(Figures 2, 3). These data demonstrate that Par3 and RGS3 are prominently expressed dur- ing RANKL-induced osteoclast differentiation of RAW264.7 cells. We suggest that Par3 and RGS3 may play some important roles in the process of the osteoclast differentiation. Secondly, ephrinB2 was also observed to be prominently expressed in RANKL-induced osteoclast differentiation of RAW264.7 cells (Figure 4). Given the C-terminal YKV motif in the intracellular domain of ephrinB ligands, it was predicted by Zhao et al. that EphB4/ephrinB2 intracellular reverse inhibitory signal mediated via interaction with PDZ domain proteins but not by tyrosine phosphorylation [5, 27]. In order to investigate the effects of reversesignaling on the molecular biology change of PDZ-domain proteins, soluble EphB4-Fc was used to stimu- late ephrinB2 because EphB4 exclusively inter- acts with ephrinB2 [28, 29]. Western blot anal- ysis and real-time RT-PCR results showed that the protein and mRNA expression level of Par3 in the EphB4-Fc treated group was higher than that in the Fc treated group (Figure 5). However, the protein and mRNA expression level of RGS3 in the EphB4-Fc treated group was lower (Figure 6). This indicated that after binding of EphB4, the downstream signal of ephrinB2 reg- ulated the expression level of Par3 and RGS3 in the different ways. However, we need to explain why ephrinB2 reversesignaling leads to the opposite expression results of the intracel- lular Par3 and RGS3? Lee et al. claimed that loss of ephrinB1 likely resulted in tight junction disruption due to increased availability of Par-6, which then forms non-functional tight junction complexes, similar to over-expression of Par-6 [16, 30]. Daar et al. furtherly analysed why loss of ephrinB1 resulted in more accessible Par-6, leading to disrupt tight junction formation . What’s more, in the Par complex, Par3 was found to associate with the Par6/αPKC hetero- dimer via the PDZ-PDZ domain interaction and ephrinB1 associates with the Par polarity com- plex (Par3/Par6/αPKC) protein. Because eph- rinB1 and ephrinB2 share a high degree of identity [32, 33], our results suggested that an in vitro interaction and negative feedback exist- ed between ephrinB2 and the Par3 protein indi- rectly. However, positive feedback existed between ephrinB2 and RGS3 protein. The results showed that expression level of RGS3 was significantly higher in the RANKL+ group compared with the RANKL- group, and down-
Within the Eph/ephrin system, the ligands and receptors that are expressed on different cells can interact in trans. Additional binding can occur, when receptors and ligands are expressed on the same cell, which is called cis interaction. For example, in axons of retinal ganglion cells Eph receptors and ephrin ligands are co-expressed and they interact on the cell surface to achieve a topographic mapping in the tectum (Hornberger et al., 1999; Suetterlin et al., 2012). However, for motor axons it has also been shown that Eph receptors and ephrins are co-expressed on the same axon, but in separate membrane domains, which prevents cis interactions. In this case, the Eph receptors and ephrin ligands signal opposing effects on the growth cone: EphAs mediate repulsion through forward signaling and ephrin As attraction through reversesignaling (Marquardt et al., 2005). Given that EphA4 is co-expressed with ephrin ligands on MGE-derived interneurons (Fig. 5B), a cis interaction could mediate the motogenic effect of EphA4 in a cell- autonomous manner. However, as mentioned above, only 3% of the ephrin A2 and EphA4 proteins were colocalized on the cell surface. Moreover, dissociated interneurons do not perform nuclear translocation without extrinsic factors, and a knockdown of ephrin A2 alone does not change the migration ability of the interneurons. Thus, it seems more likely that the interneurons are activated by neighboring cells via trans interactions rather than by cell- autonomous cis interactions. By preventing cis interactions, EphA4 forward signaling can mediate the effects of repulsive guidance cues, such as ephrin A3, A5 and B3, and at the same time EphA4 reversesignaling can trigger motogenic effects in neighboring cells via ephrin A2.
