Since opposing vasoactive effects have been reported for H2O2 depending on the vascular bed and experimental conditions, this study was performed to assess whether H2O2 acts as a vasodilator in the rat mesenteric artery and, if so, to determine the underlying mechanisms. H2O2 elicited concentration-dependent relaxation in mesenteric arteries precontracted with norepinephrine. The vasodilatory effect of H2O2 was reversed by treatment with dithiothreitol. H2O2-elicited vasodilation was significantly reduced by blocking 4-aminopyridine (4-AP)-sensitive Kv channels, but it was resistant to blockers of big-conductance Ca2+-activated K+ channels and inward rectifier K+ channels. A patch-clamp study in mesenteric arterial smooth muscle cells (MASMCs) showed that H2O2 increased Kv currents in a concentration-dependent manner. H2O2 speeded up Kv channel activation and shifted steady state activation to hyperpolarizing potentials. Similar channel activation was seen with oxidized glutathione (GSSG). The H2O2-mediated channel activation was prevented by glutathione reductase. Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester showed incorporation of glutathione (GSH) in the Kv channel proteins in the presence of H2O2. Interestingly, conditions of increased oxidative stress within MASMCs impaired the capacity of H2O2 to stimulate Kv channels. Not only was the H2O2 stimulatory effect much weaker, but the inhibitory effect of H2O2 was unmasked. These data suggest that H2O2 activates 4-AP-sensitive Kv channels, possibly through S-glutathionylation, which elicits smooth muscle relaxation in rat mesenteric arteries. Furthermore, our results support the idea that the basal redox status of MASMCs determines the response of Kv currents to H2O2.Hydrogen peroxide (HThe online version of this article (doi:10.1007/s00424-014-1513-3) contains supplementary material, which is available to authorized users. However, some ROS like hydrogen peroxide  [2O2 elicits vasodilation remain to be determined, although smooth muscle hyperpolarization seems to be required [+ channels [2+-activated (KCa), ATP-sensitive (KATP), and inward rectifier K+ (Kir) channels. The K+ channels are thought to play an important role in maintaining the membrane potential of vascular myocytes [2O2-induced smooth muscle relaxation. However, there is no general agreement on the effects of H2O2 on K+ channels in smooth muscle. Several studies have identified KCa channels as putative targets that are activated in the process of H2O2-induced vasodilation [2O2 induced a vasorelaxation through opening of ATP-dependent K+ (KATP) channels [2O2, which activates Kv channels in coronary and pulmonary vascular smooth muscles [2O2 in ductus arteriosus smooth muscles [2O2 increases Kv1.5 current for voltages < +20 mV but decreases it for high depolarizing voltages [2O2 acts as a vasodilator. Studies have reported H2O2 as a vasoconstrictor [Hr (EDHF) , a majorr (EDHF) . The celrequired . The vaschannels , 45, 49,dilation , while schannels . The Kv  muscles , 54, 57.voltages . It is sstrictor , 67 and strictor , 73, or strictor , 38. Thestrictor , 47.2O2 is a small stable molecule carrying no charge, which allows it to readily cross membranes and travel freely to targets within cells [2O2, and increasing evidence suggests that S-glutathionylation predominates in myocytes because glutathione (GSH) is the most abundant, low molecular mass, reducing equivalent [2O2 is capable of oxidizing the thiol groups of cysteine residues to form disulfide bonds with GSH (S-glutathionylation). S-glutathionylated proteins result from thiol/disulfide exchange between protein thiols and oxidized form of glutathione (GSSG) or S-glutathionylated protein. Modulation of protein activity by S-glutathionylation is a newly recognized posttranslational regulatory mechanism [S-glutathionylation induced by H2O2 [Hin cells . Variousuivalent . H2O2 isechanism , 21. Thiechanism . The Kir by H2O2 , 71, 78.2O2 relaxes rat mesenteric arteries by S-glutathionylation-dependent activation of 4-AP-sensitive Kv channels. We performed studies using an approach of combined molecular biology, electrophysiology, and isometric organ chamber mechanics. Our results show that H2O2 enhanced the activity of 4-AP-sensitive Kv channels, possibly through S-glutathionylation, leading to vasorelaxation in the mesenteric artery. We also present evidence to show that Kv channels under conditions of persistent oxidative stress were not activated, but rather inhibited by the addition of H2O2, suggesting that H2O2 may act as a vasoconstrictor under certain pathological conditions.In a previous study, we demonstrated that in mesenteric arteries, the 4-aminopyridine (4-AP)-sensitive Kv currents play a critical role in the regulation of smooth muscle resting membrane potential (Em) and vascular tone , 66. In 2PO4, 1.8 mM CaCl2, 0.5 mM MgCl2, 5 mM hydroxyethyl piperazineethanesulfonic acid (HEPES), and 11 mM glucose, adjusted to pH 7.4 with NaOH). The arteries were cut into small pieces and transferred to a digestion solution. The tissue was first digested for 15 min in Ca2+-free normal NT solution containing 1 mg/mL papain , 1 mg/mL bovine serum albumin, and 1 mg/mL dithiothreitol. The nominally Ca2+-free NT was prepared by omitting 1.8 mM CaCl2 from the NT solution. Subsequently, the tissue sample was incubated for 25 min in a second digestion solution, in which 3 mg/mL collagenase  replaced papain. Following enzyme treatment, the cells were isolated by gentle agitation with a fire-polished glass pipette in Ca2+-free NT solution.Male Sprague–Dawley (SD) rats (9–11 weeks old) were used for the experiments. All experiments were conducted in accordance with the National Institutes of Health guidelines for the care and use of animals, and the Institutional Animal Care and Use Committee of Konkuk University approved this study. Rats were euthanized by exposure to a rising concentration of carbon dioxide or exsanguinated by cutting the carotid arteries under deep ketamine–xylazine anesthesia. Single-cell suspensions of mesenteric arterial smooth muscle cells (MASMCs) were prepared as described previously . Brieflybis(aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, adjusted to pH 7.2 with KOH. Bicarbonate-buffered physiological salt solution (PSS) was used as the bath solution for the organ chamber mechanics experiments. The PSS was composed of 136.9 mM NaCl, 5.4 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 23.8 mM NaHCO3, and 0.01 mM EDTA. All chemicals, including H2O2, GSH, and GSSG, were purchased from Sigma.NT was used as the bathing solution for the patch-clamp experiments. The pipette internal solution contained 140 mM KCl, 5 mM NaCl, 5 mM MgATP, 10 mM HEPES, and 10 mM 1,2-We used the conventional whole-cell configuration of the patch-clamp technique  to recorCa (BKCa) channels. Additionally, specific activation of the Kv current was confirmed using 4-AP at the end of the experiments. Conductance–voltage (G–V) relationships were plotted using steady state current amplitudes divided by driving force . The normalized conductance was fit using Origin 6.0 software to the Boltzmann equation.Kv currents were elicited by depolarizing steps between −60 and +50 mV (200 ms duration) from a holding potential of −70 mV. Tetraethylammonium  was added to all bath solutions during the patch-clamp study to prevent activation of big-conductance K2 in 5 % CO2 to achieve pH 7.4.The mesenteric arterial rings were mounted vertically on two L-shaped stainless steel wires in a 3-mL tissue chamber for the tension measurements. One wire was attached to a micromanipulator and the other to an isometric force transducer . Changes in isometric force were digitally acquired at 1 Hz with a PowerLab data acquisition system . Resting tension was set to 1 g using the micromanipulator. After a 60-min equilibration under resting tension in a tissue chamber filled with PSS, the rings were sequentially exposed to 70 mM KCl–PSS (10 min) and PSS (15 min) three times for stabilization. The high KCl (70 mM)–PSS was prepared by replacing NaCl with equimolar KCl in PSS. Bath solutions were thermostatically controlled at 37 °C and were continuously saturated with a mixture of 95 % O2O2 for 10 min at 37 °C. The cells were then washed twice with phosphate-buffered saline (PBS) and lysed using RIPA buffer . Samples were run on an 8 % SDS–polyacrylamide nonreducing gel and then transferred to a polyvinylidene fluoride (PVDF) membrane . Rabbit primary antibodies against Kv1.2, Kv1.5, and Kv 2.1  and secondary antibodies conjugated with horseradish peroxidase were used in the Western blot . Signals were visualized using Las-4000 .Primary cultures of rat MASMCs between six and ten passages were used for Western blot. MASMCs were isolated from SD rats and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin. The cells were grown to 80 % confluence and starved in DMEM without FBS for 12 to 24 h prior to experiments. After starvation, cells were treated with H2O2 (0.1 or 10 mM) challenges for 10 min. Biotin–GSH-conjugated proteins were pulled down using Dynabeads streptavidin according to the methods provided by Invitrogen. Dynabeads streptavidin was washed thrice with PBS before conjugation with biotin. Samples were then mixed with beads and incubated at room temperature with gentle rotation for 30 min. A magnet was used to separate the biotinylated molecule–bead complex. The supernatant containing unlabeled proteins was discarded, and the pellet was resuspended, followed by washes with PBS. The biotinylated molecule–bead complex was boiled with loading buffer for 7 min for Western blotting.The culture medium was replaced with fresh medium 2 h before experiments. Biotinylated glutathione ethyl ester  was added to the medium and incubated for 1 h, followed by Ht tests were used to test for significance, and p < 0.05 was regarded as significant. We performed one-way repeated measures ANOVA and Holm–Sidak test in order to examine the statistical significance of data shown in Fig. The Origin 6.0 software  was used for data analysis. Activation kinetics was calculated by fitting the data to a single exponential decay function. The time course of current inactivation was also fit to a single exponential function. The results are shown as mean ± standard error. Paired or independent Student’s 2O2 relaxes rat mesenteric arteries. We used arterial rings without intact endothelium. The arterial rings were precontracted with norepinephrine (NE) (1 μM). H2O2 induced a concentration-dependent relaxation in the precontracted mesenteric arterial rings . These data indicate that the effect of DTT is specific for H2O2-induced relaxation and suggest that thiol groups in smooth muscle are targets of H2O2 signaling. TEA was used to assess the contribution of the K+ channels to H2O2-induced smooth muscle relaxation. At a concentration of 1 mM, TEA is reported to be relatively specific for BKCa channels and has little effect on voltage-dependent K+ channels [n = 6) did not significantly inhibit the relaxation caused by H2O2 . BaCl2 (100 μM), a blocker of Kir, did not affect the H2O2-induced relaxation either . These data suggest that 4-AP-sensitive Kv channels mediate the vasodilation by H2O2 in the mesenteric artery.We used isometric organ chamber mechanics to examine whether Hngs Fig. . We dete2O2 Fig. . The addchannels . One milher Fig. . We atteion Fig. , n = 6. 