It has been recently demonstrated that CD8+ T-lymphocyte numbers are genetically transmitted in association with the MHC class I region. The present study was designed with the objective of narrowing the region associated with the setting of CD8+ T-lymphocyte numbers in a population of C282Y homozygous hemochromatosis subjects, in whom a high prevalence of abnormally low CD8+ T-lymphocyte counts has been described.The study includes 43 C282Y homozygous subjects fully characterized both phenotypically and genotypically. Clinical characterization includes measurements of iron parameters at diagnosis (transferrin saturation and serum ferritin), total body iron stores and T-cell immunophenotyping determined by flow cytometry. Genetic characterization includes HLA class I alleles  and four additional microsatellite markers  spanning 5 Megabases in the 6p21.3 region.6/ml), in comparison with subjects carrying only one copy of those alleles (0.46 ± 0.19 × 106/ml) and subjects without any copy of those alleles . No differences were observed in CD8+ T-cell counts among control subjects carrying the same combinations of HLA-A alleles , therefore not supporting a direct effect of HLA specificity but rather an indirect association with a locus close to HLA-A. Multivariate analysis showed that the combination of the most common HLA-A alleles also have an impact on the clinical expression of HH in terms of iron stores, in males(p = 0.0009).Eighty-two extended C282Y carrying haplotypes were defined. Single-locus analysis revealed that the HLA-A region was associated with CD8+ T-cell numbers. Multivariate analysis showed that the combinations of the most common HLA-A alleles  were associated with significantly lower numbers of CD8+ T-lymphocytes . It gives additional information to better define a candidate region involved in the regulation of CD8+ T-lymphocyte numbers. A new evolutionary hypothesis concerning the inheritance of the phenotype of low CD8+ T-lymphocyte numbers associated with particular ancestral HLA haplotypes carrying the C282Y mutation and its implication on the clinical heterogeneity of HH is discussed. There are multiple regulatory mechanisms influencing peripheral blood lymphocyte numbers that are responsible for the maintenance of cell numbers in homeostasis  and revi]. An incCenter D'Etude Du Polymorphisme Humain (CEPH) families it was shown that lymphocyte counts, B cells, natural killer cells, and CD4 and CD8 counts, were all under significant genetic control, with the CD8+ T-cell counts being the most heritable trait [HFE genotyped subjects belonging to hereditary hemochromatosis (HH) families [HFE haplotypes on the setting of CD8+ T-lymphocyte numbers but not of CD4+ [HFE, are responsible for this phenotypic variation, in accordance with other studies [Studies in humans have also revealed a genetic regulation for CD4/CD8 numbers. Complex segregation analysis of the CD4/CD8 ratios in nuclear families, suggested a major recessive locus with a polygenic component and additional environmental effects for the control of CD4/CD8 ratios . The twole trait . Signifile trait . Using 4le trait . In addile trait . All thele trait . The appfamilies . A stronfamilies . Sibpair of CD4+ . Based o of CD4+ -17, we f of CD4+ . This mi of CD4+ -22 and f studies ,24.In the present study we attempt to narrow the region associated with the setting of CD8+ T-lymphocyte numbers using a population of C282Y homozygous subjects fully characterized both phenotypically and genotypically. Multivariate models were used to test the association between several genetic markers at the MHC region and CD8+ T-lymphocyte numbers, as well as the impact of these genetic markers on the severity of iron overload in HH. We discuss the hypothesis that the phenotype of low CD8+ T-lymphocyte numbers in HH is inherited in association with particular ancestral HLA haplotypes carrying the C282Y mutation.A total of 43 hereditary hemochromatosis probands, all homozygous for the C282Y mutation, identified between 1985 and 2004 and regularly followed up at the Hemochromatosis Outpatient Clinic of Santo António General Hospital (HGSA), Porto and at Predictive and Preventive Genetic Center, Porto, have been included in this study. The subjects are all Caucasian from the North of Portugal and include 32 males  and 11 females  . Total body iron stores (TBIS) were escribed . In 7 prHFE genotype and HLA-A, -B and -C genotype were available. This population was used to compare CD8+ T-lymphocyte numbers according to the HLA-A and -B class I alleles between controls and C282Y homozygous subjects. In addition, control subjects carrying the HLA-A*01 (n = 47) were genotyped for the microsatellite markers D6S2222 and D6S105. This subpopulation was used to compare the allele frequencies of these microsatellites between controls and C282Y homozygous, all carrying the HLA-A*01.Data from 264 apparently healthy unrelated subjects, previously analyzed by Cruz and co-workers , were inHFE mutations (H63D and C282Y) and were all homozygous for the C282Y mutation. HLA class I alleles  were available in all probands except the HLA-C in 3 subjects. In addition, four polymorphic markers in the MHC class I region were selected for the definition of extended HLA haplotypes. These include microsatellites spanning 5 Megabases in the 6p21.3 region, from the HLA-B to the HFE: D6S265, D6S2222, D6S105, and D6S2239  were defined in C282Y homozygous subjects by segregation in families. Haplotypes were defined from the combination of alleles inherited in blocks from each parent or from informative siblings, when any of the parents was not available. Genetic markers included in extended HLA haplotypes were aligned according to physical map of the region as follows: HLA-B, HLA-C, D6S265, HLA-A, D6S2222, D6S105, C282Y mutation, D6S2239  as described before [taq gold (Perkin-Elmer) was used to amplify specific amplicons, using genomic DNA as template. Amplification was performed, in all cases, with an initial denaturation step of 94°C (8 minutes), followed by 30 cycles at 94°C (15 sec.), 55°C (15 sec.) and 72°C (30 sec.), and a final extension step of 30 minutes at 72°C. Alleles were identified by electrophoresis on sequencing gels. The classification adopted for each microsatellite allele is based on the size (in base pairs) of each DNA fragment amplified.Biochemical parameters of iron status used in this work include determinations, at the time of the diagnosis, of TfSat and serum ferritin. These parameters are routinely determined by standard techniques at the Clinical Chemistry Laboratory of HGSA, Porto, as previously described . The T-cd before . HLA-A ad before . MicrosaGroup means were compared by the Student T-test when two groups were analyzed or by the one-way analysis of variance test (ANOVA) when more than two groups were analyzed. The Chi-square test was used to test the fitness of data to the normal distribution. Independence between categorical data and differences between allele frequencies of the genetic markers between the hemochromatosis patients and the control population were tested using the Chi-Square test.Two different sets of analyses were done in C282Y homozygous subjects to investigate the impact of the genetic markers from the 6p21.3 region, on the CD8+ T-lymphocyte numbers. First, a single-locus analysis was performed by one-way ANOVA, where the numbers of CD8+ T lymphocytes were analyzed in relation to the alleles of each genetic marker studied . Single-locus analyses were also done for CD4+ T-lymphocyte and total lymphocyte numbers. Since each subject carries two chromosomes and therefore two different allele specificities, a second set of analysis was done considering the combination of the two inherited HLA-A alleles. For this purpose a trichotomous variable denominated "HLA-A genotype" (details in the result' section), was created and its impact on the variability of CD8+ T lymphocytes was also analyzed using a stepwise multiple regression analysis. In this model, CD8+ T lymphocytes were set as dependent variable and the HLA-A genotype, age, gender and iron measures  were used as independent variables. Because serum ferritin values were not normally distributed, a log transformation was used for this variable.To investigate the effect of the "HLA-A genotype" on the severity of the disease, in terms of iron stores (TBIS), a stepwise multiple regression model was used. TBIS was set as dependent variable and the HLA-A genotype as independent variable, together with age and gender. The interaction of the variable gender with the other independent variables was tested by introducing two dummy variables in the model, corresponding to male or female gender.F and p values. The overall fit of multiple regression model is indicated by the full regression R2, and the F and p values of each variable in the model. All statistical tests were performed at the 0.05 level of significance and all p values are two-sided. Data were analyzed by Statgraphics software .The overall fit of ANOVA is indicated by 6/ml [In the group of 43 C282Y homozygous probands all had TfSat above 65% at the time of the diagnosis. There was, however, a great variability in serum ferritin levels and also in the amount of iron removed by phlebotomies (TBIS) Table . Althoug6/ml ), was 75A total of 86 HLA A-B haplotypes were defined by segregation in families from the 43 homozygous probands included in the study. Thirty-seven different HLA A-B haplotypes could be identified. These are shown in Figure HFE-C282Y, D6S2239-112  of the chromosomes  and the haplotype HLA-A*01, D6S2222-247, D6S105-150, HFE-C282Y, D6S2239-112 is also present in 13% (11/82) of the chromosomes . Haplotypes were aligned taking into account the HLA-A and then arbitrarily according to the similarity of the alleles from the several microsatellites. The predominant extended HLA haplotype carries the HLA-A*03-B*07 and is defined by: HLA-B*07, HLA-C*07, D6S265-128, HLA-A*03, D6S2222-247, D6S105-150, F = 2.36, p = 0.013, one-way ANOVA) of the HLA-A alleles on the CD8+ T-cell numbers was found. Among all HLA-A specificities, the A*03 (n = 39), A*02 (n = 16) and A*01 (n = 13) were associated with the lowest average CD8+ T-cell counts. This effect is more clearly evidenced when all chromosomes carrying HLA-A alleles other than A*03, A*02 and A*01  are pooled together in one group as illustrated in Figure F = 5.58, p = 0.0016, one-way ANOVA). No statistically significant associations were found with any of the more centromeric markers, D6S265, HLA-C and HLA-B. No significant associations were also found with the telomeric markers D6S2222, D6S105 and D6S2239, an expected result taking into consideration the high conservation of the region as evidenced by the presence of the same microsatellite alleles in the large majority of C282Y carrying chromosomes.The numbers of CD8+ T lymphocytes were analyzed in relation to single-locus HLA-A, -B, -C alleles and microsatellites D6S265, D6S2222, D6S105, D6S2239 alleles, by one-way ANOVA. A statistically significant effect  was observed when the effect of the HLA-A was tested on total lymphocyte counts (data not shown). No statistically significant associations