5-P136S), except NS5-N645D, were detected in NS2A-V83I mutants. While C-P43T+NS1-S103T+NS2A-V83I+NS3-R337K signature was fixed, NS3-I600T (n = 4) and NS5-P136S (n = 4) substitutions were detected only in two separate subsets of NS2A-V83I mutants. In addition, NS2A-V83I mutants (n = 15) possessed two fixed nucleotide substitutions in UTRs (5’UTR- T34C and 3’UTR-A19T). The second largest group possessed NS2A-L153S substitution (n = 10) of which nine isolates shared an additional substitution in the E protein (E-V164I). The third group (n = 6) was characterized by a substitution fixed in the NS2A protein (NS2A-T119N).2 = 39.016, df = 15, chi-squared and fishers’ exact p-values = <0.001), suggesting that observed substitutions had an effect on the clinical outcome of infections. There was no such association between the clinical outcome and primary/secondary infection status  and substitutions and primary/secondary infection status .None of the mutants described above  showed virus titres that were statistically different from those of non-mutants (data not shown). However, there was a statistically significant association between the clinical outcome and substitutions observed in our study  and secondary (n = 8) infections, but all DHF/DSS cases were secondary infections . InteresRelative odds of the occurrence of severe clinical outcome (DHF/DSS), given exposure to a specific mutant compared to non-mutants were calculated using the Cochran-Mantel-Haenszel test. In parallel, the confounding effect of primary/secondary infection in clinical outcome was addressed through the Breslow and Day test. Odds ratio was calculated to estimate the increased/decrease odds of the pre-defined outcome given the condition of any mutant compared to non-mutants. Data is not available (NA) for substitutions that were present only in isolates from DF cases. Odds ratio could not be calculated for NS1-V286A substitution as all DF and DHF cases due to this mutant were secondary infections.On the other hand, we observed several additional amino acid substitutions in viruses isolated from DHF  and DF  cases. Even though there was an apparent dominance of above substitutions in E and NS2A proteins in DHF and DF cases respectively, it was not able to determine any potential role of those substitutions in clinical outcome because most of them were detected in individual infections.The comparative whole genome analysis between NS2A-V83I mutants and those reported in GenBank database revealed that C-P43T, NS1-S103T, NS3-R337K and NS5-P136S substitutions were novel. Moreover, NS2A-V83I substitution was found only in two records of American Asian genotype (FJ850060 and FJ873808) from Nicaragua and was unique within the cosmopolitan genotype. Similarly, NS3-I600T substitution has been reported in only one isolate from Puerto Rico (EU482720). Nucleotide positions of 3’UTR-A19 and 5’UTR-T34 were extremely conserved residues in DENV-2 cosmopolitan clade III isolates, except for NS2A-V83I mutants. Similarly, the residue NS2A-119 is highly conserved with serine in all DENV-2 cosmopolitan clade III isolates reported before this study. The high bootstrap support gained by both NS2A-V83I (92%) and NS2A-T119N (94%) mutants in the phylogenetic analysis attested to their genetic distinction . MoreoveThe present study was carried out during a period (July 2010-January 2013) of DENV-2 dominance  in SingaFlavivirus NS2A is a 22 KDa hydrophobic protein  that intThe first group of isolates (n = 15) possessed their signature mutation at residue 83 of NS2A protein (NS2A-V83I) . AccordiAlternatively, the less virulent phenotype observed in NS2A-V83I mutants may be due to specific interactions between NS2A-V83I substitution and the rest of coexisting amino acid substitutions fixed in all isolates (C-P43T+NS1-S103T+NS3-R337K) and sub-groups (NS3-I600T+ NS5-P136S) of the mutant group. Of them, C-43 is a highly conserved non-polar, hydrophobic residue with proline in many medically important mosquito-borne flaviviruses . The delNS3-I600T, on the other hand, is a non-conservative change that resides at the C-terminal of domain III of DENV helicase . A recenet al (2011) showed that A30P substitution, together with substitutions at six other residues including 119 of NS2A protein, contributed to a dramatic reduction in the virulence and cytopathicity of West Nile virus [The second group of NS2A mutants (n = 6) that were exclusively associated with a mild disease outcome in our study, possessed NS2A-T119N substitution. NS2A-119 is a hydrophilic residue exposed within the ER lumen during DENV replication . Althougle virus . NS2A-T1le virus .Besides the coding genome, flavivirus UTRs are also important elements of RNA synthesis and virus replication . The 96-The approximately 450 nucleotide long DENV 3’UTR is divided into three domains; hyper-variable, semi-variable and conserved domains . The criOf 39 amino acid substitutions detected, 29 substitutions were found either in individual or small groups (2–3 isolates) of isolates . Of themde-novo RNA synthesis [In flaviviruses, N-terminal capsid sequence encodes for essential functional domains of virus replication such as 5’ cyclization sequence , hairpinynthesis . Of themynthesis . In addiynthesis . These oHost immune response is considered to be an important factor that shapes the clinical outcome of DENV infections. Both T- and B-cell responses play a role in this process through production of cytokines and virus neutralizing antibodies , 81. It Empirical evidence has shown that the specificity of T-cell response against DENV is broad, but primarily directed against the NS3 and NS5 proteins –91. BesiLikewise, the specificity of humoral immune response to DENV also appears broad. Antibodies to C, prM, E, NS1, NS3, NS4a and NS4b proteins have previously been detected in DENV-infected patient sera and epitope mapping has identified several immunogenic regions in respective proteins , 98. BeiOn NS1, previous studies have revealed five immuno-dominant B-cell epitopes , which bind DENV type-specific and group-specific antibodies . On C anAe. aegypti hosts, the primary vector of DENV, in order to decipher the role of DENV diversity in shaping dengue epidemiology.Our findings indicated that subtle genetic differences in a highly homogenous, monotypic DENV-2 population potentially modified the clinical outcome of our study subjects, regardless of primary and secondary infection status. Interestingly, none of the fixed genetic differences in our study isolates was specifically associated with a severe disease outcome. This observation indicated that disease progression into DHF and DSS in our patient cohort was more likely to be due to host than virus factors. However, our observations were made in relatively a small group of patients and further investigations are required, preferably in large cohorts, to determine how the observed genetic differences regulate virus replication and modulate immune response in infected individuals. It should also be noted that we did not explore variations in the strain-dependent immune reactions against different groups of virus strains within our patient cohort that can potentially influence the clinical outcome through heterotypic antibody responses , 116. MoS1 TableIsolate IDs in bold letters are dengue hemorrhagic fever (DHF) cases. Those in bold and underlined are dengue shock syndrome (DSS) cases. £Infection status is indicated in brackets; 10 = primary, 20 = secondary, DF = dengue fever.(DOC)Click here for additional data file.