2O2 activated 4-AP-sensitive Kv currents, we recorded Kv currents using the conventional whole-cell, patch-clamp technique with depolarizing voltage steps as described previously [2O2 superfusion (5 min at each concentration) increased Kv currents 2O2 activnts Fig. . Additioely Fig. . Analysi mV Fig. . This inred Fig. .Fig. 2H22O2 influences voltage dependence of activation and activation kinetics of the putative Kv current  curves of the Kv channel were significantly shifted to more negative potentials after H2O2 application  in control and 0.1, 1, and 10 mM H2O2 were 7.6 ± 1.0, 2.7 ± 1.4, −3.1 ± 1.3, and −1.7 ± 1.8 mV, respectively  for 1 h, followed by H2O2 (0.1–10 mM) challenge for 10 min, as described previously [2O2, compared to control cells  by which Hhemistry , we exameviously . Strong S-glutathionylation [n = 19) and 37.8 ± 4.3 pA/pF , respectively , and it was not significantly different from that induced by 10 mM H2O2 . GSSG also had an effect on the time course of activation  and activation kinetics  was applied to cells together with glutathione reductase. NADPH alone did not block the stimulatory effect of H2O2 on Kv currents  and 43.8 ± 3.7 pA/pF , respectively , and it was not enhanced but unaffected  or rather reduced  by subsequent bath application of 0.1 and 10 mM H2O2, respectively. These data suggest that high levels of basal H2O2 upregulates Kv currents through S-glutathionylation, which may keep acute exposure to H2O2 from regulating the Kv channels.Recent studies showed that an increase of ROS is linked to hypertension . Most enpiration , 36, wheion [2O2 , 15. Sin vessels , we hyponts Fig. . At +40 ely Fig. . Notablyhed Fig. . Summari2O2 acts as a vasodilator [2O2. For example, it may involve the BKCa channel [ATP channel [2O2-induced vasodilation through the Kv channel and proposes that S-glutathionylation underlies the stimulatory effect of H2O2 on Kv channels. Further experiments demonstrate that under oxidative conditions, Kv channels were not stimulated but rather inhibited by acute exposure to H2O2, suggesting that cellular redox status affected S-glutathionylation of the Kv channel and determined the response of the Kv channel to H2O2.It is still uncertain whether Hodilator , 80. The channel , KATP ch channel , or Kv c channel . The pre2O2 relaxed the rat mesenteric artery that was precontracted with an agonist. In contrast, H2O2 failed to relax arteries pretreated with DTT and 4-AP; 1 mM TEA and 100 μM BaCl2 did not affect the H2O2 response. The inability of H2O2 to relax arteries pretreated with 4-AP suggested that the relaxation response to H2O2 involved stimulation of the Kv channel, a mechanism that has also been suggested from patch-clamp/whole-cell studies employing other cell types [2O2 on Kv channels in MASMCs. H2O2 increased 4-AP-sensitive Kv currents in a concentration-dependent manner. This was a result of change in the voltage dependence of activation; 10 mM H2O2 shifted voltage dependence of 4-AP-sensitive Kv channel conductance by ~7.6 mV , subsequent to maximal GSSG effect, induced no further change in the Kv channel. These results suggest that S-glutathionylation of the Kv channel protein mediates the stimulatory effect of H2O2 on the Kv channel and, consequently, the vasodilatory effects in the mesenteric artery. Interestingly, conditions of increased oxidative stress within smooth muscle cells impaired the capacity of exogenous H2O2 to stimulate Kv channels , cytochrome P450 metabolites of AA such as epoxyeicosatrienoic acid, and H2O2 [2O2 was an EDHF [2O2 by hyperpolarization of the endothelium, which consequently acts on vascular smooth muscle cells, causing relaxation of blood vessels. Subsequently, several studies have verified the hypothesis that H2O2 is an EDHF in animal and human arteries [Combined with the fact that endothelial cells can produce up to 500 μM H2O2  and myoeh muscle , our rese. Sobey  suggestee. Sobey , 52, 54.and H2O2 , 42, 76.arteries , 76. Altarteries . ConsistO-glycosylation/O-GlcNAcylation) exist and are discussed elsewhere [S-glutathionylation is a major redox-mediated thiol modulation mechanism, involving the addition of a GSH moiety to the protein. Oxidative stress and ROS facilitate S-glutathionylation. Over the past few years, S-glutathionylation has been increasingly observed in many ion channels such as voltage-gated calcium channels, the ryanodine receptor, and KATP channels, all of which contribute to critical cellular functions [2O2  are important mechanisms regulating ion channel functions. One of the classical PTMs is protein phosphorylation, and a large number of ion channels are regulated by phosphorylation through protein kinase A (PKA), PKC, and other protein kinases -62, 79. lsewhere , 53, 56.unctions , 77, 78.2O2 Fig. . As deli2O2 relaxed rat mesenteric arteries by S-glutathionylation-dependent activation of Kv currents under physiological conditions. Our data suggest that S-glutathionylation of the Kv channel protein is, at least in part, an important and novel mechanism of 4-AP-sensitive Kv current activation by H2O2. Identifying the mechanisms underlying the vasoactive effects of H2O2 should increase our understanding of diseases where oxidative damage has been implicated such as in hypertension, atherosclerosis, and diabetes mellitus.In conclusion, HESM 1(PDF 164 kb)Below is the link to the electronic supplementary material.


Recent biomechanics studies have revealed distinct kinematic behavior of different lumbar segments. The mechanisms behind these segment-specific biomechanical features are unknown. This study investigated the in vivo geometric characteristics of human lumbar intervertebral discs.Magnetic resonance images of the lumbar spine of 41 young Chinese individuals were acquired. Disc geometry in the sagittal plane was measured for each subject, including the dimensions of the discs, nucleus pulposus (NP), and annulus fibrosus (AF). Segmental lordosis was also measured using the Cobb method.P < 0.05). The L5/S1 NP had a length of 21.6 ± 3.1 mm, which was significantly longer than all other levels (P < 0.05). At L4/5, the NP occupied 64.0% of the disc length, which was significantly less than the NP of the L5/S1 segment (72.4%) (P < 0.05). The anterior AF occupied 20.5% of the L4/5 disc length, which was significantly greater than that of the posterior AF (15.6%) (P < 0.05). At the L5/S1 segment, the anterior and posterior AFs were similar in length . The height to length (H/L) ratio of the L4/5 NP was 0.45 ± 0.06, which was significantly greater than all other segments (P < 0.05). There was no correlation between the NP H/L ratio and lordosis.In general, the disc length increased from upper to lower lumbar levels, except that the L4/5 and L5/S1 discs had similar lengths. The L4/5 NP had a height of 8.6 ± 1.3 mm, which was significantly higher than all other levels (Although the lengths of the lower lumbar discs were similar, the geometry of the AF and NP showed segment-dependent properties. These data may provide insight into the understanding of segment-specific biomechanics in the lower lumbar spine. The data could also provide baseline knowledge for the development of segment-specific surgical treatments of lumbar diseases. For example, lumbar disc herniation is found most often at the L5/S1 segment,6 whereas lumbar degenerative spondylolisthesis is found most often at L4/5.10 Further, segment-dependent clinical outcomes following surgical treatment of L4/5 or L5/S1 have also been reported. Microdiscectomy of L5/S1 has shown superior clinical outcomes compared with the L4/5,11 whereas total lumbar disc replacement at L4/5 has shown superior clinical outcomes compared with the L5/S1.12 However, knowledge on segment-specific physiology and clinical outcomes is limited.Lumbar degenerative disc diseases (DDDs) are often found at the lower lumbar levels.14 However, most biomechanics studies of the lumbar spine make no distinction between different segmental levels. For example, previous reports on pressure measurement and kinematics of the nucleus pulposus (NP) did not provide level-specific analysis.17 Recent kinematic studies revealed distinct motion characteristics between the L4/5 and L5/S1 segments.20 Knowledge on the mechanisms behind these segment-dependent pathologies and motion characters could be instrumental for the development of segment-dependent surgeries to treat lumbar diseases.There are various potential factors that could influence segment-dependent physiological function of the lumbar spine, including the surrounding muscles, ligaments, and vertebral geometries.Therefore, the objective of this study was to investigate the in vivo morphology of lumbar intervertebral discs (IVDs) at different segment levels using sagittal plane magnetic resonance (MR) images. Specifically, the dimensions of the NP and anterior/posterior annulus fibrosus (AF) were compared. The height to length (H/L) ratio of the NP was also calculated to investigate NP shapes. We hypothesize that the IVD has distinct geometric features at different levels of the lumbar spine.21 Any of the lumbar discs of Pfirrmann grade III–V was used for exclusion of the subject from the study.MR images of the lumbar spine were obtained from 41 young Chinese subjects  with institutional review board approval of the authors’ institution. Each subject was scanned using a 3.0 Tesla scanner  in a nonweight-bearing supine position with a spine surface coil and a T2-weighted fat-suppressed 3D SPGR sequence . Parallel sagittal images with a thickness of 4.0 mm, gap of 1.0 mm, and resolution of 256 × 256 pixels were obtained. The lumbar discs were examined using Pfirrmann classification.22Geometric measurements for each subject were taken from the T2-weighted midsagittal images using a commercial software program  Figure . The lenP < 0.05. A Newman–Keuls post hoc test was performed when a statistically significant difference was detected. NP H/L ratio and lordosis were also correlated using Pearson tests. The statistical analysis was done using the STATISTICA software version 12 .A repeated measure of 2-way analysis of variance was used to compare the disc length, NP length, AF length, NP height, H/L ratio, and segmental Cobb angle across lumbar levels. A statistical difference was achieved when P < 0.05). L4/5 had the shortest NP length (19.3 ± 2.9 mm)   . The NP H/L ratio showed a similar trend to NP height, gradually increasing from L1/2 (0.27 ± 0.06) to L4/5 (0.45 ± 0.06) before decreasing at L5/S1 (0.34 ± 0.05) .The NP height gradually increased from L1/2 (5.5 ± 1.1 mm) to L4/5 8.6 ± 1.3 mm), and then decreased at L5/S1 (7.3 ± 1.3 mm) . There was no correlation between the NP H/L ratio and segmental lordosis (R2 < 0.26).The overall lordosis of L1-S1 was 55.6 ± 9.0°. Segmental lordosis was similar at L1/2 and L2/3  before significantly increasing at each subsequent lower lumbar level  techniques demonstrated that the upper lumbar levels had more movement than the lower levels in the anterior portion of the disc, whereas the lower levels had more movement than the upper levels in the posterior portion of the disc.33 Recently, Alexander34 and Nazari35 used an open-MRI technique to investigate functional weight-bearing positions of the body (sitting and standing), and found that the in vivo deformation and migration of the lower lumbar NPs (L4/5 and L5/S1 levels) were more affected by position than the upper lumbar NPs. Most of these studies consider the NP to be a hydraulic cushion within the IVD, acting only to distribute stress evenly between vertebrae.36 Few reports have discussed segment-dependent geometrical features of the IVD.Ranu3 Numerous studies have attempted to elucidate factors for DDD such as age, body weight, physical job activities, and vertebral motion features.18 Although lumbar disc herniation has been reported more often at L5/S16 and lumbar degenerative spondylolisthesis more often at L4/5,10 surgical treatments of L4/5 or L5/S1 have also reported segment-dependent clinical outcomes.12 Recent in vivo kinematic studies have revealed distinct motion characteristics at the L4/5 and L5/S1 segments.20 However, few data has been reported on the segment-specific morphology of lumbar IVDs and its potential relationship to these segment-dependent features.Degenerative pathologies of the lumbar spine are often found at the lower levels of L4/5 and L5/S1.In our study, the L4/5 NP was found to have the greatest height and smallest length among all segments. Additionally, the anterior AF was significantly thicker than the posterior AF at the L4/5 level. The L5/S1 NP was significantly longer than all other levels, with its anterior and posterior AFs being similar in thickness. Our data also demonstrated segment-dependent NP shapes. The NP H/L ratio gradually increased from L1/2 to L4/5 and then decreased at L5/S1. The L4/5 NP showed the largest H/L ratio and was significantly greater than all other levels. There was no correlation between the NP H/L ratio and segmental lordosis in this group of subjects. Further study is warranted to examine how these geometric features may correlate with the distinct kinematic features of the lumbar spine.Several limitations of this study should be considered. First, it was limited to investigation of IVD morphology in the sagittal plane. Three-dimensional analysis would potentially provide an even better understanding of IVD geometry. Another limitation of this study is the relatively narrow age range of subjects. Future studies should consider age, sex, ethnicity, and body height/weight as study variables. Finally, IVD morphology was studied in a supine position under nonweight-bearing conditions. Future studies should investigate lumbar IVD geometry under various physiological loading conditions. Despite the above limitations, this study represents the first in vivo measurement and analysis of segment-dependent lumbar IVD morphology.In conclusion, this study investigated human lumbar IVD geometry using sagittal plane MR images and found segment-dependent properties. Notably, the NP of the L4/5 segment had the greatest height, whereas the NP of L5/S1 had the greatest length. These baseline data may provide insight into the understanding of segment-specific pathology and biomechanics in the lower lumbar spine. These data may also be instrumental for the development of segment-specific surgical treatments that are aimed to restore native spine function.