In NIH3T3 fibroblast cells, expression of full-length Sema6A induces a change in cell shape, with an increase in surface area and in membrane complexity, with numerous filopodia appar- ent. It also confers responsiveness to the extracellular domain of PlxnA2. PlxnA2 has no effect on untransfected or GFP-transfected cells but induces cellular collapse of cells transfected with full-length Sema6A. By contrast, cells transfected with a mutant form of Sema6A that does not bind PlxnA2 show the increase in cellular area and complexity but no response to PlxnA2. These data suggest that not only can Sema6A initiate reversesignaling–it can do it in two dis- tinct modes. The fact that the effects of stimulated and unstimulated signaling are qualitatively distinct, perhaps even opposite to each other, argues against the idea that constitutive signaling is merely due to over-expression. We speculate that these two modes may reflect signaling by unclustered dimers of Sema6A versus multimerised clusters induced by ligand binding.
the presence of the EphB2 receptor, which is thought to act postsynaptically by modulating glutamate receptors (Grunwald et al. 2001; Henderson et al. 2001; Contractor et al. 2002) and which was shown to associate with and to induce clustering of NMDA receptors (Figure 9). This in turn may suggest that ephrinB ligands in synaptic membranes induce the maturation of glutamatergic synapses by promoting NMDA receptor aggregation (Dalva et al. 2000). A model in which ephrin- Eph signaling influences activity-dependent processes (Ca 2+ influx) by modulating gene expression during development of synaptic connections comes from data obtained from very young cultured neurons, which showed enhanced NMDAR- mediated Ca 2+ influx and potentiated cAMP-response element binding protein (CREB)-phosphorylation and -dependent transcriptional events upon stimulation with ephrinB1 (Grunwald et al. 2001; Takasu et al. 2002). This might mechanistically occur by the recruitment and activation of SFKs, which phosphorylate certain subunits of the NMDAR (Grunwald et al. 2001; Takasu et al. 2002) Activation of SFKs is required for EphB2-mediated enhancement of Ca 2+ influx, at least in a transfected cell system (Takasu et al. 2002). These in vitro observations are consistent with findings from EphB2-deficient mice (Grunwald et al. 2001; Henderson et al. 2001) showing a reduction or even loss of hippocampal long-term potentiation and long-term depression correlated with a redistribution of a significant fraction of NMDA receptors. Interestingly, targeted expression of a kinase-deficient EphB2 receptor isoform rescued the defects in EphB2-deficient mice, suggesting that ephrinB ligand reversesignaling may be the active signaling partner at the synapse (Grunwald et al. 2001; Henderson et al. 2001).
Intriguingly, however, it has never been possible to reconstitute topographically appropriate fiber growth in vitro with these cues. Moreover, experimentally derived molecular mechanisms have failed to provide explanations as to why the mapping adapts to grossly diverse targets in some experiments, while displaying strict point-to-point specificity in others. In vitro, ephrin-A/EphA forward, as well as reverse, signaling mediate differential repulsion to retinal fibers, instead of providing topographic guidance. We argue that those responses are indicative of ephrin-A and EphA being members of a guidance system that requires two counteracting cues per axis. Experimentally, we demonstrate by introducing novel double-cue stripe assays that the simultaneous presence of both cues indeed suffices to elicit topographically appropriate guidance. The peculiar mechanism, which uses forward and reversesignaling through a single receptor/ligand combination, entails fiber/fiber interactions. We therefore propose to extend Sperry’s model to include ephrin-A/EphA-based fiber/fiber chemospecificity, eventually out-competing fiber/target interactions. By computational simulation, we show that our model is consistent with stripe assay results. More importantly, however, it not only accounts for classical in vivo evidence of point-to-point and adaptive topographic mapping, but also for the map duplication found in retinal EphA knock-in mice. Nonetheless, it is based on a single constraint of topographic growth cone navigation: the balancing of ephrin-A/EphA forward and reversesignaling.