IL17A and IL17F genes and RA.Interleukin-17 plays important role in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to examine the associations between polymorphisms in the IL17A (rs2275913) and IL17F  genes were genotyped using TaqMan genotyping assays from Life Technologies Genomic.We examined 422 RA patients and 337 subjects as a control group. Single nucleotide polymorphism (SNP) in the IL17A and IL17F genotypes and alleles between RA patients and control group. There were no significant associations between IL17A and IL17F genotypes and age of disease diagnosis rheumatoid factor, erosive disease as well as extra-articular manifestations.There were no significant differences in distribution of IL17A and IL17F gene polymorphism are not the important factors associated with susceptibility and some clinical parameters of RA in a Polish population.The results of this study suggest, that  Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with the destruction of affected joints. Numerous cytokines play the important role in the RA pathogenesis, which initiate and maintenance the inflammatory response in joints.Interleukin-17A is one member of a cytokine family consisting of six cytokines: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. Both IL-17A and IL-17F are secreted by Th17 cells and other immune cells, including innate lymphoid cells . Both ILH-17 cells to act as a bridge between innate and adaptive immune cells [IL-17A and IL-17F bind to a receptor complex consisting of two IL-17RA chains and one IL-17RC subunit. IL-17RA is highly expressed on hematopoietic cells, but also on osteoblasts, fibroblasts, endothelial cells, and epithelial cells. In humans, IL-17RA can form a heterodimer with IL-17RC that binds human IL-17A and IL-17F . All IL-ne cells .IL17 gene several polymorphisms have been detected, that may influence the expression of IL-17. The aim of this study was to examine the association between the polymorphisms in IL17A and IL17F genes and rheumatoid arthritis.The prevalence of IL-17 cells is increased in the circulation of patients with RA; these cells produce higher quantities of IL-17 after stimulation . IL-17 iWe examined 422 patients  with rheumatoid arthritis diagnosed according to the criteria of American College of Rheumatology/European League against Rheumatism . ConsentIL17A (rs2275913) and IL17F  were genotyped using TaqMan genotyping assays from Life Technologies Genomic. Fluorescence data were captured using a 7500 FAST Real-Time PCR System .DNA was extracted from 200 μL of whole blood samples using a GeneMATRIX Quick Blood DNA Purification Kit . SNPs within the 2 calculation. P < 0.05 was considered statistically significant. The power of the study to detect an association of the analyzed SNPs with presence of RA was estimated using the PS program ver. 3.0.43. The study sample size was sufficient to detect with 80 % probability the true effect size of differences in allele frequencies between groups measured as odds ratio (OR) equal to 0.736 or 1.347 for rs2275913, 0.302 or 2.106 for rs763780, 0.435 or 1.822 for rs11465553 and 0.593 or 1.543 for rs2397084.Chi-square or Fisher exact tests were used to compare genotype and allele frequencies between the study groups and to analyze associations of clinical characteristics of RA patients with genotypes. Age at onset of RA was compared between genotype groups with Kruskal-Wallis test. Haploview 4.2 software was used for haplotype analysis, D’ and rIL17A and IL17F genotypes were in Hardy-Weinberg equilibrium (HWE) and are shown in Table IL17A and IL17F genotypes and alleles between RA patients and control group.The distributions of IL17A and IL17F genotypes and age of disease diagnosis, rheumatoid factor, erosive disease as well as extra-articular manifestations  and 3 pairs in strong linkage disequlibrium  to produce vascular endothelial growth factor (VEGF) . MoreoveIn mice model of arthritis IL-17 enhanced synovial inflammation, and joint destruction . AdditioThe inhibition of IL-17 also significantly reduced bone erosion in a mouse experimental arthritis model by reducing the levels of RANKL, IL-1, and TNF-α . The bonIL17 gene polymorphisms were not widely investigated in RA patients. Nordang et al. examined the association between IL17A gene polymorphisms and RA in patients from Norway and New Zealand [So far  Zealand . A weak IL17) genotypes with age of disease onset and radiographic progression in Japanese patients with early rheumatoid arthritis (RA) [IL17 gene (rs3804513) polymorphism was associated with radiographic progression at 2 years.Furuya et al. studied the associations between human leukocyte antigen DRB1 (HLA-DRB1) shared epitope, RANK, RANK ligand, osteoprotegerin (OPG), and interleukin 17 (tis (RA) . IL17 geIL17F gene in Polish RA patients [IL17F gene were not correlated with susceptibility to RA in Polish population. However, the IL17F (rs763780) polymorphism was associated with parameters of disease activity, such as number of tender joints, HAQ score or DAS-28-CRP. Moreover, IL17F gene (rs2397084) polymorphism correlated with longer disease duration in patients with RA.Paradowska-Gorycka et al. investigated the association between RA and  polymorphism of patients . OverallIL17A and IL17F gene polymorphisms were not the factors associated with susceptibility to RA, moreover there were not the statistically significant associations between these polymorphisms and age of disease diagnosis, rheumatoid factor, joint erosions, extra-articular manifestations.In our study IL17A and IL17F gene polymorphism are not the important factors associated with susceptibility and some clinical parameters of RA in a Polish population. Nevertheless this hypothesis requires further investigations.The results of this study suggest that The study was approved by the local ethics committee, written informed consent was obtained from all subjects .Not applicable.The raw data for this study is available on request from the corresponding author.


Aeromonashydrophila strain AH-1 (serotype O11) were found to be glycosylated. Top-down mass spectrometry studies of purified polar flagellins suggested the presence of a 403 Da glycan of mass. Bottom-up mass spectrometry studies showed the polar flagellin peptides to be modified with 403 Da glycans in O-linkage. The MS fragmentation pattern of this putative glycan was similar to that of pseudaminic acid derivative. Mutants lacking the biosynthesis of pseudaminic acid (pseB and pseI homologues) were unable to produce polar flagella but no changes were observed in lateral flagella by post-transcriptional regulation of the flagellin. Complementation was achieved by reintroduction of the wild-type pseB and pseI. We compared two pathogenic features (adhesion to eukaryotic cells and biofilm production) between the wild-type strain and two kinds of mutants: mutants lacking polar flagella glycosylation and lacking the O11-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. Results suggest that polar flagella glycosylation is extremely important for A. hydrophila AH-1 adhesion to Hep-2 cells and biofilm formation. In addition, we show the importance of the polar flagella glycosylation for immune stimulation of IL-8 production via toll-“like” receptor 5 (TLR5).Polar and but not lateral flagellin proteins from  Aeromonas spp. strains are important pathogens of humans and lower vertebrates, including amphibians, reptiles, and fish [Aeromonashydrophila, A. veronii biovar veronii, or sobria are described as virulent for humans [O-antigen lipopolysaccharide (LPS) (serotype O11). This has a known chemical structure containing O-polysaccharide chains of homogeneous chain length [ca. 52,000, which forms the S layer that lies peripheral to the cell wall [Aeromonas spp. [Mesophilic and fish . Infectiand fish . Strainsr humans  and fishr humans ; these sn length . In addiell wall . The strAeromonas represents an important advantage, allowing bacteria to move towards favorable conditions or avoid unfavorable environments, and it allows it to successfully compete with other microorganisms [Aeromonas spp. constitutively express a single polar flagellum, and approximately 60% express numerous lateral flagella when grown in viscous environments or on surfaces [Aeromonas spp. are involved in adherence to both biotic and abiotic surfaces, as well as in the biofilm formation [Flagella motility in rganisms . The messurfaces ,9. Severormation .O-glycosylation), or to asparagine residues (N-glycosylation). A recent review gives an overview of O-glycosylation in bacterial systems [O-glycosylation genes involved and their location [One of the most common protein post-translational modifications is glycosylation, which for many years was thought to be a solely eukaryotic mechanism. More recently, many different protein glycosylation systems have been largely identified in all forms of life including prokaryotes. Carbohydrates are covalently attached to serine or threonine residues  in this strain. This compares with a single lateral flagellin observed in strain AH-3 [DNA probes from polar flagella region 2 of ila AH-3  allowed region 2  of AH-1 ila AH-3  allowed ain AH-3 .Both polar and lateral flagellins were purified  and theiThe MS spectrum of a purified lateral flagellin showed a multiply charged ion envelope, as shown in A. hydrophila AH-3 [These masses corresponded