However, the application of this method to analyses in later stages of brain development or in the adult brain is still difficult because the amount of injected plasmids in a cell decreases along with development due to dilution by cell proliferation and the degradation of the plasmids. Furthermore, it is difficult to exclude the influence of earlier knockdown effects.Gene knockdown analyses using the Tol2 transposon-mediated gene transfer methodology with the tetracycline-inducible gene expression technique, which allows us to maintain a transgene for a long period of time and induce the knockdown of the gene of interest. We showed that expression of the endogenous amyloid precursor protein (APP) was sharply decreased by our inducible, stably integrated knockdown system in PC12 cells. Moreover, we induced an acute insufficiency of Dab1 with our system and observed that radial migration was impaired in the developing cerebral cortex. Such inhibitory effects on radial migration were not observed without induction, indicating that our system tightly controlled the knockdown, without any expression leakage in vivo.We developed a tightly controlled conditional knockdown system using a newly constructed vector, pT2K-TBI-shRNAmir, based on a in utero electroporation gene transfer methodology. Furthermore, we can perform knockdown analyses free from the influence of undesired earlier knockdown effects.Our system enables us to investigate the brain at any of the later stages of development or in the adult by utilizing a knockdown technique with the aid of the  During the development of the cerebral cortex, neurons are generated in the ventricular zone (VZ) and then migrate outward to the cortical plate (CP), an event that is called “radial migration.” These neurons stop their movement and settle into the six layers of the cortex in an inside-out pattern, whereby early-born neurons are positioned in the deeper layers, and later-born neurons are located in the more superficial layers, migrating outward by passing the earlier born neurons In the later stage of the cortical development, just after radial migration ends, neurons that have settled in the cortex continue to elongate their axons toward their targets. In mice, layer-specific stereotyped projection patterns are established for each layer of the cortex: layer V neurons, which are born around embryonic day (E)12.5, mainly project to the subcortical regions as the subcortical projection, whereas layer II/III pyramidal neurons, which are born around E14.5, mainly project to the contralateral cortex as the callosal projection in utero electroporation gene transfer is frequently employed because it enables us to perturb the in vivo gene expression in a convenient manner compared to conventional techniques, such as making genetically modified animals in utero electroporation gene transfer, it has been revealed that various molecules are involved in the development of the cerebral cortex by controlling radial migration. One such example is an unexpected activity of the amyloid precursor protein (APP), for which involvement in Alzheimer's disease has been established, in radial migration by controlling Disabled-1 (Dab1) activity downstream Dab1 results in the malformation of the cerebral cortex in which lamination is inverted such that neurons do not keep to the inside-out pattern In such studies of cortical development, in utero electroporation gene transfer is a powerful tool for studying the events in early corticogenesis, there are some difficulties in applying this technique to the later stages of development, such as the formation of a neuronal network, which includes axon path-finding and synaptogenesis, due to the following reasons: (1) Gene expression from the injected plasmids decreases along with development due to dilution by cell proliferation and the degradation of the plasmids. (2) It is difficult to exclude the influence of earlier gene manipulation. For example, modified neuronal differentiation may influence the later neuronal network formation. Therefore, it is desirable to develop a technique that enables us to stably maintain a transgene and to induce the expression of genes of interest by design.Although Tol2, have been utilized for integrating a gene of interest into the chromosome for steady expression Tol2-flanking regions are integrated into the chromosome in the presence of transposase. Furthermore, because the Tol2 transposon, which originated in medaka fish, possesses an ability to undergo transposition in many kinds of species, including chick and mouse in ovo or in utero electroporation gene transfer in model animals for stable gene expression Up to now, transposable elements, such as Sleeping Beauty, piggyBac and Tol2-mediated gene transfer systems with the tetracycline-induced expression system maintain controllable gene expression during chick embryogenesis in vivo function of a gene of interest using a knockdown approach without the influence of earlier events.Although the tetracycline-controlled gene expression system, in which transcription is reversibly turned on or off in the presence of tetracycline, is one of the most frequently employed methods for inducible gene expression, we sometimes experienced a leakage in expression with this system in spite of the tetracycline control. A recent study showed that Tol2 system, we developed a new tool that enables us to knockdown the gene of interest at any point during development.In this study, by combining the tightly controlled inducible gene expression cassette with the Tol2 transposable vector pT2K-BI-TRE-EGFP by inserting the mir30 cassette (shRNAmir) and exchanging the promoter , an analogue of tetracycline, and activates transcription  cription . TherebyTol2 system with glial lineage-specific promoters drives the glial cell-specific expression of transgenes in the mouse cerebral cortex when transfected using in utero electroporation gene transfer We designed the knockdown cassette based on the miRNA backbone for pT2K-TBI-shRNAmir. There are some advantages in using the miRNA-based knockdown cassette: (1) Compared with the conventional stem-loop-based shRNAs, miRNA-based hairpins have been proven to exhibit lower cellular toxicity effects We examined whether the pT2K-TBI-shRNAmir vectors were integrated into the chromosomes. If the vectors were not integrated into the chromosome, these vectors were re-distributed into two daughter cells after mitosis. Eventually the number of these vectors in individual cells should become very small. In contrast, in the cells harboring such vectors in their chromosomes, these vectors should be retained after cell division. We tested the ability of this vector to integrate into the genome with APP as an inducible knockdown target. We used pT2K-TBI-shRNAmir vector as a control empty vector (mir-empty), and designed two pT2K-TBI-shRNAmir constructs, pT2K-TBI-shRNAmir-APP#2 (mir-APP#2) and pT2K-TBI-shRNAmir-APP#3 (mir-APP#3), which targeted different regions of the APP sequence.Tol2 transposase) and pCAGGS-tdTomato for the visualization of transfected cells. The cells were cloned and then subcultured every 4 to 5 days for 47 days until the expression of the transfection control, tdTomato, became undetectable. Even after 47 days of culturing, almost all of the cells still expressed EGFP upon induction by Dox, confirming that these vectors were stably integrated into the genome , pT2K-CAGGS-rtTA-M2 and pCAGGS-tdTomato were electroporated into the mouse cerebral cortex with or without the Tol2 transposase expression vector, and gene expression was induced by Dox at E15.5. Cortices were observed at P4, by which point glial cells must have experienced multiple rounds of cell divisions .Although plasmids were introduced into the neural progenitors in the VZ by nsfected . Indeed,Tol2 inducible knockdown vector and the in utero electroporation gene transfer method enables the induction of gene expression at the time of interest, it remains unclear whether the inducible knockdown is effective in vivo. We performed inducible knockdown against Dab1, a molecule that plays an important role in radial migration under the Reelin signaling pathway, in addition to APP  that utilizes drug inducible Cre-mediated knockout technology in vivo molecular mechanisms of later stages of development.Currently, the demand for the development of tools for conditional genetic manipulation, especially in a small number of neurons, is increasing. Young ICR mice were purchased from SLC . Noon on the day when the presence of a vaginal plug was confirmed was designated as embryonic day 0.5 (E0.5). For pups, the day of birth was designated as postnatal day 0 (P0). All experiments were conducted in accordance with the guidelines for animal experiments of the University of Fukui, and all efforts were made to minimize both the number of animals used and their suffering. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Fukui (Permit Number: 23012).5′-AACCTCGAGATGTCAACTGAGACAGAACTTCAAG-3′ and 5′-GCTCTCGAGCTAGCTACCGTCTTGTGGACTTATATTATC-3′. The PCR product was cloned into the XhoI sites of the pCAGGS-myc vector. Mouse full-length APP (GenBank accession number NM_007471) cDNA was cloned from E14.5 mouse brains by PCR. For pCAGGS-mAPP-FLAG, the APP cDNA was amplified by PCR with the primers 5′-AATAGATATCATGCTGCCCAGCTTGG-3′ and 5′- GAAGATATCGTTCTGCATTTGCTCAAAGAAC-3′. The PCR product was cloned into the EcoRV sites of the pCAGGS-FLAG-IRES-GFP vector. For the construction of pT2K-BI-shRNAmir, the mir-30 cassette was excised from pCAG-mir30  Mouse full-length Dab1 (GenBank accession number NM_177259) cDNA was cloned from E14.5 mouse brains by the polymerase chain reaction (PCR). For pCAGGS-myc-mDab1, the Dab1 cDNA was amplified by PCR with the primers 5′-CAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCG-3′ and 5′-CTAAAGTAGCCCCTTGAATTCCGAGGCAGTAGGCA-3′ using the following template oligonucleotides: for shRNAmir-non-target (shRNAmir-nt), ATCTCGCTTGGGCGAGAGTAAGTAGTGAAGCCACAGATGTACTTACTCTCGCCCAAGCGAGAGTGCCTACTGCCTCGGA-3′5′-TGCTGTTGACAGTGAGCG; for shRNAmir-APP#2, CGCACTAACTTGCACGACTATGTAGTGAAGCCACAGATGTACATAGTCGTGCAAGTTAGTGCTTGCCTACTGCCTCGGA-3′5′-TGCTGTTGACAGTGAGCG; for shRNAmir-APP#3, CGCTGACAAGAAGGCCGTTATCTAGTGAAGCCACAGATGTAGATAACGGCCTTCTTGTCAGCTTGCCTACTGCCTCGGA-3′5′-TGCTGTTGACAGTGAGCG; for shRNAmir-Dab1#1, CCCTCCTCCTCTTAGCACTAAATAGTGAAGCCACAGATGTATTTAGTGCTAAGAGGAGGAGGATGCCTACTGCCTCGGA-3′5′-TGCTGTTGACAGTGAGCG; for shRNAmir-Dab1#2, CAAGGATAAGCAGTGTGAACAATAGTGAAGCCACAGATGTATTGTTCACACTGCTTATCCTTTTGCCTACTGCCTCGGA-3′5′-TGCTGTTGACAGTGAGCG. The PCR product was cloned into the XhoI/EcoRI sites of the pT2K-TBI-shRNAmir vector. The underlined sequences correspond to the sense and antisense target sequences, respectively.The mir30-based shRNA was synthesized by PCR with the primers 2, 1 mM dithiothreitol, 1 mM EGTA, 0.5% Nonidet P-40) supplemented with 1% Protease Inhibitor Cocktail . After centrifugation, the supernatant was mixed with Laemmli sample buffer, boiled, and subsequently separated on SDS-polyacrylamide gels. The separated proteins were transferred to polyvinylidene difluoride membranes. After blocking for 30 min in 5% skim milk-TBST , the membranes were probed for 1 h with the primary antibodies in 5% skim milk-TBST. The membranes were then washed three times with TBST and incubated with the secondary antibodies coupled to horseradish peroxidase. Immunoblotting was visualized using an Immobilon Western Chemiluminescent HRP Substrate  on a LAS-3000 mini imaging system . The following antibodies (and dilutions) were used: the mouse anti-myc antibody (1∶1000) , anti-FLAG antibody (1∶1000) , anti-actin antibody (1∶500)  and the mouse anti-APP antibody (1∶1000) .Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with either 10% fetal bovine serum for HEK293T cells (ATCC No. CRL-2828) or 10% fetal bovine serum and 5% horse serum for PC12 cells (ATCC No. CRL-1721). Plasmid DNAs were transfected into cells using the Lipofectamine™ 2000 transfection reagent . The cells were lysed with the lysis buffer , and the number of EGFP-positive and tdTomato-positive cells were counted using NIH Image J software.in utero electroporation using previously described methods with some modifications Tol2 transposase) = 1∶5∶10∶10. After soaking the uterine horn with PBS, the head of the embryo was pinched with a forceps-type electrode , and five cycles of square electric pulses  at intervals of 950 ms were delivered using an electroporator . For the analysis of inducible expression in the cerebral cortex, the brains were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, cut coronally into 100 µm slices with a Vibratome , and imaged on a laser-scanning confocal microscope .Plasmids were transfected by 5′-AACGGTCTAGACAACAAGCTG-3′ and 5′-AGTGGTCTTGGTGTGCTGAC-3; for β-actin, 5′-ATGCTCCCCGGGCTGTAT-3′ and 5′-CATAGGAGTCCTTCTGACCCATTC-3; and for Dab1, 5′-AGCTCAAGGGTGTTGTTGCT-3′ and 5′- TGTGATGTCCTTCGCAATGT-3.pT2K-TBI-shRNAmir or pT2K-TBI-shRNAmir-Dab1#2 was electroporated with pT2K-CAGGS-rtTA-M2 into the mouse cerebral cortex at E14.5. The cells were collected at E18.5 using FACS . Doxycycline (Dox) was administrated to mice at 2 mg/ml in a 5% sucrose solution in drinking water during the experiment from E14.5 to E18.5. To make cDNA for real-time PCR from the total RNA of FACS-sorted cells, reverse transcription was performed using reverse transcriptase . Real-time PCR reactions were performed on an ABI StepOnePlus™ Real-Time PCR System  using the SYBR Green PCR Master Mix . The thermal cycling was performed using the following cycle conditions: the initial denaturation step at 95°C for 60 s, followed by 40 cycles at 95°C for 15 s, and 60°C for 60 s. The experiments were carried out in triplicate. The relative quantification of gene expression compared to a control was determined using the ΔΔCt method. Changes in gene expression were normalized to the internal control gene PRS18. The following gene-specific primers were used for real-time PCR analysis: for PRS18, Figure S1Knockdown effects of shRNA- and miRNA-based knockdown vectors against APP on the radial migration using the various promoters. APP was knocked down using the shRNA- or miRNA-based knockdown vectors ( vectors  in the c(TIF)Click here for additional data file.Table S1The nomenclature of the vectors and components.(PDF)Click here for additional data file.Text S1Supporting methods for vector constructions.(DOC)Click here for additional data file.