The immune non-recognition is often the underlying cause of failure in tumor immunotherapeutic. This is because most tumor-related antigens are poorly immunogenic, and fail to arouse an efficient immune response against cancers. Here we synthesized a novel TLR7 agonist, and developed a safe and effective immunotherapeutic vaccine by conjugating this TLR7 agonist with the pluripotency antigen OCT4.in vitro release of IL-12 and IFN-γ was observed in spleen lymphocytes. Mice were immunized with TLR7-OCT4, and the release of IFN-γ, the percentages of CD3+/CD8+ T cells and the OCT4-specific cytotoxicity rates were measured. The immunized mice were challenged with mouse embryonic carcinoma (EC), and the tumor volume and tumor weight were determined. Blood routine examination was performed to evaluate the biosafety of TLR7 agonist and TLR7-OCT4 conjugate in mice.Purified recombinant OCT4 protein was covalently linked with a novel TLR7 agonist to form a TLR7-OCT4 conjugate (T7-OCT4). After conjugation, the in vitro release of IL-12 and IFN-γ by mouse spleen lymphocytes. In addition, the release of IFN-γ, the percentages of CD3+/CD8+ T cells and the tumor-specific cytotoxicity rates in immunized mice were significantly higher. Importantly, in EC xenografted mice, immunization with T7-OCT4 conjugate decreased the growth of the tumor dramatically up to 90 %, as compared to mice immunized with OCT4 protein or TLR7 agonist alone. Furthermore, blood routine examination demonstrated that no abnormalities of the blood cells and components in the blood fluids were detected by T7-OCT4 and TLR7 agonist injections.T7-OCT4 conjugate significantly increased the Our results showed that conjugating OCT4 protein to the novel TLR7 agonist produced a vaccine which is effective and safe in preventing tumor growth in mice. Our results suggest that this type of vaccine formulation has great potentiality in preventive vaccines against OCT4 expressing tumors.The online version of this article (doi:10.1186/s12967-015-0524-y) contains supplementary material, which is available to authorized users. It functions as a key regulator of the self-renewal and pluripotency of embryonic stem (ES) cells , testis,l tumors , suggestl tumors .Testis embryonic carcinoma (EC), a highly malignant germ-cell tumor, occurs primarily in young adults. EC cells, the malignant stem cells of teratoma, exhibit a striking over-expression of the stem-related protein OCT4 . TLRs arTo date, at least 10 members of Toll-like receptors (TLRs) have been identified in human with different TLRs having different respective ligands. Of all TLRs, only TLR7/8 recognizes GU-rich short single-stranded RNA (ssRNA) as well as small synthetic molecules such as nucleoside analogues and imidazoquinolines. This creates the opportunity of screening and modifying highly efficient TLR7 agonists for the enhancing of immune response. Furthermore, TLR7/8 agonists not only activate the antigen-presenting cells (APCs), but also promote the activation of T cells and natural killer (NK) cells , 14. ActHere we showed that a conjugate of exogenous purified recombinant OCT4 protein covalently linked to a TLR7 agonist (T7-OCT4) could elicit a surprisingly strong anti-tumor effect on EC tumors by inducing tumor-specific immune response. The mechanisms by which this T7-OCT4 complex promotes immune responses were investigated.Escherichia coli (E.coli) strain BL21 and PGEX-KG-mouse-OCT4 were maintained in our lab. E. coli was regularly grown in Luria Broth (LB) media at 37 °C with shaking at 200 rpm or on media (supplemented with 100 μg/ml ampicillin LBAmp) solidified with 1.5 % agar. LLC Lewis lung cancer cells or mouse embryonic carcinoma F9 cells were maintained in our lab and cultured in Dulbecco's Modified Eagle's Medium , supplemented with 10 % fetal bovine serum , 100 μg/mL penicillin (Ameresco) and 100 μg/mL streptomycin (Ameresco). Cells were cultured at 37 °C in a humidified atmosphere with 5 % CO2. BALB/c mice at 6 weeks of age were purchased from the Medical Laboratory Animal Centre of Guangdong Province, China. The experiments were carried out in accordance with recommendations cited in the Guide for the Care and Use of SPF  Animal Center of Shenzhen University, Guangdong Province, China.2CO3 in water, stirred for 30 min, and filtered. The clear filtrate was acidified with 5 % HCl at 5-10 °C, the precipitated product was filtered and washed three times with water, dried under vacuum at 60 °C, for 4 h to obtain the final product of TLR7 agonist (T7) as white powder. The structure of T7 was determined by Nuclear Magnetic Resonance assay .The TLR7 agonist was synthesized in our laboratory, compound 5 was synthesized from 2,6-dichloropurine according to the published method , 20, TheRecombinant mouse OCT4 was produced by transfection of plasmid PGEX-KG-mouse-OCT4 into Bacterial strain BL21. The expressed protein was purified with glutathione sepharose beads  at 4 °C, and assayed using a DC protein assay kit (BioRad). The benzenecarboxylic acid group on TLR7 agonist enabled facile coupling to protein with free amino using well established conjugation chemistry. To prepare a conjugate that could be administrated into mice and conveniently monitored spectrophotometrically, we chose EDC and NHS to activate carboxylic acid as active ester. The TLR7 agonist, EDC and NHS were mixed at 1:1:1 ratio in DMSO and incubated in the dark at room temperature overnight. Then the activated TLR7 agonist was added to OCT4 protein at 40-fold molar excess in PBS, and incubated at 4-8 °C overnight. To remove excess TLR7 agonist and catalyst EDC/NHS,the reaction mixture was washed with PBS three times on 10 000 MWCO Microcon filtration device (Millipore). The lyophilized conjugate was stable for at least 6 months at −20 °C.5 cells per well. Different testing compounds were added to the cell culture medium at a final concentration ranging from 0.01 to 10 μM as indicated and incubated for 24 h. The levels of IL-12 and IFN-γ in cell culture supernatants were determined using enzyme-linked immunosorbent assay (ELISA) (eBioscience), according to the manufacturer’s instructions.Mouse spleen lymphocytes were isolated from BALB/c mice and grown in RPMI 1640 medium with 10 % FBS. Cells were seeded into 96-well plates at a density of 4 × 105 single cell suspensions from individual mice were incubated with 10 μg/ml OCT4 protein for 24 h. Then culture supernatants were collected and assayed for cytokine inductions by ELISA.All experiments were routinely performed in groups of 8 mice each. On Day 1, 14, 28 and 42, mice were administrated (i.p.) PBS, OCT4 protein (15.6 μg in PBS per mouse), TLR7 agonist (1.9 μg in PBS per mouse), or T7-OCT4 conjugate (17.5 μg). Cells were harvested from spleens on the 3rd day after the last vaccination, and 2.5 × 106 spleen lymphocytes from immunized mice  were seeded in 96-well plates and stimulated with 10 μg/mL OCT4 antigen or PBS alone every 24 h twice. Three days later, cells were collected and washed with PBST (0.05 %Tween in PBS), incubated with 5 % BSA for 1 h, stained with anti-CD4 FITC, anti-CD8 PE at 1 μg/ml final concentration on ice overnight, and then washed with PBST 3 times and immediately analyzed by flow-cytometry (Becton Dickinson).2.5 × 105) were treated with 10 μg/mL OCT4 protein and again 24 h later. Cells were maintained for 3 days in a humidified 5 % CO2 incubator at 37 °C in RPMI 1640 medium containing 5 % fetal bovine serum in 96-well plates. Before the target cells (LLC or F9 cells) were added, 100 μl of supernatants in effector cells should have been removed.Spleen from immunized mice were removed 3 days after the last injection, and single lymphocyte cell suspensions were obtained by teasing the organs through a sterile nylon mesh. Effector Cells .Target cells were prepared by incubating LLC cells or F9 cells in tissue culture for 12–16 h. Target cells were diluted in RPMI 1640 with 5 % FBS and 10 mM HEPES at the concentration of 1x10et al. [6 cells) in the mid-back region, and sacrificed 14 days post-injection of F9 cells. Tumor xenografts were surgically dissected, weighed, and measured. Tumor volume was calculated according to the equation Length × Width2/2 with the length (mm) being the longer axis of the tumor. The generation of microvessels in tumors was observed using transparent dorsal skin fold window chamber  in the affiliated hospital of Shenzhen university by an experienced clinical doctor.All data were presented as mean ± SD. The results were compared by an analysis of variance. All statistical calculations were performed with Excel software package. A p-value < 0.05 was regarded as statistically significant difference.+); and the 1H, 13C NMR were consistent with the structure assigned  was synthesized in our laboratory in six steps according to the scheme presented in Fig. TLR7 agonist (T7) could be easily coupled to proteins and peptides antigens through the amide banding commonly used in peptide chemistry. In this study, we coupled T7 to OCT4 protein directly by NHSA-EDC procedure, yielded the conjugated antigen (T7-OCT4) Fig.  which waSince the TLR7 agonist we used is not commercially available, we first evaluate the immunological activity of this TLR7 agonist and determine the optimal dose. TLR7 agonist was conjugated to small peptides M2e or MG7 (T7-M2e or T7-MG7). M2e peptide (sequence MSLLTEVETPTRNEWECRCSDSSD) is the 24 amino-acid extra-cellular domain of M2 protein (M2) which is one of the most conserved antigens of influenza A virus. MG7 peptide (KPHVHTK) is a mimic epitope recognized by specific monoclonal antibody against gastric cancer associated antigen MG7. Both M2e and MG7 peptides are poor-immunogenic, as they are short peptides with low molecular weight. After incubation of spleen-derived lymphocytes with antigen, the release of cytokine IL-12 and IFN-γ were determined by ELISA. As shown in Fig. Next, we conjugated TLR7 agonist to the purified OCT4 protein to produce T7-OCT4 conjugate and measured its effect on the IL-12 and IFN-γ released by the lymphocytes. The total amount of T7 in unconjugated and conjugated is equal. The molarity of OCT4 is 1/4 of T7 agonist since the ratio of conjugation between TLR7 and OCT4 is 4:1. As shown in Fig. in vivo tumor-prevention effect of T7-OCT4 vaccine, BALB/c mice were immunized with PBS, T7, OCT4, T7 + OCT4 (mixture of T7 and OCT4), or T7-OCT4 conjugate three times at biweekly intervals . After 35 days, mice were injected subcutaneously with embryonic carcinoma F9 cells. On day 42, one more immunization was given. Fourteen days after post-injection of F9 cells, mice were sacrificed and tumor xenografts were surgically dissected, weighed, and measured. As shown in Fig. P > 0.05). However, tumor growth was significantly inhibited by T7-OCT4 conjugate, the tumor volume and weight were dramatically decreased (P < 0.001). Using transparent dorsal skin fold window chamber, we found that tumors in T7-OCT4 conjugate treated mice showed decreased visible microvessels on Day 12 after tumor injection . Therefore, it is crucial to determine the standard haematology markers. As shown in Table To evaluate the bio-safety of T7 agonist and T7-OCT4 conjugate Research underlying the immune non-recognition involved in the failure of tumor cells removal has led to the widespread exploration of immunotherapeutic approach aiming at the development of immune-mediated tumor destruction. To date, vaccines against pancreatic cancer (telomerase peptides) , lung ca+ T cells and CD4+ T cellsin vitro and in vivo, however, immunization of OCT4 antigen alone in vivo could not efficiently inhibit the growth EC tumors, suggesting that although systemic immune response could be activated by OCT4 protein, no tumor-specific immune responses was observed in vivo during this process. It is possible that systemic immune activation by free OCT4 antigen does not create local cytokine and chemokine microenvironments