almost exactly to the predicted masses of the lateral flagellin proteins, LafA1  and LafA2 . In addition, nLC-MS/MS analyses of tryptic digests of polar flagellins showed 42% and 45% sequence coverage for LafA1 and LafA2, respectively. Manual inspection of the MS/MS data showed no evidence of glycan-related oxonium ions in peptide spectra. Taken together, these data strongly suggested that the flagellins were not post-translationally modified with carbohydrates, as had been reported with lateral flagellins of ila AH-3 .LC-MS analysis of purified polar flagellins produced a characteristic multiply charged protein ion envelope a. When dThe masses of the polar flagellin proteins predicted from the translated gene sequences are 31,413 and 31,361 Da, and neither mass was observed in the deconvoluted mass spectrum. The observed difference between the observed masses, highlighted in m/z = 999.4). The resulting MS/MS spectrum was dominated by an intense ion at m/z = 404.4. An MS3 experiment on the ion observed at m/z = 404.4 produced an MS3 which showed consecutive losses of water and a methyl group from the parent ion . The absence of asparagine residues on all sequenced glycopeptides suggests that the glycan is attached via O-linkage to serine or threonine residues.Due to the suggestion of a putative pseudaminic acid-like glycosylation of AH-1 polar flagella, genes in the biosynthetic pathway of this sugar were mutated. Using oligonucleotides 5′-TCCAGAAGGTTATCGCACT-3′ and 5′-GATGCTGGGAGCTATTACG-3′ with genomic DNA from strain AH-1, an internal DNA fragment (500 bp) corresponding to a pseB homologue was amplified . Genome rmlB mutant lacking O11-antigen LPS [The role of polar flagella glycosylation, lateral flagella, and O11-antigen LPS in the adherence to eukaryotic cells was investigated. The adhesion of several mutants to cultured monolayers of HEp-2 cells was observed. The AH-1Δigen LPS , but witpseB or I that lacked expression of flagella  showed a minimal adhesion values with a 86% reduction compared with the wild type. The same mutants expressed lateral flagella when grown in solid media, and showed an increase in their rate of adherence to Hep-2 cells (rmlB mutant lacking the O11-antigen LPS showed comparatively less reduction in adhesion (29%) compared with the reduced adhesion observed in the flagella mutants. In all the cases, complementation of mutants with the wild-type gene/s showed recovery of the wild-type adhesion values  are unable to form biofilms, with values <0.1, out of the range of detection for the assay . In all cases, biofilm formations of the mutants were fully rescued by the introduction of the wild-type genes  showed biofilm formation ability with an average ODhe assay . The AH-pe genes . MutantsA. hydrophila AH-1 polar flagellins, no IL-8 production was observed (When we stimulated HEK293-null cells (control) with purified observed .A. hydrophila AH-1polar flagellins (wild type) showed varying levels of IL-8 production in agreement with the amount of flagellin used .The degree of TLR5 activation was studied through the production of IL-8 using the HEK293 cell line, which was stably transfected with TLR5. HEK293-hTLR5 cells stimulated with purified lin used . HoweverA. hydrophila strain AH-1 (serotype O11) flagella, the constitutively expressed polar flagellum but not the inducible peritrichous lateral flagella, showed modification with a putative O-linked glycan moiety. As previously published, strain AH-3 (serotype O34) showed both flagella glycosylated. Both serotypes are among the four dominant serotypes  that are associated with gastroenteritis and septicemia in clinical studies [In the current study, we demonstrated that  studies . Mass spClostridium difficile, where the abolition of flagellin glycosylation resulted in the loss of motility [A. hydrophila strain AH-3 showed that deletion mutants of pseudaminic acid biosynthesis abolish polar and lateral flagellum formation [Aeromonas [The altered or incomplete flagellin glycosylation resulted in altered motility phenotypes. This was also observed with motility . Studiesormation . Howevereromonas , this pormlB mutant is unable to produce O11-antigen LPS [O-glycosylation and O-antigen LPS biosynthetic pathway was found in A. hydrophila. This is in contrast with studies of Pseudomonas aeruginosa, which suggested involvement of the O-antigen biosynthesis in O-glycosylation [O-antigen LPS and flagella have been described as important molecules for bacterial adherence or biofilm formation in Aeromonas [AH-1Δigen LPS ; howeversylation . In addieromonas .O-antigen LPS, as shown by adhesion to Hep-2 cells. Furthermore, the polar flagellum seems to be a determinant factor, with the lateral flagella unable to fully compensate for lack of expression of polar flagellin. This is the first study to show that polar flagellum glycosylation in Aeromonas is a determinant factor in adherence to eukaryotic cells. The results obtained in biofilm formation studies are more marked, with polar flagellum a requirement for biofilm formation, with mutants deficient in polar flagellin expression unable to form biofilms. Then, if there is any compensation from the lateral flagella to the loss of polar flagellum, it is unable to achieve the degree for biofilm formation. The O-antigen LPS mutant, able to produce either polar or lateral flagellin, was observed to form biofilms, but in a reduced capacity.Results from the current study suggest that flagella is a more important adhesion factor than A. hydrophila AH-1 polar flagellin glycosylation is important for and quantitatively related to immune stimulation of IL-8 production via TLR5. A clear reduction in IL-8 production was observed when polar non-glycosylated flagellin monomers (FlaB) were expressed in E. coli. Fish infected with pathogenic bacteria showed a significant enhanced IL-8 expression in the blood and intestine [A. hydrophila AH-1 polar flagella glycosylation in cell invasion as observed for different A. hydrophila wild-type strains [Toll-like receptors (TLRs) are major components of innate immunity. TLR5 is involved in recognizing bacterial flagellin and, after binding, it triggers the induction of pro-inflammatory cytokines such as IL-8 by the myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway . The resntestine . It is t strains .A. hydrophila strains. We show for the first time the presence of inducible lateral flagella, either with lateral flagellins glycosylated or not, in different A. hydrophila strains. We also show that different A. hydrophila strains used different glycans for polar flagellin glycosylation. Furthermore, we continue to shed light on the biological role of flagellum glycosylation, showing their implication in flagellum production, motility, adhesion to eukaryotic cells, biofilm formation, and TLR5 activation.This study supports the existence of a complex mechanism of flagella glycosylation in different E. coli strains were grown on Luria-Bertani (LB) Miller broth and LB Miller agar at 37 °C, while Aeromonas strains were grown either in tryptic soy broth (TSB) or agar (TSA) at 30 °C. When indicated, kanamycin (50 μg/mL), rifampicin (100 μg/mL), spectinomycin (50 μg/mL), and chloramphenicol (25 μg/mL) were added to the media.The bacterial strains and plasmids used in this study are listed in Standard procedures ,37,38,39flaB-J, pseB, pseI and rmlB deletion mutants A. hydrophila AH-1∆flaB-J, AH-1∆pseB, and AH-1ΔpseI, respectively, were constructed by allelic exchange as described by Milton et al. [flaB-J, pseB, and pseI were independently amplified using two sets of asymmetric PCRs. Primer pairs A-FlaB (5′-CGGGATCCAACAGTCTGCCAATGGTTC-3′) and B-FlaB (5′-CCCATCCACTAAACTTAAACAGTTAG CCTGAGCCAAAATG-3′) and C-FlaJ (5′-TGTTTAAGTTTAGTGGATGGGAGACA ACAGCTAGGGGAGTT-3′) and D-FlaJ (5′-CGGGATCCAACGTTTCACAAGCAAGA-3′) amplify DNA fragments of 581 (AB) and 637 (CD) bp for flaB-J in-frame deletion. Primer pairs A-PseB (5′-GAAGATCTGAGGACAAACAACGGATG-3′) and B-PseB (5′-CCCATCCACTAAACTTAAACATGTCTTGACCAGCATCTT-3′) and C-PseB (5′-TGTTTAAGTTTAGTGGATGGGATGAACCAGCAGGTGTTGT-3′) and D-PseB (5′-GAAGATCTAAGCTGAAGACCGTCATGT-3′) amplify DNA fragments of 759 (AB) and 574 (CD) bp for pseB in-frame deletion. Primer pairs A-PseI (5′-CGGGATCCAATGCTGGATGATGAGCAA-3′) and B-PseI (5′-CCCATCCACTAAACTTAAACAGTCAGCGGTATAGGTTTGCA-3′) and C-PseI (5′-TGTTTAAGTTTAGTGGATGGGAGACGAGGCAAAGCAGTC-3′) and D-PseI (5′-CGGGATCCTTTAACTGGCCTGGCTCTA-3′) amplify DNA fragments of 764 (AB) and 630 (CD) bp for pseI in-frame deletion. We use plasmid pDM4 [A. hydrophila AH-1ΔflaB-J, AH-1ΔpseB, and AH-1ΔpseI mutants as previously described for other mutants [The chromosomal in-frame n et al. . Brieflymid pDM4  to obtai mutants .pseB and pBAD-pseI containing the complete pseB and pseI of A. hydrophila AH-1 under the arabinose promoter (pBAD) on pBAD33 [pseB; Oligonucleotides 5′-TCCCCCGGGTTCACTTTTCACGCCTAT-3′ and 5′-GCTCTAGACTAATGCTAAAGCGACAACG-3′ generated a band of 1880 bp containing the pseI. The construction of the pBAD-pseB and pBAD-pseI plasmids was done as previously described for other genes [E. coli DH5α [Plasmids pBAD-er genes . The plaoli DH5α .A. hydrophila AH-1 was grown in TSB for the polar flagellum purification and in TSA for the isolation of lateral flagella, and the flagella purified as previously described [escribed . PurifieThe assays were performed as previously described ,22.TEM