Background: Anti TNF drugs have been widely used in rheumatoid arthritis (RA) but only 70 to 80 % of patients respond to this therapy. Exploring the mode of action of anti-TNF drugs remains important in order to improve the efficiency of the treatment and enhance our knowledge of inflammation. TNF- α exists as classical soluble cytokine as well as transmembrane protein (tmTNF- α ). Evidence suggests that tmTNF- α can induce reversesignaling. In the present study, we have explored consequences of reversesignaling in human monocytes using certolizumab pegol (CZP). Methods: Monocytes were purified from healthy blood donors and were incubated with CZP. Nuclear translocation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was evaluated by wide-field microscopy and cell fractionation. Heme oxygenase 1 (HO-1) was assessed by RT-qPCR and western blot. Monocytes were stimulated with lipopolysaccharide (LPS). IL-1 β was quantitated by RT-qPCR. Reactive oxygen species (ROS) were evaluated by flow cytometry using the H 2 DCFDA fluorescent marker.
EPHB2 in SEVs promotes endothelial tube formation. In Western blot analyses, EPHB2 protein was expressed in SEVs from multiple human HNSCC cell lines (Figure 3A). As it was expressed at the highest levels in OSC19 cells, we focused on this cell line for most further mechanistic studies. Consistent with the ability of OSC19 SEVs, but not LEVs, to induce tube formation, we detected very little EPHB2 expression in LEVs in comparison with SEVs (Figure 3A). EPHB2 can induce angiogenesis by binding to B-type ephrins on endothelial cells and inducing ephrin reversesignaling, a process that is thought to require cell-to-cell contacts (32, 55). We hypothesized that SEVs could substitute for neighboring cells and induce ephrin reversesignaling at a distance. To test this possibility, we examined phosphorylation of ephrin-B in HUVECs after incubation with OSC19 SEVs. As a positive control, we added recombinant EPHB2 (Fc-EPHB2). As shown in Figure 3B, both Fc-EPHB2 and purified OSC19 SEVs induced ephrin-B phos- phorylation in HUVECs, compared with no-treatment control. To test the role of SEV-associated EPHB2 in angiogenesis, we blocked the ability of EPHB2 to bind to ephrin-B ligands on cells by preincubating the OSC19 SEVs with recombinant ephrin-B2 protein (Fc–ephrin-B2) for 10 minutes before adding the mixture to endothelial cells (Figure 3C). Compared with control SEVs, uptake of SEVs preincubated with Fc–eph- rin-B2 by HUVEC cells was decreased (Supplemental Figure 4), suggesting that EPHB2–ephrin-B interac- tions mediate binding of a subset of SEVs with cells. Likewise, preincubation of Fc–ephrin-B2 with SEVs reduced their ability to induce HUVEC tube formation in a dose-dependent manner (Figure 3D). A similar result was observed using SEVs purified from Detroit 562 cells (Figure 3D). Recombinant Fc–ephrin-B2 protein alone had only a small effect in the opposite direction at the highest concentration.
Similarly, the induction of high levels of the anti- inflammatory cytokine IL-10 can also contribute to the therapeutic response if present in vivo. IL-10 production elicited by tmTNF reversesignaling has been reported previously  and interpreted as a potent anti-inflam- matory response. In line with this hypothesis, we found increasing concentrations of IL-10 in sera of the patients under anti-TNF treatment, although this trend did not reach statistical significance. The significant association of IL-10 serum concentrations determined at baseline with the disease activity indicates, that up-regulation of this IL-10 secretion might indeed serve as an anti-inflam- matory attenuator in the chronic autoimmune disease.
