Genetic factors play a major role in the risk for neurodevelopmental disorders such as autism spectrum disorders (ASDs) and intellectual disability (ID). The underlying genetic factors have become better understood in recent years due to advancements in next generation sequencing. These studies have uncovered a vast number of genes that are impacted by different types of mutations .Given the large volume of genetic data, analyzing each gene on its own is not a feasible approach and will take years to complete, let alone attempt to use the information to develop novel therapeutics. To make sense of independent genomic data, one approach is to determine whether multiple risk genes function in common signaling pathways that identify signaling “hubs” where risk genes converge. This approach has led to multiple pathways being implicated, such as synaptic signaling, chromatin remodeling, alternative splicing, and protein translation, among many others. In this review, we analyze recent and historical evidence indicating that multiple risk genes, including genes denoted as high-confidence and likely causal, are part of the Wingless  pathway. In the brain, Wnt signaling is an evolutionarily conserved pathway that plays an instrumental role in developing neural circuits and adult brain function.We will also review evidence that pharmacological therapies and genetic mouse models further identify abnormal Wnt signaling, particularly at the synapse, as being disrupted in ASDs and contributing to disease pathology. ASDs and other psychiatric disorders may have heritability estimates greater than 90% , suggestFMR1), Rett syndrome (MECP2), Angelman syndrome (UBE3A), and genes that cause non-syndromic forms of ASDs  have important roles during synapse development and refinement [It is no surprise that the clinical heterogeneity of ASDs can be explained, at least in part, by the large number of genetic mutations found through next-generation sequencing. The number of mutations discovered ranges in the several hundreds, according to well-known sources such as the Simons Foundation Autism Research Initiative (SFARI). There is much difficulty in trying to understand the biological etiology of ASDs when so many genes are involved. One hypothesis is that the genetic lesions disrupt specific signaling pathways during discrete time points of brain development. For example, many of the initial genetic studies identified genes involved in synapse development and refinement. This was largely based on findings that multiple genes in syndromic forms of ASDs , which upon stabilization, can exert functions at the plasma membrane or in the nucleus and can act as a transcription factor that modulates the expression of target genes  signaling. This pathway is highly studied and conserved from lower to higher organisms, where it plays a variety of roles in almost all tissues. Broadly speaking, Wnt signaling in the brain can be divided into two main pathways: (i) “canonical” signaling that results in the stabilization of the protein β-catenin . Therefore, future work needs to determine the precise mechanisms and time points during which CHD8 regulates Wnt signaling during neurodevelopment. This is important to better comprehend how prenatal brain development could be compromised in individuals with CHD8 mutations.The strongest single candidate gene for non-syndromic ASDs is chromodomain helicase DNA binding protein 8 (CHD8) –30. Therith ASDs , 31–34. ith ASDs , 34–36. ith ASDs , 38. Preith ASDs . Furtheread size , 40. Howead size . Given tCTNNB1 gene in patients with ASD using exome sequencing [β-catenin is a central player in the canonical Wnt signaling pathway and works with co-factors to initiate Wnt-dependent gene transcription Fig. . It has quencing , 29, 42.quencing . The relquencing . In contquencing . CTNNB1 from ID patients when expressed in mice have a reduced affinity for membrane-associated cadherins [β-caxtenin also functions in pathways other than canonical Wnt signaling, such as cell adhesion at the plasma membrane. β-catenin interacts with cadherins to regulate dendritic spine growth and synaptic competition during the process of postnatal period of dendritic pruning . A recenadherins . This isadherins . Therefoadherins .PTEN due to the discovery of numerous individuals with mutations [ PTEN are also at risk for macrocephaly, indicating that PTEN regulates brain size, which is thought to be an impact on certain individuals with ASD [Pten have further strengthened the notion that it is an important regulator of different neural circuits associated with ASDs and cognition. For example, an early study identified that global Pten +/- mice have impairments in social interaction behaviors [Pten in the cerebellar Purkinje cells led to impaired sociability, repetitive behavior, and deficits in motor learning. Additionally, knocking out Pten in a subset of cortical excitatory neurons results in profound synaptic signaling changes [Pten alleles expressed in inhibitory neurons cause excitatory/inhibitory imbalances [Pten (Pten +/-), and a heterozygous mutation in β-catenin suppresses the excessive cortical brain growth in Pten+/- mice [Another high-risk autism candidate gene that has roles in Wnt signaling is utations , 48–50. with ASD –59. Multehaviors . Knockin changes , while Abalances . Interes+/- mice . This inCTNNB1 within individuals with developmental delay and ID who do not have an ASD [TCF7L2 ) [TCF4, and is confused with transcription factor 4 (TCF4) as they share the same symbol. Importantly, TCF7L2 is a key player in canonical Wnt signaling as it helps to initiate gene transcriptional response when Wnt ligands bind their receptors on the membrane, and the signal is transduced to the nucleus ) , 64, whiDEAD-box helicase 3, X-linked (DDX3X) in a population of unexplained ID [A recent study identified de novo mutations in ained ID . DDX3X wained ID . Therefodisheveled genes , Dvl1 or Dvl1/3 KO mice display adult social and repetitive behavioral abnormalities, which are the core features of ASD symptoms [Mouse models provide support for Wnt signaling as a clinically relevant pathway for developmental cognitive disorders. First, modeling of high-risk  ASD genes, for example using gene knockouts (KO), offer the opportunity to determine the neural circuits and brain regions responsible for causing ASD-like behavior. Second, interrogation of other genes in the same pathway, which are not directly involved in ASD from human genetic studies, offers the opportunity to further