was performed on Formvar-coated grids and negative stained with a 2% solution of uranyl acetate pH 4.1.Mass spectrometry studies of intact flagellin proteins were carried out using 50 μL aliquots of protein-containing sample as described previously, with some modification . The purw/w) in 50 mM ammonium bicarbonate at 37 °C overnight. Protein digests were analyzed by nano-liquid chromatography MS/MS (nLC-MS/MS) using either a Q-TOF Ultima hybrid quadrupole time-of-flight MS (Waters) or an LTQ XL orbitrap MS  coupled to a nanoAcquity ultrahigh-pressure liquid chromatography system (Waters). MS/MS spectra were acquired automatically on doubly, triply, and quadruply charged ions in collision-induced dissociation (CID) mode for initial glycopeptide identification. Peak lists were automatically generated by proteinlynx software (Waters) with the following parameters: smoothing—four channels, two smooths, Savitzky-Golay mode; centroid—minimum peak width at half height of four channels, centroid top 80%. Tryptic peptides were analyzed by nano-LC-MS/MS, and spectra were searched against the National Centre for Biotechnology nonredundant database and an in-house database of sequenced flagellin proteins using mascot 2.0.6 . A peptide score of 30 and above for a top-ranked hit was taken as positive identification, with each MS/MS spectrum verified by manual inspection. MS/MS spectra not identified by MASCOT were de novo sequenced. Proteinase K digests were analyzed up nanoliquid chromatography MS/MS, using a Q-TOF Ultima, coupled to a nanoAcquity ultrahigh-pressure liquid chromatography system . The column setup and gradients did not deviate from those reported in our other studies [de novo sequenced, no database searching was employed in peptide sequence assignment.In preliminary experiments, tryptic digests of intact proteins were performed, with 50 to 200 μg of pure flagellin prepartion in solution digested with trypsin  at a ratio of 30:1  and AccuPrime . High-fidelity polymerase was used. The A. hydrophila AH-1 flaB was amplified from genomic DNA using primers PET-A3flaB-for 5′-GGTATTGAGGGTCGCATGCTTGCTGTGTGTTTACC-3′ and PET-A3flaB-rev 5′-AGAGGAGAGTTAGAGCCATTCCTTTCTTCCCAAAGC-3′, and the PCR product ligated into pET-30 Xa/LIC (Novagen), and electroporated into E. coli BL21(λDE3). The His6-FlaB protein was overexpressed and cell lysates obtained as previously reported for other proteins [For proteins .IL-8 concentration using HEK293-hTLR5 and HEK293-null cells was performed as previously described .t-test was used to compare mean values. Differences were considered significant when p-values were <0.05.Results are expressed together with the standard deviations (SD) from several experiments and Student’s


In eukaryotic genomes, about 10% of genes are arranged in a head-to-head (H2H) orientation, and the distance between the transcription start sites of each gene pair is closer than 1 kb. Two genes in an H2H pair are prone to co-express and co-function. There have been many studies on bidirectional promoters. However, the mechanism by which H2H genes are regulated at the transcriptional level still needs further clarification, especially with regard to the co-regulation of H2H pairs. In this study, we first used the Hi-C data of chromatin linkages to identify spatially interacting H2H pairs, and then integrated ChIP-seq data to compare H2H gene pairs with and without evidence of spatial interactions in terms of their binding transcription factors (TFs). Using ChIP-seq and DNase-seq data, histones and DNase associated with H2H pairs were identified. Furthermore, we looked into the connections between H2H genes in a human co-expression network.We found that i) Similar to the behaviour of two genes within an H2H pair (intra-H2H pair), a gene pair involving two distinct H2H pairs (inter-H2H pair) which interact with each other spatially, share common transcription factors (TFs); ii) TFs of intra- and inter-H2H pairs are distributed differently. Factors such as HEY1, GABP, Sin3Ak-20, POL2, E2F6, and c-MYC are essential for the bidirectional transcription of intra-H2H pairs; while factors like CTCF, BDP1, GATA2, RAD21, and POL3 play important roles in coherently regulating inter-H2H pairs; iii) H2H gene blocks are enriched with hypersensitive DNase and modified histones, which participate in active transcriptions; and iv) H2H genes tend to be highly connected compared with non-H2H genes in the human co-expression network.Our findings shed new light on the mechanism of the transcriptional regulation of H2H genes through their linear and spatial interactions. For intra-H2H gene pairs, transcription factors regulate their transcriptions through bidirectional promoters, whereas for inter-H2H gene pairs, transcription factors are likely to regulate their activities depending on the spatial interaction of H2H gene pairs. In this way, two distinctive groups of transcription factors mediate intra- and inter-H2H gene transcriptions respectively, resulting in a highly compact gene regulatory network.The online version of this article (doi:10.1186/1471-2164-15-519) contains supplementary material, which is available to authorized users. More than 10% of human adjacent protein-coding genes are divergently transcribed with their transcription start sites (TSSs) at a distance <1 kb . “Head-tIt is thought that intra-H2H gene pairs are regulated by bidirectional promoters . But whaIt has been proposed that bidirectional promoters should not be considered under an umbrella classification for one large regulatory network, nor should they be divided into thousands of gene pairs . We hypohttp://www.geneontology.org/), protein HEY1 is an RNA polymerase II core promoter sequence-specific DNA binding transcription factor. SRF is a transcription factor that binds to the serum response element (SRE). This protein regulates the activity of many immediate-early genes, and participates in cell cycle regulation, apoptosis, cell growth, and cell differentiation [In this study, there were 1447 Human H2H pairs in DBH2H in total  and 45 tntiation .Figure 2Based on the regulation profile of TFs binding to H2H gene pairs, we clustered the TF pairs hierarchically using Pearson’s correlation coefficient and Ward’s method . As showUsing the transcription factor data described in Methods, we built a binary matrix categorizing whether a given H2H gene pair is regulated by a given TF. By analyzing the correlation between each TF pair (see Methods), we identified co-occurring TF pairs which bind to the same H2H gene pairs. Then we constructed a transcription factor interaction network based on TFs’ co-occurrence.As shown in Figure Since TFs can bridge both promoters and distal cis-regulatory elements such as enhancers, insulators, and silencers while looping out of the intervening DNA , we lookUsing interacting genomic regions from Hi-C data , we mappAs shown in Table The Hi-C results indicate that intra- and inter-H2H pairs show different transcriptional regulation profiles. The TFs of H2H genes can be separated into 2 groups. One group  and calculated the number of co-expressed neighbours of each gene. The percentage of gene counts by number of co-expressed neighbours is shown in Figure It was reported that co-expressed genes tend to be co-regulated by one or more common transcription factors . Since HA systematic investigation of H2H genes, their transcription factors and the histones and DNase bound to them based on human genome Hi-C, ChIP-seq and DNase-seq data was conducted in this study. We echoed and adjusted several known properties of H2H gene organization and also provided new observations on the spatial regulation of H2H genes. We further demonstrated that H2H intra-pairs and inter-pairs are regulated by two distinct groups of transcription factors. The binding of hypersensitive DNase and the modified histones associated with active transcription may facilitate the high expression of H2H genes. Finally, we analysed the properties of H2H genes in a human co-expression network and found that H2H genes tend to be highly connected to other genes. We propose that the highly expressed H2H genes, regulated through both linear and spatial interactions, contribute to the compactness and thus the high efficiency of the entire gene regulatory network.H2H gene pair information was obtained from our previous work: DBH2H . DBH2H chttp://genome.ucsc.edu/encode/dataMatrix/encodeDataMatrixHuman.html). The data were analyzed using W-ChIPeaks (http://motif.bmi.ohio-state.edu/W-ChIPeaks) [DNase-seq data for DNase hypersensitivity and ChIP-seq data for 9 modified histones and 45 transcription factors in the K562 cell line were downloaded from UCSC (hIPeaks) , 30.We integrated the location data of human H2H blocks with genomic interaction loci from Hi-C. If a locus overlapped with a H2H block, we annotated the locus with the H2H pair. If both of the interacting loci overlapped with different H2H blocks, the two H2H pairs (an ‘inter-H2H’pair) were regarded to be spatially interacting with each other. A total of 546 pairs of interacting loci were fully annotated by H2H pairs, and among them, 105 pairs of interacting loci were annotated by different H2H pairs (‘inter-H2H’ pairs) , we used each human gene region plus its 100 bp bases before transcription start site to map to the binding sites of modified histones and DNase. We also