required to mobilize immune cells to the site of tumors, or that the free OCT4 antigen induced immune cells could not effectively recognize the epitopes of OCT4 antigen expressed in EC tumor cells.Although vaccine targeting OCT4 antigen is expected to achieve specific antitumor effect, especially on cancer stem cells and germ tumor cells. However, most tumor related antigens are poorly immunogenic, and often fail to arouse an efficient immune response against cancers. Consistently, our results also showed that although OCT4 protein alone can promote the generation of IFN-γ which are secreted by CD8Several studies have demonstrated that conjugation of Toll-like receptors (TLRs) ligands and antigens produced better immunogenicity than non-coupled antigens , 26. Forin vitro. The in vivo data showed that the T7-OCT4 vaccine can inhibit tumor growth by arousing tumor-specific immune responses. In summary, we synthesized a novel TLR7 agonist and conjugated with stemness antigen OCT4 to develop a safe and effective immunotherapeutic tumor vaccine. The vaccine is effectively against EC tumors and also expected to be useful for immunotherapy of tumor stem cells and other tumor cells which express OCT4 antigen. It is expected this vaccine will prevent the tumors from growth, spread and metastasis. Potentially, this vaccine may be of great value for the prevention of tumor generation after the transplantation of stem cells or induced pluripotent stem cells (iPS) into human body, as OCT4 is indispensible for most stem cells to generate tumors.Similarly, we designed and synthesized a novel TLR7 agonist (T7). This TLR7 agonist is small-molecule TLR7 ligands. It can functionalize many low immunogenicity antigens such as OCT4 to become potent immunostimulants by simple standardized conjugation method. The T7-OCT4 antigen serves as a model vaccine and could be used as a single immunotherapy, or in combination with other therapies for cancer prevention and treatment. Consistent to a previous study , our datLack of immune recognition is the main cause of the tumor cells escaping immune surveillance and resisting eradication by the immune system. To address this issue, many cancer immunotherapy approaches are centered on the activation of CD8 CTL that recognizes specific tumor antigens. Here, we developed a novel TLR7 agonist and conjugated OCT4 protein covalently linked to this agonist (T7-OCT4). T7-OCT4 could elicit a surprisingly strong anti-tumor effect on embryonic carcinoma (EC) by inducing tumor-specific immune response in mice. Our results demonstrated that vaccination of OCT4 antigen conjugated to the novel TLR7 agonist is effective and safe against EC tumors and other tumors which express OCT4 antigen.Additional file 1:Transparent dorsal skin fold window chamber assay.Additional file 2:Mass spectrometric analysis of TLR7 agonist.Additional file 3:Optical observation of tumor microvascular generation.


OX40 and its ligand OX40L are key components in the generation of adaptive memory response and provide necessary co-stimulatory signals for activated effector T cells. Here we investigate the dual roles of the membrane and soluble (s) forms of OX40 and OX40L in plasma and synovial fluid and their association with autoantibodies and disease activity in rheumatoid arthritis (RA).Soluble OX40 and sOX40L plasma levels were measured in treatment-naïve early RA patients (eRA) at baseline and after 3, 6, and 12 months of treatment with methotrexate and adalimumab (n = 39) and with methotrexate alone (n = 37). Adalimumab was discontinued after the first year, and patients were followed for additional 12 months. For comparison, sOX40 and sOX40L were measured in patients with chronic RA  and healthy volunteers . Membrane-bound OX40 and OX40L expression on T cells, B cells and monocytes were quantified.P <0.001) and IgM-RF (P <0.0001). The sOX40/sOX40L ratio was decreased in eRA, and a low ratio at the time of adalimumab discontinuation was associated with increased DAS28CRP and risk of flare the following year. T cells in the synovial fluid had the highest expression of OX40, while monocytes and B cells were the main expressers of OX40L in the joint.Soluble OX40 plasma levels of eRA patients were not different at the time of treatment initiation, but were significantly higher after 12 months of treatment, compared with HV or cRA patients. Soluble OX40L was significantly elevated throughout the first 12 months of treatment compared with HVs and patients with cRA. Adalimumab treatment did not influence sOX40 or sOX40L plasma levels. At baseline, sOX40L levels were strongly associated with the presence of anti-citrullinated protein antibodies (ACPA)  contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting about 0.8% of the adult population. It is characterized by synovitis and progressive destruction of the joints accompanied by multiple systemic symptoms. Autoantibodies occur in 60 to 80% of patients, suggesting a pivotal role for adaptive immune responses in the pathogenesis . This isSeveral members of the TNF superfamily play an important role for the generation of an optimal memory response; among these are OX40 and its ligand, OX40L -5. OX40 OX40 provides a co-stimulatory signal to activated effector T cells and is crucial for the generation of memory T cells and hence for the persistence of immunity . The genThe TNF superfamily is known to induce bidirectional signals and this also applies to OX40/OX40L . In addiBased on the above, we hypothesize that RA patients show signs of activity in the OX40 system. To investigate this, we measure the plasma levels of soluble OX40 and OX40L in a closely monitored cohort of early RA patients and compare with chronic RA patients and healthy volunteers, and examine the association between these soluble isoforms, autoantibodies and clinical disease parameters. We further measure the expression levels of the membrane-bound isoforms on cells from peripheral blood and synovial fluid.et al. [A sample set of ethylenediaminetetraacetic acid (EDTA)-stabilized plasma samples was obtained from a Danish randomized controlled trial (RCT), optimized treatment algorithm in early rheumatoid arthritis (OPERA) (n = 76), which included RA patients with a disease duration of less than 6 months . Sampleset al. . EDTA plA cross-sectional sample set was collected from chronic RA (cRA) patients (n = 15) including peripheral blood mononuclear cells (PBMC) with paired synovial fluid mononuclear cells (SFMC) from the outpatient clinic at Aarhus University Hospital. All cRA patients had disease duration of more than 8 years. PBMCs from HV served as a control group . In comparison we collected a cross-sectional sample set of PBMC from eRA patients with disease duration less than 6 months (n = 5) from the outpatient clinic at Aarhus University Hospital.PBMC and SFMC from EDTA-stabilized whole blood and synovial fluid, respectively, were isolated by density gradient centrifugation using Ficoll-Paque PLUS and stored at −150°C until use. Plasma was stored at −80°C until time of analysis.The studies were approved by the Danish Medical Agency (2612-3393), the Danish Data Protection Agency (2007-41-0072 and 2011-41-6863), and the Regional Ethics Committee . All patients gave written consent to participate in the study in accordance with the Declaration of Helsinki and carried out according to the principles of the International Conference on Harmonization guidelines for good clinical practice .Plasma levels of sOX40 were quantified using a sOX40 ELISA kit  in accordance with the manufacturer’s description with the following modifications: samples were diluted in sample diluent supplemented with human, murine and bovine IgG  added in excess to ensure pre-aggregation of heterophilic antibodies in the samples ; samplesA sOX40L ELISA was constructed, optimized, and validated in-house. Maxisorb ELISA plates  were coated with a monoclonal mouse anti-OX40L antibody  diluted in PBS at a concentration of 4 ug/ml and blocked with ELISA blocking buffer (10% skimmed milk powder in PBS). Samples and standards  diluted 1:2 in PBS were added in duplicate and incubated overnight at 4°C. As above, all samples were pre-incubated with human, murine and bovine IgG in excess to ensure pre-aggregation of heterophilic antibodies. A biotinylated monoclonal mouse anti-OX40L  was used as detection antibody at a concentration of 1 ug/ml in PBS. The minimal detectable concentration was 1.16 ng/ml for sOX40L.Both ELISAs were spiked with variable concentrations of sOX40 and sOX40L, respectively. Recovery was >80% in all cases (data not shown). Furthermore, the samples were tested for the presence of sOX40/sOX40L dimers by adding the detection antibody from the sOX40 ELISA; this gave blank readings (data not shown). The cutoff of both ELISAs was calculated as the average value of blanks plus two SDs of the blanks. Values below the cutoffs were assigned the value of the cutoff.For this study, paired SFMC and PBMC samples from cRAs n = 15) and PBMC samples from eRA (n = 5) and HVs (n = 10) were analyzed for the expression of OX40 and sOX40L. Surface staining was performed on unstimulated cells using mouse monoclonal antibodies against OX40 , OX40L , CD4 , CD45RO , CD14 , and CD19 . All antibodies were titrated to the optimal working concentrations. All samples were blocked for unspecific binding by preincubation with 10% heat-inactivated murine serum (in-house production). Gating of OX40 and OX40L was done using fluorescence minus-one (FMO) controls combined with the relevant isotype controls (FMO/iso) matched for species, IgG subtype, fluorochrome and concentration. All flow cytometric analyses were performed within 24 hours using the LSRFortessa with Diva 7 software (BD Biosciences) and FlowJo software . For gating strategy, please see Additional file 5 and PBMU-test. Data were correlated using Spearman’s Rho. All data are expressed as median and IQR and 25th to 75th percentiles, unless otherwise specified. In all tests, the level of significance was a two-sided P-value <0.05.Statistical analyses were performed using GraphPad Prism 6.0 for Mac . All data were analyzed by non-parametric statistics. Paired data were analyzed using the Wilcoxon signed-rank test. Non-paired data were analyzed by the Mann-Whitney P <0.001), and sOX40 levels at 12 months were also significantly higher than those of cRA patients (5.97 pg/ml (5.97 pg/ml to 10.03 pg/ml) and HV (5.97 pg/ml (5.97 pg/ml to 17.34 pg/ml))  at baseline to 11.47 pg/ml (7.69 pg/ml to 13.68 pg/ml) at 12 months (P <0.05) and a final increase to 15.10 ng/ml (2.09 ng/ml to 61.61 ng/ml) at 12 months , and at baseline and 12 months when compared with cRA patients (1.59 ng/ml (1.16 ng/ml to 3.37 ng/ml) (both P <0.01)). Comparison of the two treatment groups ; ADA, n = 39) showed that there was no significant difference in their sOX40 and sOX40L plasma levels during the 12-month treatment period; thus, anti-TNF treatment seemed not to affect the shedding of the two proteins (data not shown).Plasma levels of sOX40L in eRA patients were significantly elevated throughout the first treatment year with a bimodal concentration curve where the highest levels were reached upon treatment initiation (18.30 ng/ml (2.59 ng/ml to 57.54 ng/ml)). This was followed by an 83% decrease after 3 months (3.06 ng/ml (1.16 ng/ml to 28.26 ng/ml), P <0.0001). Similarly, we observed sOX40L in the ACPA-positive group to have 23.2 ng/ml (3.6 ng/ml to 73.2 ng/ml) versus 2.7 ng/ml (1.2 ng/ml to 6.4 ng/ml) in the ACPA-negative group (P <0.001). This association with IgM-RF and ACPA was still present with sOX40L plasma levels measured at 3 months after treatment initiation (both P <0.01), but not after 12 months. Plasma levels of sOX40L in the IgM-RF-positive group was 22 ng/ml (2.68 ng/ml to 76.53 ng/ml) and 6.85 (1.82 ng/ml to 29.17 ng/ml) in the IgM-RF-negative group 12 months after treatment initiation, and