were found for total lymphocyte counts with any of the other markers used. No statistically significant effect was found on the numbers of CD4+ T cells and any of the genetic markers tested (data not shown).A statistically significant result (F = 20.56 and p = 0.0001). The R2 for the full regression was 0.37. Iron related variables did not enter the model . Gender and age also did not enter the model . Due to the fact that there are only two subjects without any copy of the most common HLA-A alleles and these two had the highest CD8+ numbers, a new regression was run where the HLA-A genotype variable was analyzed only with the groups of subjects with either two copies of the most common HLA-A alleles (n = 27) or with only one copy of those alleles (n = 14). A statistically significant effect of the HLA-A genotype on CD8+ T-cell numbers was maintained (F = 7.98 and p = 0.0082).To further investigate the association found between particular HLA-A alleles in chromosomes carrying the C282Y mutation and the number of CD8+ T cells, C282Y homozygous subjects were divided according to the combination of the two inherited HLA-A alleles. This created a trichotomous variable, from now on denominated "HLA-A genotype", dividing C282Y homozygous probands in the following groups: 1.subjects with two copies of the most common HLA-A alleles ; 2.subjects with only one copy of the most common HLA-A alleles (n = 14) and 3.subjects without any copy of those alleles (n = 2). A stepwise multiple regression analysis was run, where the number of CD8+ T lymphocytes was set as dependent variable and the HLA-A genotype, iron measures , age and gender (dummy variable), were set as independent variables. The only variable significantly affecting CD8+ T-cell numbers was the HLA-A genotype (6/ml) and subjects without any copy of the most common HLA-A alleles presented the highest average value (0.79 ± 0.15 × 106/ml). Subjects with only one copy of the most common HLA-A alleles presented intermediate average CD8+ T-cell numbers (0.46 ± 0.19 × 106/ml).The effect of the HLA-A genotype on CD8+ T lymphocytes is illustrated in Figure 6/ml, n = 65), control subjects carrying only one copy of these alleles  and subjects without any copy of those alleles , therefore not supporting a direct effect of the HLA specificity on the results. In the previously described control population [p < 0.00001). For the microsatellite D6S105, although the allele 150 was the most common in both groups, its allele frequency was significantly lower (p = 0.0001) in controls  than in C282Y homozygous .The results described in C282Y homozygous probands confirm an association of the HLA-A region with the setting of CD8+ T lymphocytes. Since CD8+ T lymphocytes are known to expand in the context of MHC class I molecules, it is plausible to consider that the HLA specificity itself could be responsible for the results observed. To test this hypothesis, we examined retrospectively the effect of the combinations of HLA-A alleles on CD8+ T-cell numbers in a previously studied control population, not carrying the C282Y mutation . Controlpulation , subjectF = 17.29 and F = 7.16, respectively). The R2 for the full regression was 0.306 and the global significance level was 0.0009. This result is illustrated by the correlation between HLA-A genotype and residual values of TBIS on age in males, as shown in Figure F = 16.54 and F = 7.58, respectively). The R2 for the full regression is similar . This result shows that the effect of the HLA-A region on the severity of iron stores may be confounded by the number of CD8+ T lymphocytes.In face of the results showing an impact of the HLA-A region on the setting of the CD8+ T-cell numbers and the previously described association of low CD8+ T cells with a more severe expression of iron overload ,17, we rThe present study was designed to further investigate our previous results showing that CD8+ T-lymphocyte numbers are under the control of genes localized at the MHC region. An attempt to narrow the candidate region is made, using as a model 43 HH subjects, C282Y homozygous, in whom a high proportion of subjects have low CD8 + T-cell numbers. The results provide evidence supporting an inextricable link between extended HLA haplotypes, CD8+ T-lymphocyte numbers and severity of iron overload in HH.6/ml). The highest value of CD8+ T-cell numbers was observed in the subjects without any copy of the most common HLA-A alleles (0.79 ± 0.15 × 106/ml). The most attractive hypothesis to explain these results is the existence of a genetic trait associated with low CD8+ T-lymphocyte numbers localized in the HLA-A region. In this case, the trait would be in linkage with haplotypes carrying the A*03, A*02 or A*01 in the C282Y background. In a previous report, Pratiwi and collaborators [HFE (14 kilobases telomeric) and at D6S105, located about 200 kilobases away from the D6S2222 [Single-locus analysis of the several genetic markers of the MHC region in chromosomes carrying the C282Y mutation showed that CD8+ T-lymphocyte numbers were associated with the HLA-A region. No significant association was found with the more telomeric markers, spanning from the microsatellites D6S2222 to D6S2239. This region is well conserved in all haplotypes, independently of CD8+ T-cell numbers. Centromerically to HLA-A, a high allele diversity is observed and no association was found with CD8+ T-cell numbers  declare that they have no competing interests.HFE genotyping. RL performed the T-cell immunophenotyping. AG and CSC performed the microsatellite genotyping and some of the HLA genotyping. HA is responsible for the HLA genotyping and contributed to the interpretation of the genetic data. All authors read and approved the final manuscript.EC and GP conceived and designed the study, diagnosed and treated the hemochromatosis patients, compiled their clinical data, contribute to the interpretation of data and wrote the manuscript. Additionally, EC analyzed the data and performed the statistical analysis related to hemochromatosis patients and family members; compiled data and performed statistical analysis related to the control population. JV participated in the design of the study and contributed to the statistical analysis and its interpretation and to the writing of the manuscript. SA performed the The pre-publication history for this paper can be accessed here:


Cellular immunity plays a crucial role in cytomegalovirus (CMV) infection and substantial populations of CMV-specific T cells accumulate throughout life. However, although CMV infection occurs during childhood, relatively little is know about the typical quantity and quality of T cell responses in pediatric populations.One thousand and thirty-six people  were examined for HLA typing. All of 1036 subjects were tested for HLA-A2 antigen. Of 1036 subjects, 887 were also tested for HLA-A23, 24 antigens. In addition, 50 elderly people  were also tested for HLA-A2 antigen. We analyzed the CD8+ T cell responses to CMV, comparing these to responses in children and young. The frequencies, phenotype and function CD8+ T cells for two imunodominant epitopes from pp65 were measured.We observed consistently high frequency and phenotypically "mature"  CMV-specific CD8+ T cell responses in children, including those studied in the first year of life. These CD8+ T cells retained functionality across all age groups, and showed evidence of memory "inflation" only in later adult life.CMV consistently elicits a very strong CD8+ T cell response in infants and large pools of CMV specific CD8+ T cells are maintained throughout childhood. The presence of CMV may considerably mould the CD8+ T cell compartment over time, but the relative frequencies of CMV-specific cells do not show the evidence of a population-level increase during childhood and adulthood. This contrast with the marked expansion ("inflation") of such CD8+ T cells in older adults. This study indicates that large scale analysis of peptide specific T cell responses in infants is readily possible. The robust nature of the responses observed suggests vaccine strategies aimed at priming and boosting CD8+ T cells against major pathogens  could be successful in this age-group. Human cytomegalovirus (HCMV) is a widespread β-herpesvirus that infects 40% to 100% of adults worldwide ,3. In deViral infection is usually controlled by a range of innate and adaptive immune effector mechanisms. In particular, virus-specific CD8+ cells play a crucial role in the control of HCMV replication during both acute and chronic phases -8. StronThe "phenotype" of virus-specific CD8+ T cells can be described using a few distinct subsets based on cell surface markers . CD27 and CD28 are T cell costimulatory molecules, which are differentially expressed on CD8+ T cells under different conditions of stimulation over time -13. NaïvLittle detail is available on the frequencies, phenotype and function of antiviral CD8+ T cells in pediatric populations. It was previously considered that infants may fail to mount Th1 type responses of a quality similar to those seen in adults, although emerging data from smaller studies has led to a revision of these ideas. A recent study showed that a mature and functional CD8+ T cell response to HCMV was observed in specific cases of infection of fetuses and newborns . HoweverIn this study, a large cross-sectional analysis was performed to clarify the status of HCMV specific CD8+ T cells in infants and children. In order to quantify HLA-restricted HCMV-specific CD8+ T cells, we used human leukocytic antigen (HLA)-A2 and HLA-A24 tetrameric complexes (tetramers) loaded with the immunodominant peptide derived from HCMV structural protein pp65. In addition, effector functions of HCMV-specific CD8+ T cells in children were assessed using IFN-γ enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining (ICS) for IFN-γ-producing cells. Furthermore, we evaluated the differentiation status using cell surface markers  to clarify the phenotype of the HCMV-specific CD8+ T cells and any changes over time. We show that the effector functions of HCMV-specific CD8+ cell in infants are as strong as those in young adults. Moreover, the phenotype of HCMV-positive CD8+ T cells is well differentiated in infants and young children.Between March and September 2003, blood samples were taken from patients who attended pediatric clinics at National Defense Medical College , International University Health and Welfare , and Hokkaido University  due to various diseases. Informed consent was taken from them or their parents. One thousand and thirty-six people  were enrolled in this study. All of 1036 subjects were tested for HLA-A2 antigen. Of 1036 subjects, 887  were also tested for HLA-A23, 24 antigens. In addition, 50 elderly volunteers  were also enrolled in this study. None of them was examined for HCMV sero -status.Anti-HLA-A2-fluorescein isothiocyanate (FITC), anti-CD8-peridinin chlorophyll protein (PerCP), anti-CD27-FITC, anti-CD28-:allophycocyanin (APC), anti-CD45RA-FITC, anti-CD45RO-APC, anti-IFN-γ-FITC mAbs, and isotype control Abs were purchased from Becton Dickinson PharMingen . Anti-HLA-A23, 24 mAbs were purchased from One Lambda . Goat