compared TF similarities between interacting inter-H2H pairs and random inter-pairs to study the characteristics of their spatial interactions. Here, the TF similarity between an inter-gene pair is represented as the ratio of the shared TFs to the total TFs binding to the same inter-gene pair.We obtained a binary regulation matrix of transcription factors and H2H genes after completing the above analysis  of gene expression profiles and a relative correlation index: mutual rank (MR) for each gene . Mutual Functional annotation for H2H genes. The function enrichment results for H2H genes using DAVID are listed in the table. (XLS 182 KB)Additional file 1:Inter-H2H pairs with spatial interaction. Using interacting genomic regions from Hi-C data, 105 inter-H2H pairs with spatial interactions were identified (see the first sheet). And the detailed H2H pair information is also provided on another sheet. (XLS 370 KB)Additional file 2:The distribution of the average number of co-expressed neighbours of the randomized genes in the co-expression network. The red line represents the mean number of co-expressed neighbors of H2H genes and the black curve is the distribution of the average number of co-expressed neighbors of randomized genes. (PNG 29 KB)Additional file 3:A binary matrix of TFs regulating H2H gene. A binary regulation matrix of transcription factors (TFs) and H2H genes is provided in sheet 1. Row names are H2H gene pairs, column names are TFs. The value of the matrix indicates whether a transcription factor is binding to an H2H gene pair. 1 represents a transcription factor binding to an H2H pair, 0 represents a transcription factor not binding to an H2H pair. (XLS 467 KB)Additional file 4:


Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D) form of eIF4E, but not phospho-dead (S209A) eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin), starvation , and oxidative stress (arsenite). De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins . We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents  Regulation of protein synthesis has recently been linked to a central role in cancer development and malignant progression. The eukaryotic translation initiation factor (eIF) 4E mediates association of the eIF4F complex  with the 5'-methylated guanosine cap structure of mRNA and is an essential and rate-limiting factor of canonical protein synthesis initiation , 2. eIF4In vitro, eIF4E induces malignant transformation through the regulation of multiple cell features, such as by increasing cell proliferation, cell survival, and anchorage-independence [C-MYC, BCL2, FGF2, and BIRC5 (survivin), referred to as eIF4E-sensitive mRNAs [eIF4E is an oncogene with prognostic value in various human cancers, including head and neck squamous cell carcinoma and breast cancer –8. In vipendence , 9, 10. ve mRNAs . HoweverPhosphorylation of eIF4E at Ser-209 by the kinases Mnk1 and Mnk2 in response to mitogens, tumor promoters, and growth factors –15 is crin vivo models. In the Eu-myc mouse lymphoma model [et al. for example, who used mouse NIH3T3 cells in an anchorage-independent soft agar assay, only observed an effect upon overexpression of wild type eIF4E but not the phosphomimetic mutant eIF4E-S209D [The oncogenic features mediated through eIF4E phosphorylation have also been analyzed in ma model , 20, 21,ma model . In PTENma model . It shou4E-S209D .Various cytoplasmic bodies are formed when cells are stressed, such as stress granules (SGs) and processing bodies . Although eIF4E and 4E-T have been described as components of both SGs and PBs, the role of eIF4E phosphorylation in the formation of these intracellular structures has not been sufficiently studied. Curiously, arsenite treatment induces both phosphorylation of eIF4E and assembly of PBs and SGs, and results in physically associated SG-PB structures . SGs seqThe relationship between the phosphorylation of eIF4E and cellular stress, survival, and malignant progression remains to be studied in detail. Here, we demonstrate that phosphorylation of eIF4E at Ser-209 increases resistance to cellular stress, including oxidative stress (arsenite), starvation (glucose/glutamine), and cytotoxic stress (cisplatin), leading to enhanced cell survival and subsequent cell recovery, proliferation, and tumor progression. This process appears to act through an interaction with 4E-T and a qualitative regulation of protein synthesis, processes that could potentially be exploited therapeutically to target certain cancers.Phosphorylation of eIF4E Ser-209 by Mnk1/2 is known to affect cell proliferation and tumor malignancy . To furtIn an anchorage-dependent clonogenic assay, however, expression of S209D (and to a lesser extent S209A) clearly increased both colony size and number in MDA-MB-231 malignant breast cancer cells as well as in non-tumorigenic HaCaT keratinocytes , consistSince clonogenic assays subject cells to stress conditions, we reasoned that phosphorylation of eIF4E confers a positive survival advantage during and/or after stress situations. We therefore treated the cells before plating for 90 minutes with arsenite, a well-known inducer of oxidative stress . As expe2 for 90 minutes); cisplatin (33 μM CDDP for 3 hours), a DNA-damaging chemotherapeutic agent; and nutrient starvation (lack of glucose and glutamate for 24 hours). Cell viability and recovery after restoration to normal culture conditions was measured at 24, 48, and 72 hours. As seen in For a more detailed analysis, we assessed proliferation and viability after exposing cells to a variety of stress signals: arsenite  and SGs (TIA-1). In cells overexpressing the S209D mutant, endogenous 4E-T formed novel cytoplasmic bodies that colocalized with the PB marker DCP1A, but not the SG marker TIA-1, confirming previous results that 4E-T can localize to PBs  an increased interaction between eIF4E and 4E-T in wild type cells with phosphorylated eIF4E. From these observations, we conclude that phosphorylation of eIF4E is implicated in some aspects controlling its binding with 4E-T and that this complex is required for the formation of de novo cytoplasmic bodies.To confirm the direct eIF4E/4E-T interaction, we immunoprecipitated exogenous eIF4E with an anti-Myc antibody in MDA-MB-231 cells. Strikingly, eIF4E-S209D, but not-S209A, was able to pull down 4E-T under normal conditions, suggesting that phosphorylation of eIF4E increases its affinity for or proximity to 4E-T . To corrWe speculated that the association between 4E-T and phosphorylated eIF4E might regulate resistance to stress. We therefore analyzed the role of the eIF4E/4E-T complex in MDA-MB-231 cells by knocking down 4E-T and by using eIF4E mutants unable to bind 4E-T. 4E-T protein levels were downregulated by over 80% following transfection with shRNA plasmids targeting 4E-T . KnockdoAs eIF4E is a rate-limiting regulator of mRNA translation, we sought to analyze changes in mRNA translation and protein synthesis in response to stress. We thus performed polysome profiling to assess overall mRNA translation in MDA-MB-231 cells. Under normal conditions, both phosphomimetic and phospho-dead forms of eIF4E, as well as control GFP-expressing cells, displayed the same polysome profile. Two hours after arsenite treatment, however, the S209D-expressing cells, in comparison to those expressing S209A and GFP, showed an increase in the 80S peak, indicating a rapid impediment to translation. Twelve hours after stress, the 80S peak in S209D-expressing cells was maintained, whereas the S209A- and GFP-expressing cells now displayed an even higher 80S peak . These rde novo transcription. As seen in To validate whether phosphorylation of eIF4E mediates a selective synthesis of proteins under stress , we scrTo validate this further, we performed Q-PCR analysis of mRNA isolated from polysomal mRNA isolated before or after arsenite treatment. As seen in in vitro cellular stress, including oxidative stress, DNA-damaging agents, and nutrient starvation. Indeed, stress appears to be one of the major inducers of eIF4E phosphorylation. This result has profound implications for cancer therapy. Cellular stress is frequently encountered by tumor cells during cancer progression, with sources of stress that include lack of nutrients, cellular crowding, and hypoxia, as well as that encountered during therapeutic interventions, such as during chemo- and radiotherapy. Our results indicate that inhibiting eIF4E phosphorylation is therefore a therapeutic option worth evaluating. Indeed, there are already safe and orally available drugs, such as cercosporamide, that inhibit eIF4E phosphorylation via Mnk1/2 kinases. Our in vitro data indicate that blocking eIF4E phosphorylation with CGP 57380 is indeed sufficient to reduce cellular resistance to stress and chemotherapeutic reagents such as cisplatin. Although non-specific effects of CGP 57380 is reported, we believe that our additional experiments using the S209D mutant, Mnk1/2 KO MEFs and Mnk1 shRNA knockdown, all support the idea that CGP 57380 acts via the Mnk1/2-eIF4E pathway fairly specifically and may be useful as a tool to sensitize tumour cells to anti-cancer treatments.Recently, control of protein synthesis has emerged as an important concept in the regulation of cancer development, cancer prognosis, and therapeutic response , 36–40. How eIF4E regulates stress resistance remains to be studied