support that pathway in ASD pathophysiology. For example, while no ASD-specific genetic mutations have been identified in the symptoms –69. Thisglycogen synthase kinase 3 (GSK3) α and β, which is a negative regulator of canonical Wnt signaling and also plays important roles directly at the synapse  mice display behaviors that resemble wild type (WT) mice treated with lithium, a drug that is used to treat bipolar disorder [Gsk-3β in excitatory neurons leads to anxiolytic and pro-social effects [In addition to Dvl1/3, recent studies have highlighted that conditional or complete KO mouse models of other genes involved in Wnt signaling support the pathway being involved in ASD-like phenotypes. One of the best-studied genes is disorder , demonst effects , suggest effects , 74. Fra effects , 76, and effects –82. Furt effects , 84. Of disrupted in schizophrenia 1 (DISC1). While the genetic evidence linking DISC1 to developmental cognitive disorders is not strong, the multiple cellular and mouse models of Disc1 perturbation has led to important findings linking Wnt signaling to abnormal neurodevelopment. For example, a landmark study initially described DISC1 as an inhibitor of GSK-3β, similar to the actions of lithium, demonstrating that DISC1 positively regulates canonical Wnt signaling [Disc1 demonstrate that DISC1 perturbation causes significant neurodevelopmental phenotypes, including cognitive defects and psychiatric-like behavioral manifestations [Another well-studied gene in relation to ASD and psychiatric disorders is ignaling , which hignaling –89. Sevestations –94.AnkyrinG (Ank3), which was found to possess a genome-wide significant signal in bipolar disorder [Ank3 heterozygous mice possess behavioral phenotypes such as reduced anxiety and increased motivation for reward, which can be corrected by modulating Wnt signaling through GSK-3β [Ank3, other recently characterized genes in mice also support a role for Wnt signaling in neurodevelopmental disorders. DIX domain containing 1 (DIXDC1) is a positive regulator of Wnt signaling and neurogenesis through binding to DISC1 [Dixdc1 KO mouse displayed behaviors associated with neuropsychiatric disorders such as abnormal startle reflex and reduced social interaction [There are other known regulators of Wnt signaling that when disrupted leads to neurocognitive and neurodevelopmental phenotypes. A recent example is disorder , 96. Ankdisorder . It has disorder . Interesdisorder . Ank3 heh GSK-3β , demonstto DISC1  and a Dieraction . The beheraction , 102.adenomatous polyposis coli (APC), which is a critical component of the destruction complex in the canonical Wnt pathway and is important for neural plasticity, learning, and memory in mice. A conditional Apc KO mouse showed increased synaptic spine density, elevated frequency of miniature excitatory postsynaptic potentials (mEPSPs), enhanced long-term potentiation (LTP), and ASD-like behaviors  [Prickle2 mouse model demonstrates its importance in ASD-related neural circuits and behavior [Prickle2 KO mouse has previously been shown to be more sensitive to seizures and also shows reduced dendrite branching, synapse number, and postsynaptic density (PSD) size, as well as behavioral abnormalities . Although the involvement of Prickle2 implicates non-canonical Wnt signaling, the phenotypes associated with the KO mouse demonstrate that multiple aspects of Wnt signaling  are important for the establishment of neural circuits that are disrupted in ASD. While the studies on APC and Prickle2 do not directly implicate abnormal Wnt signaling, we speculate that these mice would have alterations in this pathway due to the importance of these molecules in Wnt signaling in neural cells. Third, a recent study identified that rare missense variants in the Wnt1 gene discovered in ASD patients show abnormal activation of the Wnt signaling pathway, providing evidence that subtle changes to the coding sequence of Wnt signaling molecules alter biological signaling [There are three other Wnt signaling-related genes that have been characterized in mice that lend further support to the involvement of Wnt signaling in developmental cognitive disorders see Fig. . The firnterest) . Second,behavior , 105. Prbehavior . The Priignaling . TogetheIn addition to animal models, there are two specific drug-induced models that implicate Wnt signaling. The first is valproic acid (VPA), which is thought to increase the risk for ASD through exposure to a pregnant woman during prenatal development . The admThere is only one FDA approved for ASDs, which is used to treat irritability associated with ASDs , demonstrating the urgent need to find new medications. Many of the medications used are “off-label”  and do not treat the core symptoms, and can have very strong side effects. While these medications have multiple modes of molecular action, interestingly, many impact Wnt signaling. For example, haloperidol  is known to inhibit dopamine receptors, thereby increasing GSK3β inhibition through Akt activation , which i, since embryonic brain development is most affected by such genetic mutations, posing ethical issues for pre-diagnosis therapies.Many times the medications tested in pilot clinical trials for neurological disorders are failed drugs from cancer trials. There are many drugs developed and tested as modulators of Wnt signaling in the cancer field that could potentially be repurposed for developmental cognitive disorders. In cases where a reduction in Wnt signaling is thought to underlie the pathology of the disorder, usage of compounds that elevated canonical Wnt signaling could be applied. An example of this is GSK-3β inhibitors that have failed in cancer trials but may be effective for ASDs and ID . In cases where elevated Wnt signaling is thought to contribute to disease pathology, there are many potential options to inhibit canonical Wnt signaling using chemicals Fig.  that inhThe goal towards better understanding and treating developmental cognitive disorders is a difficult road and will require a multifaceted research and clinical approach to be successful. In this review, we present evidence that one such signaling pathway that may be central to disease pathogenesis and treatment is the Wnt signaling network. Ongoing and future genetic studies will need to determine the strength of association of this pathway with disease; however, given the medications and drugs that target this pathway currently available, this presents an opportunity for new clinical trials in the near future.