Stroke severity is worsened by recruitment of inflammatory immune cells into the brain. This process depends in part on T cell activation, in which the B7 family of co-stimulatory molecules plays a pivotal role. Previous studies demonstrated more severe infarcts in mice lacking programmed death-1 (PD-1), a member of the B7 family, thus implicating PD-1 as a key factor in limiting stroke severity. The purpose of this study was to determine if this protective effect of PD-1 involves either of its ligands, PD-L1 or PD-L2.-/-) mice undergoing 60 minutes of middle cerebral artery occlusion (MCAO) followed by 96 hours of reperfusion and compared to wild-type (WT) C57BL/6J mice.Central nervous system (CNS) inflammation and infarct volume were evaluated in male PD-L1 and PD-L2 knockout . Suppressor T cells (IL-10-producing CD8+CD122+ T cells) trafficked to the brain in PD-L1-/- mice and there was decreased expression of CD80 on splenic antigen-presenting cells (APCs) as compared to the WT and PD-L2-/- mice.PD-L1-/- mice suggests an overriding interaction leading to T cell activation. Conversely, low CD80 expression by APCs, along with increased PD-1 and PD-L2 expression in PD-L1-/- mice suggests alternative T cell signaling pathways, leading to a suppressor phenotype. These results suggest that agents (for example antibodies) that can target and neutralize PD-L1/2 may have therapeutic potential for treatment of human stroke.Our novel observations are the first to implicate PD-L1 involvement in worsening outcome of experimental stroke. The presence of suppressor T cells in the right MCAO-inflicted hemisphere in mice lacking PD-L1 implicates these cells as possible key contributors for controlling adverse effects of ischemia. Increased expression of CD80 on APCs in WT and PD-L2 Stroke is the fourth most common cause of death and a leading cause of disability in the United States alone. It is increasingly accepted that human stroke does not just create a single organ injury but induces a complex interaction between the central nervous system (CNS) and peripheral immune system. Experimental stroke in mice induces a biphasic response in the peripheral immune system characterized by an initial activation phase 6 to 22 hours)  followed2 hours  + and CD8+ T lymphocytes, microglia, and macrophages [+ and CD8+ T cells, natural killer T (NKT) cells, B cells, monocytes, and some dendritic cell (DC) subsets [The activation of T cells via the various antigen-presenting cells (APCs) forms an integral part of the inflammatory process. This process gains importance especially since the inflammatory cells in ischemic stroke include CD4rophages . Program subsets . PD-L1 a subsets  and they subsets , it is a subsets ,14-18. E subsets ,13,19-21-/-) versus wild-type (WT) male mice after 96 hours of reperfusion, cortical, striatal, and total infarct volumes were significantly larger in PD-1-/- versus WT with substantial recruitment of inflammatory cells from the periphery into the CNS [-/- and PD-L2-/- male mice, on a C57BL/6 background, were subjected to 60 minutes of MCAO followed by 96 hours of reperfusion and infarct volumes and immunological parameters were compared to similarly treated WT mice. Our results clearly demonstrate that PD-L1-/- and PD-L2-/- mice had lower total infarct volumes compared to WT mice. The immune parameters matched the stroke outcome in that the PD-L1-/- and to a lesser extent PD-L2-/- had reduced levels of proinflammatory activated microglia and/or infiltrating monocytes and CD4+ T cells in the ischemic hemispheres. There was a reduction in ischemia-related splenic atrophy accompanied by lower activation status of splenic T cells and monocytes in the absence of the PD-L1, suggesting a pathogenic rather than a regulatory role for both PD-Ls. Also, suppressor T cells (IL-10-producing CD8+CD122+ T cells) trafficked to the brain in PD-L1-/- mice possibly acting as key contributors of immunoregulation. Thus, these results clearly establish a role for PD-L1 and to a lesser extent PD-L2 in increasing infarct volumes in experimental stroke, making them potential targets for future immunotherapy.Our previous work demonstrated an increased expression of PD-1 on brain macrophages and microglial cells and PD-L1/2 on B cells from the spleen, blood, and CNS in mice after induction of experimental stroke by middle cerebral artery occlusion (MCAO). Thus, when MCAO was compared in PD-1 knockout  and PD-L2-deficient (PD-L2-/-) mice on a C57BL/6 background were gifts from Indira Guleria, PhD  and Arlene Sharpe, PhD , respectively. Based on Dr. Guleria’s and Sharpe’s recommendations and previous publications [PD-L1-deficient  according to the manufacturer’s instructions. For preparation of inflammatory cells from the brain, each mouse was perfused transcardially with 30 mL saline to exclude blood cells, the forebrain was dissected from the cerebellum and suspended in RPMI-1640 medium, and the suspension was digested with type IV collagenase  and DNase I  at 37°C for 45 minutes in a shaker at 180 times per minute. Inflammatory cells were isolated by 37% to 70% Percoll density gradient centrifugation as previously described . The cel3 and 2% FCS). After incubation with mAb and washing with staining buffer, propidium iodide (PI) was added to identify dead cells. The Foxp3 staining kit (eBioscience) was used according to the manufacturer’s protocol as previously described [Individual performing FACS analysis was not blinded to genotype. Anti-mouse antibodies CD19 , CD45 , CD11b , MHCII , CD4 , CD8 , CD69 , CD122 , PD-1 , PD-L1 , and PD-L2  were used for the study. Single-cell suspensions were washed with staining medium , with PMA (50 ng/mL), ionomycin (500 ng/mL), and Brefeldin A  for 4 hours. For intracellular IL-10 detection, a modification was followed for the immunofluorescence staining protocol [6 cells/mL) in complete medium and cultured with LPS (10 μg/mL) in addition to PMA (50 ng/mL), ionomycin (500 ng/mL), and Brefeldin A (10 μg/mL)  for 4 hours. Fc receptors were blocked with anti-FcRmAb  before cell surface staining, fixed, and permeabilized with the Fixation/Permeabilization buffer (eBioscience) according to the manufacturer’s instructions. Permeabilized cells were washed with 1X Permeabilization Buffer (eBioscience) and stained with anti-TNF-α , anti-INF-γ , or APC-conjugated anti-IL-10 mAb . Isotype matched mAb served as negative controls to demonstrate specificity and to establish background TNF-α, IFN-γ, and IL-10 staining levels.Intracellular staining was visualized using a published immunofluorescence protocol . Brieflyprotocol . Briefly-/-, and PD-L2-/-) in median neurological deficit score. Post-hoc Dunn’s test was used for all pairwise comparisons against the control (WT) group following rank-based ANOVA as treatment group sizes were unequal. The multiple comparisons on ranks did not include an adjustment for ties. Statistical significance was P <0.05. Statistical analyses were performed using SigmaStat Statistical Software, Version 3.1 . For flow data analysis and representation of three and more groups, the one-way ANOVA followed by post-hoc Tukey’s test was applied. For all tests, P values ≤0.05 were considered statistically significant. All values are reported as mean ± SEM. Significant differences are denoted as *P ≤0.05; **P ≤0.01; ***P ≤0.001.Infarct volume data are presented as mean ± SEM. Differences in cortical, striatal, and hemispheric  infarct volume were determined by one-way analysis of variance (ANOVA) with post-hoc Newman-Keuls test. A Kruskal-Wallis ANOVA on ranks was conducted to evaluate differences among the three experimental groups  or PD-L2  reduced cortical infarct volume when compared to male WT mice (50 ± 3%) , but not PD-L2 , decreased infarct volume in comparison to male WT mice (69 ± 8%) , striatal infarct volume did differ between these two strains (P = 0.040)  or PD-L2  reduced hemispheric infarct volume. We also observed that hemispheric infarct volume was smaller in PD-L1-/- versus PD-L2-/- mice . Representative cerebral sections from WT, PD-L1-/-, and PD-L2-/- mice are shown in Figure Genetic deletion of either PD-L1  were greater than would be expected by chance at 1 hour (P = 0.013), 24 hours (P = 0.044), 48 hours (P = 0.031), and 96 hours (P = 0.020) of reperfusion, but not at 72 hours of reperfusion (P = 0.062) (Table P <0.05) was observed between WT and PD-L1-/- groups at 1 hour and 24 hours of reperfusion  in the total number of viable leukocytes as compared to the unaffected  hemisphere  of leukocytes in their ipsilateral hemispheres compared to the contralateral side, there was no difference in the numbers of viable leukocytes between the ipsilateral and contralateral hemispheres of PD-L1-/- mice. The PD-L1-/- mice demonstrated significantly lower cell infiltration in the ischemic hemisphere as compared to WT mice (P ≤0.05). However, there was no difference in leukocyte numbers between the ischemic brains of the PD-L1-/- and PD-L2-/- mice  hemisphere in the WT mice had a significant increase  and PD-L2-/- mice (P ≤0.05) had significantly higher numbers of microglia and/or infiltrating monocytes/macrophages as compared to their respective non-ischemic halves, while there was no increase in these cells in the ischemic brains of the PD-L1-/- mice. The ischemic hemispheres of the PD-L1-/- mice, in fact, had significantly lower numbers of CD11b+CD45high population as compared to the ischemic halves of WT (P ≤0.001) and PD-L2-/- (P ≤0.05) mice  and PDL2-/- mice (P ≤0.01) as compared to their non-ischemic halves, there were no differences in the PD-L1-/- mice post-MCAO. The ischemic hemispheres of the PD-L1-/- had significantly lower CD4+ T cells (P ≤0.05) as compared to WT mice  as compared to both WT and PD-L2-/- mice, but also significantly more (P ≤0.05) in the left hemisphere of the PD-L1-/- mice as compared to the PD-L2-/- mice post-MCAO  and also as compared to the ischemic hemisphere of PD-L1-/- mice (P <0.05). The ischemic hemispheres of the PD-L2-/- mice also had a significantly higher percentage of TNF-α-producing CD3+ T cells as compared to its contralateral side (P ≤0.05) and as compared to the ischemic hemisphere of PD-L1-/- mice (P ≤0.01)  (Table -/- mice were significantly higher than those of the MCAO-induced WT mice (P <0.05). While the spleens of PD-L2-/- underwent an approximately 80% reduction from their sham state, this change was not significant when compared to the viable splenic cell numbers from either MCAO-induced WT or PD-L1-/- mice , B cells, and monocytes were enumerated. As previously determined [+ T cells and CD8+ T cells, with a reduction in the B cell percentages (Table + and CD8+) in both the MCAO-induced PD-L1-/-, and in the MCAO-induced PD-L2-/- as compared to their sham-treated counterparts (Table + and CD8+) in the spleens of MCAO-induced PD-L1-/- mice were significantly higher than those in the MCAO-subjected WT, with no significant difference between the WT and PD-L2-/- cell composition . Thus, these results clearly demonstrate that the presence of PD-L1 contributes more to MCAO-induced cell death and less proliferative responses than PD-L2 after 96 hours of ischemic stroke in mice.At this point, it was unclear whether increased accumulation of CD4+ T cells and CD8+ T cells, and of major histocompatibility complex (MHC) class II by CD11b+ monocytes in the spleens of MCAO-induced WT, PD-L1-/-, and PD-L2-/- mice. CD69 is an ‘activation marker’ that is rapidly induced on mature T cells after stimulation through the TCR. As shown in Figures -/- mice after MCAO had significantly decreased percentages of CD69+CD4+ (P ≤0.05) and CD69+CD8+ (P ≤0.05) T cells as compared to the MCAO-induced WT mice. The activation states of T cells (CD4+ and CD8+) in the PD-L2-/- mice were slightly reduced as compared to the WT mice after MCAO, but not significantly. Similarly, the expression of MHC class II on monocytes was significantly reduced (P ≤0.05) in the PD-L1-/- mice  for CD4+ and CD8+ T cells, and also to the PD-L2-/- mice (P ≤0.05) for CD4+ T cells and (P ≤0.01) for CD8+ T cells. However, there were no differences in the expression of PD-1 among strains of the sham-treated mice. Our earlier studies demonstrated that MCAO induced a threefold increase in the percentage of Foxp3+CD4+ Tregs in the spleens as compared to sham or naive mice [+CD4+ T cells were assessed in each of the three mouse strains, post-MCAO. The PD-L-deficient mice demonstrated a trend towards lower expression of Foxp3+CD4+ T cells although these levels were not significant as compared to those in the WT mice at 96 hours post-MCAO  and the PD-L2-/- (P ≤0.05) mice is shown in Figure +CD122+ T cells from the spleens, low infarcts in the PD-L1-/- mice and increased numbers of total CD8+ T cells in the ischemic hemispheres of PD-L1-/- mice, we speculated a plausible exit of these suppressors from the spleens and their entry into the ischemic hemisphere of the brains. Upon further characterizing the CD8+ T cells in the brains for the presence of the suppressor CD8+ T cell sub-population, there was indeed a significant increase in the percentage of CD8+CD122+ T cells in the ischemic hemispheres of the PD-L1-/- mice as compared to those found in the ischemic hemispheres of the WT mice (P ≤0.05) after 96 hours of reperfusion  sub-population known as CD8 T cells . CD8+CD1oduction . A signiWT P ≤0.0 and the WT P ≤0.0 and the + DCs in the PD-L1-/- mice, both as compared to the WT (P ≤0.05) and PD-L2-/- (P ≤0.05) mice, and by the CD11b+ monocytes (P ≤0.01)   in the WT mice post-MCAO as compared to the shams. In the PD-L1-/- and PD-L2-/- mice, CD80 expression on CD11b+ cells (P ≤0.01) and CD80 expression on CD11c+ cells increased significantly (P ≤0.001), respectively, post-MCAO, as compared to their sham-treated counterparts. However, the PD-L1-/- mice, as demonstrated in Figure P ≤0.05), there was no difference in the PD-L1 expression between the WT and PD-L2-/- mice after MCAO, with no difference between the groups in sham conditions. However, there was indeed an increase in the total PD-L2 expression in the PD-L1-/- mice as compared to the WT mice (P ≤0.001) post-MCAO  besides PD-1, and PD-L1/CD80 interaction also delivers inhibitory signals in T cells ,32. Henc) Figure B and CD1) Figure C as compO Figure E, making-/- mice which were subjected to 60 minutes of MCAO followed by 96 hours of reperfusion. These studies demonstrated a critical role for PD-1 in limiting functional and histological damage after MCAO [-/- mice, was selected as a starting point of discerning the effects of interaction between PD-1 and its ligands in ischemic stroke.Ischemic stroke induces neurological deficits in almost one-third of patients, leading to increased mortality and long-term functional disability ,34. Althter MCAO . However-/- mice. The PD-1/PD-L pathway is recognized to control peripheral T cell tolerance in several ways. This pathway can limit the initial phase of activation and expansion of self-reactive T cells, and restrict self-reactive T cell effector function. The PD-1/PD-L interactions also play a role in inhibiting expansion of naive self-reactive T cells and/or their differentiation into effector T cells [+ T cell proliferation was enhanced and immunological tolerance was broken, as mice developed spontaneous diabetes [-/- DCs was demonstrated where intracerebral microinjections resulted in amelioration of subsequent experimental autoimmune encephalomyelitis (EAE) [+ Treg recruitment into the CNS, suggesting that the lack of DC-derived PD-L1 allows for the development (or recruitment) of regulatory CD8+ T cells (CD122+) in the CNS. Thus, by ‘inhibiting the inhibitors’, DC/APC expression of PD-L1 at the site of inflammation leads to exacerbated autoimmunity [+ subpopulation from the periphery post-MCAO in the PD-L1-/- mice, only to find its accumulation in the ischemic half of the brains.In our current study, we hypothesized a critical role for the PD-Ls in exacerbating ischemic stroke. However, as clearly demonstrated in Figures  T cells . Thus, P T cells ,41, its  T cells . However T cells . As a rediabetes . In yet diabetes , an unexis (EAE) . Furtherimmunity . Our fin+ Tregs. Thus, as demonstrated in Figure -/- mice might suggest an overriding CD80/CD28 interaction leading to T cell activation. Conversely, low CD80 expression in PD-L1-/- mice and the increase in the total PD-L2 expression in the PD-L1-/- mice as compared to the WT mice makes a plausible case for a PD-1/PD-L2 co-inhibitory interaction in the absence of PD-L1.A number of lines of evidence have suggested a receptor for PD-L1 (B7-H1) or PD-L2 (B7-DC), aside from PD-1. CD80 (B7-1) has recently been identified as a binding partner for PD-L1 . SurfaceHowever, one also needs to keep in mind that the broad distribution of PD-L1 expression on hematopoietic and parenchymal cells suggests one or both cell types may be central to limiting autoimmune disease during multiple phases of the immune responses. Due to its broad expression pattern in lymphoid and non-lymphoid organs, the PD-L1/PD-1 pathway has been suggested to play a crucial role for the maintenance of immune tolerance ,46. In f-/-, PD-L2-/-, and WT mice. Second, individuals performing infarct volume and immunological assessments were not blinded to genotype. This potentially could have led to subjective bias in assessing results. However, this seems unlikely given that our observed results suggested a pathogenic rather than the expected regulatory role for both PD-Ls. Lastly, differences in striatal infarct volumes between PD-L1-/- and PD-L2-/- mice could be due in part to altered glial proliferative responses as suggested by earlier studies [The present study has some limitations with regards to outcome evaluations. First, we used a neurological deficit scoring system to evaluate each animal. While this scoring system is sensitive enough to confirm ischemia and consequent ischemic injury in each animal, it may not be sensitive enough for evaluating subtle changes in functional outcomes due to experimental manipulations or treatments. Future follow-up studies will use more sophisticated and sensitive behavioral measures to evaluate functional outcomes at different time points following ischemic insult in PD-L1 studies . Evaluat-/- and PD-L2-/- mice have smaller total infarct volume compared to WT mice. Improved stroke outcome was accompanied by significant reduction in the brain infiltrating cells as well as their proinflammatory states in the PD-L1-/- and to a lesser extent PD-L2-/- mice as compared to the WT mice. Our study also demonstrated a novel connection between the ischemic lesion in the brain and evolving inflammatory changes in distant peripheral immune cell populations. Reduction in infarct volumes promoted reduction in ischemia-related splenic atrophy accompanied by lower activated states of the splenic T cells and monocytes in the absence of the PD-L1, suggesting a pathogenic rather than a regulatory role for both PD-Ls. Suppressor T cells (IL-10-producing CD8+CD122+ T cells) trafficked to the brain in PD-L1-/- mice and their presence in the right MCAO-inflicted hemisphere of the PD-L1-/- mice implicates them as possible key contributors in controlling further adverse effects of ischemia. There was increased expression of CD80 on splenic APCs in WT and PD-L2-/- mice, suggesting an overriding interaction leading to T cell activation. Conversely, low CD80 expression by the APCs in PD-L1-/- mice suggests suppression possibly via PD-1/PD-L2 interactions. Our novel observations are the first to implicate PD-L1 in enhancing the severity of experimental stroke. These results suggest that agents (for example antibodies) that can target and neutralize PD-L1/2 may have therapeutic potential for treatment of human stroke.In summary, the current study conclusively demonstrates for the first time that PD-L12-β-ME: 2-β-mercaptoethanol; ANOVA: analysis of variance; APC: antigen-presenting cell; CNS: central nervous system; CTLA-4: cytotoxic T lymphocyte antigen 4; DC: dendritic cell; EAE: experimental autoimmune encephalomyelitis; DNase I: deoxyribonuclease I; FACS: fluorescence-activated cell sorting; FCS: fetal calf serum; Foxp3: forkhead box P3; IFN: interferon; IL: interleukin; LDF: laser Doppler flowmetry; LPS: lipopolysaccharide; mAb: monoclonal antibodies; MCAO: middle cerebral artery occlusion; MHC: major histocompatibility complex; NIH: National Institutes of Health; NKT: natural killer T; PBS: phosphate buffered saline; PD-1: programmed death-1; PD-L: programmed death-1 ligand; PD-L1: programmed death-1 ligand 1; PD-L2: programmed death-1 ligand 2; PI: propidium iodide; PMA: phorbol 12-myristate 13-acetate; RPMI: Roswell Park Memorial Institute; SCID: severe combined immunodeficiency; SEM: standard error of the mean; TCR: T cell receptor; TNF: tumor necrosis factor; Treg: regulatory T cell; WT: wild-type.The authors declare that they have no competing financial interests.SB performed and interpreted the immunology experiments, carried out statistical analyses, prepared graphics, and wrote the manuscript. YC performed the MCAO procedures, carried out statistical analyses, prepared the graphics, and wrote the methods and results for infarct volume data. AAV critiqued and edited the manuscript. SJM directed study design and data analysis of the MCAO experiments and edited the manuscript. HO directed the overall study, designed and supervised the immunological studies and data analysis, and edited the manuscript. All authors read and approved the final version of the manuscript.