no longer significant. Similarly, there was no difference in sOX40L between the ACPA-positive group (16.01 ng/ml (1.72 ng/ml to 60.0 ng/ml)) and the ACPA-negative group (12.0 ng/ml (2.79 ng/ml to 130 ng/ml)) after 12 months of treatment. Plasma levels of sOX40L did not at any time point correlate with disease activity parameters or CRP ) and CRP. We divided the eRA patients into IgM-RF and ACPA positive and negative at the time of treatment initiation. Plasma levels of sOX40L were strongly associated with the presence of IgM-RF and ACPA at baseline. In the IgM-RF-positive group sOX40L was 28.0 ng/ml (7.84 ng/ml to 75.48 ng/ml) versus 1.7 ng/ml (1.2 ng/ml to 3.4 ng/ml) in the IgM-RF-negative group . We did not observe the same correlation in the Placebo group . We also examined any association with disease flare and identified four patients with flare. They had a significantly decreased sOX40/sOX40L ratio compared with patients without flare .It has been suggested that like other members of the TNF superfamily, sOX40 and sOX40L function antagonistically to their membrane-bound forms. We therefore calculated the sOX40/sOX40L ratio to determine this axis in our cohort of eRA patients and correlated it with core disease parameters. Because sOX40 was measured in pg/ml and sOX40L was measured in ng/ml, the ratio was multiplied by a factor of 1,000. The sOX40/sOX40L ratio was significantly decreased in eRA compared with cRA throughout the study period . OX40+ cells were significantly higher on CD4 + CD45RO + T cells of cRA PBMC compared with HV PBMCs (P <0.05). OX40+ B cells and monocytes were only present in low numbers.We investigated which cells express the membrane-bound isoforms. PBMC from eRA (n = 5), and paired SFMC and PBMC from cRA (n = 15) were examined for expression of OX40 and OX40L by T cells, B cells, and monocytes. As expected, OX40 was mainly expressed by CD4 + CD45RO + T cells in the inflamed joint compared with peripheral blood Figure . The higP <0.05 and P <0.001). The percentage of OX40L expressing B cells from cRA PBMC was significantly increased compared with HV (P <0.01). The percentage of B cells expressing OX40L in PBMCs from eRA (16.7% (8.7% to 34.8%)) was also significantly increased compared with both cRA and HV . Generally, a higher percentage of monocytes were OX40L + compared with B cells. A higher percentage of monocytes from eRA PBMCs were OX40L + (80.7% (36.1% to 98.5%)) compared with monocytes from HV PBMCs (23.1% (2.0% to 43.6%)) (P <0.05). As mentioned above, only 5 eRA PBMC samples were analyzed, and the results should therefore be interpreted with caution.OX40L was primarily expressed by B cells and monocytes, which was consistent with the fact that OX40L is expressed mostly on antigen-presenting cells. OX40L expressing B cells were significantly increased in SFMC from cRA patients (10.3% (4.3% to 47.3%)) as compared with cRA PBMC (4.5% (2.7% to 10.6%)) and HV PBMC (1.2% (0.5% to 3.2%))  remission , this waThe potential of the OX40 system in generating immunological memory is particularly interesting in RA. Like other studies, the OPERA study showed that RA patients who have achieved remission can discontinue anti-TNF therapy if intensively treated in the early phase of disease ,24. ThisThe importance of the imbalance between sOX40 and sOX40L is supported by the association between the sOX40/sOX40L ratio and disease activity after adalimumab discontinuation. A low ratio was significantly associated with a higher DAS28CRP score after 3 months. However, defined flare (DAS28CRP >1.2 if DAS28CRP <3.2 or >0.6 if DAS28CRP ≥3.2 ) was onlWe hypothesize that the modulation of RA disease processes through the mechanisms exercised by the sOX40 and sOX40L molecules could take two forms. Soluble OX40 possibly works as a decoy receptor and blocks sOX40L or membrane-bound OX40L from binding to membrane-bound OX40. As a result, it inhibits proliferation and survival signals for the cells. Previous studies have shown that sOX40 mimics the anti-inflammatory function of inducible T-regulatory (Treg) cells and thereby suppresses the autoreactive T cells . By contIn summary, we found that sOX40 and sOX40L levels were changed in eRA and that sOX40L was associated with IgM-RF and ACPA. Furthermore, sOX40/sOX40L ratio was associated with increase in DAS28CRP after adalimumab discontinuation. In addition, the number of cells expressing membrane-bound OX40 and OX40L was increased in RA. Overall, our findings suggest that the OX40 system is important for the formation of key autoantibodies in RA and possibly associated with future disease activities.


KDM5B is a jmjc domain-containing histone demethylase which remove tri-, di-, and monomethyl groups from histone H3 lysine 4 (H3K4). KDM5B has been determined as an oncogene in many malignancies. However, its expression and role in hepatocellular carcinoma (HCC) remain unknown.We detected the expression of KDM5B in HCC tissues and cell lines. Cell proliferation was performed to reveal the role of KDM5B depletion on HCC cells both in vivo and in vitro. Flow cytometry was used to analyze the cell cycle and chip analysis was conducted to determine the direct target of KDM5B.KDM5B is frequently up-regulated in HCC specimens compared with adjacent normal tissues and its expression level was significantly correlated with tumor size, TNM stage, and Edmondson grade. Moreover, Kaplan-Meier survival analysis showed that patients with high levels of KDM5B expression had a relatively poor prognosis. Knockdown of KDM5B notably inhibits HCC cell proliferation both in vivo and in vitro via arresting the cell cycle at G1/S phase partly through up-regulation of p15 and p27. Further molecular mechanism study indicates that silencing of KDM5B promotes p15 and p27 expression by increasing histone H3K4 trimethylation in their promoters.KDM5B could be a potentially therapeutic target, which provides a rationale for the development of histone demethylase inhibitors as a strategy against HCC.The online version of this article (doi:10.1186/s13046-016-0311-5) contains supplementary material, which is available to authorized users. Mounting evidence indicates that epigenetic alterations contribute significantly to the initiation and progression of multiple human malignancies, including hepatocellular carcinoma –3. As anLiver cancer is the sixth most common malignant disease worldwide but the third most frequent cause of cancer-related death, with only 5 % of patients surviving more than 5 years. In fact, about half of these cases and deaths occur in China. Hepatocellular carcinoma (HCC) represents the major histological subtype of primary liver cancer, accounting for 70 to 85 % of the total liver cancer worldwide, and its molecular etiology is heterogeneous . NumerouKDM5B is a jmjc domain-containing histone demethylase, which belongs to KDM5 family. KDM5B was first identified and characterized in 1999 when Lu found its overexpression in human breast-cancer cell lines and primary breast carcinomas . MammaliWe are particularly interested in KDM5B because we found that it was the most significantly up-regulated histone demethylase among 27 histone demethylase family members by comparing their expression profiles between HCC and their adjacent normal liver tissues using public database. Then we determined whether the highly expressed KDM5B could contribute to the pathogenesis of HCC. In the present work, we demonstrate that KDM5B is frequently up-regulated in HCC specimens and cell lines. Knockdown of KDM5B significantly inhibits cellular proliferation of HCC cells and arrests cell cycle progression at the G1/S-phase. Moreover, the effect of KDM5B knockdown on HCC cell proliferation is mediated through upregulation of the cyclin-dependent kinase inhibitors, p15 and p27. Furthermore, our molecular mechanism study indicates that silencing of KDM5B promotes p15 and p27 expression by increasing histone H3K4 trimethylation in their promoters. Collectively, our findings provide new insight into the molecular pathogenesis of HCC and KDM5B may constitute a promising novel therapeutic target for blocking progression of HCC.All HCC specimens were obtained from HCC patients who underwent surgical resection with informed consent from May 2008 to October 2011. Both the tumor and adjacent normal tissue were collected from each patient and were frozen at −80 °C until processed. All patients had negative histories of exposure to either chemotherapy or radiotherapy prior to the surgical operation. The diagnosis of HCC was validated by two individual pathologists. The use of human tissues was approved by the institutional ethics committee of Nanjing Medical University.The human HCC cell lines  were obtained from the Chinese National Human Genome Center at Shanghai. Cells were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10 % FBS  and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) at an atmosphere of 5 % CO2.Total RNA was extracted from tissue or cell culture samples using TRIZOL reagent (Invitrogen) following the manufacturer’s protocol and was reverse transcribed into cDNA with a M-MLV reverse transcriptase kit . Quantitative real-time PCR was performed using the Thermal Cycler Dice Detection System with the SYBR Premix Ex Taq™ . All samples were done in triplicate and the housekeeping gene β-actin was used as an endogenous control. The following primers were used to specially amplify the KDM5B gene and β-actin. For KDM5B, forward: 5-GGTGAGCCAAAAACCTGGTA-3, and reverse, 5-AATCACAAACTCCCCAGCAC-3; For β-actin: forward: 5-AGAGCCTCGCCTTTGCCGATCC-3, and reverse, 5-CTGGGCCTCGTCGCCCACATA-3.KDM5B-specific small interference RNAs (siRNAs) were designed and chemically synthesized  to suppress endogenous KDM5B expression in HCC cells: si-370, 5-CACGUAUCCAGAGACUGAAUGAAdTdT-3; si-2531, 5-GGAUGCAGAGAAGUGUGCCUCUGdTdT-3.In addition, si-NC was also synthesized to act as a negative control: si-NC, 5-UUCUCCGAACGUGUCACGUdTdT-3.The oligonucleotides encoding short hairpin RNAs (shRNAs) for the continuous knockdown of endogenous KDM5B were synthezised and inserted into pSUPER : sh-370, 5-GATCCCCCACGTATCCAGAGACTGAATGAATTCAAGAGAAATTCATTCAGTCTCTGGATACGTGTTTTTGGAAA-3; sh-2531, 5-GATCCCCGGATGCAGAGAAGTGTGCCTCTGTTCAAGAGACAGAGGCACACTTCTGCATCCTTTTTGGAAA-3; sh-NC containing irrelevant nucleotides was served as a negative control: sh-NC, 5-GATCCCCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTGGAAA-3. All constructs were fully sequenced. The cells were then transfected with siRNA or shRNA using lipofectamine 2000  according to the manufacturer’s instructions at cell density of 30-50 %.Transiently transfected HCC cells were seeded at a density of 2000–5000 cells/well into 96-well plates and cultured for a week. Cell growth was examined by Cell Counting Kit-8  according to the manufacturer’s instructions. The absorbance value at a wave length of 450 nm was used as an indicator of cell viability.Transfected HCC cells were plated at a density of 10,000–50,000 cells/10-cm plate and maintained in the medium with addition of 0.8 mg/ml G418  for colony formation. After 3 weeks, colonies stained with 0.1 % crystal violet were counted and photographed.For the soft agar colony formation assay, 2000–5000 transfected cells were plated and grown in the 24-well plate with the medium containing 1 % base agar and 0.5 % top agar. After 3 weeks, all colonies were counted and photographed under a dissecting microscope.Animal care and euthanasia were approved by the Nanjing medical University animal studies committee. Fifteen BALB/c nude mice were randomly divided into three groups including the negative control group and two KDM5B knockdown groups. Hep3B cells transfected with sh-KDM5B or negative control were selected under 0.8 mg/ml G418  for 3 weeks. Then the stably transfected cells were harvested at a concentration of 1 × 107 cells/ml. Of the suspending cells, 0.1 ml was inoculated subcutaneously