Appropriate level of Dab2 expression is essential for fate determination of mesodermal and megakaryocytic differentiation. Dab2 is also shown to regulate cell-cell and cell-fibrinogen adhesion, integrin αIIbβ3 activation, fibrinogen uptake, and intracellular signaling of the megakaryocytic cells. In human platelets, p82 is the sole Dab2 isoform present in the cytoplasm and α-granules. Dab2 is released from the α-granules and forms two pools of Dab2 on the outer surface of the platelet plasma membrane, one at the sulfatide-bound and the other at integrin αIIbβ3-bound forms. The balance between these two pools of Dab2 controls the extent of clotting reaction, platelet-fibrinogen interactions and outside-in signaling. In murine platelets, p59 is the only Dab2 isoform and is required for platelet aggregation, fibrinogen uptake, RhoA-ROCK activation, adenosine diphosphate release and integrin αIIbβ3 activation stimulated by low concentration of thrombin. As a result, the bleeding time is prolonged and thrombus formation is impaired for the megakaryocyte lineage-restricted Dab2 deficient mouse. Although discrepancies of Dab2 function and isoform expression are noted between human and murine platelets, the studies up-to-date define Dab2 playing a pivotal role in integrin signaling and platelet activation. With the new tools such as CRISPR and TALEN in the generation of genetically modified animals, the progress in gaining new insights into the functions of Dab2 in megakaryocyte and platelet biology is expected to accelerate.Multiple functions of platelets in various physiological and pathological conditions have prompted considerable attention on understanding how platelets are generated and activated. Of the adaptor proteins that are expressed in megakaryocytes and platelets, Disabled-2 (Dab2) has been demonstrated in the past decades as a key regulator of platelet signaling. Dab2 has two alternative splicing isoforms p82 and p59. However, the mode of Dab2’s action remains to be clearly defined. In this review, we highlight the current understanding of Dab2 expression and function in megakaryocytic differentiation, platelet activation and integrin signaling. Accordingly, Dab2 is upregulated when the human K562 cells, human CD34 The crute (ADP) . Collagete (ADP) . Other sectively , 6. The ectively . DespiteAdaptor protein is a type of proteins mediating protein-protein and protein-lipid interactions. It has been clearly demonstrated that adaptor proteins are essential for coupling membrane receptors to intracellular signaling pathways and the assembly of signaling scaffolds within the cells. Many adaptor proteins expressed in the platelets are involved in inside-out and outside-in signaling of integrin during platelet activation . Disabledab2 gene is located at the chromosome 5p13 and was first identified by Mok et al. as the tumor suppressor gene of the ovary cancer in 1994 [dab2 as the differentially expressed gene that was up-regulated in the castrated rat prostate [2), and a subset of receptors such as integrin, low density lipoprotein receptor, megalin and related receptors that contain the non-tyrosine-phosphorylated NPXY motif [Human  in 1994 . The mou in 1994 . In 1998prostate . BecauseXY motif –21. The XY motif –26. By iXY motif , 27–36. XY motif , 37–39.FDab2 is a phosphoprotein with several phosphoryation sites having been identified. Dab2 is phosphorylated at serine residues in murine macrophage cell line in response to mitogenic stimulation by CSF-1 . Dab2-Se+ hematopoietic pluripotent stem cells, and murine embryonic stem cells (ESCs) are undergone megakaryocytic differentiation  mice have been generated by using the Cre-loxP transgenic system driven by the platelet factor 4 promoter to elucidate Dab2 functions in vivo [-/- platelets, when stimulated by low concentrations of thrombin, are defective in platelet aggregation, clot retraction, and spreading on fibrinogen. The functional imperfection of Dab2-/- platelets is related to the defective responses to thrombin-induced RhoA-ROCKII and Akt-mTOR activation, ADP release and integrin αIIbβ3 activation [-/- mice. These findings are consistent with the perception that protease-activated receptors (PAR) 3- and PAR4-deficient mice, despite eliciting a partial decrease in thrombin-induced platelet aggregation, are impaired in hemostasis and are protected against thrombosis [Megakaryocyte/platelet lineage-restricted Dab2 knockout (Dab2 in vivo . Dab2-/-tivation . Althougrombosis , 51. TheThe study linking Dab2 functions with megakaryocytic differentiation, platelet signaling and integrin activation was first reported 15 years ago . The stuDab2 is known as a phosphoprotein in a variety of cellular processes , 40–43. Multiple functions of platelets in hemostasis, thrombosis, immunology, cancer progression, microorganism infections call for considerable attention on understanding how the activated platelets transmit intracellular signal to elicit their roles in different biological responses. Extensive evidence from the studies in the past decades demonstrates that Dab2 is a key regulator of platelet signaling, in particular, the endocytosis and the activation of thrombin-stimulated inside-out signaling of platelet integrin. With the complicated nature of the signaling network within megakaryocytes and platelets, the definitive roles of Dab2 in megakaryocytic differentiation, platelet activation and integrin signaling remain to be explored further.