antenna lobe in a graded fashion with the highest concentration in the dorsolateral region. This pattern is established by the PNs themselves and the PNs with the highest level of Sema-1a target to the most dorsolateral glomeruli. Loss of Sema-1a leads to dendrite and axon mistargeting, which can be rescued cell autonomously with full-length Sema-1a. A Sema-1a transgene without the cytoplasmic domain fails to rescue these defects in either dendrites or axons. Overexpression of Sema-1a in PNs results in a directional shift toward the dorsolateral zone of the antenna lobe (Komiyama et al., 2007). This targeting is achieved through the repulsive action of Sema- 1a reversesignaling in PNs in response to an opposing gradient of the secreted Sema-2s. Sema-2a and Sema-2b are expressed in overlapping patterns and function redundantly to repel Sema-1a expressing PNs. Sema- 2a and Sema-2b single mutants lack PN defects, yet the double mutants display significant mistargeting errors. This interaction is likely to be indirect since physical interactions between Sema-1a and Sema-2a or Sema-2b could not be demonstrated. However, Sema-1a can bind in vivo to tissue
ephrinB2 (1:2000, clone number EPR10072(B), Abcam, Cambridge, UK), phospho-ephrinB2 (Tyr324/329, 1:500, Cell Signaling Technology, Danvers, MA, USA), EphB4 (1: 200; Santa Cruz Biotechnology, Dallas, TX, USA), phospho-EphB4 (1:1000; Signalway Antibody, College Park, MD, USA), EphB1 (1:50; Affinity Biosciences, Changzhou, Jiangsu, China), EphB2 (1:50; Affinity Biosciences, Chang- zhou, Jiangsu, China), or β-actin (1:3000, Beyotime, Shang- hai, China) overnight at 4 °C. After washing with PBST, the membranes were incubated with horseradish peroxidase- conjugated secondary antibodies (Proteintech, Wuhan, Hubei, China) for 2 h at room temperature. Protein blots were detected using a chemiluminescence kit (NCM Bio- tech, Suzhou, Jiangsu, China) and Tanon 4500 Immunode- tection System (Tanon, Shanghai, China). Gray values were analyzed by ImageJ (Rawak Software, Germany).
44 ephrinB ligands (ephrinB1-3) span the membrane and have a short, highly conserved cytoplasmic tail. Unlike the Eph receptors, ephrin ligands do not have an intrinsic catalytic domain and must therefore transduce their signal with the help of cytoplasmic proteins. The cytoplasmic tail of ephrinB ligands contains five conserved tyrosines, that serve as potential phosphorylation sites, and a PDZ-binding motif ( Figure 4-12, a ). EphrinB- activation leads to the rapid phosphorylation of their cytoplasmic tyrosine residues (Bruckner et al., 1997), three of which are characterized as major phosphorylation sites (Kalo et al., 2001). The rapid phosphorylation is known to be mediated by Src family kinases (SFKs) that become activated and are recruited to ephrin clusters upon Eph receptor binding (Palmer et al., 2002). Cytoplasmic proteins can interact with ephrinB ligands via SH2 domains that recognize phosphorylated tyrosines. So far, the only SH2- domain protein that has been found to interact with tyrosine-phosphorylated ephrinB molecules is the growth-factor-receptor-bound protein 4 (Grb4). Grb4 is a SH2/SH3 domain-containing adaptor protein. Its association with activated ephrinBs leads to the disassembly of F-actin-containing stress fibers and increased FAK activation (Cowan and Henkemeyer, 2001). Similar to forward signaling through Eph receptors, the regulation of cytoskeletal rearrangement seems to be one of the major effects of ephrinB-reversesignaling. However, the signaling induced by rapid tyrosine phosphorylation is a short- lived event. With delayed kinetics, the cytoplasmic PDZ-domain-containing protein tyrosine phosphatase PTP-BL is recruited to activated ephrinB ligands thereby terminating the signal by ligand and SFK dephosphorylation. In contrast, PDZ-interactions were shown to persist (Palmer et al., 2002). Based on these findings, a switch-model for ephrinB-reversesignaling has been established distinguishing the fast, but short-lived phosphotyrosine-dependent signaling from the delayed, but persistent, PDZ-dependent signaling ( Figure 4-12, (b) ).