Burkholderia ambifaria AMMD (SadA) stereoselectively catalyzes the C3-hydroxylation of N-substituted branched-chain or aromatic L-amino acids, especially N-succinyl-L-leucine, coupled with the conversion of α-ketoglutarate to succinate and CO2. To elucidate the structural basis of the substrate specificity and stereoselective hydroxylation, we determined the crystal structures of the SadA.Zn(II) and SadA.Zn(II).α-KG complexes at 1.77 Å and 1.98 Å resolutions, respectively. SadA adopted a double-stranded β-helix fold at the core of the structure. In addition, an HXD/EXnH motif in the active site coordinated a Zn(II) as a substitute for Fe(II). The α-KG molecule also coordinated Zn(II) in a bidentate manner via its 1-carboxylate and 2-oxo groups. Based on the SadA.Zn(II).α-KG structure and mutation analyses, we constructed substrate-binding models with N-succinyl-L-leucine and N-succinyl-L-phenylalanine, which provided new insight into the substrate specificity. The results will be useful for the rational design of SadA variants aimed at the recognition of various N-succinyl L-amino acids.A novel dioxygenase from  S,3R,4S)-4-hydroxyisoleucine has insulinotropic and anti-obesity effects and seems to have potential for the treatment of diabetes cis-4-hydroxy-L-proline has been clinically evaluated as an anticancer drug The hydroxy amino acids, which are components of glycopeptide antibiotics, cyclodepsipeptides and collagen, have many physiological activities. Some hydroxy amino acids can also be used as precursors in the asymmetric synthesis of pharmaceuticals 2. This family of enzymes possesses a common protein fold, which is called the double-stranded β-helix (DSBH) fold, as the core of the structure, and an HXD/EXnH motif in the active site to coordinate the Fe(II) cofactor The hydroxylation of amino acids is catalyzed by the ferrous [Fe(II)]- and α-ketoglutarate (α-KG)-dependent dioxygenases. These enzymes can also hydroxylate proteins, nucleic acids, lipids and small molecules Burkholderia ambifaria AMMD. This enzyme is useful as a novel biocatalyst for the (R)-selective hydroxylation at the C-3 position of N-substituted branched-chain L-amino acids, especially N-succinyl-L-leucine (NSLeu), to produce N-succinyl--3-hydroxyleucine (NSHLeu) with >99% stereoselectivity -3-hydroxyleucine is a promising material for the preparation of certain cyclic depsipeptides which function as platelet aggregation inhibitors and is also a component of the antibiotic lysobactin N-formyl-L-leucine, N-acetyl L-leucine and N-carbamyl-L-leucine were also recognized as substrates by SadA, whereas the activities toward them were low (2–22%) compared with that toward NSLeu. SadA showed almost the same activity for several kinds of N-substituted branched-chain L-amino acids, N-succinyl-L-valine, N-succinyl-L-isoleucine and NSLeu (data not shown). In addition, SadA is the first characterized Fe(II)- and α-KG-dependent dioxygenase that catalyzes N-substituted aromatic L-amino acids, especially N-succinyl-L-phenylalanine (NSPhe), although its activity toward NSPhe is lower than that toward NSLeu (data not shown). Thus, SadA has the potential for widely producing C3-hydroxylated amino acids with various types of branched chain or aromatic ring.SadA is a member of the dioxygenase family from ectivity  [10]. , which gives us only the Fe(II)-binding HXD/EXnH motif as an enzymatic property. Although a few Fe(II)/α-KG-dependent dioxygenases are known to hydroxylate free amino acids Escherichia coli Rosetta(DE3) cells (Novagen) harboring the pQE80 vector (QIAGEN) with full-length SadA gene were grown in Lysogeny Broth medium and incubated at 310 K until the OD600 reached 0.6–0.8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM and the culture was then further incubated at 298 K overnight. After harvesting, the cells were disrupted by sonication in the resuspending buffer  and the cell debris was removed by centrifugation. SadA was trapped on Ni-NTA Superflow resin (QIAGEN). After washing, the protein was eluted and further purified by using Resource Q . The solution containing purified SadA was concentrated to 15 mg ml−1 in 20 mM Tris-HCl (pH 8.0), 1 mM DTT for crystallization.The SeMet) were performed as reported previously SadA was crystallized using the sitting-drop vapor diffusion method without any metals such as Zn(II) added to the solution during purification and crystallization. The crystals were obtained by mixing 1.0 µl protein solution with 1.0 µl reservoir solution consisting of 0.1 M CHES (pH 9.5) and 30% (w/v) PEG 3,000 at 293 K. The purification and crystallization of selenomethionine-substituted SadA  and SadA.Zn(II).α-KG complex crystals were collected on the AR-NW12A and AR-NE3A beamlines at Photon Factory , respectively. For phasing by single-wavelength anomalous dispersion (SAD) of selenium atoms, we collected the X-ray diffraction data of SadASeMet was obtained by SAD using the CNS program suite SeMet structure as the initial model. Manual rebuilding and refinement were performed with COOT and REFMAC5, respectively. The data-collection and processing statistics are summarized in The initial phase of SadAN-succinyl-L-phenylalanine (NSPhe) was also docked in the structure of the SadA.Zn(II).α-KG complex in the same way. The final model was obtained by perturbing the MD minimized model based on activity analyses.The initial model of SadA.Zn(II).α-KG.NSLeu was constructed using the Molecule Builder of the molecular operating environment . The initial model was minimized by employing the Merck Molecular Force Field 94x (MMFF94x). The substrate-binding site of the SadA structure was detected using the Alpha Site Finder in MOE. For NSLeu, 250 conformations were generated using the default LowModeMD search parameters. Ligand docking simulations were performed using the ASEDock program of MOE. The molecular dynamics (MD) simulations were performed using MMFF94x with the Nose-Poincare-Anderson (NPA) algorithm and the generalized Born method. The distance and relative position between Zn(II) and C3 of NSLeu were fixed to 3.0–3.5 Å Site-directed mutageneses were performed by PCR with a QuikChange kit  and pQE80-SadA plasmid as a template 4·7 H2O, 10 mM L-ascorbate, 50 mM Tris-HCl buffer (pH 8.0), and 1 mg ml−1 purified SadA was used. The reaction was allowed to proceed at 30°C for 2 h and was terminated by the addition of 20 mM EDTA. The N-succinyl group of the products was desuccinylated by adding a 1/50 vol of 6 M HCl to the reacted solution and heating at 105°C for 1 h. After neutralization with NaOH, the hydroxy amino acids in the reaction mixture were derivatized with AccQ Tag  according to the manufacturer’s instructions. The derivatives were analyzed using an Alliance 2695 high-performance liquid chromatography (HPLC) system (Waters) equipped with a fluorescence detector. An XBridge C18 column  was used for separation at 40°C. All measurements were performed in triplicate.The activity assay was performed as described previously −1 and the metal content of the enzyme was determined by inductively coupled plasma atomic emission spectrophotometer (ICP-AES).The wild-type enzyme was expressed and purified as described above. Protein concentrations were about 23 mg ml10 helix, and possessed the DSBH fold at its core  and SadA.Zn(II).α-KG were determined at 1.77 Å and 1.98 Å resolutions, respectively. The electron density maps of residues Ser60–Thr74 and Ala148–Phe152 were not observed in either of the structures. The structure of SadA.Zn(II) contained 11 β-strands, 6 α-helices and one 3its core , which wA-Cys101B and two salt bridges of Lys171–Asp87 (3.4 Å) and Asp105–Arg102 (3.2 and 3.7 Å)  B–D. Thed 3.7 Å) E. Moreod 3.7 Å) F. ThesenH motif. The electron density map of metals can be observed in the active site. We have performed crystallization and soaking experiments with Fe(II) under aerobic or anaerobic conditions, but failed to obtain the crystal with Fe(II). The data from inductively coupled plasma atomic emission spectroscopy (ICP-AES) showed that the concentration of Zn(II) was about 14-fold higher than that of Fe(II) in the SadA solution (In the SadA.Zn(II).α-KG structure, the active site is surrounded by the loop of β4-β5 and the β9 strand. The structure possesses a conserved HXD/EXsolution ; therefonH motif. This water would be displaced by O2 in the course of the catalytic reaction.On the other hand, only one α-KG molecule is clearly observed in chain A of the SadA.Zn(II).α-KG structure . The α-KN-oxalylglycine  and NSLeu under aerobic or anaerobic conditions, but failed to obtain the complex structure. The SadA.Zn(II).α-KG structure has a deep cavity that is large enough to accommodate the substrate . We also predicted and verified the residues related to substrate recognition around the active site by biochemical analyses. Although we do not obtain the crystal structure of the complex of SadA with an N-substituted branched-chain L-amino acid, substrate-binding model analyses combined with the activity assays of various mutants suggest how SadA binds its substrates. SadA showed a different activity toward N-substituted branched-chain L-amino acids. Based on the constructed model, we predict that the binding site of the N-succinyl group is located in an electropositive-rich cavity by the formation of salt bridges with the side chains of Arg83, Arg163 and Arg203. Consistent with the proposed binding mode of the N-succinyl group above, the R83A, R163A and R203A mutants showed reduced hydroxylation activities (N-succinyl branched-chain L-amino acids compared with other N-substituted branched-chain L-amino acids (10), which have no additional negatively-charged substituent.In this study, we determined the crystal structures of SadA.Zn(II) and SadA.Zn(II).α-KG. SadA is the first enzyme shown to catalyze the hydroxylation of the tivities A. ThereN-succinyl amino acid probably are formed with the main chain of Gly79 and the phenyl ring of Phe261 .The atomic coordinate and structure factor (code: 3W20 and 3W21) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ  and conalbumin (75 kDa) were used to create the calibration curve (dotted lines). A single peak corresponding to a dimer was observed. The scale at the bottom indicates the elution volume.(TIF)Click here for additional data file.Figure S20−Fc electron density map of α-KG and Zn(II) contoured at 1.0 sigma. The HXD/EXnH motif is shown as white sticks.2F(TIF)Click here for additional data file.Figure S3−1), red for negative (−5kTe−1) and white for neutral. The black ellipse indicates the predicted substrate-binding pocket. The residues which are related to substrate binding are shown as green sticks.Electrostatic surface potential as displayed in blue for positive (5 kTe(TIF)Click here for additional data file.Table S1Primer used for the construction of SadA mutants.(DOC)Click here for additional data file.Table S2Metal analysis of SadA.(DOC)Click here for additional data file.


Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein.The two-step transposition pathway of insertion sequences of the IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily.A soluble and active IS2 transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level.Intractably insoluble transposases, such as the IS The critical residues here (in bold) are, (i) the optional hydrophobics (ho), V35 in helix 1 and H39 and V41 in the turn  and  and as many as 57 residues from the N-terminus. PSIPRED secondary structure analyses of the TPases of all other prototypes of the principal subgroups of the IS3 family show three helices whose positions are similar to those shown for IS2 (data not shown).These results are in general accord with, and extend the work of, Prere et al. , Hu et a GCG3'. Primer B - lower strand: 5'CGC[TGGATTTGCCCCTATATTTCCAGACATCTGTTATCACTTAA]GGATCC3'.Annealed 50-mer oligonucleotides containing the 41 bp IRR sequence were used in all but one of the EMSA experiments Figure . The upp32P-ATP. Primer A - 5'GCTGACTTGACGGGACGGGGATCC[TTAAGTGATAACAGATGTCTGGAAATATAGGGGCAAATCCA]ATCGACCTGCAGGCATATAAGC3'. Primer B - 5'GCTTATATGCCTGCAGGTCGAT[TGGATTTGCCCCTATATTTCCAGACATCTGTTATCACTTAA]GGATCCCCGTCCCGTCAAGTCAGC3'.Reactions shown in Figure 32P-ATP. The reaction was incubated at 37°C for 30 minutes and heat-killed at 90°C for 5 minutes. A 100-μL annealing reaction contained 10 ρmol and 13 ρmol of the labeled and unlabeled strands respectively, 20 mM Tris-Cl pH 8.0 and 100 mM NaCl. The reaction was placed in a boiling water bath, cooled to 65°C, held there for 15 minutes and allowed to cool to room temperature.A 20 μL labeling reaction contained 40 units of T4 polynucleotide kinase in 1X T4 polynucleotide kinase reaction buffer (New England Biolabs), 20 μM of the primer (upper strand) and 50 μCi of γBinding of the TPase to its cognate DNA was carried out for 30 minutes at room temperature (20°C) in a 15-uL reaction mixture of 20 mM Tris-Cl pH 8.0, 1 mM ethylenediaminetetraacetic acid, 5.0 μg/mL calf thymus DNA, 10 nM of the radioactively labeled annealed primers and 0.13 μM of the partially purified preparation of the OrfAB-GFP fusion protein. Reactions were separated on 5% native polyacrylamide gels at 4°C for an average of 450 volt hours (Vhrs)  and h2 when less hydrophobic residues are present or the conserved residues are only found in fewer than 80% of the sequences. Three different algorithms were used for secondary structure predictions: the PSIPRED server[The ExPASy SWISS PROT translation toolkit  of the Sent tool ,109,110  [et al.  and RezsED server using thED server from theED server). A PCOIED server,58 was uED server from theCb: carbenicillin; CAS: catalytic active site; CD: catalytic domain; EMSA: electrophoretic mobility shift assay; E-10: extended-10 promoter; F-8: figure-of-eight; GFP: green fluorescent protein; IRR/IRL: right and left inverted repeats; IS: insertion sequences; IR: inverted repeat; kb: kilobases; kDa: kiloDaltons; LB: lysogeny broth; LZ: leucine zipper; MCJ: minicircle junction; NaCl: sodium chloride; OD: optical density; orf: open reading frame; PCR: polymerase chain reaction; RISF: TPase/retroviral integrase superfamily; RSV: Rous sarcoma virus; SC: synaptic complex; Tpase: transposase; Tris-Cl: tris(hydroxymethyl)amino methane; Vhr: volt hour.The authors declare that they have no competing interests.in silico determination of the amino acid substitutions in the mutant strains. SA carried out all cloning experiments involving the creation of plasmids with the orfAB mutations. RS performed the PCR and PCR-based mutagenesis experiments. JA carried out all of the lacZ papillation experiments. LAL designed the study and provided facilities and funding. LAL and MA wrote the manuscript. All authors have read and approved the final manuscript.PTU created the fusion construct, carried out the overexpression and protein purification experiments, the secondary structure analysis and


HLA-B*51 (B*51), is the strongest known genetic risk factor for BD, however the gene immediately centromeric to HLA-B, MICA, has also been implicated in BD. Because of strong linkage disequilibrium (LD) between HLA-B and MICA, their respective contributions to BD susceptibility have been debated. A recent report has proposed that B*51 is not a BD susceptibility allele, and several studies have identified B*51-independent association signals within the MHC.Family studies support a genetic contribution to Behçet's disease (BD), with a sibling recurrence-risk ratio of 11-52. The class I MHC molecule, B*51 and BD, and to test for B*51-independent genetic variation within the MHC that influences BD susceptibility.To clarify the relationship between HLA-B/MICA region with Haploview. We performed SNP imputation of the MHC using IMPUTE2 and the 1000 Genomes Phase 1 dataset. We inferred classical HLA types and their amino acids using SNP2HLA. Association testing and regression analyses were performed using SNPTEST and SNP & Variation Suite 7.Using Illumina Human 370CNV SNP genotypes in a Turkish collection of 1244 BD patients and 1303 geographically-matched healthy subjects, we examined SNP haplotypes and LD patterns across the B*51(+) HLA-B/MICA haplotype that was strongly associated with BD . A B*51(-) version of the same haplotype occurred at equal frequencies in cases and controls, demonstrating that B*51 is essential to the risk haplotype. Further, we found that rs2848713, a variant on the MICA end of the haplotype, conferred additional risk of BD in B*51(+) individuals. Through imputation, we generated a set of 32,689 imputed SNPs. The 2 most strongly associated SNPs were 4.8Kb centromeric of HLA-B (pmin=1.4E-50), but no SNP was more strongly associated with BD than was B*51 itself (p=1.3E-55). Conditioning on B*51 revealed an association near HLA-A (pmin=5.4E-9), and upon adding a representative HLA-A SNP to the regression model, we detected residual association centromeric of HLA-B (p=1.5E-5). Analysis of imputed HLA types supported these findings. In addition to the association of BD with B*51 (p=2.2E-55), sequential regression of imputed HLA types identified associations of HLA-A*03 (p=1E-8), HLA-C*0701 (p=9.5E-4), and HLA-B*15 (p=1.2E-4) with BD. Stepwise forward regression of imputed HLA-B amino acids identified 6 HLA-B residues that together fully accounted for the regional association at HLA-B.We identified a B*51 as the strongest risk factor of BD. We have provided strong evidence opposing a B*51-independent role for MICA variants in BD susceptibility. We have identified significant effects of HLA-A*03 and HLA-C*0701, which protect against BD, and HLA-B*15, which confers risk of BD. We have identified a group of HLA-B amino acids, most of which reside in the antigen binding groove, that together account for the entire association signal at the HLA-B locus.This study affirms None declared


GB virus C (GBV-C) or hepatitis G virus (HGV) is an enveloped, RNA positive-stranded flavivirus-like particle. E2 envelope protein of GBV-C plays an important role in virus entry into the cytosol, genotyping and as a marker for diagnosing GBV-C infections. Also, there is discussion on relations between E2 protein and gp41 protein of HIV. The purposes of our study are to multi aspect molecular evaluation of GB virus C E2 protein from its characteristics, mutations, structures and antigenicity which would help to new directions for future researches.Briefly, steps followed here were; retrieving reference sequences of E2 protein, entropy plot evaluation for finding the mutational /conservative regions, analyzing potential Glycosylation, Phosphorylation and Palmitoylation sites, prediction of primary, secondary and tertiary structures, then amino acid distributions and transmembrane topology, prediction of T and B cell epitopes, and finally visualization of epitopes and variations regions in 3D structure.Based on the entropy plot, 3 hypervariable regions (HVR) observed along E2 protein located in residues 133-135, 256-260 and 279-281. Analyzing primary structure of protein sequence revealed basic nature, instability, and low hydrophilicity of this protein. Transmembrane topology prediction showed that residues 257-270 presented outside, while residues 234- 256 and 271-293 were transmembrane regions. Just one N-glycosylation site, 5 potential phosphorylated peptides and two palmitoylation were found. Secondary structure revealed that this protein has 6 α-helix, 12 β-strand 17 Coil structures. Prediction of T-cell epitopes based on HLA-A*02:01 showed that epitope NH3-LLLDFVFVL-COOH is the best antigen icepitope. Comparative analysis for consensus B-cell epitopes regarding transmembrane topology, based on physico-chemical and machine learning approaches revealed that residue 231- 296 (NH2- EARLVPLILLLLWWWVNQLAVLGLPAVEAAVAGEVFAGPALSWCLGLPVVSMILGLANLVLYFRWL-COOH) is most effective and probable B cell epitope for E2 protein.The comprehensive analysis of a protein with important roles has never been easy, and in case of E2 envelope glycoprotein of HGV, there is no much data on its molecular and immunological features, clinical significance and its pathogenic potential in hepatitis or any other GBV-C related diseases. So, results of the present study may explain some structural, physiological and immunological functions of this protein in GBV-C, as well as designing new diagnostic kits and besides, help to better understandingE2 protein characteristic and other members of Flavivirus family, especially HCV. In 1995 and 1996, different isolates of the same new enveloped, RNA positive-stranded flavivirus-like particles with a genomic size of about 9.3 Kb, were isolated by two independent research groups, which named GB virus C (GBV-C) and hepatitis G virus (HGV), respectively. This RNA contains an open reading frame (ORF) which encodes polyprotein with about 2900 amino acids length. By viral/host proteases the polyprotein of GB virus C is cleaved into structural proteins  and nonstructural proteins  (There are evidences on reducing HCV-related liver morbidity associated with GB virus C (GBV-C) and inhibitory effect of GB virus C on HCV/HIV viremia, survival, a lower mortality rate, slower disease progression in patients with coinfection and also, GBV-C could play role as a predictor for hospital acquired infection , 8. InteE2 envelope protein of GB virus C plays role in virus entry into the cytosol, genotyping , the ideNowadays, viral-related bioinformatics analysis tools are powerful approaches to predict molecular features such as similarity, glycosylation/phosphorylation/ Palmitoylation sites, epitope recognition, protein primary secondary/ tertiary structures of proteins encoded in viral genomes .One of the branches of bioinformatics is Immunoinformatics or computational immunology which has emerged recently as an important field in the analysis, immune function modeling and prediction of both B and T cell epitopes, novel vaccines designing and allergenicity analysis , 14.Glycoprotein glycosylation characteristics are known to be in association with changes of virulence, cellular tropism in enzymes, and survival of viruses . PalmitoDifferent structure prediction approaches with different reliability simplify the discovery process in biology, and provide a structural framework for new hypotheses. They were also continuously developed and evaluated , 21. UndThe purposes of our study are to multi aspect molecular evaluation of GB virus C E2 protein from its characteristics, mutations, structures and antigenicity. These valuable information would help to new directions for future research such as designing diagnostic kits and help to better understanding