Phase matching is shown to provide a tunable gate that helps discriminate entangled states of light generated by four-wave mixing (FWM) in optical fibers against uncorrelated photons originating from Raman scattering. Two types of such gates are discussed. Phase-matching gates of the first type are possible in the normal dispersion regime, where FWM sidebands can be widely tuned by high-order dispersion management, enhancing the ratio of the entangled-photon output to the Raman noise. The photon-entanglement gates of the second type are created by dual-pump cross-phase-modulation-induced FWM sideband generation and can be tuned by group-velocity mismatch of the pump fields. Four-wave mixing (FWM) in optical fibers provides a compact, tunable, and efficient source of quantum states of light123512345Raman scattering has long been recognized as a major physical factor that limits the performance of fiber-optic sources of quantum states of light1616In a broader context of classical nonlinear optics, the interplay between FWM and Raman scattering gives rise to a vast variety of nonlinear-optical field evolution scenarios. In optical fibers, FWM effects have been shown1921231830Here, we demonstrate that the Raman noise can be radically reduced in fiber-optic FWM-based photon entanglement generation through carefully tailored phase matching, which provides a tunable gate that helps discriminate entangled photon pairs against uncorrelated photons originating from Raman scattering. Two types of phase-matching gates will be considered. Phase-matching gates of the first type are possible, as shown below in this paper, in the normal dispersion regime, where FWM sidebands can be widely tuned by high-order dispersion management, enhancing the ratio of the entangled-photon output to the SpRS noise. The photon-entanglement gates of the second type are created by dual-pump cross-phase-modulation-induced FWM sideband generation and can be tuned by group-velocity mismatch of the pump fields.ωp = ωs + ωa FWM process where two pump photons of the same frequency, ωp, give rise to idler and signal photons  with frequencies ωs and ωa. In the undepleted-pump approximation, the Hamiltonian HFWM that describes all the FWM processes coupling these fields is quadraticaj (j = s and a for the Stokes and anti-Stokes fields), defined in such a way as to satisfy the commutation relations aj, can be written in the input–output form asμ(z) and ν(z) being the transfer functions.We consider a generic 2ωp1 = ω2p = ωp and 2ωp = ωs + ωa, and the FWM HamiltonianP0 is the peak power of the pump, γ is the Kerr nonlinearity coefficient, δ = Δβ/2 + γP0, Δβ = βs + βa − 2βp, and βp, βs, and βa are the propagation constants of the pump, Stokes, and anti-Stokes fields, the solution for aj reduces to12In the case when both pump photons are taken from the same pump field, μ(z) = cos(κz) + i(δ/κ)sin(κz), ν(z) = i(γP0/κ)sin(κz),κ2 = δ2 − (γP0)2.where aj and nj(z)〉 = |ν(z)|2. For a two-mode input vacuum state With cn = (μ*)−1(ν/μ*)n, we find IsIa = 〈ns〉〈na〉 = |ν|4, leading to the following expression for the cross-correlation coefficient: ρ0(z) = |μ(z)|2/|ν(z)|2.Quantum correlations between the Stokes and anti-Stokes photons are quantified in terms of the cross-correlation coefficientaj and mjgR(Ω) is the Raman gain. With the Raman effect added, the spectral density Sj(z) of the Stokes (j = s) and anti-Stokes (j = a) photon flux η = 1 + fR(R1 + R2 − 1), fR is the Raman fraction of the nonlinear refractive index, R1 and R2 are the Fourier transforms of the isotropic and anisotropic parts of the Raman response, such that gR = 2γfRIm(R1 + R2), ra(z) = nth(Ωa), rs(z) = −nth(Ωs) − 1, Ωj = ωj − ωp, nth(Ω) = [exp(ħ|Ω|/θ) − 1]−1 is the thermal photon number, θ = kBT, kB is the Boltzmann constant, and T is the temperature.The Raman effect is included in the model of FWM sideband generation through the inertial part of the nonlinear-optical responsenth(Ω). As a result, the Raman noise decreases correlations between the Stokes and anti-Stokes photons. The degree of this correlation degradation, however, strongly depends on the phase mismatch Δβ. This dependence, as shown below in this paper, helps discriminate entangled states of light generated by FWM against uncorrelated photons originating from Raman scattering.Unlike FWM, which can generate strongly correlated Stokes and anti-Stokes photon pairs as a part of the two-mode squeezed-state FWM output of β| ≫ γP0. In this regime, the spectral density of the photon-pair flux is given by Sj(z) = Fj(z) + SR, with the FWM part of the flux, βz/2) = [sin(Δβz/2)]/(Δβz/2) phase-mismatch factor. When |Δβz| ≫ 1 and γP0z is kept small, γP0z ≪ 1, to avoid an excessive degradation of photon-pair correlations as dictated by ρ0(z) = |μ(z)|2/|ν(z)|2, the Raman noise dominates over the FWM photon-pair flux, giving rise to uncorrelated Stokes and anti-Stokes photons with ρ(z) ≪ 1.To understand the significance of phase matching for correlated photon-pair generation, we first consider the case of large phase mismatch, |Δδ is small, FWM sidebands are strongly coupled, giving rise to correlated Stokes and anti-Stokes photons. Moreover, the FWM parametric gain is at its maximum at δ = 0, providing the highest efficiency of FWM sideband generation. Within the FWM parametric gain band, i.e., for δ < γP0, the solutions for aj and μ(z) = cosh(gz) + i(δ/g)sinh(gz), g2 = (γP0)2 − δ2. At the center of the FWM parametric gain band, δ = 0, the two-mode squeezed-state output is In the opposite limit, when γP0z ≪ 1 and δ = 0 regime is given by17The cross-correlation coefficient of Raman-noise-contaminated Stokes and anti-Stokes photon pairs in the fR ≪ 1, ρ0(z) written in the same approximation, i.e., with δ = 0 and γP0z ≪ 1, leading to ρ0(z) ≈ (γP0z)−2. The choice of the nonlinear phase φnl = γP0z is thus a tradeoff between the photon flux Ij, which increases with φnl as |ν(z)|2, and the correlation between the Stokes and anti-Stokes photons, which decreases with φnl even in the absence of the Raman noise as |μ(z)|2/|ν(z)|2.When the Raman noise is negligible, V = ρ/(ρ + 2) of a two-photon interference pattern, which can be measured, e.g., with the use of an unbalanced Mach–Zehnder interferometer37V plotted as a function of the frequency Ω/(2π) = (ω − ωp)/(2π) for Stokes and anti-Stokes photons generated through pure FWM with fR = 0 (red line), as well as through FWM with the Raman noise (blue line). For the highest efficiency of photon-pair generation, FWM is assumed to be ideally phase-matched in both cases, δ = 0. The nonlinear phase shift is kept small, γP0z = 0.1, to provide a low-nj output, which helps avoid an excessive degradation of photon-pair correlations. Parameters of the Raman noise are chosen in such a way as to mimic the Raman effect in silica fibers17fR = 0.18 and the peak Raman gain gR0 = 6.2 10–12 cm/W.We quantify the time–energy entanglementδ = 0 is achieved, it is instructive to isolate the spectral density of the Raman noise15SR = sRP0z, where sR(Ω) = |gR|[nth(Ω) + H(−Ω)] and H(Ω) is the Heaviside step function. sR(Ω) for fused silica