in detail, although we have uncovered several possible mechanisms. S209D appears to cause a rapid (within 2 hours) translational block in the 80S complex  after stress, followed by a qualitative regulation of synthesis of certain proteins involved in resistance to apoptosis and proliferation, such as cyclin D1, Mcl-1, and MMP3, in line with previous reports , 38, 41.The association of eIF4E and phospho-eIF4E with stress resistance has been described in several biochemical settings in the literature. For example, Ser-209 of eIF4E is phosphorylated in response to different types of cyto- or genotoxic stresses , as wellin vivo knockdown of eIF4E [in vitro and in vivo studies have shown that knocking down eIF4E enhances sensitivity to anticancer agents, including cisplatin and doxorubicin [in vivo and prevent lung metastasis in a melanoma model [Finally, the biological role of eIF4E in tumors and its inhibition as a therapeutic target have already been proposed. A phase I clinical trial recently reported mild side-effects, without clinical response, probably due to inefficient of eIF4E . Other aof eIF4E . In addiorubicin , 44. Imporubicin . Interesma model . In thisma model .In summary, because phosphorylation of eIF4E is associated with resistance to cellular stress, with tumor development in mice models, and worse prognosis in human tumors, the use of specific inhibitors of eIF4E phosphorylation may be a promising approach in cancer treatment. We propose combining eIF4E phosphorylation inhibition with other therapeutic approaches, with our data supporting the rationale for combining inhibitors of eIF4E phosphorylation with cisplatin or other DNA-damaging agents. Importantly, lack of eIF4E phosphorylation is not associated with anomalies in mice development and some inhibitors of Mnk1/2, such as cercosporamide, are already employed in several clinical settings to treat diseases, without major clinical side-effects. Moreover, inhibition of this phospho-eIF4E/4E-T complex could be a valuable approach for the development of novel therapeutic strategies.Vall d’Hebron Institut de Recerca ethical committee (PR(SC)92/2011). MDA-MB-231 cells were treated overnight with 40 μM of MNK inhibitor .Immortalized keratinocytes, HeLa cells , Middlesex, UK), the breast carcinoma cell lines MDA-MB-231  and MDA-MB-468  , and mou2 for 90 minutes, 33 μM cisplatin (CDDP) for 3 hours, or nutrient starvation (glucose and glutamine deprivation) for 24 hours. After stress was induced, the medium was changed to normal medium.Either 2,000 (MEF WT and MEF KO) or 5,000 cells/well (HaCaT and MDA-MB-231) were seeded in 96-well plates and exposed to different types of stress: 250 μM NaAsOStudent’s t-test has been done for all the statistical analysis in triplicate experiments, accepting statistical significance a P-value less than 0.05. P-value is shown in each graph.pLPCX-S209A-eIF4E, pLPCX-S209D-eIF4E, pLPCx-W73A, pLPCX-W73AS209D, and pLPKo-sh4E-T constructs were kindly provided by Dr. N. Sonenberg . Both eIF4E mutants contain one Myc tag. Human pMV7.2-sheIF4E3UTR construct were kindly provided by Dr. Sonia V Del Rincón .Retroviral production and infection were essentially performed as previously described  except tTen thousand cells were plated in 100-mm plates and cultured for 12 days prior to fixation with 4% paraformaldehyde . After fixation, cells were washed with phosphate-buffered saline (PBS) and stained with 0.1% crystal violet (Sigma) for 30 minutes, rinsed thoroughly, and dried. Each cell type was plated in duplicate for each experiment.MTT  was added to the medium to a final concentration of 0.5 mg/mL and incubated for 4 hours at 37°C. The medium was then removed and 0.2 mL DMSO was added. Absorbance was measured at 590 nm by using a Synergy spectrophotometer (Biotek). Readings were taken 0, 24, 48, 72, and 96 hours after cell treatment.2 for 90 minutes. Then 100 μL Caspase-Glo 3/7 reagent was added to each well and the cells were incubated at room temperature for 1 hour. Luminescence was measured in the Synergy Mx Monochromator-Based Multi-Mode Microplate Reader.To measure caspase-3 and -7 activity, the Caspase-Glo 3/7 Assay (Promega) was used. Five thousand cells in 200 uL were seeded in white-walled 96-well plates and treated with 250 μM NaAsOCells were plated in 24-well plates, fixed for 30 minutes with 4% formaldehyde in PBS, blocked with 5% bovine serum albumin (BSA)/PBS, and permeabilized with 0.5% Triton X-100 in PBS for 1 hour. Cells were incubated with primary antibodies for 2 hours at room temperature and washed three times with PBS both before and after incubation with Alexa Fluor 488- or 594-conjugated secondary antibodies  for 1 hour at room temperature. Cells were mounted in 50% glycerol in PBS. Images were taken with an Espectral FV1000 Olympus confocal microscope.Western blotting was performed as previously described . The priCells were harvested in lysis buffer . Lysed cells were incubated with anti-Myc tag antibody (diluted 1:2000), eIF4E antibody (diluted 1:500), or 4E-T antibody (diluted 1:500) overnight at 4°C. The following day Protein G Sepharose 4 Fast Flow  was added and the solution was gently mixed for 1 hour at 4°C. Samples were centrifuged at 3000 rpm for 3 minutes. The pellet was washed 3 times with 1 mL lysis buffer and resuspended in 50 μL sample buffer . The suspension was heated to 95°C for 5 minutes and centrifuged at 3000 rpm for 3 minutes to remove the beads.Cells were lysed in lysis buffer . Cell extracts were incubated overnight with anti-Myc tag primary antibody (diluted 1:2000). 7-methyl-GTP-Sepharose or control Sepharose  was added for 1 hour at 4°C under constant shaking. Beads were washed three times with lysis buffer and absorbed proteins were eluted in SDS-PAGE sample buffer.2, 5.0 mM Tris, pH 7.4, 1.0% Triton X-100, 1.0% Na-deoxycholate, a protease inhibitor mix, 100 U/mL RNase OUT, and 100 μg/mL cycloheximide). Cell extracts were analyzed in a 10%–50% sucrose gradient . Extracts were centrifuged in an SW41Ti rotor for 2 hours and 30 minutes at 37000 rpm at 4°C. Gradients were sampled by using an ISCO UV detector.For polysome analysis, 400 mg of cell lysate was frozen in liquid nitrogen and then resuspended in 1 mL of polysome extraction lysis buffer Click here for additional data file.S2 FigMTT assays in MDA-MB-468 and HeLa cell lines after arsenite treatment and nutrient starvation indicated significantly faster recovery after stress in cells expressing eIF4E-S209D than in those expressing-S209A or GFP. * = P<0.05 and ** = P<0.01 compared to control, n = 3.(TIF)Click here for additional data file.S3 FigA, MDA-MB-231 stably expressing either GFP or S209A or S209D mutants of eIF4E were transfected with a short hairpin plasmid targeting the endogenous 3’UTR of eIF4E. Western blot clearly shows knock-down of endogenous eIF4E without affecting exogenous eIF4E. B, MTT assays after arsenite treatment suggest reduced endogenous eIF4E significantly reduces the recovery capacity after arsenite treatment. * = P<0.05, ** = P<0.01 and *** = P<0.001 compared to control, n = 3. C, overexpression of S209D completely rescues cells and allow recovery from arsenite and CDDP. S209A also moderately improves recovery in the context of reduced endogenous eIF4E and arsenite treatment, perhaps as it may substitute for some functions of endogenous (unphosphorylated eIF4E).(TIF)Click here for additional data file.S4 FigA, MTT assay in MEF Mnk1/2 KO with and without CGP57380 treatment and with arsenite treatment indicates that the effect of CGP 57380 is mainly due to inhibition of the Mnk1/2 pathway. B, Mnk1 depletion in normal conditions in MDA-MB-231 using a lentiviral shMnk1 construct. C, MTT assay in MDA-MB-231 pre-treated with arsenite. Both shMnk1 knockdown and CGP 57380 treatment displayed a similar reduction in recovery after arsenite treatment, inhibiting the recovery.(TIF)Click here for additional data file.S5 FigA, Higher magnification of immunofluorescence analysis of MDA-MB-231 expressing S209D (20X and 40X). 4E-T antibody Sigma HPA001619 B, Immunofluorescence analysis of HaCaT cells expressing either S209A or S209D mutants of eIF4E under normal conditions indicated the specific spontaneous formation of cytoplasmic bodies in S209D-expressing cells. These bodies partially colocalized with 4E-T. 4E-T antibody Cell Signaling 2297, unspecific staining is observed in the nucleus. C, Immunofluorescence analysis of MDA-MB-231 after arsenite treatment to increase the levels of endogenous peIF4E, colocalization of peIF4E with 4E-T in cytoplasmic bodies. 4E-T antibody Abnova H00056478.(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 FigMDA-MB-231 stably expressing either GFP or S209A or S209D mutants of eIF4E were cotransfected with sh4E-T. Endogenous 4E-T levels were reduced in all three cases.(TIF)Click here for additional data file.S8 FigImmunoprecipitation assays with anti-Myc-tag antibodies against eIF4E-W73A and—W73A/S209D confirmed previous findings that the W73A mutation prevents the direct interaction with 4E-T.(TIF)Click here for additional data file.S9 FigA, immunoprecipitation of endogenous peIF4E in MDA-MB-231. Two hours after arsenite treatment there are no binding of 4E-T to peIF4E. B, two hours after arsenite treatment in MDA-MB-231 and HaCaT cell lines, eIF4E-S209D mutant realize the binding with 4E-T.(TIF)Click here for additional data file.