similarities or differences of biological features of GB C virus in comparison with other members of the Flavivirus family, especially Hepatitis C virus (HCV). The interplay between experimental and computational biology has enormous benefits and providing invaluable Information in many different areas of the sciences.Complete putative E2 (Accession number (AC)NP_803203) of GB virus C/Hepatitis G virus mentioned as a reference sequence in National Center for Biotechnology Information (NCBI) Databases (http://ncbi.nlm.nih.gov/) was retrieved. In bioinformatics analyzing a reference sequence (RefSeq) is mostly preferred causes that well annotated and nucleotide sequence  and its protein products are available and reliable.We retrieved 100 sequences of E2 protein of GB virus C from NCBI by direct searching. Obtained sequences were aligned, analyzed and trimmed in Bioedit 7.7.9 software. Subsequently, short sequences and areas with ambiguous alignment were excluded. Then, Entropy values (Hx) were measured. This analysis measures variation at each amino acid position in the set of aligned sequences. Results are shown in The primary protein structure of E2 , Isoelectric point (pI) and amino acid distribution) was arranged in We used NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc) and NetPhos 2.0 server (http://www.cbs.dtu.dk/services/NetPhos.) to predict N-Glycosylation and Phosphorylation sites in E2 protein. These two servers are both taking advantage of artificial neural networks (ANN) for this prediction. NetNGlyc 1.0 server examines the sequence context of Asn-Xaa-Ser/Thr sequences and the NetPhos 2.0 server predicts serine, threonine and tyrosine phosphorylation sites. Palmitoylation sites were predicted with the medium threshold frequency by using services at http://csspalm.biocuckoo.org/prediction.php, in particular CSS-Palm 2.0 software.The secondary structure of the protein was evaluated by using bioinformatics tools available on the website; http://npsa-pbil.ibcp.fr. The method of GOR4 (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_ gor4.html) was used to identify the alpha helices, beta strands, and coil residues. As we could not find any matches in SWISS-PROT for E2 to analyze functional and structural motifs, we used SCRATCH suite (http://www.igb.uci.edu/) combines machine learning methods, evolutionary information, fragment libraries and energy functions to predict protein structural features and tertiary structures. The 3D model is visualized by the Swiss-Pdb Viewer software.IEDB (Immuno Epitope Database) server website (http://tools.immuneepitope.org/mhci/) provides access to predictions of peptide binding to MHC class I molecules.It estimates IC50 values for peptides binding to specific MHC molecules. List box for selecting the prediction method allows to use different MHC class I binding prediction methods such as Artificial Neural Networks (ANN), Stabilized Matrix Method (SMM), SMM with a Peptide MHC Binding Energy Covariance matrix (SMMPMBEC), Scoring Matrices derived from Combinatorial Peptide Libraries (Comblib_Sidney2008), Consensus method , and NetMHCpan. HLA-A*0201 is the most frequent allele and also the first human HLA allele for which peptide binding prediction was developed . TherefoE2 protein antigenicity prediction was checked based on hydrophobicity, assessment of solvent accessibility regions, flexibility, secondary structure (Beta-Turn prediction), and Kolaskar and Tongaonkar method . KolaskaSeveral methods using machine learning approaches have been introduced. The hybrid method applied in this study is composed of hidden Markov model, Feed forward and recurrent neural network, subsequence kernel based SVM and SVM which are used in BepiPred , ABCPredFinally, we compared all the analyses mentioned above to interpret unique molecular features and Immunoinformatics of this protein. Also, the predicted B-cell epitopes were evaluated whether they were present in outer transmembrane regions, using TMHMM results. Epitopes exposed on the surface of the membrane were selected and subjected to further analysis. Moreover, variations represented in entropy plot were checked in 3D model.The quality evaluation of the modeled structure is an essential step in homology modeling. The geometric estimation of the modeled 3D structure (tertiary structure) was performed using the Ramachandran plot (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php). Ramachandran plots is The two-dimensional (2D) scatter plots of φ, ψ  which tests whether the model structure is stereo-chemically stable and the number of outliers . The ploBased on the entropy plot, 3 hyper variable regions (HVR) observed along E2 protein that located in residues 133-135, 256-260 and 279-281. HVR are regions in sequence with highest variation in different isolates of virus. Besides, highest conservation observed at amino acids 152-168 and 183-248. Residue 256-260 is located in outer membrane region of E2 protein (see 4.2.), and this variability may help GB virus C to escape immune response of its host.Summarized obtained data from Expasy ProtParam tool presented in An average length of protein sequence and molecular weight of constructs were mentioned in the In physicochemical analysis, it was revealed that the most abundant amino acid residues were glutamic and glycine. Distribution of amino acid frequency in E2 protein showed that hydrophobic residues are more frequent than hydrophilic residues, and also, negative R-group to positive R-group . So, mosAnalysis of transmembrane topology using the TMHMM online server found that residues 257-270 presented outside while residues 234- 256 and 271-293 were transmembrane region, and residues 1- 233 and 294-312 were inside the core region of the protein . Also, tJust one N-glycosylation site (residue 73) was found in E2 protein of GB virus C . PotentiTo account for the possible Palmitoylation sites we applied CSS-PALM 3.0 software by choosing medium threshold . ResultsAs it shown in Finally calculating Coils (Beta turns) revealed 17 coil region in E2 structure. Outer membrane region predicted by TMHMM online server has α-helix (dominant structure), small β-strand as well as coil structure.Transmembrane regions have α-helix predominantly.The predicted epitopes were evaluated for their immunogenicity, and epitopes found to be immunogen in nature were introduced as major immunogenic epitopes for T CD8+-cell . EpitopeIn Antigenicity (immunogenicity) prediction plot of E2  protein B-cell epitope prediction based on machine learning approaches were performed usingBCPRED server, where criteria were set to have 75% specificity and ABCpred 65.93% accuracy with fixed length of 20 and 16 amino acids .Higher sPrediction of B-cell epitopes regarding transmembrane topology , based on physico-chemical properties and machine learning approaches showed that this protein has different regions with potential of immunogenicity, but machine learning method by BCPREDS (specificity 80%) and ABCpred specificity (85%) could not predict epitopes in range of 257-270 (outer membrane region of protein). These servers had a consensus epitope in approximate region of 230-253 that is in transmembrane region based on TMHMM server prediction. In physico-chemical approaches the best performance was seen by Kolaskar-Tongaonkar algorithm in which a part of epitope Residue 231- 296 (fragment of NH2- EARLVPLILLLLWWWVNQLAVLGLPAVEAAVAGEVFAGPALSWCLGLPVVSMILGLANLVLYFRWL-COOH) was located in outer and transmembrane of E2 protein . These e3D model of the E2 protein with a total number of 310 amino acids was validated using the Ramachandran plot. Assessment of the plot  revealedHere we provided deep insight into the computational molecular features and Immunoinformatics characteristic of E2 protein of GBV-C/HGV by using various bioinformatics techniques.GBV-C and HGV are closely related isolates of the same virus, with more than 95 percent sequence homology . GBV-C aRNA virus genomes (due to the lack of proofreading ability of their RNA-dependent RNA polymerase) have special potential to undergo mutation at high frequencies, and under selective pressures rapidly generate populations of viral variants. Such variability helps to evading of virus from clearance by both T- and B-cell immunity .Three different HVR  observed along E2 protein. Besides, residue HVR2256-260 located in outer membrane region of E2 protein. Different researchers suggest that HCV hypervariable region 1 (HVR1) is located in a spanning of 27–31 (or 25-30 in some reports) residues at E2 glycoprotein which is the main target of the anti-HCV neutralizing response and hence plays an important role in providing viral persistence , 42. SubSequence analysis of the transmembrane topology of HCV E2 in details and its importance are widely discussed . These sFinding modifications sites, patterns and number of important viral protein such as; N-glycosylation, palmitoylation, phosphorylation etc. have an enormous effects on foldings, entry functions, viral transportation/replication/assembly, infectivity, pathogenicity, immunogenicity as well as it may explain different virulence between different isolates of a virus and also viral genus .In residue 73, N-glycosylation site was found in E2 protein of GB virus C. In case of HCV the ectodomain of envelope glycoproteins E2 has high modification by N-linked glycans and defined 11 potential glycosylation sites , 50, thaAlso, there are reports on in-silico evaluation of glycosylation, phosphorylation and palmitoylation in other viral proteins such as S1 protein from Infectious Bronchitis Virus (IBV), and they finally interpreted that there is differences in number and location of mentioned properties between isolates but most of the glycosylation, phosphorylation and Palmitoylation sites were conserved within specific genotypes . These cStructure prediction approaches have been continuously developed and they greatly accelerated and simplified discovery of biological features of macromolecule and provided a structural framework for novel and innovative hypotheses. It might notice that different methods have different reliability, and this subject has to be taken into account while using their results and compare the prediction with an experimental result . Six α-hThe data extracted from the three-dimensional structure of a protein is essential for understanding and solving the details of its molecular function, and gives valuable knowledge for the development of effective rational strategies for experiments such as findings disease related mutations, site directed mutagenesis, or vaccine and drug design based on protein structure  . In thisThe predicted epitopes for T CD8+-cell  with higBy comparative analysis of B-cell epitopes between physico-chemical and machine learning approaches regarding 3D/secondary structure and outer membrane region, the best performance was seen by Kolaskar-Tongaonkar algorithm. This epitope was residue 231- 296 (fragment of NH3-EARLVPLILLLLWWWVNQLAVLGLPAVEAAVAGEVFAGPALSWCLGLPVVSMILGLANLVLYFRWL-COOH) . So, thiThe comprehensive analysis of a protein with important roles has never been easy, especially when we attempt to make statements from different aspects about this protein. In case of E2 envelope glycoprotein of HGV, there is no much data on its molecular and immunological features, clinical significance and its pathogenic potential in hepatitis or any other GBV-C related diseases. So, results of the present study may explain some of its structural, physiological and immunological functions in GBV-C virus, as well as help to better understanding E2 protein potential of other members of Flavivirus family, especially HCV.