at T = 300 K with the Raman gain profile gR(Ω) as specified by Stolen et al.sR(Ω) is symmetric with respect to Ω = 0, sR(Ωs) ≈ sR(Ωa), only for low Ω (|Ω|/(2π) < 1 THz for T = 300 K in π) < kBT/ħ. In this region, nth ∝ 1/|Ω|, giving rise to constant low-frequency noise. For |Ω|/(2π) > kBT/ħ, the Stokes sideband is much more prone to the Raman noise than its anti-Stokes counterpart. The sR(Ω) profile features a broad peak at Ω/(2π) ≈ 13 THz (π) > 20–30 THz.To understand the influence of Raman scattering on quantum correlations between the Stokes and anti-Stokes photons as a function of the frequency at which phase matching ≈ 13 THz , rollingV. In V parameter as a function of the frequency Ω/(2π) for the phase-matched 2ωp = ωs + ωa, δ = 0, and γP0z = 0.1 FWM process with (blue line) and without (red line) the Raman effect. As one would expect from the spectral profile of sR(Ω), for low Ω, the Raman noise dramatically reduces the entanglement of the Stokes and anti-Stokes photons. Indeed, for Ω/(2π) ranging from approximately 1 to 15 THz, the two-photon interference fringe visibility is very low, V < 0.1. In this range, Raman scattering imposes severe limitations on fiber sources of quantum states of light.This behavior of the spectral density of the Raman noise is crucial for understanding the properties of the time–energy entanglement of the Stokes and anti-Stokes photons as quantified by the fringe visibility π) > 20–30 THz, the time–energy entanglement of the Stokes and anti-Stokes photons becomes stronger, approaching, for Ω/(2π) > 35–40 THz, the Stokes–anti-Stokes entanglement in pure phase-matched FWM  with the approximate solution Ωpm ≈ pm ≈ Ω0 provides a highly accurate prediction for the frequency of phase matching everywhere in the anomalous-GVD range except a narrow region near the zero-GVD frequency ωz, which corresponds to lc = π/|2δ| for 2ωp = ωs + ωa FWM with P0 = 27 W calculated as a function of the pump frequency and the Stokes/anti-Stokes wavelengths λs,a = 2πc/ωs,a. As an important universal tendency, the FWM phase-matching maps and, hence, the maps of the FWM gain look drastically different for the normal- and anomalous-GVD regions 1/2, the upper bound of the parametric gain band is given by Parametric sideband generation is now possible when δ = 0 equation  ≈ 16. The thermal photon number is exponentially small in this regime, nth ≈ exp(−ħ|Ω|/θ), providing a strong suppression of the Raman noise in the photon-pair output.As can be seen in ≈ 40 THz . The two≈ 96 THz . For a fω1,2 ± Ω around the central frequency ωk  of each of the pump fields. The domains of this XPM-induced parametric gain and their central frequencies Ω0 are defined by the dispersion equation4243In dual-pump FWM, cross-phase modulation (XPM) tends to induce energy transfer from one of the pump fields to the sidebands of the other pump18Pk, ωk, uk, and βkp are the peak power, the central frequency, the group velocity, and the propagation constants of the first (k = 1) and second (k = 2) pump fields, and γk is the nonlinear coefficient at the frequency ωk.where ω1,2 ± Ω is controlled by the gain g = 2 ImK, which can be found by solving the quartic k = 1, 2. Each of these solutions is equivalent to the solution of The buildup of XPM-induced sidebands g calculated by numerically solving σ for the conditions of experiments44β21 ≈ −0.115 ps2/m, while the second-harmonic pump lies in the region of normal GVD, with β22 ≈ 0.016 ps2/m. The nonlinear coupling constant is γ1P1 + γ2P2 ≈ 5 cm−1.In a more general scenario, Θ ≠ 0, the two pump fields and their sidebands are coupled by XPM. Both the gain bands and the gain controlling the buildup of XPM-induced sidebands can be tuned in this scheme by varying the frequencies and the peak powers of both pump fields, as well as by tailoring fiber dispersion and nonlinearity. As a typical example, σ) map in The g. The phase-matching condition for the ω1 + ω2 = (ω1 + Ω) + (ω2 − Ω) XPM-coupled FWM sideband generation is then written asWith this goal in mind, we set The solution to this equation isγ(P1 + P2) ≪ σ2/|β21 + β22|, In the case of low pump peak powers, 2γ(P1 + P2) = 0 and β21 ≈ β22 = β2, 20pm ≈ σ/|β2|. In a more general case of nonzero, but low nonlinearity, σ2/|β21 + β22| ≫ γ(P1 + P2) ≠ 0 and β21 ≠ β22, the frequency shift of the considered parametric gain band, as can be seen from σ. In particular, for the parameters of the fiber and the pump in β21 + β22| ≈ 0.1 ps2/m and 2γ|β21 + β22|(P1 + P2) ≈ 100 ps2/m2, the approximation of σ > 10 ps/m. Specifically, for σ ≈ 30 ps/m, pm/(2π) ≈ 100 THz, which agrees very well with numerical calculations in π) ≈ 100 THz in a fiber at T ≈ 25 °C, ħ|Ω|/(kBT) ≈ 16, and the thermal photon number is exponentially small, nth ≈ exp(−ħ|Ω|/θ), leading to a strong suppression of the Raman noise in the photon-pair output.With β21 + β22|/2 ≈ 0.01 ps2/m, σ ≈ 2.5 ps/m, and γ(P1 + P2) ≪ 2σ2/|β21 + β22|, the maximum gain of XPM-induced sideband generation in dual-pump FWm is achieved at Ωpm ≈ 40 THz. At this frequency, the time–energy photon-pair entanglement parameter, V ≈ 0.91, is more than an order of magnitude higher  ≪ σ2/|β21 + β22|, the frequency shift of the high-frequency gain band in XPM-induced FWM sideband generation, as can be seen from Notably, with nth(Ω), the manifold of FWM processes in orthogonal polarization modes of the fiber give rise to strongly correlated Stokes and anti-Stokes photon pairs, enabling the generation of multipartite entanglement. Indeed, when the peak power of both pump fields in dual-pump FWM is P0 and the input is a four-mode vacuum state, qj  of Stokes and anti-Stokes  vacuum fields, the FWM-sideband four-mode output in the gz ≪ 1 regime, as shown in the earlier workη = 1 − iγP0z, Θ = iγP0z/(2η), and et al.In its general, polarization-nondegenerate version, the dual-pump FWM scheme considered in this section gives rise to multiple sideband pairs, which can be coupled to each other by the Kerr-type optical nonlinearityWe have shown that phase matching can provide a tunable gate that helps discriminate entangled states of light generated by four-wave mixing in optical fibers against uncorrelated photons originating from Raman scattering. Two types of such gates are discussed. Phase-matching gates of the first type are