Livestock that consume locoweed exhibit multiple neurological symptoms, including dispirited behavior, staggered gait, trembling, ataxia, impaired reproductive function and cellular vacuolar degeneration of multiple tissues due to toxicity from plant-derived alkaloids such as swainsonine.0 and F1 mice by intraperitoneal injection before, during and after pregnancy at the following doses: 0.525 mg/kg BW(I), 0.2625 mg/kg BW(II), 0.175 mg/kg BW(III) and 0 mg/kg BW(IV). Hemosiderin deposits were observed the lamina propria of endometrium in uterus and the red pulp of spleen. Ovary corpus lutea counts in F0 mice were higher in swainsonine-treated mice compared to control mice. Indirect bilirubin content and reticulocyte numbers were increased in swainsonine-treated F0 and F1 generation mice compared to control group (P < 0.05). Lactate dehydrogenase, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase content in F0-I and F0-II mice were significantly increased compared with F0-IV group mice (P < 0.05). Red blood cells, hemoglobin and mean corpuscular hemoglobin levels were significantly decreased in F0 and F1 mice compared with the control group (P < 0.05).Swainsonine was administered to FSwainsonine exerts effects on estrus period and reproductive ability, and offspring of dams dosed with swainsonine were affected in-utero or from nursing. Damage to liver, uterus and spleen, as well as hematological changes, are observable before neurological symptoms present. Astragalus spp and Oxytropis spp. containing the toxic indolizidine alkaloid swainsonine [Astragalus and Oxytropis species that contain swainsonine are found on multiple continents, and have poisoned animals in South America and Asia [0-I: 0.525 mg/kg BW; F0-II: 0.2625 mg/kg BW; F0-III: 0.175 mg/kg BW and F0-IV: 0 mg/kg BW). After treatment with swainsonine for two weeks, female mice were mated to untreated male mice, and pups were kept with dams for one month. We sacrificed dams and offspring and observed swainsonine toxicity effects on internal organs via histopathological analysis as well as altered hematological and blood biochemical parameters in both parent and offspring mice.Locoweeds are perennial herbaceous plants of the insonine . Locoisminsonine . Swainsoinsonine . Astragaand Asia ,4. Earlyand Asia -8. Thereand Asia . In thisAll extracts were collected using column chromatography, which was placed on the thin layer plate using the capillary sample. Figure 0 and F1 mice revealed no marked changes (data not shown).Examination of heart, lung and kidney of treated F0 swainsonine-treated mice displayed few differences compared with their controls, with cellular infiltrates consisting mostly of inflammatory cells, neutrophils and granulocytes in F0-I, F0-II and F0-III mice. No histopathological differences were noted between F1-I, F1-II, F1-III and F1-IV control mice . Lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) content of F0-I and F0-II mice were significantly increased compared with F0-IV controls (P < 0.05). Furthermore, indirect bilirubin (IBIL) level of F1-I, F1-II and F1-III mice were significantly increased compared with F1-IV controls (P < 0.05). Examination of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) content in swainsonine-treated F1 mice compared with controls revealed no statistically significant differences (Table Indirect bilirubin (IBIL) content of F0 dams revealed that WBCs in F0-I, F0-II and F0-III treatment groups were not significantly different from F0-IV controls (P > 0.05). RBCs, and levels of Hb, HCT, PLT, and MCH in F0-I, F0-II and F0-III treatment groups were significantly decreased compared with F0-IV controls (P < 0.05). MCV and reticulocyte levels in F0-I, F0-II and F0-III treatment groups were significantly increased compared with F0-IV controls (P < 0.05). Examination of F1 mice revealed that reticulocytes in F1-I, F1-II and F1-III treatment groups were significantly increased compared with F1-IV controls (p < 0.05). WBCs counts in F1-I, F1-II and F1-III treatment groups were not significantly different from F1-IV controls (P > 0.05). RBCs, Hb and MCH levels in F1-I, F1-II and F1-III treatment groups were significantly decreased compared with F1-IV controls (P < 0.05). HCT levels in F1-I group mice were significantly decreased compared with F1-IV control mice (P < 0.05), and MCV levels in F1-I and F1-II mice were significantly increased compared with F1-IV control mice (P < 0.05) (Table Examination of F5) Table .Table 20 and F1 mice, and a large amount of hemosiderin deposition in uterus in F0 mice. When animals are fed a dose of swainsonine arrive to a certain time, the vacuolar degeneration of pathological change will show in the internal organs [Swainsonine, a trihydroxy indolizidine alkaloid, is the main toxin in locoweed. The structure of the swainsonine cation is similar to the structure of mannose, and it has a higher affinity than mannose for mannosidase . Swainsol organs . Therefol organs . We posi0 mice. Alterations in liver weight and histopathological changes in liver of swainsonine- treated mice were slight. Liver from swainsonine-treated mice showed cellular infiltrates consisting mostly of inflammatory cells and neutrophil granulocytes. Significant increase of liver weight and significant alterations in levels of AST, ALT and ALP in plasma may indicate hepatic injury in F0 mice given swainsonine. The elevations of ALT, AST and ALP observed in swainsonine-treated mice may, in part, be due to the hepatic hypertrophic effect of swainsonine and/or may also represent borderline chronic liver toxicity [0 mice. However, no significant differences in biochemical markers were found between treatment and control mice F1 mice. This is consistent with the lack of histopathological changes in liver of F1 mice.The experiment results showed that two ways were not significantly different between irrigation and intraperitoneal injection by Liu Tianya . Therefotoxicity ,17. Incrtoxicity . In the 0 and F1 mice, namely expansion of red pulp with vascular congestion. Furthermore, the endometrium of the uterus displayed notable deposition of hemosiderin granules in a swainsonine-treated dose-dependent manner in F0 mice. The molecular weight of swainsonine is small enough to penetrate the placental barrier and expose offspring in-utero. A major function of the spleen is to remove aged and damaged erythrocytes from the blood [1 and F0 mice include reduction of RBCs, reduction in levels of Hb, HCT, PLT and MCH, as well as an increase in the number of reticulocytes, suggesting the development of anemia [0 and F1 mice given swainsonine. The increase of IBIL further indicates that swainsonine could be damaging red blood cells. When organs bleed, red blood cells are phagocytized by macrophages and degraded by lysosomes; Fe3+ of hemoglobin from lysed red blood cells can combine with protein to form hemosiderin. Because we observed decreased RBCs, and decreased levels of Hb, MCH and MCV as well as an increase in reticulocytes, we suspect that our dose levels of swainsonine may lead to anemia.The present study identifies important histological alterations in the spleen in Fhe blood . Excess he blood . Toxic ef anemia . Signifi0 mice was heavily damaged. This was characterized by the presence of hemosiderin deposits in the lamina propria of endometrium in uterus of F0 mice in this study. In ovary, F0-I and F0-II mice displayed decreased numbers of primordial and primary follicles compared to F0-IV controls. In addition, F0-I and F0-II mice displayed increased size and number of corpus lutea compared to F0-III and F0-IV. The lesions in ovary and uterus were dose-dependently observed in F0-I, F0-II and F0-III treatment groups. However, F1 did not display notable histopathological changes in the uterus and ovary. Swainsonine easily accumulates in uterus at high concentrations, which may impair uterus and ovary function and cause toxicity. In the present study, the uterus suffered noticeable damage, which led to a decline in the rate of conception, an increase in the rate of abortion and increases in stillborn births. It is suspected that significant early embryonic loss occurs in cattle and sheep grazing locoweed, and there are documented effects of swainsonine on oocyte maturation, fertilization and subsequent embryonic implantation and development [Swainsonine is water-soluble and rapidly distributed to many parts of the body. In previous studies, swainsonine concentrations varied widely in various tissues and organs of sheep that had ingested locoweed -24. In tBased on sub-chronic toxicity results, our data establishes effects of swainsonine on reproductive toxicity in a mouse model. In addition, we found that swainsonine can cause hematological changes and lesions in spleen, uterus, ovary and liver. Furthermore, we provide evidence of trans-generational swainsonine toxicity through