anti-mouse IgG-FITC Abs were purchased from Immunotech . The HLA-A*0201-restricted HCMV-specific CTL epitope pp65 495–503 (NLVPMVATV) and HLA-A*2402 restricted HCMV-specific CTL epitope pp65 328–336 (QYDPVAALF) both previously identified , were puHLA class I-peptide tetramers were synthesized as described previously . BrieflyDetection of HLA-A2 and HLA-A24 was performed by flow cytometry using the HLA-A2 and HLA-A23, 24 mAb. In brief, 20 μL of EDTA or heparinized whole blood was stained with anti-HLA-A2-FITC and HLA-A23, 24 mAb for 20 minutes at 4°C respectively. After staining with anti-HLA-A2-FITC mAb, red blood cells were lysed using FACS lysis . Whole blood stained with anti-HLA-A23, 24 mAb was washed with PBS. After the incubation for 20 minutes at 4°C with FITC-labeled mouse IgG, it was washed again with PBS and lysed using FACS lysis solution (Becton Dickinson Sciences).50 μL of whole blood was stained with tetramer for 15 minutes at 37°C, followed by addition of titrated Abs  directed against surface molecules and incubation for 20 minutes at 4°C. Samples were lysed as described above.To determine the frequencies of IFN-γ-producing peptide-specific T cells in peripheral blood, intracellular staining was conducted on fresh whole blood. Frequencies of IFN-γ-producing peptide-specific T cells were quantified after stimulation with the CMV HLA-A2 peptide , according to the protocol of the supplier . Briefly, heparinized whole blood were stimulated with peptide  for 6h at 37°C in the presence of Brefeldin (Sigma-Aldrich) at 10 μL/ml. Whole blood was stained with anti-CD8-PerCP Ab for 20 minutes at room temperature. Whole blood was lysed and fixed using FACS lysis solution (Becton Dickinson PharMingen), permeabilized using FACS permeabilization buffer (Beckton Dickinson), and then stained with FITC-labeled IFN-γ or FITC labeled IgG-isotype control mAb.Cell were stored in Cell Fix buffer (Becton Dickinson Immunocytometry systems) at 4°C until analysis. Samples were analyzed with a FACS Calibur (Becton Dickinson Bioscience) using CellQuest software (Becton Dickinson Bioscience), after compensation was checked using freshly stained PBMCs.6 PBMC.PBMCs were isolated from heparinized venous blood by density gradient sedimentation using Ficoll-Hypaque . Fresh PBMCs were plated in 96-well polyvinylidene plates  that have been precoated with 0.5 μg/ml anti-IFN-γ-mAb . The peptides were added in a volume of 10 μl and then PBMCs were added at 50,000–100,000 cells/well in a volume of 190 μl. The final concentration of HLA-A2 peptide (NLVPMVATV) was 5 μM. The plates were incubated overnight at 37°C, 5% CO2 and washed PBS before addition of the second, biotinylated anti-IFN-γ mAb (Mabtech) at 0.5 μg/ml and incubated at room temperature for 100 min. After washing, streptavidin-conjugated alkaline phosphatase (Mabtech) was added at room temperature for 40 minutes. Individual cytokine-producing cells were detected as dark spots after 20-minutes reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium using an alkaline phosphatase-conjugated substrate . The number of specific T cells was calculated by subtracting the negative control values and expressed as either the number of spot-forming units per 10p value of 0.05 or less was considered to indicate statistical significance. For nonparametric statistical analyses, Spearman rank correlation test were used. All p values were 2-tailed and considered significant if <0.05. Nonparametric Mann-Whitney U tests were used for data pairs and Kruskal-Wallis tests were used for group data. Results were presented using Prism .Frequency distributions were compared using the Yates corrected chi-square test or Fisher's exact test. A Of 1036 subjects, 448 (43%) were positive for HLA-A2. Of 887 subjects, 519 (58%) people were positive for HLA-A23, 24, of which 76 (15%) were also positive for HLA-A2. Because it is well established that HLA-A23 antigen is very rarely detected in Japanese populations [Initially, a cross-sectional analysis of HCMV-specific CD8+ T cell frequency was performed. Analysis of HLA-A2 negative and HLA-A23, 24 negative samples provided a cut-off for detection of 0.05% and 0.04% of CD8, respectively, as previously obtained  . These findings suggest that HLA type did not influence the immunodominance using tetramer assay.Seventy-six individuals were positive for both HLA-A2 and HLA-A24. Of 76 subjects, 17 were positive for both HCMV-A2 and -A24 tetramer staining. The mean frequency of HCMV-specific CD8+ T cells in HLA-A2 and HLA-A24 individuals was 1.09 and 0.09, respectively. Of 76 subjects, 12 and 8 were positive for HCMV-A2 and -A24 tetramer staining, respectively. The remainings were negative for trtramer staining. There was no significant difference in positive rate of HCMV tetramer between two alleles and one allele . We also performed ICS for IFN-γ for HLA-A2+ subjects . A comparison of the ICS and tetramer assays is shown in Figure p = 0.001). The results of ELISPOT assays in HLA-A2 subjects were stratified by age and shown in Figure 6 cells, and although this varied slightly with age there was no statistically significant difference between groups. These data indicate that a functional CD8+ T cell response to HCMV consistently develops in early life and is sustained throughout childhood.To evaluate the effector function of the HCMV-specific cells, we performed IFN-γ ELISPOT assays and compared these with tetramer staining. Fifty-six HLA-A2+ subjects  and 17 HLA-A24+ subjects  were selected for ELISPOT assays. A comparison of the ELISPOT assay and tetramer assay is shown in Figure To identify the differentiation status of HCMV-specific CD8+ T cells in individual groups, we studied the expression of CD28, CD27, CD45RA, and CD45RO on peripheral blood tetramer+ CD8+ cells using four-color flow cytometry. Both HLA-A2+ and HLA-A24+ subjects were included in this analysis, the results of which are shown in Table Table In this study, we adapted immunological techniques which have been widely applied to the study of adults to a large pediatric population of over 1000 children. The aim of the study was to define the quantity and quality of antiviral responses in early infection and relate any changes in these to age. Long term longitudinal studies of CMV specific populations are difficult to perform in healthy pediatric populations; nevertheless, using similar large scale cross sectional approaches we have previously observed a clearly dynamic host-virus relationship in adult infection ,25.The pattern of age-related accumulation observed in this cross-sectional analysis revealed strong responses in the first year of life, consistent with acquisition of infection during this period. The acquisition of cellular immunity can be regarded as a good marker for viral infection, at a time when serological responses might be confounded by the presence of maternally transmitted antibody.In particular, we evaluated in detail the frequencies of specific CD8+ T cells during the first four years of life Table , 4. The In this study, responsiveness in both ICS and ELISPOT assays was significantly correlated with tetramer staining. High frequencies of HCMV specific CD8+ cells were observed in even in 0–2 year-old infants, and, although the number of infants examined was relatively small, these findings suggest that infants infected with HCMV have a functional CD8+ T cell response to HCMV similar to that in adults. Moreover, all of these infants but one, who were positive for HCMV tetramer under 1-year-old, were asymptomatic in this study. Those observations are consistent with a recent smaller study which showed that newborns with congenital HCMV infection were asymptomatic and had functional and mature HCMV-specific CD8+ T cells ,29. A reBased on an analysis of CD28/CD27 expression, the stage of "maturation" or "differentiation" of HCMV-specific CD8+ T cells can be defined. CD28+ CD27+ T cells are considered to be naïve cells or early-differentiated T cells, progressing to CD28-CD27- T cells, thought to be fully differentiated T cells -14. We hNaïve CD8+ T cells express CD45RA, and this is uniformly expressed in cord blood. While antigen-experienced CD8+ T cells initially express CD45RO, re-expression of CD45RA may occur and this has been described as a state of late or terminal differentiation – although longitudinal studies of single cells have not been performed. According to this scheme, it was expected that a subset of CD45RA+CD45RO- tetramer+ cells would accumulate with age. In contrast, the CD45RA+CD45RO- subset was predominant in all of the age-groups, even in the youngest children and the proportion did not change through life. There are two possibilities to explain these observations. First, this state may be driven by continuous interaction with antigen. However, this hypothesis is not entirely consistent with the results of CD28/CD27 subsets of this study, in which CD28-CD27- "late" differentiated cells, were predominant in only the elderly. Alternatively, the levels of CD45 isoforms expression correlate poorly with the stages of antigen-driven T cell differentiation. This is supported by a previous study, which showed that CD45RA expression was not correlated to the differentiation phenotypes (28). Expression of other cell surface markers, such as CCR7, CD57 or CD85j might correlate more closely with the evolution of T cell responses over time -37.In this study, HLA-A2+ subjects were selected by flow cytometry using the HLA-A2 mAb. However, the HLA-A2 molecular type of Japanese is classified into three subtypes. HLA-A*0201, A*0206, and A*0207 representing 10%, 10%, and 3%, of Japanese, respectively . BecauseCMV sero-status of subjects was not evaluated in this study. We reported that 77% of HCMV-seropositive HLA-A2 subjects were positive for CMV tetramer . These fIn previous study , we showIn conclusion, the maintenance of large populations of HCMV-specific CD8+ T cells was observed throughout childhood, with substantial responses to infection in early infancy. Overall major age-related changes in tetramer+ CD8+ T cells were not observed in the first two decades of life and tetramer+ CD8+ cells of young infants showed a mature phenotype and function similar to that in adults. These data provide novel insight into the relative maturity of the infant antiviral response, and the impact of CMV on the childhood immune system. Additionally, they demonstrate the technical feasibility of detailed cellular immunological studies in pediatric populations, undertaken at a large scale as may be applicable in other childhood infections and vaccine programs.HCMV, human cytomegalovirusMCMV, murine cytomegalovirusTetramer, tetrameric complexesELISPOT, enzyme-linked immunospotICS, intracellular cytokine stainingHLA, human leukocytic antigenFITC, fluorescein isothiocyanatePerCP, peridinin chlorophyll proteinAPC, allophycocyaninPE, phycoerythrinPBMCs, peripheral mononuclear cellsHK, SS, JN, ML, AV and PK conceived and designed the experiments. HK performed the experiments. HK, AI, TS, TF, SN, and HN participated in data collection. HK and JN analysed the data. HK and PK wrote this paper.The author(s) declare that they have no competing interests.


Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid.Clinical isolates of the gastric pathogen H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity.The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori.These studies demonstrated that  Helicobacter pylori, a Gram negative bacterium, is a highly host-adapted gastric pathogen that has been implicated in a wide spectrum of diseases ranging from gastritis to adenocarcinoma  was used for cloning rocF genes.H. pylori and E. coli were harvested in 0.9% NaCl and sonicated in an ice-bath . Following centrifugation , the supernatants were retained on ice until measurement of arginase activity. Protein concentrations were determined by the bicinchoninic acid method using bovine serum albumin as a standard .Arginase activity was measured using a colorimetric arginase assay developed and validated previously . The extUrease activities were measured using the phenol-hypochlorite method as described previously .2, 0.05% (w/v) sodium azide, and 1 mM MgCl2, pH 10.4). To develop the blot, 3-indoxyl phosphate (10 μg/ml) and nitroblue tetrazolium (100 μg/ml) in glycine buffer were used.Bacterial proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% resolving gel and 10 μg of protein loaded per lane. These electrophoresed proteins were then transferred to methanol-treated polyvinylidene difluoride membrane using the Trans-blot cell transfer system (Bio-Rad). The blots were subsequently blocked in 5% (w/v) nonfat dry milk in Tris-buffered saline containing 0.5% (v/v) Tween-20 (TBST-1), and incubated with the primary RocF antiserum  for 2 h . After t4, 0.20–0.25 mM dNTPs, 200 pmol of each primer, 2.5 U of Pfx polymerase (Invitrogen) and 10× Pfx polymerase buffer (Invitrogen). E. coli was transformed by the heat shock CaCl2 method.Plasmid DNA was extracted by column chromatography (Qiagen) or by alkaline lysis . RestricrocF gene from clinical isolates and laboratory-adapted strains was PCR-amplified using the primers RocF-F27  and RocF-R28 . The conditions for PCR amplification were set at 94°C for 5 minutes;  × 30; 68°C for 5 minutes; 4°C overnight. After amplification, the PCR products (1118 bp) were analyzed by gel electrophoresis and purified via Qiaquick DNA extraction (Qiagen), and then cloned into pBS predigested with EcoRV. Following an overnight ligation, E. coli DH5α was transformed by the calcium chloride method and the colonies screened via the blue-white method using X-gal [The ng X-gal .rocF gene was PCR-amplified as described above and cloned directly into pCR2.1 in E. coli strain Top10 by electroporation, according to manufacturer's guidelines (Invitrogen).The ClaI and PstI restriction endonucleases to confirm presence of the insert. The rocF inserts were sequenced using T3  and T7-1  by the Sanger dideoxynucleotide method at Iowa State University DNA Sequencing and Synthesis Facility. The sequence data have been deposited into the GenBank database as accession numbers EF126010 to EF126032.Plasmid DNA from white transformants was purified by Qiagen column chromatography and was digested with rocF gene of clinical isolates and laboratory-adapted strains were analyzed by BLAST searches, sequence alignments, and sequence translations at the National Center for Biotechnology Information website [The sequences of the  website . Amino a website . Multi-s website ,63 and f website . The nei website ,66.rocF, harboring the wild-type rocF gene from H. pylori strain 26695, was mutated using the Quik Change kit following the manufacturer's instructions . The primer sequences were as follows: 1) I174M: DM141-RocFI174M-F1 5'GGAGGGTTAGAAATGGATCCTAAATGTTTG3'; DM142-RocFI174M-R1 5'CAAACATTTAGGATCCATTTCTAACCCTCC3' ; 2) S232I: DM143-RocFS232I-F1 5'GATATTATTTATCTCATTTTGGATTTGGAC3'; DM144-RocFS232I-R1 5'GTCCAAATCCAAAATGAGATAAATAATATC3';Plasmid pQE30-3) D257N: DM266-D257NrocF-F1 5'GGCTGAGTTTTAATGAACTCAAGCAATTACTGG3'; DM267-D257NrocF-R1 5'CCAGTAATTGCTTGAGTTCATTAAAACTCAGCC3';ggatccATGATTTTAGTAGGATTAGAAGCAGAG; BamHI site underlined; non-rocF sequence in lower case); 5) RocF-R8: gcctgcagAGTAACTCCTTGCAAAAGA GTGCTTC; PstI site underlined; non-rocF sequence in lower case). All constructs were confirmed by sequence analyses.4) RocF-F6: gcH. pylori strains, conducted some of the arginase Western blots, and PCR amplified, cloned and sequenced the rocF gene from about half the strains; ELW conducted some of the arginase Western blots, conducted some of the arginase assays from E. coli and H. pylori strains, and PCR amplified, cloned and sequenced some of the arginase genes; MLL conducted the experiments on strain-specific arginase regulation and overexpression and provided detailed editorial comments on the manuscript; EFH conducted arginase activity in F-12 broth cultures; SB conducted arginase assays on H. pylori strains; JRB conducted some of the arginase assays from E. coli and H. pylori; DS conducted the site-directed mutagenesis of arginase and conducted the related arginase and Western blot assays; GLM assisted with writing the manuscript, critically analyzed the data and provided numerous strains for the study; DJM conceived of and designed the study, directed the execution of the study, conducted the phylogenetic analyses, conducted the urease assays, prepared some of the figures, and edited later drafts of the manuscript. All authors read and approved the final manuscript.JGH co-designed the study, drafted the manuscript, collected data on arginase activity in some H. pylori strains used in this study.Table S1. Characteristics and arginase activity of Click here for fileH. pyloriFig. S1. Urease activity of clinical isolates of . H. pylori strains were grown on Campylobacter blood agar for 48 h and measured for urease activity using the phenol hypochlorite method. Urease activity is shown as nmol ammonium per min per mg protein ± standard deviation. A. Urease activity of 12 clinical isolates. B. Comparison of urease and arginase activities from the same 12 clinical isolates. There was no correlation between the arginase activity and the urease activity of the strains.Click here for fileE. coli. Fig. S2. Variation of arginase activity in Arginase activity was assessed in the genetically stable background of E. coli in order to eliminate the genetic variability of other H. pylori loci as a compounding factor. Arginase activity in E. coli containing rocF genes from different H. pylori strains. The rocF genes from 20 H. pylori strains were cloned into pBS and the arginase activities from plasmid-bearing E. coli clones were measured. The graph shows the average arginase activity (pmol L-Orn/min/mg protein) ± standard deviation of one experiment representative of three. Plasmid names include the H. pylori strain name from which the rocF gene was derived. For example, pJH254-1 carries rocF from H. pylori strain J254, pJH17 carries rocF from strain HPDJM17, and procFB1 carries rocF from strain B1.Click here for file


Purpose. The functions of vascular endothelial growth factor (VEGF) in scar formation after trabeculectomy were investigated in a human Tenon fibroblast cell line from glaucoma patients using lentivirus-mediated VEGF shRNA. Methods. Human Tenon fibroblast (HTF) cells were isolated from scar tissue of glaucoma patients during secondary surgery. Lentivirus-VEGF-shRNA was constructed and transfected into HTF cells. Subsequently, VEGF mRNA and protein expression were analyzed using quantitative RT-PCR and western blotting, respectively, and the effects of VEGF knockdown were analyzed. The inhibition of HTF proliferation was monitored according to total cell numbers using ScanArray. Results. Both mRNA and protein levels of VEGF were reduced by lentivirus-mediated VEGF-shRNA, and proliferation of HTF cells was inhibited. Conclusions. Primary cultures of human Tenon fibroblast (HTF) were established, and proliferation was decreased following inhibition of VEGF. VEGF may be a suitable therapeutic target for reducing scar tissue formation in glaucoma patients after filtration surgery. Glaucoma is a common cause of blindness, and filtration surgery remains the most effective therapy for reducing intraocular pressure (IOP). Bleb scarring is the leading cause of surgical failure , and cor in vitro delivery of VEGF. This suggested that Tenon fibroblasts are direct targets of VEGF during scar formation after filtration surgery [Vascular endothelial growth factor (VEGF) plays an important role in scar formation. In a previous study, we showed that VEGF levels were significantly increased in the aqueous humor of the anterior chamber of primary open angular glaucomas (POAG) in patients and rabbits at surgery. Expression of the VEGF receptors VEGFR-1 (Flt-a) and VEGFR-2 (KDR/Flk-1) in Tenon fibroblasts was increased and fibroblast proliferation was stimulated following surgery . Moreove surgery , indicat surgery .The use of anti-VEGF agents to control scar formation after trabeculectomy remains controversial –12. How RNA interference (RNAi) has emerged as a powerful tool to induce loss-of-function phenotypes by posttranscriptional silencing of gene expression and has been used to suppress VEGF-induced retinal neovascularization . In the The tenets of the Declaration of Helsinki were upheld, approval was granted by the IRB committee, and informed consent was obtained for all human experiments.Scar tissues were obtained from eight bleb-scarring patients who had secondary surgeries after trabeculectomy. There was no difference in terms of harvesting HTF between the patients with the primary and secondary surgeries. Primary cells were cultured as tissue adherence explants. For series quality control, observations of cell morphology, immunofluorescence cell validation using mouse antihuman vimentin monoclonal antibody , mycoplasma testing (Huyuan Biotech), and stability testing were performed and growth curves, proliferation, and population doubling times were determined in isolated primary cells. Primary HTF cells were mycoplasma free and no significant changes in cell morphology or growth rates were observed between passage 5 and passage 15 cells.shRNA-1: 5′-CCGGGCGCAAGAAATCCCGGTATAACTCGAGTTATACCGGGATTTCTTGCGCTTTTT-3′shRNA-2: 5′-CCGGGACGTGTAAATGTTCCTGCAACTCGAGTTGCAGGAACATTTACACGTCTTTTT-3′shRNA-3: 5′-CCGGATGCGGATCAAACCTCACCAACTCGAGTTGGTGAGGTTTGATCCGCATTTTTT-3′shRNA-4: 5′-CCGGCAAGATCCGCAGACGTGTAAACTCGAGTTTACACGTCTGCGGATCTTGTTTTTT-3′shRNA-5: 5′-CCGGCAAGATCCGCAGACGTGTAAACTCGAGTTTACACGTCTGCGGATCTTGTTTTTT-3′Positive control miR-214 was designed according to the methods described in Yang et al. [Five VEGF probes were designed and synthesized based on the released sequence from RNAi Codex as follows:g et al. . SubsequRecombinant VEGF-shRNA plasmids were transiently transfected into HTF cells, and shRNAs were screened using a pSLIK miRNA-based vector that lacked VEGF interference sequences as a negative control.μg/mL and the cells were incubated for at least 72 h as necessary. The cells were harvested at indicated times after induction, and cell extracts were prepared using RNeasy Kits (Qiagen) for qRT-PCR or using RIPA buffer for western blotting. Standard protocols were used for qRT-PCR and western blot analyses.Both transiently and stably infected cell lines were grown in 6-well plates with no selection of drug for validation using qRT-PCR and western blotting. DOX was added to cell cultures at a final contraction of 1 All five VEGF shRNAs reduced VEGF mRNA expression in HTF cells, and shRNA-2 and shRNA-3a reduced expression to 41.0% and 36.5%, respectively, and were selected for subsequent VEGF functional knockdown experiments.5 TU/mL.Lenti-neo-GFP with the same backbone as pSLIK-VEGF-shRNA was used as the control titer. Viruses with recombinant target sequences and Lenti-neo-GFP were produced in parallel. Virus titers were determined using FACs analysis, and lentiviral titers of pSLIK-neo-GFP were 1.3 × 102 and 95% air. HTF cells were infected with lentivirus-shRNAs at MOI = 1. At 96 h after transfection, 1 μg/mL of DOX was added to the cultures to induce the transcription of shRNA.Primary HTF cells were cultured in Dulbecco's Modified Eagle Medium containing high glucose (Gibco BRL) and 10% fetal bovine serum. Cells were incubated in a humidified incubator at 37°C in 5% COβ-actin . Peroxidase-conjugated secondary antibodies were used as secondary detection reagents with an enhanced chemiluminescence kit . X-ray films were developed to visualize chemiluminescence signals.At four days after induction with DOX, transfected HTF cells were collected and lysed in buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 1% NP-40, and protease inhibitors . Total protein in extracts was measured using the Bradford method. Aliquots of total proteins were resolved using SDS polyacrylamide gel electrophoresis and were then transferred onto immobilon P membrane using an SD semidry transfer apparatus according to the manufacturer's instructions. Membranes were incubated with polyclonal antihuman VEGF antibody  and monoclonal antihuman HTF cells were seeded into 96-black well plates at 30,000 cells per well. Cells were then infected with lentiviru-shRNA-2 and shRNA-3. After four days of induction with DOX, cell quantities were determined using ArrayScan and curves were calculated using PRISMBIOLAB.Interference efficiencies were compared between various transfected VEGF-shRNA plasmid constructs in cultured HTF cells. VEGF mRNA levels were measured using quantitative RT-PCR.“Naïve” indicates relative VEGF mRNA expression in nontransfected controls and vehicle indicates that of samples transfected with vehicle only. With the exception of shRNA-4, all VEGF-shRNAs significantly reduced VEGF mRNA levels, and VEGF-shRNA-2 and VEGF-shRNA-3 inhibited expression to 41.0% and 36.5%, respectively, and were selected for functional analyses; see Lentivirus-carrying VEGF-shRNA-2 and VEGF-shRNA-3 were transfected into cultured HTF cells. VEGF protein expression was then investigated using western blotting, which showed reduced VEGF protein expression in HTF cells transfected with lentivirus VEGF-shRNA .In these experiments, shRNA-2 and shRNA-3 significantly knocked down VEGF protein expression after 96 h of DOX treatment.Lentivirus-VEGF was transfected into cultured HTF cells. After 96 h, DOX was added to the culture medium to induce the transcription of shRNA.Numbers of vehicle transfected HTF cells did not significantly differ from those of naïve HTF cells. However, total numbers of VEGF-shRNA-2 and VEGF-shRNA-3 transfected HTF cells were suppressed, particularly with VEGF-shRNA-3; see Filtration surgery remains the most effective choice for glaucoma patients. However, excessive scarring remains a leading cause of surgical failure –21. Anti in vitro. Accordingly, bevacizumab has been administered to inhibit Tenon fibroblast proliferation but requires repetitive injections [Among wound healing factors, angiogenesis plays a key role and VEGF is a key factor because it stimulates angiogenesis, inflammation, and fibrosis at operative sites in glaucoma patients. Several VEGF isoforms and three high affinity VEGF receptors have been identified . In partjections . The maijections –33. Howejections , 35.In the present study, we demonstrated that inhibition of VEGF expression using lentivirus-mediated shRNA suppresses the proliferation of human Tenon fibroblast cells. These observations may reflect binding of VEGF to VEGFR-2, leading to activation of the MAPK/ERK pathway, which is a key signaling pathway of cell proliferation, differentiation, and apoptosis. VEGFR-2 dimerization activates the intracellular serine/threonine kinase Raf, the dual specificity MAPK kinase (MEK), and the MAPK-ERK pathway upon stimulation, leading to increased DNA synthesis and cell proliferation. In addition, inhibition of ERK phosphorylation limits proliferation of Tenon fibroblasts, indicating that VEGF stimulates the proliferation of Tenon fibroblasts via the MAPK/ERK pathway. Phosphorylated ERK is translocated into the nucleus and phosphorylates growth factors, such as c-fos, c-Jun, c-myc, and ATF2, which lead to increased nucleotide synthesis, activated transcription and translation, and enhanced cell cycle progression , 37. VEGWe used lentivirus-mediated VEGF-shRNA to suppress VEGF mRNA expression. Previous studies of RNAi for VEGF show inhibition of neovascularization in ocular angiogenic diseases and demonstrate significant specific inhibition of VEGF by siRNAs , 39. How in vivo siRNA delivery are another obstacle to siRNA therapies. However, proprietary modifications of siRNA QPI-1007 structure and chemistry have been reported, with similar drug efficacy and reduced off-target effects [Efficiency and specificity of delivery represent a significant obstacle to therapeutic RNAi. In the present study, a lentivirus-mediated VEGF-shRNA system was used for the following reasons: lentiviruses efficiently transfect cells , lentivi effects .The main surgical failure of using MMC or 5-FU is bleb leaking, which is followed by complications as infections and hypotony-related maculopathy. In the previous study, we observed the postoperative effect of inhibiting VEGF in rabbit model. In addition, no bleb leakages were found. In the future study, we will research on lentivirus VEGF-shRNA inhibiting scarring after trabeculectomy in animal models. The comparative research will be also designed in antiproliferative effects between shRNA and MMC.In summary, lentivirus VEGF-shRNA was successfully used to knock down VEGF expression in Tenon fibroblasts from patient glaucomas, and fibroblast proliferation was suppressed. Subsequent inhibition of ERK phosphorylation had a similar effect, indicating that VEGF stimulates Tenon fibroblast proliferation via the MAPK/ERK pathway. RNAi technology may produce an adjunct agent that improves outcomes of glaucoma filtration surgery, especially secondary surgery for patient with postoperative scar formation.