into either side of the posterior flank of the 4–6-week-old BALB/C nude mice. Xenografts in each group were observed periodically after injection. Nude mice were killed by cervical dislocation after 3 weeks and the xenografts were peeled off subcutaneously. The weight of the xenografts in each group was compared and used for further analysis.Flow cytometry was performed to analyze the cell cycle. Serum starvation was used to induce cell cycle synchronization before cell transfection. At 48 h after transfection with KDM5B siRNAs duplexes, cells were harvested as single cell suspensions. After fixation in 70 % ethanol for 1 h at −20 °C, cells were washed and resuspended in PBS, followed by incubation with propidium iodide (10 mg/ml) and RNase A (10 mg/ml) for 30 min at 4 °C. FACSCalibur fow cytometer, CellQuest  was used to calculate the percentage of cells in each phase.Total protein was extracted using cell lysis buffer containing 50 mM Tris–HCl (pH 6.8), 2 % SDS, 10 % 2-mercaptoethanol, 10 % glycerol, and protease inhibitor cocktail . Protein samples (20 mg) were resolved using SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with 5 % milk in TBS for 2 h at room temperature. After incubation with the appropriate primary antibody overnight at 4 °C, membranes were washed and incubated with the IRDye 800cW or 680RD secondary antibodies in TBST for 2 h at room temperature. Then the protein bands of interest were visualized with the Odyssey system . The specific antibodies used in our study were as follows: anti-KDM5B , anti-p15 , anti-p27 , anti-H3K4me2 , anti-H3K4me3 , anti-total-H3 , and anti-β-actin .The ChIP assays were performed by using EZ-ChIP KIT according to the manufacturer’s instruction . The control and KDM5B siRNA-treated Hep3B and Focus cells were treated with formaldehyde and incubated for 10 min to generate DNA-protein cross-links. Then the cell lysates were sonicated to generate chromatin fragments of 200–1000 bp. The antibodies against KDM5B and trimethylated H3-K4 (H3-K4me3) were used to precipitate DNA fragments bound by their corresponding elements. The protein-DNA complex was collected with protein A Sepharose beads , eluted, and reverse cross-linked. Following treatment with Protease K , the samples were extracted with phenol-chloroform and precipitated with ethanol. The recovered DNA was resuspended in TE buffer and used for the PCR amplification.p-values were calculated, and a probability level of less than 0.05 was considered to be statistically significant.All statistical data were carried out using Statistical Program for Social Sciences 18.0 software  and presented with Graphpad prism 5.0 . The significance of differences between groups was evaluated with Student’s t-test and χ2 test as appropriate. DFS and OS rates were calculated by the Kaplan-Meier method with the log-rank test applied for comparison. Two-sided To explore a histone demethylase with oncogenic characteristics for HCC, We first analyzed expression profiles of a number of histone demethylase genes in a subset of HCC samples by using public GEO Profiles database (GSE 25097) and found that KDM5B was the most obvious up-regulated histone demethylase with at least 2-fold up-regulation in 40 % HCC specimens Fig. . To verin = 50) and low expression group (n = 50), median was used as cut-off value. Significant correlations were found between KDM5B expression and tumor size, TNM stage, and Edmondson grade, suggesting that KDM5B might have a stimulatory role in the progression of HCC (Table P = 0.0005)   Fig. . Likewis06) Fig. . These rTo determine whether KDM5B is necessary for the proliferation of HCC cells, we used chemically synthesised siRNAs and constructed the corresponding shRNA plasmids to knockdown endogenous KDM5B in 2 HCC cell lines (Hep3B and Focus) with relatively high KDM5B level. The efficient inhibition of KDM5B expression in siRNA-treated cells was verified using quantitative real-time PCR Fig. . As expeTo further determine the effect of KDM5B knockdown on tumorigenicity in vivo, we performed a subcutaneous xenograft tumor model in nude mice. Hep-3B cells were transfected with pSUPER-shRNA plasmids, then these cells were harvested and injected into the flanks of nude mice. The diameters of the tumors were measured every 3 days. As expected, pSUPER-sh370 and pSUPER-sh2531 were able to significantly suppress tumorigenicity, resulting in obvious reductions in tumor weight and volume compared to the negative controls pSUPER-shNC Fig. . All theTo further assess the mechanism of growth suppression induced by KDM5B knockdown, we performed a flow cytometry to analyze the cell cycle status of cancer cells of cells with or without KDM5B depletion. The results showed that the fraction of HCC cells at the G1 phase was significantly higher in cells treated with siKDM5B than in those treated with control siRNAs, while the proportion in S phase was remarkably decreased Fig.  and b. TThe regulation of G1-S phase transition is governed by cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CDKIs). So we detected whether the expression of these genes was altered by KDM5B knockdown in HCC cells using quantitative real-time PCR. Interestingly, KDM5B depletion notably increased the mRNA expression of p15 and p27 in the Hep3B cell line. In contrast to p15 and p27, expression of other genes such as p14 and p16 were inconsistently or weakly affected by KDM5B knockdown. Similar results were obtained in the Focus cell line, indicating that KDM5B regulates p15 and p27 mRNA expression in multiple HCC cell lines Fig. . In addiSince p15 and p27 expression were regulated by KDM5B at both mRNA and protein levels, we speculated whether these 2 genes are direct targets of KDM5B. Chromatin immunoprecipitation assay was thus performed to determine an association of KDM5B with p15 and p27 promoters. In the cells treated with control siRNA, KDM5B occupancy on the promoter region of the 2 genes was readily detectable. In contrast, knocking down KDM5B abolished its association with these promoter sequences Fig.  and b. MTo determine whether p15 and p27 are key mediators of KDM5B’s function in cellular proliferation, we assessed the colony formation when both KDM5B and p15 or p27 were simultaneously silenced via RNAi Fig. . InteresHepatocellular carcinoma (HCC) is a worldwide malignancy with a high mobility and mortality, especially in China. The long-term survival rate for HCC remains poor despite advanced therapeutic modalities and strategies such as surgery, chemotherapy and radiotherapy. Therefore, better understanding of the pathogenesis of HCC and exploring novel therapeutic approaches against drug targets are urgently needed.Emerging evidence has indicated that a number of histone methylation modifiers are dysregulated in tumors and are critical for oncogenic phenotypes –38. JumoThe effect of KDM5B inhibition on cell proliferation is not cell type or context-specific, since we observed similar phenotypic changes in two different HCC cell lines (Hep3B and Focus) upon KDM5B knockdown. Analysis of cell cycle distribution revealed that KDM5B depletion led to an obvious arrest at G0/G1 phase. Moreover, results of FACS assays and senescence-associated (SA) β-galactosidase  analysis excluded the possibility that KDM5B knockdown affected HCC cell proliferation through apoptosis or senescence. Therefore, we decided to lay emphasis on some molecules involved in the regulation of G1-S phase transition. P15, a member of the INK4 family of cyclin-dependent kinase inhibitors, is capable of inducing cell cycle arrest in G1 phase and has been identified as a tumor suppressor, as well as the other two INK4 family members p16 and p14 , 40. P27In conclusion, our results suggest that KDM5B is overexpressed in the vast majority of HCC specimens and that its depletion leads to highly impaired clonogenesis and cell cycle arrest through up-regulation of p15 and p27. Therefore, KDM5B could be a potentially therapeutic target, which provides a rationale for the development of histone demethylase inhibitors as a strategy against HCC.


RIZ1) as an anticancer domain and its ability to induce apoptosis in esophageal squamous cell carcinoma (ESCC) cells. The TE13 ESCC cell line was transfected with pcDNA3.1(+) eukaryotic expression vectors bearing the open reading frames of either the human RIZ1 gene or the PR domain, and the mRNA and protein expression levels were then detected using quantitative reverse transcription polymerase chain reaction and western blotting, respectively. The rate of apoptosis was determined by flow cytometry and the cell invasion ability was determined by an invasion assay. RIZ1 and the PR domain induced apoptosis and reduced the cell invasion ability (P<0.01). These findings indicate that the RIZ1 gene possesses anticancer activity in the PR domain, which may be important in inhibiting the development of ESCC.The present study examined the role of the PRDI-BF1-RIZ (PR) domain of tumor suppressor retinoblastoma protein-interacting zinc finger gene 1 ( RIZ) gene was identified by Bird  domain, whereas RIZ2 does not; the sequences of the two proteins are otherwise identical  H2O supplemented with 10% fetal bovine serum , 1X L-glutamine (2 mm), 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were incubated at 37°C in a 5% CO2 humidified incubator.The TE13 human esophageal squamous cell carcinoma (ESCC) cell line was purchased from American Type Culture Collection . The cells were cultured in RPMI-1640  containing 4.76 g HEPES, 2.0 g NaCO6-1×107 cells. The RNA pellets were resuspended in diethylpyrocarbonate-treated H2O. The total RNA concentrations were quantified using an ultraviolet (UV) spectrophotometer .RNA was isolated from the cells using TRIzol  according to the manufacturer’s instructions, and 1 ml TRIzol was added to 5×10RT reactions were performed to generate cDNA using 2 μg RNA, Moloney murine leukemia virus reverse transcriptase, ribonuclease inhibitor and a dNTP mixture , according to the manufacturer’s instructions. Semi-quantitative, RT-polymerase chain reaction (PCR) was conducted using the cDNA templates.RIZ1 mRNA sequence published by the National Center for Biotechnology Information (NCBI), the 5,157 bp protein-coding region is located between base pairs 857 and 6,013. Due to the amplicon size, the open reading frame was divided into five sections, termed A603, A1200, B, C and D. The following primers were designed for the five RIZ1 sections, hereafter referred to as amplicons, using Primer Premier 5.0 software : A603 forward, 5′-GTGGCTAGCATGAATCAGAACACTACTG-3′ and reverse, 5′-TTGGCTAGCAGAGGTGAAATCTGGCTC-3′; A1200 forward, 5′-TGGCTGCGATATGTGAATTG-3′ and reverse, 5′-CTCTACGCTGATGCCGTCTC-3′; B forward, 5′-GCTGATGGCAAAGCATCTG-3′ and reverse, 5′-AATTCCTTGCCTTCAGAGTCAC-3′; C forward, 5′-TCAAAGAAAGTCATTCAGTGC-3′ and reverse, 5′-CGGTGATGGTACTGAAATG-3′; and D forward, 5′-GCCTCAATCAGCATTACC-3′ and reverse, 5′-GTCTACTCTTTGAAGAATGGTC-3′. PCR was conducted in 50-μl reactions containing 5 μl 10X KOD buffer, 5 μl 2 mm dNTPs, 3 μl 25 mm MgSO4, 2 μl of each forward and reverse primer, 1 μl cDNA, 1 μl KOD-Plus-Ver. 2 polymerase  and ddH2O. Each reaction required the following specific conditions in accordance with the melting temperature and size of each amplicon: Initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 98°C for 10 sec, annealing  and extension at 72°C for 1 min, and a final extension at 72°C for 10 min.According to the NheI and BamHI endonuclease sites, respectively, and three protective base pairs. The downstream primer also contained a termination codon. Initial denaturation was performed at 94°C for 3 min, followed by 35 cycles of denaturation at 98°C for 10 sec, annealing at 60°C for 1 min and final extension at 72°C for 10 min. Subsequently, 0.3 μl Easy Taq  was added to append a polyA tail at the end of the PCR products, turning the blunt end into a sticky end. This was followed by