E. coli. We showed the efficient purification of Fc-III tagged proteins by immobilized non-native IgG-Fc and the detection of the cellular locations of fusion proteins by fluorescent-conjugated IgG-Fc. Our results prove that Fc-III tagged protein expression system is a simple and efficient tool for protein purification and detection and is a useful addition to the biochemistry and proteomics toolbox.In the present work, we developed the Fc-III tagged protein expression system for protein purification and detection. The Fc-III sequence encodes for a 13 residue peptide and this peptide is cyclized by disulfide bond formation when the fusion protein is expressed. The Fc-III-fusion proteins selectively bind to immunoglobulin Fc domains (IgG-Fc) expressed from  Protein tagging involves genetically grafting a peptide sequence onto a recombinant protein. These fusion proteins have various applications in molecular biology such as affinity purification, protein detection, and epitope presentation Staphylococcus aureus has a high affinity (10 nM) to the constant region (Fc) of immunoglobulin G (IgG) 2, in which two Cys residues form a disulfide bond to make the cyclized peptide. The synthetic Fc-III peptide binds to the surface of the IgG-Fc domain by mimicking the protein-protein binding interface of PrA for the hinge region on the IgG-Fc. The affinity with a Kd value of 185 nM for the binding of the Fc-III peptide to IgG-Fc depends on both the Cys-Cys disulfide bridge and the C-terminal amide group et al. to competitively displace affinity-purified Protein A fusion proteins and protein complexes from IgG-Sepharose with biotinylated Fc-III In the present study, we engineered an Fc-III tagged fusion protein expression system. The Fc-III peptide is a mimic of Protein A, a commonly used affinity tag. The genetically expressed Protein A (PrA) fusion proteins from GACTGTGCATGGCATCTTGGAGAACTCGTATGGTGTACTGGAATGTCCCATCACTGGGG; r3: CATGGTATATCTCCTTCTTAAAGTTAAAC), and (2) C-terminal insertion . The codons of the Fc-III peptide are underlined. The gene of CK (creatine kinase) was bought from Addgene, and was cloned into pET21a (Novagen). Various primers were used to construct HisTag fused CK, N-terminal Fc-III tagged CK and C-terminal Fc-III tagged CK. The gene of CD38 was cloned from the mRNA by RT-PCR from the Raji cell line (purchased from ATCC), which was then sub-cloned into eukaryotic plasmid pcDNA3.1B (Invitrogen). The Fc-III peptide was added onto the N-terminus of CD38, and the primers are the followings: F5: GACTGTGCATGGCATCTTGGAGAACTCGTATGGTGTACTGCCAACTGCGAGTTCAGCCATG; and r3: AAGCTTAACTAGCCAGC.The gene of CA (carbonic anhydrase) was a gift from Zhou HM’s laboratory at Tsinghua University and was cloned into the expression vector pET28a (Novagen). The primers of the inserting Fc-III peptide were the followings: (1) N-terminal insertion (F5: E. Coli BL21(ED3)-pLysS strain  and purified. Briefly, for Fc-III tagged CA and CK, the cell lysates were incubated with IgG-beads  or immobilized IgG-Fc sepharose beads for 1 hour at 4°C, and were eluted with HAc-NH4Ac (pH3.4); for other His-tagged proteins, Ni-FF (Qiagen) columns were used, and proteins were eluted with 250 mM imidazole in PBS (pH8.0). For biochemical analysis, the samples were dialyzed and loaded onto Sephacryl S300 column. The purity of the recombinant protein was characterized by SDS-PAGE. All the protein concentrations were determined using Bradford method with BSA as the standard.CA-II, Fc-III tagged CA-II, IgG-Fc and different CK proteins were expressed in Protein bands of interest were excised from the SDS PAGE and digested by trypsin with or without prior reduction and alkylation in 50 mM ammonium bicarbonate at 37°C overnight. The peptides were extracted twice with 1% trifluoroacetic acid in 50% acetonitrile aqueous solution for 30 min. The extractions were then centrifuged in a speedvac to reduce the volume.For LC-MS/MS analysis, the digestion product was separated by a 65 min gradient elution at a flow rate 0.250 µL/min with the EASY-nLCII™ integrated nano-HPLC system  which is directly interfaced with the Thermo LTQ-Orbitrap mass spectrometer. The analytical column was a home-made fused silica capillary column  packed with C-18 resin . Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The LTQ-Orbitrap mass spectrometer was operated in the data-dependent acquisition mode using the Xcalibur 2.0.7 software and there is a single full-scan mass spectrum in the Orbitrap  followed by 20 data-dependent MS/MS scans in the ion trap at 35% normalized collision energy. The MS/MS spectra from each LC-MS/MS run were searched against the selected database using an in-house Mascot or Proteome Discovery searching algorithm.All the protein samples were dialyzed into 50 mM PBS (pH 7.8). For protein complex detection, Fc-III tagged or His-tagged CA were incubated with IgG-Fc for half an hour at room temperature, and then samples were centrifuged at 13 000 g and 100 µl sample was injected onto Superdex HR200 column and analyzed with the ÄKTA purifier 10 .2 atmosphere. After transfected with CD38 recombinant plasmid for 24 h, cells were fixed with 4% formaldehyde and permeabilised in 0.2% Triton X-100. The fixed cells were washed with PBS for three times, and then treated with 0.04 mg/ml Rhodamine B labeled IgG-Fc. Cells were stained with 1 µg/ml Hoechst 33342 for cell nucleus. After the washes, samples were analyzed by Zeiss LSM 710 (Carl Zeiss) Confocal Microscope with excitation wavelengths for Hoechst 33342 and Rhodamine B.Human liver carcinoma HUH-7 cells (purchased from ATCC) were cultured on coverslips at 37°C in DMEM medium with 10% FBS under 5% CO2-126 reagent was used to label the tagged proteins binding to the affinity matrix, and TMT2-127 reagent was for the proteins left in the flow-through fraction. The ratio of the TMT2-126 labeled peptide to the TMT2-127 labeled peptides was calculated by the Proteome Discovery Software. The relative contribution from 13C isotope was deducted based on the manufacture’s protocol.The purification efficiency was quantified by chemical labeling with TMT reagents  according to the manufacture’s instruction et alThe CA activity was determined using the Esterase activity assay as described by Pocker GACTGTGCATGGCATCTTGGAGAACTCGTATGGTGTACT) onto either the 5′ or 3′ end of the human carbonic anhydrase II (hCAII) that was cloned into pET-28a plasmid. CA has been widely used as the model system to study protein folding and enzymatic reaction. CA is broadly distributed among most of the tissues of animals and green plants and reversibly catalyzes the rapid interconversion of CO2 and H2O to HCO3− and H+. The plasmid was introduced into E. coli T7 protein expression system for protein expression. The expressed protein was separated on the 1D SDS PAGE and a gel band corresponding to the expressed 32 kD Fc-III fusion proteins was observed. For finding out if the Fc-III in the fusion protein was in its cyclized form, the gel band was excised and digested with or without reduction and alkylation. The mass spectra of the tryptic peptides containing Fc-III are displayed in m/z701.30 in m/z 730.32 corresponding to the carboxymethylated Fc-III-containing peptide ion was observed in m/z 730.32 precisely matched to the b ions or y ions of the carboxymethylated Fc-III-containing peptide. This shows that the expressed fusion protein has a cyclized Fc-III peptide.To generate Fc-III fusion protein, we appended the DNA sequence of Fc-III . E. coli expressed IgG-Fc is a homodimer as shown in Previous studies showed that cyclized Fc-III peptide as the mimic of PrA or PrG tightly binds to the hinge region on the Fc fragment of human IgG. Using the IgG immobilized sepharose beads, we were able to affinity purify the Fc-III fusion protein for carbonyl anhydrase (data not shown). To evaluate the binding of the Fc-III tagged fusion protein to E. coli expressed IgG-Fc is a dimer formed by inter-chain disulfide bond, in consistent with SEC analysis  and non-reduced (Lane 6–9) conditions, respectively. Lane 1&6 for His-tagged CA; Lane 2&7 for Fc-III-tagged CA; Lane 3&8 for IgG-Fc purified from E. coli; and Lane 4&9 for the mixture of Fc-III tagged CA and IgG-Fc. Lane 5 is the marker indicated the molecular weight. Noting that most expressed Fc-III tagged CA were monomers and most expressed IgG-Fc are dimers.(TIF)Click here for additional data file.Figure S2Rhodamine B-labeled IgG-Fc was generated by a reaction of Rhodamine B succinimidyl ester with IgG-Fc. To prove the labeling efficiency, in-gel fluorescence images of IgG-Fc labeled with rhodamine B succinimidyl ester was acquired with the exciting wavelength at 572 nm. Lane 1, 0.4 mg/ml IgG-Fc; Lane 2, 0.8 mg/ml IgG-Fc; Lane 3, 0.8 mg/ml IgG-Fc reacted with rhodamine B for 4 h; Lane 4, 0.4 mg/ml IgG-Fc reacted with rhodamine B overnight; Lane 5, 0.8 mg/ml IgG-Fc reacted with Rhodamine B overnight.(TIF)Click here for additional data file.Figure S3The MS/MS spectra of 10 TMT-labeled tryptic peptides from CA. Each MS/MS spectrum shows the relative intensity of two major fragments at m/z 126 and 127, corresponding to the TMT2-126-labeled and the TMT2-127-labeled peptides, respectively. The intensity ratio of these two fragments represents the relative quantity of the targeted protein on the affinity matrix and in the flow through fraction. The 13C contribution of TMT2-126 was used to calculate the purification efficiency. (A–J) 10 peptides from His-tagged CA; (A–J) 10 peptides from Fc-III tagged CA.(TIF)Click here for additional data file.


Necrosis, a type of cell death accompanied by the rupture of the plasma membrane, promotes tumor progression and aggressiveness by releasing the pro-inflammatory and angiogenic cytokine high mobility group box 1. It is commonly found in the core region of solid tumors due to hypoxia and glucose depletion (GD) resulting from insufficient vascularization. Thus, metabolic stress-induced necrosis has important clinical implications for tumor development; however, its regulatory mechanisms have been poorly investigated.in vitro model of solid tumors and of human tumors. Snail short hairpin (sh) RNA inhibited metabolic stress-induced necrosis in two-dimensional cell culture and in multicellular tumor spheroid system. Snail shRNA-mediated necrosis inhibition appeared to be linked to its ability to suppress metabolic stress-induced mitochondrial ROS production, loss of mitochondrial membrane potential, and mitochondrial permeability transition, which are the primary events that trigger necrosis.Here, we show that the transcription factor Snail, a key regulator of epithelial-mesenchymal transition, is induced in a reactive oxygen species (ROS)-dependent manner in both two-dimensional culture of cancer cells, including A549, HepG2, and MDA-MB-231, in response to GD and the inner regions of a multicellular tumor spheroid system, an Taken together, our findings demonstrate that Snail is implicated in metabolic stress-induced necrosis, providing a new function for Snail in tumor progression. Unlike tumor-suppressive apoptosis or autophagic cell death, necrosis has been implicated in tumor progression and aggressiveness by releasing a nuclear protein, high mobility group box 1 (HMGB1), that is normally involved in DNA bending and acts as a transcriptional regulator in nuclei but exerts tumor-promoting cytokine and angiogenic activities when released into the extracellular space during necrosis in vivo. The resistance to gamma radiation-induced apoptosis caused by Snail is associated with the inhibition of PTEN (phosphatase and tensin homolog) phosphatase in vivo tumor growth Snail is a zinc finger transcription factor that induces the epithelial-mesenchymal transition (EMT) by directly repressing E-cadherin expression. It can be induced by many kinds of tumor-stimulating cytokines, such as transforming growth factor (TGF) β, Wnt, Notch, and hedgehog, in many human invasive carcinomas in vitroIn this study, we tried to identify the molecules that are involved in necrosis. Previously we demonstrated that GD, one of the stresses that causes metabolic stress in tumors A549, MCF-7, MDA-MB-231, MDA-MB-361, HepG2, HCT116, and HeLa cells were obtained from American Type Culture Collection, maintained in RPMI-1640 or DMEM containing 10% FBS, and treated with GD or chemicals as described previously Western blotting were performed as described previously using the following antibodies: Snail  http://www.genocheck.com. Data analysis was carried out using GeneSpring GX 7.3 (Agilent technologies). The values were normalized using the LOWESS algorithm.Hybridization to microarrays was performed to screen the differentially expressed genes using Operon Human Whole 35 K Oligo chips ; a complete listing of the genes on