possible in the normal dispersion regime, where FWM sidebands can be widely tuned by high-order dispersion management, enhancing the ratio of the entangled-photon output to the Raman noise. The photon-entanglement gates of the second type are created by dual-pump cross-phase-modulation-induced FWM sideband generation and can be tuned by group-velocity mismatch of the pump fields.How to cite this article: Zheltikov, A. M. Phase matching as a gate for photon entanglement. Sci. Rep.7, 46115; doi: 10.1038/srep46115 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Prdm14 overexpression in EpiLCs induced their conversion to ESC-like cells even in the absence of leukemia inhibitory factor in adherent culture. This was impaired by the loss of Kruppel-like factor 2 and ten-eleven translocation (TET) proteins. Furthermore, PRDM14 recruited OCT3/4 to the enhancer regions of naive pluripotency genes via TET-base excision repair-mediated demethylation. Our results provide evidence that PRDM14 establishes a transcriptional network for naive pluripotency via active DNA demethylation.Primordial germ cells (PGCs) are specified from epiblast cells in mice. Genes associated with naive pluripotency are repressed in the transition from inner cell mass to epiblast cells, followed by upregulation after PGC specification. However, the molecular mechanisms underlying the reactivation of pluripotency genes are poorly characterized. Here, we exploited the in vitro differentiation of epiblast-like cells (EpiLCs) from embryonic stem cells (ESCs) to elucidate the molecular and epigenetic functions of PR domain-containing 14 (PRDM14). We found that   •PRDM14 converts from EpiLCs to ESCs in adherent culture•EpiLC to ESC conversion by PRMD14 depends on KLF2 and TET-base excision repair pathway•PRDM14 enhances the recruitments of OCT3/4 at pluripotent genes In this article, Seki and colleagues exploit an in vitro differentiation system of epiblast-like cells (EpiLCs) from embryonic stem cells (ESCs) to investigate the function of PRDM14. Overexpression of PRDM14 in EpiLCs induces the conversion from EpiLCs to ESCs through active DNA demethylation-mediated OCT3/4 recruitment at pluripotent genes in adherent culture. Nanog, Kruppel-like factor 2 (Klf2), and T cell leukemia/lymphoma 1 (Tcl1), which are regulated by the transcription factors B lymphocyte-induced maturation protein 1 (BLIMP1), PR domain-containing 14 (PRDM14), and transcription factor AP-2 gamma (TFAP2C)  produces a totipotent zygote. Since all cells harbor the same genetic code, differentiation depends on the epigenetic state of each cell; germ cells reprogram epigenetic information during their specification, development, and maturation to acquire toti- or pluripotency. Germ cells in mice are specified from proximal epiblast cells that lack expression of some subsets of pluripotency genes such as (TFAP2C) . Specifi(TFAP2C) . Migrati(TFAP2C) . DNA dem(TFAP2C) . CpG met(TFAP2C) . The for(TFAP2C) . PGCs us(TFAP2C) . Global (TFAP2C) .Prdm14 in the ICM is required for priming of the epiblast cells for proper somatic differentiation  of blastocysts at embryonic day 3.5 (E3.5) and reactivated in specifying PGCs at approximately E6.25 . Knockountiation , and thantiation . Furtherntiation . KO and  to PGCs . InteresPrdm14 expression is controlled by doxycycline (Dox) to reveal the molecular and epigenetic function of PRDM14 during the transition from epiblast cells to PGCs in mice. We showed that inducing Prdm14 expression in EpiLCs in adherent culture promotes pluripotency and self-renewal, resulting in conversion from EpiLCs to ESC-like cells (ESCLCs).Here, we exploited adherent culture of EpiLCs in which Prdm14 is expressed in proximal epiblast cells at around E6.5 that constitute PGC precursors in mice. In three-dimensional aggregate culture of EpiLCs, PRDM14 has been shown to be sufficient to induce PGCLCs , which is the basic medium used for PGCLC induction  activity to a degree similar to that of ESCs, which was associated with the reactivation of pluripotency genes . The number of AP-positive colonies was higher for EpiLCs overexpressing PRDM14 than for control EpiLCs , and DNA methylation  genes were enriched in clusters 2 and 4, respectively , and T-box 3 (Tbx3) were upregulated by PRDM14 in EpiLCs but not in EpiSCs , helicase lymphoid-specific (Hells), and suppressor of variegation 3–9 homolog 1 (Suv39h1)—were downregulated by PRDM14 only in EpiLCs , EpiLCs + PRDM14, and EpiLCs (d4). Unsupervised hierarchical clustering revealed that the global gene expression profile of EpiLCs + PRDM14 was more similar to that of ESCs than that of EpiLCs A. Scatten EpiLCs B. Unsupen EpiLCs C. Genes ectively C and 2D.ectively . To idenectively . Critican EpiSCs E, suggesn EpiLCs A, which Nanog, Klf2, and Tcl1) and of germ cell character  (Blimp1 (Prdm1), a critical determinant of germ cell specification  and stella-eCFP (SC) (BVSC), and monitored the fluorescence of mVENUS and eCFP in different culture conditions (Blimp1 is upregulated by the mesoderm marker gene T (Brachyury)  . The difology 1) . We compfication   . We thernduction G, suggesnd Foxd3 , which and Foxd3 B.Klf2 in the PRDM14-associated acquisition of pluripotency, we generated Klf2 KO ESCs harboring a Dox-inducible Prdm14 expression cassette using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system  and Klf2 KO using qRT-PCR analysis. We plotted the relative Ct values of qRT-PCR data (EpiLCs + PRDM14 − EpiLCs [d2]) for WT (y axis) and Klf2 KO (x axis). Consistent impairment of activation of pluripotent-associated genes by PRDM14 was observed, whereas the repression of differentiation markers by PRDM14 was relatively maintained in Klf2 KO EpiLCs  ESC line with a Dox-inducible Prdm14 cassette  and poly(ADP-ribose) polymerase (CRT 004876) abrogated the reversion of EpiLCs to ESCLCs, similar to the Tet1/Tet2 KD phenotype -qPCR. Since ChIP-grade anti-PRDM14 antibody was unavailable, we detected the binding of exogenous FLAG-PRDM14 in EpiLCs using an anti-FLAG antibody , we analyzed Prdm14 and Klf2 expression as well as PRDM14 and OCT3/4 binding, and 5mC and 5hmC distribution at the two Klf2 enhancers every 6 hr after Prdm14 induction in EpiLCs. Prdm14 expression was detected starting 18 hr after induction; this was