placental barrier and milk. Spleen, heart, liver, lung, kidney, uterus and ovary were among the organs affected in offspring of dams given swainsonine. Large amounts of hemosiderin deposition in uterus and spleen were observed in the parent generation. We present evidence that hemosiderin deposition may preclude vacuolar degeneration in some tissues of mice given swainsonine. Alterations in hematological and histopathological parameters suggest a link to anemia and decreases in reproduction ability. Our data suggest that anemia and organ-specific hemosiderin deposition followed by destruction of red blood cells are clinical features of swainsonine-treated mice. However, further research is needed to elucidate specific mechanisms of swainsonine toxicity.Rattus norvegicus mice were supplied by the Animal Center of the Fourth Military Medical University. During the experiment, mice were housed individually in polypropylene cages with laboratory grade pine shavings as bedding. Mice were maintained in a controlled environment with temperature maintained between 19-25°C, relative humidity maintained between 40-70%, >8 air changes/hour, and with a 12:12-h light: dark cycle. The experimental procedures were in accordance with the Ethical Principles  in Animal Research adopted by the China College of Animal Experimentation and were approved by the College of Veterinary Medicine- Northwest A&F University.Female Oxytropis kansuensis was collected from the grassland in Tianzhu city, Gansu province in July 2011. The plants were then taxonomically identified by Zhao Bao-Yu, College of Veterinary Medicine, Northwest A and F University, China. The plants were subsequently dried in the shade, finely ground and comminuted.The aerial portion of Oxytropis kansuensis was conducted as previously described [The extraction and analysis method of swainsonine from escribed .2O2/10% acetic anhydride in EtOH/Ehrlich’s reagent was the chromogenic agent.Thin-layer chromatography (TLC) detection was performed on silica gel G precoated plates with the developing solvents chloroform:methanol:ammonia:water , chloroform:methanol:ammonia:water , and methanol: ethylacetate: ammonia  and modified potassium heptaiodobismuthate reagent or H2O2 (heated for 10 min in an oven at 115°C), a spray of 10% acetic anhydride in dehydrated alcohol (heated at the same temperature until the smell of acetic anhydride disappeared) and finally a spray of Ehrilich’s reagent (heated for 15 min at 120°C). The color of the spots in each plate was recorded, and the Rf was determined [The extracts were dissolved in methanol, spotted onto the GF254 silica gel G precoated plates. The plates were developed with an ascendant run after saturation with the mobile phase in a s glass chamber for 5–10 min. The plates were dried when the mobile phase was 10 mm from the front edge of the plates. The plates were stained successively with a spray of H0-I: 0.525 mg/kg BW; F0-II: 0.2625 mg/kg BW; F0-III: 0.175 mg/kg BW and F0-IV: 0 mg/kg BW). All mice were administered swainsonine by intraperitoneal injection 14 days before the mating period followed by re-administration every three days. After this pre-mating period, the treated mice were transferred to the home cage of a male in the same group and cohabited on a 1:1 basis until achievement of successful mating. During the mating period, mice were examined daily for presence of vaginal plugs, and a vaginal plug was considered evidence of successful mating. Pregnant dams continued to receive swainsonine every three days via intraperitoneal injection throughout parturition and the lactation. Upon weaning of four-week-old pups (F1), the dams (F0) were sacrificed, and the liver, kidney, heart, spleen, lung, uterus and ovary were collected. In total, F0 mice were given swainsonine for six to eight weeks in the whole experiment.Female mice  were divided into four equal groups of 10 mice  of treated dams were selected from each of the four treatment groups . The F1 offspring were not treated with swainsonine, however, the dams continued to be dosed while nursing their F1 pups. The F1 offspring were then sacrificed after approximately 1 month of nursing. The liver, kidney, heart, spleen, lung, uterus, and ovary were collected.Female offspring , red blood corpuscles (RBCs), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), blood platelets (PLTs), mean corpuscular hemoglobin (MCH) and reticulocyte counts were determined by automatic hematological analyzer, MEK-8222 K .Blood was collected when mice were sacrificed. Lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and indirect bilirubin (IBIL) were quantitated using the Beckman Synchron CX7 Delta Chemistry Analyzer .p < 0.05 were considered significant. The data are presented as the group mean values ± SD (standard deviation).The statistical software “Statistical Product and Service Solutions” (SPSS V11.3) was used to determine statistically significant differences between treatment groups and the control group. A one-way ANOVA was used to evaluate the homogeneity of the data, and a least squared differences model or Dunnett’s multiple comparison test were then used. Values of


Citrus sinensis) and B-intolerant ‘Sour pummelo’ (Citrus grandis) leaves to B-toxicity. Using two-dimensional gel electrophoresis (2-DE) technique, we identified 50 and 45 protein species with a fold change of more than 1.5 and a P-value of less than 0.05 from B-toxic C. sinensis and C. grandis leaves. These B-toxicity-responsive protein species were mainly involved in carbohydrate and energy metabolism, antioxidation and detoxification, stress responses, coenzyme biosynthesis, protein and amino acid metabolism, signal transduction, cell transport, cytoskeleton, nucleotide metabolism, and cell cycle and DNA processing. A detailed analysis of this data may be obtained from Sang et al. (J. Proteomics 114 (2015))Here, we provide the data from a comparative proteomics approach used to investigate the response of boron (B)-tolerant ‘Xuegan’ ( Specifications tableValue of data•Data provided B-toxicity-induced changes in leaf protein profiles of two citrus differing in B-tolerance.•B-toxicity-responsive protein species in leaves differed between the two citrus species.•The integrated proteomic analysis provided a better insight into the tolerant mechanism of citrus plants to B-toxicity at the translational level.1Citrus sinensis) and B-intolerant ‘Sour pummelo’ (Citrus grandis) seedlings were used to investigate the effects of B-toxicity on leaf proteomics. Plant culture and B treatments were performed as described previously C. sinensis and C. grandis grown in 6 L pots (two seedlings per pot) containing river sand in a greenhouse under natural photoperiod were irrigated every other day until dripping with nutrient solution containing 10 μM (control) or 400 μM (B-toxic) H3BO3 for 15 weeks. There were 20 pots per treatment in a completely randomized design. At the end of the experiment, fully expanded leaves collected from B-toxic and control plants at noon under full sun were immediately frozen in liquid N2. Frozen samples were stored at −80 °C until extraction.Boron (B)-tolerant ‘Xuegan’  were mixed as a biological replicate. There were three biological replicates for each treatment. Proteins from control and B-toxic frozen leaves were extracted according to Yang et al. 3P-value of less than 0.05 and a fold change of more than 1.5. Based on the two criteria, we obtained 32 up-regulated and 28 down-regulated protein spots from B-toxic C. sinensis leaves, and 31 upregulated and 31 down-regulated protein spots from B-toxic C. grandis leaves. All these differentially abundant protein spots in B-toxic C. sinensis and C. grandis leaves are presented in 2-DE was performed according to You et al. 4P<0.05) and accepted. Bioinformatics analysis of proteins was performed according to Yang et al. C. sinensis and C. grandis leaves. A detailed data could be obtained from All theses differentially abundant protein spots were excised from the colloidal Coomassie Brilliant Blue stained gels and subjected to MS analysis, which was performed on an AB SCIEX 5800 TOF/TOF. All acquired spectra of samples were processed using TOF/TOF ExplorerTM Software (AB SCIEX) in a default mode. The data were searched by GPS Explorer (Version 3.6) with the search engine MASCOT . Protein spots with scores greater than 75 were considered statistically significant (5C. sinensis and C. grandis leaves (S-transferase (GST) GST1 (S17), ribulose bisphosphate carboxylase activase (S39) and ribosomal protein L12 (G50), demonstrating that most of these differentially abundant protein species were regulated in the transcriptional level.To understand the correlation between gene and protein expression levels, we used qRT-PCR to assay the transcript levels of genes encoding 14 differentially expressed protein species in B-toxic s leaves . The tra