this microarray is available at the following web site: Data deposition: The Affymetrix microarray data have been deposited in the Gene Expression Omnibus (GEO) database (GEO accession no. GSE24271).Plasmid pEGFP-CuZnSOD  that was constructed by inserting the CuZnSOD open reading frame into plasmid pEGFP  were transfected into A549 cells using jetPEI (Polyplus transfection); stable cells were then selected with 0.5 mg/ml Geneticin  according to the manufacturer's instructions.GCGAGCTGCAGGACTCTAATTCAAGAGATTAGAGTCCTGCAGCTCGCTTTTTA-3′5′-GATCCCC and GCGAGCTG CAGGACTCTAATCTCTTGAATTAGAGTCCTGCAGCTCGCGGG-3′5′-AGCTTAAAAA (Accession No. NM_005985) GCGAGTGGTTCTTCTGCGCTATTCAAGAGATAGCGCAGAAGAACCACTCGCTTTTTA-3′5′-GATCCCC and GCGAGTGG TTCTTCTGCGCTATCTCTTGAATAGCGCAGAAGAACCACT CGCGGG-3′5′-AGC TTAAAAA) that were inserted into the HindIII and BglII sites of pSUPER.gfp/neo ; the human Snail target sequence is underlined. Control shRNA was generated from annealed oligonucleotides (AATTCTCCGAACGTGTCACGTTTCA AGAGAACGTGACACGTTCGGAGAATTTTTTTA-3′5′-GATCCCC and AATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAATTGGG-3′5′-AGCTTAAAAA) pSUPER-Snail shRNA was generated from two different annealed oligonucleotides , dihydroethidium , and MitoTracker Red CM-H2XRos , respectively, by fluorescence microscopy. ΔΨm was analyzed using JC-1. Cells were incubated with 5 mg/ml JC-1 (in DMSO) for 15 min at room temperature in darkness. After centrifugation , cells were washed with 4°C PBS twice, resuspended in 0.5 ml PBS, and analyzed by fluorescence microscopy. ΔΨm was also detected by rhodamine 123. Cells were loaded with 5 µM rhodamine 123 for 30 min. The cells were then washed three times with PBS. Rhodamine 123 was excited at 488 nm and emission was detected at 525 nm. mPT pore opening was assessed by CoQC staining procedures according to the method of Petronilli et al. (1999) 2 for the final 15 min of the incubation. To detect the mitochondrial distribution, 50 nM MitoTracker CM-H2XRos were added during calcein loading. Calcein fluorescence was excited at 488 nm and emitted at 515 nm, and MitoTracker Red CM-H2XRos was excited at 579 nm and emitted at 599 nm.HO/PI staining was performed as described previously 2-controlled chamber on the heating stage of a Zeiss Axio Observer. D1 microscope equipped with an Axiocam MRm monochrome digital camera was used to take photographs every 5 min for an observation period of 5 h . Fluorescence intensity was analyzed with Axiovision LE software (Release 4.8 version).For live cell imaging, cells were grown in culture dishes with a glass bottom. For time-lapse studies, they were placed in a temperature- and CO4, dehydrated in graded ethanol, and embedded in Epon mixture. Sections were prepared with an ultra-microtome (MT-7000), mounted on copper grids, and counterstained with uranyl acetate and lead citrate. Photographs were taken using an electron microscope (HITACHI H-7600).For TEM, collected cells were fixed in 2.5% glutaraldehyde with 0.1 M cacodylate buffer (pH 7.2) for 2 h at 4°C, washed twice with cold PBS, post-fixed in OsOIHC was performed on 4-µm sections of formalin-fixed, paraffin-embedded tissues. Sections were deparaffinized in xylene and graded alcohol. Antigen retrieval was performed by autoclaving for 15 min. After incubation with blocking solution for 30 min, sections were incubated with an anti-Snail antibody MCF-7, MDA-MB-231, and MDA-MB-361 cells were seeded at a density of 400 cells in 200 µl medium into 1.2% agarose-precoated 96-well plates. After 3 days of culture, 100 µl of medium was replaced with fresh medium every 2 days. To determine the MTS growth, diameters of spheroids were measured every day. MTSs were harvested and fixed in formalin. After processing into wax blocks, the spheroids were sectioned, and stained with hematoxylin and eosin, stained with HO/PI, or incubated with an anti-Snail antibody as described above.et al.MTSs were dissociated into subpopulations of cells from different locations in the spheroid as described by LaRue t-test and P<0.05 was considered statistically significant.All experiments were independently performed at least three times. Data were analyzed by the Student's As demonstrated previously Previously, we showed that in A549 cells, phorbol-12-myristate-13-acetate (PMA), a PKC activator, prevented GD-induced necrosis and switched the cell death mode to apoptosis by inhibiting ROS production, possibly by inducing manganese superoxide dismutase (MnSOD) expression and preventing GD-induced downregulation of copper-zinc superoxide dismutase (CuZnSOD) Real-time quantitative PCR confirmed the induction of Snail by GD in A549 (26.5-fold), HepG2 (7.9-fold), and MDA-MB-231 cells (13.7-fold), but not in HeLa and HCT116 cells that underwent apoptosis upon GD . RT-PCR in vitro model of solid tumors et al.We examined Snail expression using MTSs, an Next, we examine the changes in Snail expression during MTS culture. Snail level was increased with extended MTS culture ; 2.6-folin situ, the cells in the inner regions experience hypoxic and GD stresses. Thus, these results further support that Snail is induced by metabolic stress.We examined the patterns of Snail protein in human tumors, including metastatic colonic carcinoma in liver, colonic adenocarcinoma, and pulmonary adenocarcinoma, by IHC . The resWe investigated whether Snail is functionally linked to GD-induced necrosis in A549, HepG2, and MDA-MB-231 cell lines by specific transcript knockdown with short hairpin RNA (shRNA). We employed two different shRNA oligonucleotides; one (target 1) is a 19-mer shRNA oligonucleotide directed to the N-terminal region of the first zing finger of human Snail mRNA sequences  Transcript knock-down by stable Snail shRNA significantly blocked GD-induced necrosis in MDA-MB-231, HepG2, and A549 cells, as revealed by a prominent decrease in the population of cells that had intact pink nuclei in HO/PI staining  and S3. Snail induces EMT in cancer cells Continued spheroid growth leads to the formation of necrotic core due to microenvironmental stresses including deprivation of oxygen and nutrients. HO/PI staining using total cells obtained from MTSs revealed the occurrence of necrosis (29.8±3.6%) at 9 day culture of control shRNA MCF-7 MTSs . We examin vivo tumor growth.We also observed that stable Snail silencing in MCF-7 MTSs suppressed the growth of the MCF-7 MTSs , support2-, and intracellular H2O2  in GD-induced cytotoxicity lar H2O2  and S4, ular ROS , indicatWe also monitored the ΔΨm of cells with JC-1, a mitochondria-specific and lipophilic cationic fluorescence dye. Upon GD treatment, red J-aggregate fluorescence was progressively lost, and cytoplasmic diffusion of green monomer fluorescence was detected; Snail shRNA inhibited the effects of this GD-induced decline in ΔΨm  and S6. Cobalt-quenched calcein (CoQC) measurement was used to determine mPT. Upon GD treatment, calcein fluorescence was lost following the opening of the mPT pore, and Snail shRNA prevented this mPT pore opening  and S6.Our results show that Snail may regulate mitochondrial ROS production, loss of ΔΨm, and mPT upon metabolic stress, leading to potentially significant mitochondrial and cellular injury.2- is produced mostly at Complex I or Complex III of the electron transport chain even under normal conditions 2- generator)-induced necrosis  or menadione  (data not shown). PI3K/Akt-mediated inhibition of GSK3β has been demonstrated to be responsible for Snail expression by ROS such as H2O2The transcriptional factor Snail is induced in response to several kinds of tumor-promoting cytokines and growth factors, such as TGFβ and Wnt in many human carcinomas and regulates epithelial-mesenchymal transition, which has essential role(s) in tumor invasion and progression d tumors , and in d tumors , supportd tumors . SimilarIn addition to its expression in the inner regions (F3 and F4) of MTS system, Snail expression was also detected in the F2 region of 7 and 8 day MTSs . BecauseWe showed that Snail shRNA inhibited metabolic stress-induced increase in PI-positive cell population, HMGB1 release, and LDH release in two-dimensional cell culture , indicatSnail knock-down also inhibited MTS growth . Snail h2- is known to mediate GD-induced cytotoxicity and cell death Mitochondrial OHow does Snail control mitochondrial ROS production in response to GD? Mitochondrial dysfunction has been linked to the induction of necrosis. Tumor cells have been shown to have abnormal mitochondrial structure and DNA integrity and high rates of mtDNA mutations Our results showed that the GD-induced expression of Snail is ROS-dependent , and theIn solid tumors, cells that are not adapted to metabolic constraints such as hypoxia and GD die by necrosis as ‘a reparative cell death’ that could promote tumor progression by releasing the tumor-promoting cytokine HMGB1 Figure S1Induction of Snail during metabolic stress-induced necrosis. (A) A549, HepG2, MDA-MB-231, HeLa, and HCT116 cells were exposed to GD medium for 12 h, and the cells were stained with HO/PI and observed under a fluorescence microscope. Arrow indicates apoptotic cells, * indicates necrotizing cells.(TIF)Click here for additional data file.Figure S2Snail interference is no effect in the level of endogenous Slug mRNA. MDA-MB-231 cells that were stably transfected with control or Snail shRNA analyzed by real-time PCR for Snail, Slug and GAPDH. Values are normalized to GAPDH. Results are expressed as mean ± SE from three independent experiments. #P<0.05; ##P<0.01 versus control shRNA.(TIF)Click here for additional data file.Figure S3Snail shRNA prevents metabolic stress-induced necrosis. MDA-MB-231, HepG2, and A549 cells that were stably transfected with control or Snail shRNA were cultured in normal growth medium or GD medium for 12 h, stained with HO/PI, and observed by fluorescence microscopy.(TIF)Click here for additional data file.Figure S4Snail shRNA prevents metabolic stress-induced ROS production. MDA-MB-231 cells that were stably transfected with control or Snail shRNA were exposed to GD medium for 3 h or 6 h, and mitochondrial ROS and O2- and intracellular H2O2 production was measured using the MitoTracker Red CM-H2XRos, HE, and DCFH-DA, respectively, under a confocal microscope .(TIF)Click here for additional data file.Figure S5Snail shRNA prevents metabolic stress-induced mitochondrial ROS. MDA-MB-231 control and Snail shRNA stable cells that were treated with 50 nm MitoTracker Red CM-H2XRos were exposed to GD medium and observed by time-lapse microscopy . Fluorescence images were taken every 5 min for 5 h, and fluorescence intensity was analyzed with Axiovision LE software (Release 4.8 version). Values represent average of total cellular MitoTracker Red CM-H2XRos fluorescence expressed as relative fluorescence intensity at 0 min.(TIF)Click here for additional data file.Figure S6Snail shRNA prevents metabolic stress-induced loss of ΔΨm and mPT. (A) MDA-MB-231 cells stably transfected with control or Snail shRNA were incubated with GD for the indicated times and then treated with 5 mg/ml JC-1 for 15 min, and observed by fluorescence microscopy. (B) MDA-MB231 cells that were transiently transfected with control or Snail shRNA were incubated in normal medium or GD medium for 9 h and loaded with 0.5 µM calcein AM and 5 mM CoCl2 for the final 15 min of the incubation. To detect cytoplasmic mitochondrial distribution, 50 nM MitoTracker CMX-ROS were added during calcein loading. Calcein fluorescence was excited at 488 nm and emitted at 515 nm, MitoTracker Red CMX-ROS was excited at 579 nm, and emitted at 599 nm, and the cells were observed by fluorescence microscopy.(TIF)Click here for additional data file.Figure S7The effects of Snail on antioxidant expression and menadione-induced necrosis. (A) The Snail tet-inducible cell line MCF-7 was treated with DOX for 48 h or 72 h and then analyzed by Western blotting with antibodies against Snail, CuZnSOD, MnSOD, catalase, and α-tubulin. (B) MDA-MB-231 cells that were stably transfected with control and Snail shRNA were cultured in normal growth medium in the presence or absence of 100 µM and 250 µM menadione, stained with HO/PI, and observed under a fluorescence microscope. Results are expressed as mean ± SE from 500 to 800 cells per treatment group and from three independent experiments.(TIF)Click here for additional data file.Table S1Primer sequences for RT-PCR and real time PCR.(TIF)Click here for additional data file.Table S2Statistical analysis of HO-PI staining results .(TIF)Click here for additional data file.Table S3Statistical analysis of immunohistochemistry results .(TIF)Click here for additional data file.Table S4Statistical analysis of immunohistochemistry results in solid tumors .(TIF)Click here for additional data file.