followed by increased PRDM14 binding at the two enhancers at 24 hr  followed by deep sequencing (ChIP-seq) in ESCs, EpiLCs, and EpiLCs expressing PRDM14, and identified 18,093, 8,136, and 13,882 OCT3/4 peaks, respectively, using the MACS2 callpeak . Next, tn EpiLCs B. An oven EpiLCs  revealeded genes C and 6D.ed genes E. To deted genes F. 5mC leT and Blimp1 , and reproductive homeobox 6/9 (Rhox6/9) was normal in Tet1/Tet2 KD EpiLCs. These data are consistent with the phenotype of Tet1/Tet2 double KO (DKO) PGCs, and demonstrate normal PGC specification and development  and spalt-like transcription factor 4  enhancers were unchanged. Interestingly, the reactivation of Rif1 and Sall4 by PRDM14 was impaired in Tet1/Tet2 double KD EpiLCs  transferase to target chromatin, which leads to its activation via O-GlucNAcylation of histone H2B  showed low expression in human ESCs/induced PSCs (iPSCs). Although expression of Prdm14 is closely associated with naive pluripotency in mice, primed human ESCs/iPSCs, primitive streak-like cells, iMeLCs, and 4i ESCs consistently express PRDM14 at moderate levels. Therefore, the specific upregulation of Prdm14 observed during the transition from pluripotent cells to PGCs in mice does not occur in human PGC specification. The present study has clearly demonstrated that PRDM14 governs the transcriptional network for pluripotency in the absence of BLIMP1 in mice. In contrast to early mouse embryos, epiblast cells in human early embryos need to maintain pluripotency for longer than 10 days until the emergence of PGCs. Furthermore, primitive streak-like cells, which have a high capacity for PGC specification, retain pluripotency markers including NANOG and PRDM14  . For insd PRDM14 . On thisd PRDM14 . Howeverntiation , implyinPrdm14 KO fibroblasts failed to be converted to iPSCs in the absence of Nanog and Sox2 reactivation under standard ESC culture conditions  containing 10% fetal calf serum (Invitrogen), 1 mM glutamine (Wako), nonessential amino acid (Wako), and 0.1 mM 2-mercaptoethanol (Wako). The culture was supplemented with LIF (Wako) in the absence of feeder cells.6 EpiLCs expressing PRDM14, cultured without LIF, were injected subcutaneously into both the flanks of female nude mice. After 30 days, the tumors were excised, embedded in paraffin, and sectioned into 4-μm-thick slices. H&E staining was done according to the standard protocol. Markers for the three germ layers were monitored with immunofluorescence with antibodies: α-AFP , α-SMA , and α-β-tubulin . These experiments were conducted according to the Kwansei Gakuin University Regulation for Animal experimentation.Approximately 5 × 10Prdm14-IRES-Neo, PB-CA-rtTA Adv, pCAG-Pbase, and pGG131 (pCAG-DsRed-IRES-Hygro). The cells were grown in selective medium containing 1.5 μg/mL puromycin (Sigma-Aldrich) and 200 μg/mL hygromycin B (Wako) for 7 days  were generated from pLKO.1-puro  or pLKO.1-blast . The Tet1 and Tet2 shRNAs were adapted for pLKO.1 plasmids. The RNAi target sequences are presented in http://www.addgene.org/). pLKO.1 plasmids were co-transfected with pCMV-VSV-G  and pCMV-dR8.2 dvpr  into HEK293T cells. Lentivirus-containing supernatants were centrifuge-filtered through a 0.45-μm filter. Clarified supernatants were combined using the Lenti-X Concentrator (Clontech) according to the manufacturer's protocol. The mixture was centrifuged, after which the supernatant was carefully removed and the pellet resuspended in DMEM.For lentivirus transduction, subconfluent iP14 ESCs were incubated with lentivirus-containing DMEM supplemented with 8 μg/mL polybrene (Sigma-Aldrich) for 24 hr. After changing the spent medium with fresh medium, cells were selected with 2 μg/mL puromycin (Sigma-Aldrich) or 20 μg/mL blasticidin S hydrochloride . Knockdown efficiency was analyzed by qRT-PCR and western blotting.Klf2 guide RNA was adapted for and generated from the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid . The Klf2 guide RNA sequences are presented in 5) with or without doxycycline in GK15 medium on a dish coated with 16.7 μg/mL human plasma fibronectin. For the colony formation assay for AP-positive cells, ESCs, EpiLCs, and EpiLCs induced with or without PRDM14 were dissociated by TrypLE Select (Invitrogen). Cells (1.0 × 104) were cultured under standard ESC culture conditions. After culture for 3 days, the cells were stained for AP activity. The aggregate culture of EpiLCs was performed as previously reported , 0.4 μM PD0325901 (Wako), and LIF in a dish coated with 0.01% poly-L-ornithine (Millipore) and 10 ng/mL laminin (BD Biosciences). Induction of EpiLCs was performed as described previously . EpiLCs reported . In brieTotal RNA was extracted using TRIzol (Invitrogen). The ReverTra Ace qPCR RT kit (Toyobo) was used for cDNA synthesis according to the manufacturer's instructions. Subsequently, cDNA was used as a template for qPCR with the Thunderbird SYBR qPCR Mix (Toyobo) and gene-specific primers  on a LigCells were lysed by boiling in SDS sample buffer (Wako). The total proteins from lysed cells were denatured by 2-mercaptoethanol and were then applied to polyacrylamide-SDS gels. The proteins were separated on polyacrylamide-SDS gels, blotted on polyvinylidene fluoride membrane, and probed using the following primary antibodies: α-PRDM14 , α-tubulin , α-histone H3 , α-FLAG , α-KLF2 , α-TET1 , α-TET2 , and α-OCT3/4 . Following the primary antibody reaction, the membrane was incubated with secondary horseradish peroxidase-coupled antibodies. Detection was achieved using the Luminata Forte Western HRP Substrate (Millipore). For immunoprecipitation analysis, extracts containing a protease inhibitor cocktail (Roche) were incubated with anti-PRDM14 antibody or anti-OCT3/4 antibody for 2 hr at 4°C, and captured with protein A beads. Protein complexes were washed with wash buffer , and eluted by boiling with SDS sample buffer.N.O. performed most experiments and analyzed data. Y. Suwa, N.S., M.K., H.K., and A.N. performed some experiments. O.N. and M.K. contributed ChIP-seq analysis. G.N. established ESC cell line. Y. Seki designed and performed experiments. M.T. contributed to the immunofluorescence study of teratoma tissue. N.O. and Y. Seki wrote the manuscript. Y. Suwa, O.N., and N.S. contributed equally to this work.