Pseudomonasaeruginosa phage PaP1, we performed steady-state and pre-steady-state kinetic analyses of nucleotide incorporation opposite 8-oxoG by Gp90 D234A that lacks exonuclease activities on ssDNA and dsDNA substrates. Gp90 D234A could bypass 8-oxoG in an error-free manner, preferentially incorporate dCTP opposite 8-oxoG, and yield similar misincorporation frequency to unmodified G. Gp90 D234A could extend beyond C:8-oxoG or A:8-oxoG base pairs with the same efficiency. dCTP incorporation opposite G and dCTP or dATP incorporation opposite 8-oxoG showed fast burst phases. The burst of incorporation efficiency  is decreased as dCTP:G > dCTP:8-oxoG > dATP:8-oxoG. The presence of 8-oxoG in DNA does not affect its binding to Gp90 D234A in a binary complex but it does affect it in a ternary complex with dNTP and Mg2+, and dATP misincorporation opposite 8-oxoG further weakens the binding of Gp90 D234A to DNA. This study reveals Gp90 D234A can bypass 8-oxoG in an error-free manner, providing further understanding in DNA replication encountering oxidation lesion for P.aeruginosa phage PaP1.As one of the most common forms of oxidative DNA damage, 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) generally leads to G:C to T:A mutagenesis. To study DNA replication encountering 8-oxoG by the sole DNA polymerase (Gp90) of  Accurate synthesis of DNA is of great importance for genomic integrity in all forms of life. DNA replication is generally performed by DNA polymerases with high fidelity. Accurate DNA replication is under constant threat, which are formed within the genome . DNA damLesion tolerance is achieved, in part, by special translesion synthesis (TLS) polymerases, which are able to bypass lesions during DNA replication . CompareBacillus stearothermophilus DNA Polymerase I are capable of bypassing 8-oxoG in a mostly error-free manner  ATP was obtained from PerkinElmer Life Sciences . Bio-spin columns were obtained from Bio-Rad . Phage PaP1 was propagated and extracted, and its genomic DNA was extracted and purified as described previously [Mutagenesis was performed using a QuikChange site-directed mutagenesis kit . Oligonucleotides were synthesized by Midland Certified Reagent Co . All unlabeled dNTPs and T4 polynucleotide kinases were obtained from Amersham Bio-sciences . Ni-NTA mini-spin columns were purchased from GE Healthcare . [γ-eviously ,22. OtheEscherichia coli polymerase I [E60A:sense, 5′-GTAGTAGCCGCCCACGGCGGTAACATTCTGGCGTTCTAC-3′;antisense, 5′-GCC GTGGGCGGCTACTACGACACAGTGATGACTGTAGCT-3′.D137A:sense, 5′-ATTAACTTCGCCCTTATGTCGATGAAGCTTGTGGAAGATATG-3′;antisense, 5′-CGACATAAGGGCGAAGTTAATCATGTTGTGAGCCACTACGCG-3′.D234A:sense, 5′-TGTATCTATGCCGTAAAGGCGAACACCGCTGTATGGCACTGG-3′;antisense, 5′-CGCCTTTACGGCATAGATACAGTAGTAAAGCATATCGGCTGC-3′.The DNA sequences were confirmed by sequence analysis prior to bacterial expression. Wild-type and three mutants were expressed in E. coli A307 (DE3) cells, followed by purification through a HisTrapTM FF column  as described previously [Glu-60, Asp-137 and Asp-234 were predicted as the potential exonuclease active residues of Gp90 by alignment of the sequences of Gp90, T7 DNA polymerase, and merase I . These reviously .32P-labeled 27-mer ssDNA or 32P-labeled 27-mer/62-mer primer/template dsDNA, respectively, in a buffer containing 40 mM Tris-HCl (pH 7.5), 30 mM Mg2+ and 10 mM DTT at 37 °C. After 0.5, 1, 2, or 5 min, reactions were terminated with a quench solution containing 20 mM EDTA, 95% formamide (v/v), bromphenol blue, and xylene cyanol. The samples were then separated on a 20% polyacrylamide (w/v)/7 M urea gel. Products were visualized and quantified using a phosphorimaging screen and Quantity OneTM software  [The ssDNA or dsDNA exonuclease activities were determined by mixing 10 nM each of DNA polymerase with 20 nM CA, USA) .32P-labeled 27-mer primer, annealed to 62-mer template oligonucleotide, was extended in the presence of all four dNTPs in a buffer containing 40 mM Tris-HCl (pH 7.5), 30 mM Mg2+, 10 mM DTT, and 50 mM potassium glutamate at 37 °C. The reactions were initiated by mixing 20 nM DNA substrates with 10 nM Gp90 or Gp90 mutants and 350 μM each of dNTP for 0.5, 1, 2, or 5 min. Reactions were terminated by a quench solution containing 20 mM EDTA, 95% formamide (v/v), bromphenol blue, and xylene cyanol. The samples were then separated on a 20% polyacrylamide (w/v)/7 M urea gel. Products were visualized and quantified using a phosphorimaging screen and Quantity OneTM software. Primer extension beyond 8-oxoG were performed similarly by mixing 20 nM DNA substrates containing 8-oxoG with 10 nM Gp90 or Gp90 D234A and 350 μM each of dNTP and reacted for 0.5, 1, 2, or 5 min.A 32P-labeled 27-mer/62-mer and 32P-labeled 28-mer/62-mer dsDNA substrates, respectively  using GraphPad Prism Version 6.0  to determine kcat and Km values [kcat/Km) of incorrect dNTP by that of the correct dCTP.Steady-state kinetic analysis of single-base incorporation and next-base extension by Gp90 D234A were performed using ectively . The molm values . The mis32P-labeled 27-mer/62-mer primer/template dsDNA substrate and 160 nM Gp90 D234A mixtures with an equal volume of 2 mM dNTP and 60 mM Mg2+ complex, incubated for a varied time (0.005–60 s) at 37 °C, and then quenched with 0.6 M EDTA. Substrate and product DNA were separated by electrophoresis on a 20% polyacrylamide (w/v)/7 M urea gel. The products were then visualized using phosphorimaging and quantified using Quantity OneTM software. The product and time were fit to Equation (1), corresponding dNTP incorporation in the first binding phase and the subsequent steady-state phase:A is the amount of active complex formed in the first binding phase, nM; kp is the dNTP incorporation rate in the first binding phase, s−1; kss is the rate of steady-state dNTP incorporation, s−1; and t is time, s−1.Rapid chemical quench experiments were performed using a model RQF-3 KinTek Quench Flow Apparatus  with 50 mM Tris-HCl (pH 7.5) buffer in the drive syringes . Reactio2+, and incubated for a varied time. The product amount and time were fit to Equation (2), corresponding dNTP incorporation only in the first binding phase:A is the amount of active complex formed in the first binding phase, nM; kobs is the dNTP incorporation rate in the first binding phase (burst rate), s−1; and t is time, s−1.Nucleotide incorporation rates in the first binding phase could also be determined by rapidly mixing 400 nM Gp90 D234A and 200 nM DNA with an equal volume of different concentrations of dNTP and 60 mM Mgkobs) and concentrations of dNTP were fit to hyperbolic Equation (3) to obtain kpol and Kd,dNTP values:kpol is the maximal rate of dNTP incorporation, s−1; and Kd,dNTP is the equilibrium dissociation constant for dNTP in the burst phase, μM [Burst rates  and a template (5′-TGAATTCTAAT GTAGTATAGTAATCCGCTCTATCGGACGTATCGTCACCATAACTCTGTAGC-3′) was coupled to a streptavidin (SA) chip. The Cdd (double deoxycytosine) at the 3′-end of primercan stop DNA polymerization. A varying concentration of Gp90 or Gp90 D234A (20-600 nM) was flowed over the chip in a buffer containing 40 mM Tris-HCl (pH 7.5), 10 mM DTT, and 50 mM potassium glutamate at a flow rate of 10 µL/min at room temperature. In a control flow cell, biotin was used instead of the biotinylated DNA to compensate for background. The chip surface was regenerated by injection of 1 M NaCl solution at a flow rate of 100 μL/min. The binding signal was fitted to Equation (4) using a steady-state model provided by BIA evaluation 3.0.2 computational software (Biacore). The dissociation constants Kd were calculated using the steady-state average RU.Y is the response signal corresponding to the binding, RU; B is the concentration of protein, nM; RUmax is the maximal binding amount, RU; and Kd is the dissociation constant, nM. All experiments were carried out three times, and standard errors were derived using Prism 6.0 software.Surface plasmon resonance analysis was performed by using a Biacore-3000 instrument  . An anne2 and 1 mM dCTP or dATP. In the presence of Mg2+ and dNTP, the active site of DNA polymerase would locate at the 3′-end of primer where dNTP is paired or mispaired opposite G or 8-oxoG in the template strand. The binding affinities of the polymerase to DNA were determined by the same methods as described above.The similar binding assays were also performed in the presence of 30 mM MgClE. coli polymerase I [32P-labeled 27-mer ssDNA  for each single dNTP incorporation opposite G or 8-oxoG by Gp90 D234A were measured  for the next-base extension beyond G or 8-oxoG by Gp90 D234A  . In the Excess molar concentration of DNA, compared with polymerase, was used to determine the rates of dNTP incorporation opposite G or 8-oxoG by Gp90 D234A in burst and linear steady-state phases . Among fkpol) and the apparent dissociation constants of dNTP from the Gp90-DNA-dNTP ternary complex  were estimated by fitting the burst rates against dNTP concentrations to Equation (3)  was reduced by 59-fold compared with dCTP incorporation opposite G. The efficiency of dATP incorporation opposite 8-oxoG was further reduced by 188-fold compared with dCTP incorporation opposite 8-oxoG mainly because of the 115-fold reduction in kpol.dCTP incorporation opposite G and dCTP or dATP incorporation opposite 8-oxoG exhibited fast burst phases. Moreover, the maximal rates of nucleotide incorporation (tion (3) . kpol ofKd,DNA) between DNA and DNA polymerase were determined by surface plasmon resonance to determine whether 8-oxoG affects the binding affinity of Gp90 D234A to DNA [Kd,DNA was obtained by fitting the observed response signals against protein concentrations to Equation (4) using the steady-state model. In the absence of dNTP and Mg2+, DNA polymerase randomly bound to DNA to form a binary complex. The binding of polymerase to DNA containing G or 8-oxoG exhibited a similar Kd of 108 and 116 nM, respectively  was three-fold higher than that of the G complex (11 nM) in the presence of dCTP and Mg2+, indicating that 8-oxoG in the template reduced the binding of Gp90 to DNA by three-fold. In the presence of dATP and Mg2+, Kd,DNA of 8-oxoG complex was further increased to 45 nM, showing that the presence of incorrect dATP further reduces the binding affinity of Gp90 D234A to DNA.In the presence of dNTP and Mgand Mg2+ . TherefoE. coli DNA polymerase I and Asp-174 in T7 DNA polymerase based on sequence alignment analysis. Furthermore, Gp90 D234A was used as