a final extension step at 72°C for 30 min.The PR domain primers were as follows: Forward, 5′-GTGGCTAGCATGAATCAGAACACTACTG-3′ and reverse, 5′-TTGGGATCCTCAAGAGGTGAAATCTG-3′. The 5′ terminal of the forward and reverse primers contained The quality of the amplified products was analyzed on 12 g/l agarose gels using a UV spectrophotometer and the RT-PCR products were sequenced.RIZ1 and PR domain amplicons were inserted into Trans1-T1 Phage Resistant vectors  that were subsequently transformed into Trans1-T1 Phage Resistant competent cells, and plated on agar containing ampicillin and X-gal. White colonies were selected for further analysis. Subsequent to expansion of the selected bacterial colonies, plasmid DNA was extracted by alkaline lysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The RIZ1 and PR domain amplicons were digested from plasmids containing the correct insert and were ligated into the pcDNA3.1(+) eukaryotic expression vector (Invitrogen Life Technologies). Insertion was verified by restriction enzyme digestion followed by sequencing.The amplicons were extracted from the agarose gels using the Tiangel Midi Purification kit  according to the manufacturer’s instructions. The five ne lysis . Restric5 cells/well in 2 ml media, and then incubated at 37°C to 90–95% confluence. After 24 h, the media was replaced with complete serum- or antibiotic-free RPMI-1640 in preparation for transfection. Ultra pure pcDNA3.1(+)/RIZ1 and pcDNA3.1(+)/PR domain plasmid DNA was extracted using a HighPure Mini Plasmid kit (Tiangen). A liposome-mediated method . The primers for the genes of interest (10μM) were as follows: RIZ1/PR domain forward, 5′-AATCAGAACACTACTGAGCCTGT-3′ and reverse, 5′-ACCAATCCGGGTCTTGTCAAC-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-GGGTGGAATCATATTGGAAC-3′. The reactions were conducted using an CFX96 Real-time PCR System  according to the manufacturer’s instructions. Briefly, initial denaturation was performed at 95°C for 2 min, followed by 45 cycles of denaturation at 95°C for 15 sec, annealing for the RIZ1/PR domain at 63°C for 15 sec or for GAPDH at 60°C for 15 sec, and extension at 72°C for 40 sec, followed by the production of thermal melting curves. Each sample for each gene was conducted in triplicate.The TE13 cells transfected with pcDNA3.1(+)/RIZ1 were homogenized in radioimmunoprecipitation buffer  and the protein concentrations were determined using a bicinchoninic acid protein assay kit . Cell lysates (30 μg) were separated by 8% SDS-PAGE, transferred to nitrocellulose membranes  and immunoblotted with the indicated antibodies overnight in the Orbital Shaker  at 4°C. The monoclonal mouse anti-human RIZ1/PR domain antibodies, monoclonal mouse anti-human β-actin primary antibody and secondary polyclonal goat anti-mouse polyclonal antibody were all obtained from Abcam .Bands were visualized using a PowerLook scanner  and quantified using ImageQuant software . The relative expression levels of RIZ1 and the PR domain were calculated as the gray values of RIZ1, the PR domain and β-actin. Untransfected and empty vector-transfected TE13 cells served as negative controls.5 cells/well and allowed to attach for 12 h. The cells were then transfected with either pcDNA3.1(+)/RIZ1 or pcDNA3.1(+)/PR domain and harvested after 24 h. A total of ~1×105 cells were washed with cold phosphate-buffered saline  for each test. Cells were suspended in 1 ml 1X binding buffer using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Analysis Kit . Next, the cells were centrifuged  at 300 × g for 10 min at room temperature and the supernatant was removed. The cells were then resuspended in 1 ml 1X binding buffer and the cell concentration was adjusted to 1×106 cells/ml. A total of 100 μl cell suspension was used for analysis. Next, 5 μl Annexin V-FITC staining solution was added and the tube was kept in the dark at room temperature for 10 min. A total of 5 μl propidium iodide staining solution  was added and the tube was kept in the dark at room temperature for 5 min. Finally, 500 μl PBS was added to the cells and vortexed gently for 1 h. The cells were then analyzed using a BD FACSAria II cell sorter . Untransfected and empty vector-transfected TE13 cells served as negative controls.To investigate the effect of overexpression of RIZ1 or the PR domain on apoptosis, TE13 cells were seeded in six-well plates at a density of 2×106 cells/ml in serum-free media, was added to each insert. The invasion assay was then incubated for 24 h in a tissue culture incubator. Using a cotton-tipped swab, the non-invading cells were then gently removed, and the ECMatrix gel was also removed from the interior of the inserts. The invasive cells on the lower surface of the membrane were stained by dipping the inserts in the crystal violet staining solution for 20 minutes and then dipping the inserts in a beaker of water three times to rinse. The cells were counted by capturing images of the membrane through the microscope .To study the invasion ability using a cell invasion assay kit . The invasion chamber was allowed to adjust to room temperature in a tissue culture hood. Media containing 10% FBS (500 μl) was added to the lower chamber and 300 μl of cell suspension, containing 1.0×10−averageΔΔCT × 100%. Student’s t-test and one-way analysis of variance (ANOVA) were employed to examine parametric data. The χ2 test was used for statistical analysis of the group comparisons and to compare enumerated data. P<0.05 was considered to indicate a statistically significant difference.Statistical analysis was conducted using SPSS 18.0 software . The data are presented as the mean ± SD. The quantitative PCR results are shown as 2RIZ1 and the PR domain, recombinant plasmids were generated to enable ectopic overexpression of RIZ1 (To overexpress  of RIZ1  and the  of RIZ1  in TE13 The RIZ1 and PR domain mRNA and protein expression levels in TE13 cells transfected with pcDNA3.1(+)/RIZ1 or pcDNA3.1(+)/PR were significantly higher compared with untransfected and empty vector-transfected cells (negative control groups) P<0.05; . No statThe apoptotic rates were significantly higher in the cells transfected with pcDNA3.1(+)/RIZ1 or the pcDNA3.1(+)/PR domain compared with the untransfected and empty vector-transfected TE13 cells P<0.01; .The Matrigel invasion ability of the TE13 cells transfected with pcDNA3.1(+)/RIZ1 or pcDNA3.1(+)/PR domain was significantly reduced compared with the invasion ability of the negative controls P<0.05; .RIZ1 (The silencing of tumor suppressor genes (TSGs) by genetic and epigenetic pathways is recognized as important in human carcinogenesis ,8. In adRIZ1 .RIZ gene encodes two protein products of different lengths, RIZ1 and RIZ2. As a TSG, RIZ1 contains the PR or Suvar3–9, Enhancer-of-zeste, Trithorax domain, but RIZ2 lacks this domain. A number of studies have investigated the underlying mechanism of RIZ1 gene inactivation, which has been shown to include genetic and epigenetic changes /PR domain to investigate whether the domain could be expressed independently and whether it influenced apoptosis or invasion. Quantitative PCR and western blotting determined that cells transfected with the recombinant plasmid successfully expressed RIZ1 and the PR domain. Flow cytometry revealed that transfection with either the PR domain or RIZ1 inhibited cell proliferation. The cell invasion assay revealed that the invasion ability was significantly reduced in the TE13 cells transfected with pcDNA3.1(+)/RIZ1 or pcDNA3.1(+)/PR-domain (P<0.05). These findings indicate that the PR domain of RIZ1 exerts anticancer activity in ESCC. In conclusion, further study regarding the mechanism of action of the RIZ1 tumor suppressor gene and the PR domain may reveal the underlying mechanism of anticancer function, and thus may lead to the development of novel biomarkers for early diagnosis and prognostic evaluation in ESCC.


Cdk1 provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms.Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs): homologous recombination (HR) and nonhomologous end-joining (NHEJ). DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1) activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2  •Cdk1 phosphorylates the core NHEJ factor Xlf1 in fission yeastCdc2•Phosphorylation of Xlf1 inhibits nonhomologous end-joining (NHEJ)•Cells with phospho-null Xlf1 have elevated levels of NHEJ repair•Cdk1 regulation of Xlf1 is lostNHEJ repair can predominate over HR when Cdc2 Repair of DNA double-strand breaks (DSBs) by homologous recombination is activated by the cell cycle kinase Cdk1. Hentges et al. now find that the NHEJ factor Xlf1 is phosphorylated by Cdk1 and that this modification restrains end-joining in cycling cells. Removal of this regulation alters DSB pathway selection in vivo. The ability to repair DNA damage is critically important for the preservation of genomic integrity. DNA double-strand breaks (DSBs) can be repaired by two different cellular pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ) . HR proc1 phase and HR restricted to the G2 and S phases of the cell cycle . The C-terminal region of Xlf1 has two sites, T180 and S192, that conform to [ST]-P-x-[KR], a consensus motif for phosphorylation by the Cdc2Cdk1 kinase. These potential phosphorylation sites are conserved in other fission yeasts  and mutated Xlf1 proteins were incubated with mammalian Cdk1 kinase  caused a significant reduction of the phosphoband of Xlf1 in Phos-tag western blots, which could be further reduced by phosphatase treatment  to synchronize GFP-xlf1 cultures in G1. A phospho-Xlf1 species was not detectable in G1-arrested cells  displayed a ∼2.5-fold increase in end-joining compared to WT cells (xlf1.EE showed a moderate decrease in plasmid end-joining. The religation levels were unaffected by the nature of the ends (blunt or overhangs) in xlf1 mutants. These results indicated that an inability to phosphorylate Xlf1 leads to increased NHEJ activity and supports the hypothesis that Cdc2Cdk1 phosphorylation of Xlf1 inhibits NHEJ.As NHEJ is most active in G in S/G2 , we predsmid DNA . LeucineWT cells A. In conctp1+. Deleting ctp1 had no effect on plasmid religation levels -induced DSBs in Xlf1 phospho-null strains. A key event in the cellular response to DNA damage is the activation of DNA damage checkpoints that arrest progress in the cell cycle to ensure that DSBs can be repaired. We monitored the phosphorylation status of Chk1 kinase, a marker of Gtivation , in respcultures C. We alsrad52-GFP and nmt41-xlf1.AA rad52-GFP cells with 50 Gy and monitored the formation and persistence of Rad52-GFP foci using live-cell imaging. While Rad52 foci were observed in most cells with both WT xlf1 and nmt41-xlf1.AA, foci formation was slower in nmt41-xlf1.AA  . However, overexpression using the medium-level lf1 (WT) . The sen deleted , revealiinviable  and S2.Cdk1 on chromosomal DSBs. Chromosome end fusions can be generated by NHEJ in cells with unprotected telomeres, such as taz1d  and S/G2 (log-phase) cultures in which telomeres were unprotected due to taz1+ deletion. Chromosome fusions were detected in taz1d cells in G1 but not in S/G2 cells  in the xlf1.AA phosphomutant require inactivation of HR indicates crosstalk between NHEJ and HR. To verify if a similar effect could be identified in the processing of plasmid DNA ends by the two DSB repair pathways, we designed an assay in which the levels of HR and NHEJ can be assessed in parallel. Cells were electroporated with two different linearized plasmids. One plasmid, containing WT leu1+ but no ARS, could be integrated at the leu1-32 locus via an HR-dependent mechanism, giving rise to Leu+ colonies. The second plasmid, containing the antibiotic resistance gene hphR and an S. pombe ARS, could be recircularized by NHEJ and stably maintained, giving rise to hygromycin-resistant colonies. Hygromycin selection was applied after 2 hr. Cells were transformed