pathway. Interestingly, the crosstalk between Corticos- teroids and cardioprotection pathway and Insulin signal- ing pathway is mediated by AKT according to our pathway-based network. Previous study also has shown that Calcium signaling pathway is associated with dopa- mine-induced cortical neuron apoptosis which is consid- ered as an important mechanism in SCZ pathogenesis . Meanwhile, Actions of Nitric Oxide in the Heart, another pathway for both SCZ and T2D, is a crosstalk between Calcium signaling pathway and Insulin signal- ing pathway either. Previous study indicated that Nitric oxide was involved in pathophysiology of SCZ . IL-10 Anti-inflammatory signaling pathway is an immune-related pathway. Accumulated evidence from epidemiological, clinical and animal studies suggests that immune-related pathway may play a key role in the development of mental diseases including SCZ and mood disorders [69,70]. IL-10 Anti-inflammatory Signal- ing Pathway has been reported previously to be involved in pathophysiology of SCZ  and T2D , respec- tively. Therefore, the above evidence suggests that IL-10 Anti-inflammatory signaling pathway may be involved in the pathogenetic association between SCZ and T2D. In another perspective, due to inflammation contributes to injury or enhances CNS vulnerability, and acute inflam- mation can also be shifted to a chronic inflammatory state and adversely affect brain development, therefore, through efficient anti-inflammatory and reparative pro- cesses, inflammation may resolve without any harmful effects on the brain. Alternatively, intervention of TNF- a, before the progressive loss of beta cell function, may yield promising results in the treatment of T2D. Since IL-10 is a cytokine with potent anti-inflammatory prop- erties, it represses the expression of inflammatory cyto- kines such as TNF- a , IL-6 and IL-1 by activated macrophages. The anti-inflammatory actions of IL-10 may be therapeutically useful by intervention of TNF- a , IL-1 or IL-6 to avoid inflammatory response, then to decrease the CNS vulnerability, further to reduce the chance to trigger T2D.
Abstract. In this paper Reverse Wiener index, Reverse Detour Wiener index, Reverse Circular Wiener index Reverse Harary index, Reverse Detour Harary index, Reverse Circular Harary index, Reverse Reciprocal Wiener index, Reverse Detour Reciprocal Wiener index, Reverse Circular Reciprocal Wiener index, Reverse Hyper Wiener index, Reverse, Detour Hyper Wiener index, Reverse Circular Hyper Wiener index, Reverse Gutman Wiener index, Reverse Detour Gutman Wiener index, Reverse circular Gutman Wiener index are defined using the diameter and Detour diameter of a graph and the above indices have been estimated for the Cartesian product P 2 and C n .
Functional annotation for significant modules is determined using ConsensusPathDB . We query genes in the top-scoring modules for overrepresentation in curated pathways. Canonical cancer pathways and pathways associated with hallmarks of cancer are enriched in each cancer dataset: cell-cycle control, DNA replication/repair, cellular adhesion/migration, apoptosis, angiogenesis, evasion of the immune response and immortality. A summary of the statistics and representative pathways for the top- scoring modules is presented in Table 2. BC modules are highly enriched with cell cycle control, growth signaling, focal adhesion, and angiogenesis control genes. A number of BC modules are also annotated with progesterone, estrogen and steroid hormone signaling; and levels of these hormones are known to correlate with BC risk. In HCC, detoxifying pathways including cytochrome P450, UBR, HSD detoxifying pathways and fatty acid metabolism, are among the most enriched pathways. Inflammation and deregulation of liver-related detoxifying pathways are frequent markers of carcinogenic toxicity, oxidative stress, and tumorigenesis. Chronic inflammation and the immune response are associated with adenoma formation in the colon; and several related path- ways are over-represented in CCA, including chemokine, cytokine, T-cell receptor, fatty acid metabolism, and intestinal immunity. Wnt signaling is a key pathway in early stages of colorectal cancer and is enriched in CCA modules. Amino acid synthesis and metabolism pathways, associated with stability of DNA replication and repair, are over- represented across all three cancer types, although most notably in HCC.
The RLRFE (reverse logistic recycling flow equilibrium) problem as a flow equilibrium problem from a system wide policy-making perspective, focusing particularly on equilibrium in situations in which market price and recycling channel flows are coupled interactions and input-output recycled material flows at each agent are not balanced. They propose a three-loop nested diagonalization method in which asymmetric link interactions are gradually relaxed to achieve the equilibrium solution (Kara et al., 2007)
However, with the Reverse DTML-Conversion model, all steps with respect to length (i.e. one dimension distance) , mass and litre S.I units are in multiples of ten (10). All steps with respect to Area (i.e. two dimension distance) are in multiples of hundreds (100), all steps with respect to volume (i.e. three dimension distance) are in multiples of thousands (i.e. 1000), and all steps with respect to time are in multiples of sixty (60). All units on the number line started with zero mark through ten steps, hundred steps, thousand step and sixty steps each, and also in place of the basic unit for both higher units and smaller as in metric mania and the stair-step have been replaced with the smallest unit and the highest unit on a straight line in ten steps, hundred steps, thousand step and sixty steps.