exonuclease-deficient DNA polymerase for kinetic analysis in this work.Gp90 is the sole DNA polymerase responsible for DNA replication in PaP1 . The exo−4 to 10−5 for G and 8-oxoG  is lower than that of dCTP. Similarly, human Y-family DNA polymerase κ also shows a fast burst phase for dATP incorporation opposite 8-oxoG [For most DNA polymerases, incorporation of a correct dNTP shows a biphasic character; whereas misincorporation shows only a linear steady-state phase. Unexpectedly, dCTP or dATP incorporation opposite 8-oxoG by Gp90 D234A shows a biphasic character , althouge 8-oxoG . T7 DNA e 8-oxoG . However2+, Gp90 D234A randomly binds to DNA to form a binary complex. The similar Kd,DNA values (2+, DNA polymerase is prone to bind at the 3′-end of the primer strand and form a ternary complex [Kd,DNA values of ternary complexes compared with binary complexes indicate that the presence of dNTP and Mg2+ stabilizes the binding of polymerase to DNA for G and 8-oxoG. Notably, the Kd,DNA value of 8-oxoG ternary complex was three-fold higher than that of G ternary complex in the presence of dCTP and Mg2+, indicating that 8-oxoG in the template reduces the binding of Gp90 D234A to DNA (2+, the misincorporation further weakens the binding affinity of Gp90 D234A to DNA.The physical binding of Gp90 D234A to DNA containing G or 8-oxoG was measured. In the absence of dNTP or MgA values  show tha complex . The lowA to DNA . In the S. solfataricus DNA polymerase Dpo4 catalyzes 8-oxoG bypass efficiently and accurately. Crystal structures reveal the potential role of Arg332 in stabilizing the anti-conformation of 8-oxoG through the hydrogen bond or ion-dipole pair, which results in an increased enzymatic efficiency for dCTP insertion and a less favorable formation of a Hoogsteen pair between 8-oxoG and dATP [Since the crystal structures of PaP1 DNA polymerase in complex with DNA containing 8-oxoG are not available, crystal structures of other DNA polymerases may provide insight in how Gp90 bypasses 8-oxoG. and dATP . DNA poland dATP .−4 to 10−5. Misincorporation opposite G inhibits its subsequent extension, whereas misincorporation opposite 8-oxoG does not inhibit its subsequent extension. dCTP incorporation opposite G and dCTP or dATP incorporation opposite 8-oxoG show a fast burst phase, indicating that the incorporation step is faster than the subsequent dissociation of polymerase from DNA. The burst incorporation efficiency is decreased in the following order: dCTP:G > dCTP:8-oxoG > dATP:8-oxoG. 8-oxoG in the template does not affect the binding of Gp90 D234A to DNA in the binary complex, in contrast to that in the ternary complex in the presence of dCTP and Mg2+. dATP misincorporation opposite 8-oxoG further weakens the binding affinity of Gp90 D234A to DNA. This study reveals that Gp90 D234A can ensure error-free bypass of 8-oxoG, providing an understanding of the DNA replication while encountering oxidation lesions for P. aeruginosa phage PaP1.In this work, we investigated the steady-state and pre-steady-state kinetics of nucleotide incorporation opposite G or 8-oxoG using Gp90 D234A, which has eliminated the ssDNA and dsDNA exonuclease activities. Among four dNTPs, dCTP was preferentially incorporated opposite G or 8-oxoG, exhibiting similar misincorporation frequencies of 10


Despite the remarkable progress to fight against breast cancer, metastasis remains the dominant cause of treatment failure and recurrence. Therefore, control of invasiveness potential of breast cancer cells is crucial. Accumulating evidences suggest Aryl hydrocarbon receptor (Ahr), a helix-loop-helix transcription factor, as a promising target to control migration and invasion in breast cancer cells. Thus, an Ahr-based exploration was performed to identify a new Ahr agonist with inhibitory potentials on cancer cell motility.in vitro using the MDA-MB-231 and T47D cell lines. The silencing RNAs were transfected into cells by electroporation. Expressions of microRNAs (miRNAs) and coding genes were quantified by real-time PCR, and the protein levels were detected by western blot.For prediction of potential interactions between Ahr and candidate molecules, bioinformatics analysis was carried out. The interaction of the selected ligand with Ahr and its effects on migration and invasion were examined in silico and in vitro results identified Flavipin as a novel Ahr agonist. It induces formation of Ahr/Ahr nuclear translocator (Arnt) heterodimer to promote the expression of cytochrome P450 family 1 subfamily A member 1 (Cyp1a1). Migration and invasion of MDA-MB-231 and T47D cells were inhibited with Flavipin treatment in an Ahr-dependent fashion. Interestingly, Flavipin suppressed the pro-metastatic factor SRY-related HMG-box4 (Sox4) by inducing miR-212/132 cluster. Moreover, Flavipin inhibited growth and adhesion of both cell lines by suppressing gene expressions of B-cell lymphoma 2 (Bcl2) and integrinα4 (ITGA4).The Taken together, the results introduce Flavipin as a novel Ahr agonist, and provide first evidences on its inhibitory effects on cancer cell motility, suggesting Flavipin as a candidate to control cell invasiveness in breast cancer patients. Breast cancer is a dominant cause of cancer-related death among cancer patients worldwide . DespiteThe Aryl hydrocarbon receptor (Ahr) is a heterodimeric protein belongs to the helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors. It is activated by wide range of natural and synthetic molecules. Upon ligation, Ahr heterodimerizes with the Ahr nuclear translocator (Arnt) to modulate cell functions through downstream genes such as cytochrome P450 (Cyp) , 10. RecImportantly, Ahr has emerged as a potential therapeutic strategy to control motility of breast cancer cells including migration, invasiveness and metastasis. For example, omeprazole-activated Ahr based down-regulation of matrix metalloproteinase-9 (MMP-9) and C-X-C chemokine receptor 4 (CXCR4) suppresses invasion and metastasis of MDA-MB-231 cells . The 3,3Flavonoids are a wide range of natural compounds that have been intensively studied for their anticancer properties , 19. Intin silico and in vitro approach to examine a potential interaction between Flavipin and Ahr in relation to breast cancer. For the first time, we introduced Flavipin as a novel Ahr agonist, and we provided evidences for its inhibitory activity on migration and invasion of ER-negative and ER-positive breast cancer cell lines. In addition, we suggested a precise molecular explanation of the reported biological effects.Herein, we applied an http://www.scripps.edu/mb/olson/doc/autodock). The 3D chemical structure of Ahr PAS-A (PDB ID: 4M4X) was retrieved from the Protein Data Bank (http://www.pdb.org), and that of Flavipin (CID_3083587), Epicorazines A (CID_ 57383998), Epicorazines B (CID_ 73891006) and Lonchocarpol A (CID_124035) were retrieved from Pubchem compound database (http://www.ncbi.nlm.nih.gov/pccompound). The active sites of the target protein were identified using Q-site Finder. Ligands docked to the receptor were reflected as a rigid body, and receptor was considered as flexible factor. The results were evaluated and sorted based on predicted binding energy.Potential interactions between Ahr and candidate ligands including Flavipin, Epicorazines A, Epicorazines B and Lonchocarpol A were examined by means of docking analysis using Autodock tools (ADT) v1.5.4 and Autodock v4.2 program (https://www.ncbi.nlm.nih.gov/protein/). The 3D structure of Ahr PAS-B was predicted based on human Arnt PAS-B (PDB ID: 2B02) obtained from Protein Data Bank as described earlier [http://www.ps2.life.nctu.edu.tw/). The developed 3D model was validated for stereochemical quality using SAVES software; https://services.mbi.ucla.edu/SAVES/, and for superimposition with human Arnt PAS-B using SALIGN software . Finally the predicted 3D structure was visualized by UCSF Chimera (http://www.cgl.ucsf.edu/chimera/).The Ahr molecule is composed of PAS-A and PAS-B domains. Due to unavailability of the 3D structure of human Ahr PAS-B, a model was predicted and docked against Flavipin. Peptide sequence of human Ahr PAS-B (NP_001612.1) was retrieved from NCBI . Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 (F12) containing fetal bovine serum (FBS)  and 1% antibiotic antimycotic agent  was used for maintaining the cells (complete medium). Cells were incubated in humidified incubator at 37°C and 5% CO4 in a medium containing 2.5% charcoal-stripped FBS and seeded in 96-well plate, and then treated for 48 h with DMSO or Flavipin. The T47D medium contained 10 nmol/L β-estradiol . Cell proliferation was quantified calorimetrically at 540 nm using Cell-counting Kit-8  following manufacturer’s instructions. For cell adhesion, 5×105 cells were seeded in 6-well plate and incubated overnight in complete medium to attach, then washed with PBS to remove the non-adherent cells. Cells were then treated with DMSO or Flavipin in the charcoal-stripped medium for 48 h, and washed twice with PBS before microscopic examination.The MDA-MB-231 and T47D cells were suspended at 2.0×104 cells were suspended in 200 μL serum-free medium containing either DMSO or Flavipin and seeded in the trans-wells, and 750 μL complete medium was placed in the lower wells. After 24 h incubation, the cells on the trans-wells were fixed with 10% formaldehyde and permeabilized with methanol then stained with Giemsa. Cells with spread-out shape on the lower side of the trans-well were counted in 4 microscopic fields.For migration assay, the cells were seeded in 6-well plate in a complete medium and allowed to attach overnight. The media were then changed to DMEM/F12 with 2.5% charcoal-stripped FBS containing DMSO or Flavipin for 24 h. Around 70% cell confluency, a line was made at the central axis of the wells using pipette tip, the wells were then gently washed and treated again with either DMSO- or Flavipin-containing medium for another 24 h. The T47D medium was supplemented with E2. The invasion assay was performed using a matrigel-coated Boyden chamber  as previously described . BrieflyTotal RNA was extracted using RNeasy extraction kit  and cDNA was prepared using reverse transcriptase. The DNA amplification was carried out in ViiA7 real-time PCR system with the conditions of 50°C for 2 min and 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min. The TaqMan® gene expression assays of Cyp1a1 (Hs01054797_g1), B-cell lymphoma 2 , Sox4 (Hs00268388_s1), integrinα4 , and TaqMan® microRNA assay of hsa-miR-132 (ID: 00457) and hsa-miR-212 (ID: 00515) were used. The GAPDH (Hs02758991_g1) was used as an internal control for protein-coding genes, and RNU6B (ID: 001093) was used for miRNAs. Relative expressions were calculated using the ΔΔCt method. Machines, kits and reagents for cDNA synthesis and real-time PCR were purchased from Applied Biosystems, NY, USA.http://imagej.nih.gov/ij/download.html [Cells were lysed using RIPA lysis buffer system (Santa Cruz Biotechnology), and then fractionated using SDS-page. The proteins of Cyp1a1, Arnt, Bcl2, Sox4, ITGA4 and β-actin were detected using their specific rabbit polyclonal antibodies (Santa Cruz Biotechnology). Band intensities were quantified using ImageJ v.1.48 software oad.html .The silencing RNA for Ahr (siAhr) and non-specific control (siNS)  were transfected at 75 nmol/L, and pWPXL-Sox4 plasmid  or empty plasmid were transfected at 200 μg. The oligonucleotides were transfected into cell lines using 4D-nucleofector device and cell-specific transfection kits . Transfected cells were allowed to recover for 6 h, and then the transfection medium was changed to a complete DMEM/F12 medium with DMSO or Flavipin. Transfection efficiency was confirmed by real-time PCR and western blot assays.Data are shown as mean ± SD from representative experiment studied in triplicates out of three independent experiments produced similar results. The analysis of variance (ANOVA) test was used to analyze the statistical significance. P<0.05 was considered significant.Four natural compounds from flavonoids family, which have never been investigated for their potential anticancer properties, were chosen to examine their potential interaction with Ahr. These include Flavipin, Lonchocarpol A, Epicorazines A and Epicorazines B. The preliminary analysis using docking approach revealed that Flavipin and Lonchocarpol A significantly interact with Ahr PAS-A with binding energy of -4.63 and -4.35 kcal/mol, respectively . The FlaThe PAS-B domain is vital for interaction of Ahr with its ligands. Therefore, the 3D structure of human Ahr PAS-B domain was predicted. The primary structure properties, amino acid composition, Ramachandran analysis and plot, and G-factor parameters are presented in When Ahr is ligated, it typically heterodimerizes with Arnt to promote the transcription of the P450 downstream genes. The Cyp1a1 is a member of the P450 family that is often used as an indicator of Ahr activation. Therefore, we first examined whether Flavipin induces gene expression of Cyp1a1 in breast cancer cells. The mRNA and protein quantifications presented in The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent Ahr agonist that binds with high affinity. Therefore, we compared the effect of Flavipin and TCDD on Cyp1a1 gene expression. There were no significant differences in the mRNA levels of Cyp1a1 in both breast cancer cell lines , but the50 values. Quantification of cell proliferation of MDA-MB-231 and T47D cells 48 h after Flavipin treatment showed that the values of IC50 of Flavipin were 235.4 and 225.1 μmol/L, respectively , adhesion (ITGA4) and pro-metastatic (Sox4) markers. As shown in The Ahr is a unique environmentally responsive transcription factor activated by a wide range of exogenous aromatic hydrocarbons. Extensive evidences have clearly indicated the modulatory role of the ligand-activated Ahr in different types of cancers –28. In bin silico and in vitro means [in vitro approach, we identified Flavipin as a novel potent Ahr agonist. Docking analysis of the interaction between Flavipin and Ahr PAS-A revealed formation of eight hydrogen bonds and binding of Phe115, Leu116 and Ala119 residues of Ahr molecule. These hydrophobic amino acid residues are critical for the Ahr-ligand interaction due to their vital role at the Ahr PAS-A domain and dimerization process [Previous studies have identified new Ahr ligands that exhibit biological activities using an ro means , 31. App process . The pre process . It has  process , 35, 14.Unlike most of other flavonoids, few studies have investigated the biological activities of Flavipin showing its antibacterial and antifungal properties , 36. In in vitro and in vivo [The findings of the current study indicated that Flavipin exerts inhibitory effects on migration of the MDA-MB-231 and T47D cells. In lines, the suppressive effects of certain Ahr ligands on migration of breast cancer cells are documented , 14. The in vivo , 43. In  in vivo , 44. TheRecent studies have shown that ligand-activated Ahr suppresses invasiveness of different cancer types , 14. In Some studies have suggested correlation between endogenous ligand-activated Ahr and breast cancer progression including growth and cell motility and several mechanisms have been proposed , 49. It In conclusion, current study introduced Flavipin as a novel Ahr agonist, and provided evidences on its inhibitory activities on growth and motility of breast cancer cells. In addition, it provided mechanistic elucidation for the reported antitumor properties, suggesting Flavipin as a fascinating candidate to control metastasis in breast cancer patients.S1 FigLonchophorcol A binds to Ahr at Thr264 and Ala119. The yellow-dotted lines indicates the hydrogen bonds.(JPG)Click here for additional data file.S2 Fig(A) Superimposition of predicted 3D structure of Ahr PAS-B with the Arnt PAS-B. (B) The Ramachandran plot of predicted Ahr PAS-B and Arnt PAS-B.(TIF)Click here for additional data file.S3 FigThe T47D and MDA-MB-231 cells were seeded in 6-well plate and incubated overnight in complete medium to attach, then washed with PBS to remove the non-adherent cells. Cells were then treated with DMSO or Flavipin (200 μmol/L) in the charcoal-stripped medium for 48 h, and washed two times with PBS before microscopic examination. The T47D medium contained 10 nmol/L E2.(TIF)Click here for additional data file.S4 FigThe T47D and MDA-MB-231 cells were seeded in 10 cm plate and incubated overnight in complete medium to attach, then treated with DMSO or Flavipin (200 μmol/L) in the charcoal-stripped medium for 48 h. The T47D medium contained 10 nmol/L E2. (A) ITGA4 mRNA. The mRNA was quantified by real-time PCR using specific probe. (B) ITGA4 protein. The cells were then lysed and ITGA4 protein was quantified using western blotting method using specific antibodies. Band intensities were quantified using ImageJ software. Data are shown as mean ± SD from representative experiment studied in triplicates. *P<0.05.(TIF)Click here for additional data file.S5 FigSox4-expressing or empty vector and incubated for 6 h to recover. The cell were seeded in 6-well plate in a complete medium and allowed to attach overnight, then treated with DMSO or Flavipin (200 μmol/L) in the charcoal-stripped medium for 24 h. A line was made at the central axis of the wells. The T47D medium was supplemented with 10 nmol/L E2.(TIF)Click here for additional data file.S6 FigThe cells were transfected by electroporation with Sox4-expressing or empty vector and incubated for 6 h to recover. Cell invasion was studied using Boyden chamber. The cells were suspended in serum-free medium and placed in the trans-well, the lower well contained complete medium. Invaded cells were counted in four microscopic field. Data are shown as mean ± SD from representative experiment studied in triplicates. *P<0.05.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Mycobacterium spp. recorded to date in the Protegen database. We found that the extracellular protein 85B displayed the most robust antigenicity among the proteins identified. Computational tools for identifying T-cell epitopes predicted an epitope, 181-QQFIYAGSLSALLDP-195, that could bind to at least 13 major histocompatibility complexes, revealing the promiscuous nature of the epitope. Molecular docking simulation demonstrated that the epitope could bind to the binding groove of MHC II and MHC I molecules by several hydrogen bonds. Molecular docking analysis further revealed that the epitope had a distinctive binding pattern to all DRB1 and A and B series of MHC molecules and presented almost no polymorphism in its binding site. Moreover, using “Allele Frequency Database,” we checked the frequency of HLA alleles in the worldwide population and found a higher frequency of both class I and II HLA alleles in individuals living in TB-endemic regions. Our results indicate that the identified peptide might be a universal candidate to produce an efficient epitope-based vaccine for TB.Tuberculosis (TB) is a reemerging disease that remains as a leading cause of morbidity and mortality in humans. To identify and characterize a T-cell epitope suitable for vaccine design, we have utilized the Vaxign server to assess all antigenic proteins of Mycobacterium tuberculosis in 2011, resulting in an estimated 1.4 million people dying from the disease , HLA-DRB1∗0901 [Uniprot:Q0R326], HLA-B39 [Uniprot:P30475], and HLA-B58 [Uniprot:P10319] were constructed based on the experimental structures of HLA-DR1 beta chain [PDB:1AQD-b], DRB1-1 beta chain [PDB:3PGD-b], HLA-B∗1402 alpha chain [PDB:3BXN-a], and HLA-B∗5701 alpha chain [PDB:3VRI-a], respectively. The amino acid sequence homology of HLA-DRB1∗0701, HLA-DRB1∗0901, HLA-B39, and HLA-B58 with HLA-DR1, DRB1-1, HLA-B∗1402, and HLA-B∗5701 is 89.31%, 87.90%, 97%, and 98%, correspondingly. To evaluate how much the structural models deviate from the template, the root-mean-square deviation (RMSD) value between the template and the homology model superposed was determined by the Internal Coordinate Mechanics method based computer program Molsoft Internal Coordinate Mechanics- (ICM-) pro 3.5 [∗0101 [Uniprot:P04229], HLA-DRB1∗0404 [Uniprot:P13760], HLA-DPB1∗0101 [Uniprot:P04440], and HLA-DQB1∗0301 [Uniprot:P01920] were 1A6A-a, 1SJE-b, 1D5 M-b, 3LQZ-b, and 1JK8-b, respectively. MHC I HLA supertypes A2, A24, B8, B27, and B44 were retrieved from PDB. PDB ID for experimental structures of HLA supertype A2 [Uniprot:P01892], A24 [Uniprot:P05534], B8 [Uniprot:P30460], B27 [Uniprot:P03989], and B44 [Uniprot:P30481] were 1HHI-a, 3I6L-a, 1AGB-a, 10GT-a, and 1SYS-a, respectively.The 3D structure of epitopes was built using PyMol molecule builder, as described previously . There whome.do)  for the eviously . The hom pro 3.5 . The ste pro 3.5   and MHC I  molecules. Molecular docking of the epitope was performed against the aforementioned 6 MHC II and 5 MHC I molecules. Ionizable group