in parallel; the frequency of leu1+ colonies and hygromycin-resistant colonies were determined in relation to an uncut plasmid control as a measure of HR and NHEJ activity, respectively. Deletion of xlf1+ decreased plasmid religation ∼50 fold , suggesting that disabling the phosphorylation of Xlf1 leads to inhibition of HR. In addition, while deletion of ctp1+ by itself had no significant impact on NHEJ, deletion of ctp1+ in an xlf1.AA mutant led to a much larger increase in plasmid religation than in HR-competent xlf1.AA. Together, these observations provide further evidence that phospho-null xlf1.AA affects the levels of HR.The observation that chromosome end ligations  and broken with glass beads. Cleared lysate containing 15 μg total protein was incubated for 2 hr at 4°C with 15 μl GFP-Trap A magnetic beads (Chromotek) per immunoprecipitation. Beads were washed with lysis buffer, washed three times in PMP buffer  plus MnCl2, resuspended in 100 μl, divided into two, and either mock treated or incubated with 800 units of lambda phosphatase (NEB) at 30°C for 30 min. Beads were resuspended in Laemmli buffer and boiled. Samples were separated by SDS-PAGE on 12% gels containing 25 μM Phos-tag (AAL-107 Wako) and 50 μM MnCl2. Prior to transfer onto polyvinylidene fluoride, gels were incubated for 10 min in transfer buffer with 1 mM EDTA and then without EDTA. GFP-Xlf1 was detected using anti-GFP antibody .A total of 108 WT S. pombe cells resuspended in 400 μl of HB buffer  broken using glass beads. Per kinase reaction, cell lysate containing 1 mg total cellular protein was mixed with 40 μl of p13suc1 agarose conjugate (Millipore), incubated with rotation at 4°C for 3 hr, washed three times with HB buffer, and then washed once with kinase buffer . For each kinase assay reaction, beads were then mixed in a total volume of 15 μl containing 1.5 μg purified protein (either recombinant 6His-Xlf1 or histone H1), 20 μM ATP, and 5μCi γ-32P-ATP, all diluted in kinase buffer. The same method was used with Cdk1 (NEB). The kinase reaction was allowed to proceed for 10 min at room temperature and then was stopped by adding SDS-PAGE loading buffer and heated to 90°C for 5 min. Protein was separated on 15% gels and subjected to Coomassie staining and autoradiography.Active Cdc2 enzyme was isolated from a lysate of 5 × 10chk1 and rad52 strains. Experimental work using Taz1d was designed by M.G.F. and C.C.R. C.C.R carried out and made strains needed for these taz1 experiments. P.H. carried out all other experimental work and strain production.P.H. and A.J.D. designed the key experiments and wrote the manuscript with advice from M.G.F. and C.C.R. H.W. designed and carried out hygromycin plasmid assays and spore survivals and made


Type A Acute Aortic Dissection (AAAD) is a highly deadly disease. Management of AAAD suspicion is extremely important in order to gain time and increase the likelihood of survival.The aim of this study is to review our experience based on our local algorithm developed for the assessment and management of patients with AAAD suspicion over the last 8 years.All patients who underwent an emergency surgery for AAAD between 2007 and 2014 following our algorithm were assessed (Table). Their clinical situation at admission, evaluation, operative and postoperative data were evaluated retrospectively. Continuous variables were expressed as mean ± standard deviation; categorical variables were shown as frequency and percentage. A total of 68 patients were included during the study period. The mean age was 61 ± 13 years, with 42 men (65%). Supracoronary ascending aorta replacement was the primary surgical procedure . The mean cardiopulmonary bypass, cross clamp and circulatory arrest times were 3.2 ± 1.6, 2.1 ± 1.2 and 0.4 ± 0.2 hours. Acute renal failure , re-operation  and pneumonia  were the main postoperative complications. Mean intensive care unit stay and hospitalization times were 5.8 ± 6.2 and 26.5 ± 53.5 days. There were 14 perioperative deaths (21%) and 18 hospital deaths (26%). The 6-year survival was 67.5%.Our institutional experience in managing AAAD is comparable to results from other centers. Standardizing management using an algorithm is important to gain time for rapid decision-making and having successful outcomes in centers with limited volume.


Moreover, fission yeast cells with Chp1 loop mutations have a defect in Chp1 recruitment and heterochromatin formation. This study reveals the structural basis for heterochromatic silencing and suggests that chromodomains could read histone code in the H3 tail and the nucleosome core, which would provide an additional layer of regulation.To maintain genome stability, cells pack large portions of their genome into silent chromatin or heterochromatin. Histone H3 lysine 9 methylation, a hallmark of heterochromatin, is recognized by conserved readers called chromodomains. But how chromodomains interact with their actual binding partner, the H3K9 methylated nucleosome, remains elusive. We have determined the structure of a nucleosome trimethylated at lysine 9 of histone H3 (H3K9me3 Nucleosome) in a complex with the chromodomain of Chp1, a protein required for RNA interference-dependent heterochromatin formation in fission yeast. The cryo-electron microscopy structure reveals that the chromodomain of Chp1 binds the histone H3 lysine 9 methylated tail and the core of the nucleosome, primarily histones H3 and H2B. Mutations in chromodomain of Chp1 loops, which interact with the nucleosome core, abolished this interaction  To maintain genome stability, cells pack large portions of their genome into silent chromatin or heterochromatin. Heterochromatin is required for centromere formation, gene silencing, repression of recombination and maintenance of telomere stability. A class of histone methyltransferases, the Su(var) family, specifically deposits the histone H3 lysine K9 methylation (H3K9me), which is recognized by the chromodomain (CD)-containing Heterochromatin Protein 1 (HP1) family of proteins. HP1 proteins bind to H3K9me nucleosomes and mediate chromatin compaction and transcriptional gene silencing . MutatioIn fission yeast small RNAs guide the RNA induced transcriptional silencing (RITS) complex to centromeric repeats to induce H3K9 methylation and heterochromatin formation . In the Schizosaccharomyces pombe, four chromodomain proteins are involved in heterochromatin formation and transcriptional gene silencing: Chp1, Chp2, Clr4 and Swi6. The chromodomain of Chp1 (Chp1CD) has the highest affinity for the H3K9me peptide and is essential for tethering the RITS complex to centromeric region and for heterochromatin establishment . Al. Al10]. In Chp1CD-H3K9me3Nucleosome cryo-EM map we observe three contacts between Chp1CD and the nucleosome core. These contacts are formed by the two loops of Chp1CD β-sheet and by the tip of the α-helix . Chp1CD We can also trace a density stretching from Chp1CD density to the DNA and then along the DNA to the site where H3 tail exits the core of the nucleosome . The denS. pombe and other eukaryotes, whereas N33 and N35 are conserved only in a subset of chromodomains  that are in proximity of the H3 loop. LOOP1A construct has R31S mutation in addition to N33A and N35A. The Chp1CD LOOP2B construct has W49A, Y50A and D51A mutated, whereas the Chp1CD LOOP2A has a N52A point mutation . In our odomains . The resrevisiae  and inteWe purified the above described Chp1CD mutants  and confIn vitro binding assays reveal that wild-type Chp1CD can weakly bind to the nucleosome core even in the absence of histone tails (We generated nucleosomes without histone tails (tailless) to determine whether Chp1CD mutations specifically abolish interaction with the core of the nucleosome . In vitrne tails . Chp1CD ne tails . These dne tails .As expected, wild-type Chp1CD had the highest affinity for H3K9me3Nucleosomes in our binding assays. When LOOP1B and 2B were mutated, we observed a strong reduction in binding to H3K9me3Nucleosomes by pulldown assay . AlthougAlthough Chp1CD LOOP1B/2B had no defect in binding to RNA , RNA binin vitro binding assays indicate that Chp1CD, after recognition of H3K9me tail, interacts with the core of the nucleosome and this interaction increases the stability of the complex and promotes interaction with RNA.Both, the cryo-EM structure and chp1 genes into chp1Δ fission yeast cells. All chp1 constructs were expressed at similar level  in regions of the nucleosome core that are interacting with Chp1CD are posttraslationaly modified in human cells -Histone H3 (1–21)-GGK(Biotin) peptide (Eurogentec) was incubated with 15 μl of streptavidin agarose resin (Invitrogen) in 20 m−1.For MST SUMO tag was removed from Chp1CD constructs with Ulp1 protease. In total, 100 μg of wild-type and mutant Chp1CD were fluorescently labeled using the MO-L003 Monolith Protein Labeling Kit BLUE-NHS (Amine Reactive) according to manufacturer's instructions . A 1:1 Fluorescence: Protein ratio was estimated by using the Nanodrop1000 software ‘Proteins and Labels’ feature and each Chp1 Chromodomain was run on a 15% SDS acrylamide gel to normalize concentrations to 0.1 mg mlM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM DTT and 0.05% Tween-20. For H3K9me3Nucleosomes-binding assays glycerol was added to 10% final concentraction. For H3K9me3 assays these dilutions were used for measurements: 0 nM, 9 nM, 13 nM, 20 nM, 31 nM, 46 nM, 70 nM, 105 nM, 158 nM, 237 nM, 355 nM, 530 μM, 800 μM, 1.2 μM, 1.8 μM. For H3K9me3Nucleosome assay we used following dilutions for measurements: 0 nM, 18 nM, 27 nM, 41 nM, 62 nM, 93 nM, 140 nM, 210 nM, 316 nM, 474 nM, 711 nM, 1.06 μM, 1.2 μM, 1.4 μM, 1.6 μM, 2.4 μM.Reactions were assembled in 20 μl with 300 ng of fluorescent Chp1CD, and increasing quantities of [Lys(Me3)9]-Histone H3 (1–21)-GGK(Biotin) peptide (Eurogentec) or H3K9me3Nucleosomes, in a buffer containing 10 mMST runs were performed using standard treated capillaries (Nanotemper Cat#K002) on the NT.115 Monolith instrument. All measurements were performed using 80% LED and 40% MST power, with 30 s Laser On time and 5 s Laser Off time. For each experiment, five single measurements were performed.y axis.Data were analyzed with the GraphPad Prism software version 6.00  and the SigmaPlot software version 13.0 . For the peptide-binding assays, with the curves showing a distinct sigmoid trend, we fitted the Richard´s five Parameter Logistic Asymmetric Sigmoidal equation and automatically calculated the Kd. For the H3K9me3Nucleosome-binding assays, as the raw data trend was not anymore sigmoid for all proteins analyzed, a third order polynomial (cubic) equation was used for fitting and comparing the raw data points of wild-type Chp1CD and the different mutants. Kd was calculated based on the ‘Interpolation’ feature of the GraphPad prism software, using the 50% bound/unbound value on the M HEPES pH 7.5, 75 mM KCl, 0.5 mM DTT . Fe. Fe46]. Unsupervised particle classification was performed by random seeding with the complete density maps without focused classification in SPIDER software package. Attempts to use focused classification resulted in a strong bias and noise overfitting in regions of interest. Therefore, we did not to use focused classification to reduce bias. In initial classification done in SPIDER, classes C0–C6 and N0–N5 were backprojected using angles from C0 and N0 maps and this classes were not fully refined. Here we just wanted to select classes that had additional density bound to the nucleosome. Particles generating maps with different Chp1CD densities were separated and further classified. Approximately 20 rounds of random seeding classifications were performed until we have partitioned particles into five different groups . ClassesLocal resolution was calculated using ResMap software  final s. For ChpMolecular models were built using Chimera software package using rigid body fitting of crystal structures . The fit