of cationic residues in neutral or mildly acidic compartment were transformed into the protonated states and ICM default partial atomic charges were set. ICM pocket finder macro was used for pocket selection, and after receptor positioning, the ICM method based molecular docking was done, as described previously [The 3D structure of the Ag85B T-cell epitope built by the PyMol molecule builder was subjected to molecular docking simulation with MHC I and II molecules by Molsoft ICM-pro 3.5. The Ag85B T-cell epitope could bind to MHC II . Although IvAgNP is a viral antigenic protein, we used it as a positive control to cross check the in silico approaches used here and determine whether they can identify the epitopes previously reported . The epi M. tuberculosis. Among the 24 antigenic proteins, 11 are extracellular, five are cytoplasmic, and one is a cell wall component. The localization of the remaining seven proteins is unknown. Five proteins, namely, Ag85B, Ag85A, ESAT-6, pepA, and PPE18, have been extensively studied and used as subunit vaccines in animal models [ M. tuberculosis [∗0101 with an affinity of 4.5 nM. Notably, an affinity below 50 nM in the NetMHCII 2.2 server is considered as strong binding. Upon further analysis, this epitope showed high affinity to a large number of MHC alleles . The most promiscuous epitope, 301-IDPFRLLQNSQVYSL-315, was found against HLA-DRB1∗0101 with an affinity of 3.4 nM . A previously identified epitope, 380-ELRSRYWAIRTRSGG-393 was determined to be not promiscuous [∗0101 with a significant affinity of 9 nM (data not shown). The binding capability of Ag85B with MHC II molecules was further analyzed with ProPred and SYFPEITHI web servers. Each of these approaches predicted several epitopes that showed an ability to bind to many MHC II molecules. The 9-mer of the strongest predicted epitope mentioned above was identified as one of the top five epitopes in this analysis  of pathogenic influenza virus and 73 nonidentical sequences were aligned to find the conservation of the control epitope and the strongest promiscuous epitope . Figure  S1 shows the percentage of identity in amino acid level of the predicted strongest epitope was 100, 93, and 86 in 6, 42, and 25 influenza A viruses, respectively. The level of identity was 86.75% in all influenza A virus.To predict the universal effectiveness of the epitope, we retrieved all 150 Ag85B sequences  of pathogenicium spp. . This an75H115N17O23). Analysis with the ProtParam computer program revealed that the instability index was 32.93. This result indicates a high stability of the epitope since a ProtParam instability index smaller than 40 is considered to be stable. However, the ProtParam instability index of the positive control epitope and the predicted strongest promiscuous epitope of IvAgNP was 76.04 and 46.69, respectively, indicating that these epitopes are unstable.Physicochemical properties of the epitope include a molecular weight of 1622.8 Da with 230 atoms . As HLA-DRA does not have polymorphisms in the peptide binding region, we performed molecular docking simulation between the epitope and HLA-DRA . However, docking analysis showed that the epitope 380-ELRSRYWAIRTRSG-393 in IvAgNP bound within the grooves of only HLA-DRB1∗0101 through 10 hydrogen bonds with a binding energy of −191.05 kcal/mol (data not shown).Molecular docking was performed to analyze the interaction of the epitope with MHC molecules. Docking showed that the protrusion of the side chains of the epitope bound in cavities within the groove of MHC II HLA-DRB1M method . Molecul Figures . Docking HLA-DRA . DockingDocking of the 9-mers of the predicted epitope was performed with MHC I molecules. Docking analysis showed that the 9-mer epitope 183-FIYAGSLSA-191 bound to HLA-A2 through eight hydrogen bonds with a binding energy of −162.01 kcal/mol . Due to this specificity, these vaccines can avoid immune responses induced by unfavorable epitopes of the antigen, thereby preventing complications . Along w T-cells . Computa T-cells , 61. Thr M. tuberculosis could bind to several MHC II and MHC I molecules and thus revealed its promiscuous nature [Analysis with Vaxign revealed that Ag85B was more antigenic than other immunogenic mycobacterial proteins. Results of analyses with NetMHCII 2.2, SYFPEITHI, ProPred, and NetCTL 1.2 servers confirmed that the predicted epitope from Ag85B ofs nature , 57. Epis nature –71. In a M. tuberculosis epitopes bind to DR antigens [ DRB1 gene was extensively studied because the expression level of DRB1 proteins is typically fivefold greater than that of DRB3, DRB4, or DRB5 gene [It is important to recognize the inherent limitations of epitope prediction programs. No epitope prediction program is absolutely accurate, and there is substantial discrepancy among the epitopes predicted by each program , 73. We antigens . In addiRB5 gene .NetCTL 1.2 is an integrative approach to CTL epitope prediction that predicts MHC I binding, TAP transport efficiency, and proteasomal cleavage , 62. The Mycobacterium spp. If the proposed epitope sequence is highly variable among the Ag85B, the protective efficacy of Ag85B as an epitope-based vaccine might be compromised on the global stage. Our investigation showed that the proposed epitope had 100% identity among MTBC and 87–93% identity among the remaining 40% of Mycobacterium spp., defining it as a universal epitope  present in 10 Ag85B sequences is substituted by aspartic acid and asparagine in M. avium and M. intracellulare, respectively. Isoleucine (I184 in M. tuberculosis-1 H37Rv) present in 12 Ag85B sequences is substituted by valine in M. intracellulare. Leucine (L192 in M. tuberculosis-1 H37Rv) present in 14 Ag85B sequences is substituted by methionine in M. kansasii. Glutamine has strongly similar properties (roughly the same size and the same hydropathy) as aspartic acid and asparagine, while isoleucine is similar to valine [ Mycobacterium spp. may not alter the properties of the epitope to change its universality. ProtParam analysis of the epitope with the substituted amino acid(s) to mimic the polymorphism in M. avium, M. intracellulare, and M. kansasii showed almost the same half-life and instability index as that of the predicted epitope (data not shown). Thus, comparative proteomics of the pathogen population from a greater range of geographic origins stands as an additional useful tool for preclinical evaluation of new vaccine targets.Recently, the incidence of Nontuberculous Mycobacterial (NTM) infections has surpassed the incidence of TB infections in developed countries –86. Alth disease , 84, 90.o valine . TherefoThe Ag85B T-cell epitope formed a number of significant hydrogen bonds with the binding groove of each of the MHC II and MHC I molecules  strong binding affinity of the epitope with MHC I and II molecules, (iv) physicochemical properties of the epitope, (v) molecular docking simulation, and (vi) coverage of HLA alleles in TB-endemic countries, which supports the notion that the proposed epitope might be a novel universal efficient epitope to produce peptide or epitope-based vaccines against TB. While biochemical analysis remains necessary to validate the interaction between the epitope and the MHC molecules through elucidation of immunity induction, most of the approaches have been validated by applying them on a positive control protein, IvAgNP.Here, we report (i) high percentage of identity of the epitope in all Ag85B sequences, (ii) adhesin capability and antigen probability of Ag85B fromAdditional file 1-Table S1: Binding of epitopes of IvAgNP from Influenza virus against MHC II alleles.Additional file 2-Table S2: Binding affinity of predicted strongest epitope 301-IDPFRLLQNSQVYSL-315 from IvAgNP to MHC alleles.Mycobacterium pathogenic strains found in protein database.Additional file 3-Table S3: Accession numbers of antigenic Ag85B sequences of  Additional file 4-Figure S1: Multiple alignment of protein sequences of IvAgNP from Influenza virus.Additional file 5-Table S4: Hydrogen Bonds and binding statistics of epitope 181-QQFIYAGSLSALLDP-195 from Ag85B with MHC II complexes.Additional file 6-Figure S2: Docking of predicted strongest epitope 301-IDPFRLLQNSQVYSL-315 from IvAgNP with HLA-DRB1∗0101 (A), DRB1∗0901 (B), DRB1∗0404 (C) and DRB1∗0701 (D).Additional file 7-Table S5: Hydrogen Bonds and binding statistics of predicted strongest epitope 301-IDPFRLLQNSQVYSL-315 from IvAgNP with MHC II complexes.Additional file 8-Table S6: Hydrogen Bonds and binding statistics of epitope 181-QQFIYAGSLSALLDP-195 from Ag85B with MHC I complexes.Additional file 9-Figure S3: Docking of control epitope 380-ELRSRYWAIRTRSG-393 from IvAgNP with HLA-B8 (A) and B27 (B).Additional file 10-Table S7: Hydrogen Bonds and binding statistics of control epitope 380-ELRSRYWAIRTRSG-393 from IvAgNP with MHC I complexes.Additional file 11-Figure S4: Docking of predicted strongest epitope 301-IDPFRLLQNSQVYSL-315 from IvAgNP with HLA-A2 (A), A3 (B), B27 (C), B39 (D) and B58 (E).Additional file 12-Table S8: Hydrogen Bonds and binding statistics of predicted strongest epitope 301-IDPFRLLQNSQVYSL-315 from IvAgNP with MHC I complexes.Additional file 13-Figure S5: Multiple alignments of amino acid sequences of HLA-A and HLA-B series.


Type III CRISPR-Cas systems can provide redundancy to counteract viral escape from type I systems. Published 17, August 2017M. mediterranea CPR1 as a cas6 instead.A typographical error on our part was identified in Figure 6B. We erroneously labeled one cmr6 gene in the type III-B CRISPR-Cas system in The corrected figure 6 is shown here:The originally published figure 6 is shown for reference:The language in the following sentence of the Discussion describing this type III-B CRISPR-Cas system has been amended to account for this correction.Original text:M. mediterranea CPR1 encodes two diverse Cas6-family proteins  that may enable the system to independently process pre-crRNAs from different CRISPR-Cas systems.In addition, the ability to process type I pre-crRNAs might be carried within some type III operons; for example, the type III-B system in Corrected text:M. mediterranea CPR1 encodes a divergent Cas6-family protein  that may enable the system to independently process pre-crRNAs from different CRISPR-Cas systems.In addition, the ability to process type I pre-crRNAs might be carried within some type III systems; for example, the type III-B system in The article has been corrected accordingly.