Biological rhythms lie at the center of regulatory schemes that control many aspects of living systems. At the cellular level, meaningful responses to external stimuli depend on propagation and quenching of a signal to maintain vigilance for subsequent stimulation or changes that serve to shape and modulate the response. The hypothalamus–pituitary–gonad endocrine axis that controls reproductive development and function relies on control through rhythmic stimulation. Central to this axis is the pulsatile stimulation of the gonadotropes by hypothalamic neurons through episodic release of the neuropeptide gonadotropin-releasing hormone. Alterations in pulsatile stimulation of the gonadotropes result in differential synthesis and secretion of the gonadotropins LH and FSH and changes in the expression of their respective hormone subunit genes. The requirement to amplify signals arising from activation of the gonadotropin-releasing hormone (GnRH) receptor and to rapidly quench the resultant signal to preserve an adaptive response suggests the need for rapid activation and feedback control operating at the level of intracellular signaling. Emerging data suggest that reactive oxygen species (ROS) can fulfill this role in the GnRH receptor signaling through activation of MAP kinase signaling cascades, control of negative feedback, and participation in the secretory process. Results obtained in gonadotrope cell lines or other cell models indicate that ROS can participate in each of these regulatory cascades. We discuss the potential advantage of reactive oxygen signaling for modulating the gonadotrope response to GnRH stimulation and the potential mechanisms for this action. These observations suggest further targets of study for regulation in the gonadotrope. The fundamental role of pulsatile stimulation of gonadotropes by the hypothalamic neuropeptide GnRH, or GnRH-I in maintaining function of the hypothalamic–pituitary–gonad (HPG) axis is one of the earliest principal findings after discovery of the hormone , 2. StudLhb, this is the pairing of the immediate-early Egr1 family of transcriptional activators with the Nab1/2 family of repressors. Transient frequency and amplitude-dependent stimulation of Egr1 expression is countered by pulse-insensitive expression of Nab1/2, establishing a high-pass filter that requires sustained stimulation to overcome suppression (in vivo studies and mixed primary pituitary culture in rats and in αT3-1 cells that do not express gonadotropin β-subunit genes (Fshb prefers low frequencies for promoter activator (c-Fos and c-Jun) upregulation, and high frequencies lead to upregulation of Fshb promoter inhibitors, such as Skil and Tgif1  and diacylglycerol (DAG) production. IP3 mobilizes Ca2+ from intracellular stores and influx via L-type voltage-gated Ca2+ channels. The mobilization of Ca2+ is associated with initiation of the secretory response and fusion of secretory granules with the extracellular membrane. In a related signaling branch, DAG along with Ca2+ activates multiple PKC isozymes, including the conventional isoforms PKCα, PKCβII, the novel isoforms PKCδ and PKCε, and the atypical PKCζ in αT3-1 and LβT2 cells  family, which are targets of activation by intracellular kinases or elevated intracellular Ca2+ . Exposur2+ , collectElevated ROS is associated with MAPK activation in multiple cell types . InsulinLhb and Fshb transcription   . The cysr (EGFR) . But demr (EGFR) –60. An ar (EGFR) .Fshb and Lhb transcription can be regulated through ROS.In addition to activation of MAP kinase pathways through positive regulation of signaling cascades, ROS may also play a central role in promoting MAP kinase phospho-activation through inactivation of negative feedback. Dual-specificity protein phosphatases  serve a primary role as negative feedback regulators of MAP kinase signaling through dephosphorylation of activated MAP kinases Figure  61). DU. DU61). Srxn1 gene expression is proportionally induced by increasing pulsatile and tonic GnRH stimulation  . Translas active , 35. Tratability , 70. Pul kinases . Althougαq/11 signaling component gene expression. MiR132/212 regulates Fshb mRNA expression and secretion via SIRT1 deacetylation in gonadotropes (αq/11 pathway (On a broad time scale, ROS may regulate gonadotropins through regulation of gene expression and sensitivity to GnRH through microRNA modulation of gonadotropin and Gdotropes . MiR-7a2dotropes , 73. Fur pathway . MicroRN pathway . The ROS2+ released from intracellular stores. A rise of intracellular Ca2+ regulates several steps of exocytosis; including, vesicle priming and fusion to the plasma membrane (2+ increase with IP3 stimulation is necessary for gonadotropin exocytosis (2+ also induces ROS production by DUOX activation (2+ to enzymatic ROS generation by DUOX. Thrombin promotes Ca2+ influx in smooth muscle cells by NOX-derived ROS activation of L-type calcium channels (3 receptor activity and Ca2+ release in skeletal muscle (2+ and ROS signaling in exocytosis.Hormone secretion by exocytosis in endocrine cells is triggered by Camembrane . Localizocytosis . Increastivation  and bothtivation , 79. Thichannels . Also, il muscle . In gonal muscle  and suggInterestingly, ROS in the form of nitric oxide may play an important role in maturation and regulation of the hypothalamus in concert with pituitary ROS. Nitric oxide production in the hypothalamus elicits GnRH secretion and expression of GnRH mRNA is modulated through miR-200 and miR-155 expression before puberty by controlling the nitric oxide-sensitive regulators CEBPB and ZEB1 , 83. Thevia NOX/DUOX (Energy balance has a profound influence on reproductive fitness and operation of the HPG axis. The critical fat hypothesis suggests that an optimal level of body fat is permissive to menarche and may be necessary for optimal operation of the HPG axis , 85. AdiNOX/DUOX . This suThe emerging evidence that ROS are integrated into multiple cell signaling cascades has led to appreciation as an important signaling molecule. In gonadotropes, it appears that enzymatic ROS plays a role in GnRH receptor signaling. The rapid and transient nature of ROS signaling is well-suited for the episodic pattern of GnRH stimulation and ROS signaling can contribute to both rapid activation and rapid resolution of activated signaling cascades. Further studies are needed to confirm or disprove the role of PRDX and SRXN1 in resolution of MAPK activation. It is also necessary to examine the impact of FFA-induced ROS on cell stress and to confirm the potential role of enzymatic ROS on L-type calcium stimulation in gonadotrope cells. The integration of ROS in GnRH receptor signaling provides an opportunity for other ROS sources to impact gonadotrope function at the cellular level. Incorporating ROS into our view of GnRH signaling is likely to yield useful insight into the mechanism of pulse interpretation and integration of stress signaling into the reproductive axis.TT authored the manuscript and prepared figures. MA produced new data presented in Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In vitro studies suggest involvement of intracellular signaling through mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and mammalian target of rapamycin (mTOR). However, anabolic signaling responses to bone loading using a whole animal in vivo model have not been studied in detail. Therefore, we examined mechanically-induced signaling events at five time points from 0 to 24 hours after loading using the rat in vivo ulna end-loading model. Western blot analysis of bone for MAPK’s, PI3K/Akt, and mTOR signaling, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to estimate gene expression of calcitonin gene-related protein alpha (CGRP-α), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), c-jun, and c-fos in dorsal root ganglion (DRG) of the brachial intumescence were performed. There was a significant increase in signaling through MAPK’s including extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) in loaded limbs at 15 minutes after mechanical loading. Ulna loading did not significantly influence expression of the genes of interest in DRG neurons. Bone signaling and DRG gene expression from the loaded and contralateral limbs was correlated . However, bone signaling did not correlate with expression of the genes of interest in DRG neurons. These results suggest that signaling through the MAPK pathway may be involved in load-induced bone formation in vivo. Further characterization of the molecular events involved in regulation of bone adaptation is needed to understand the timing and impact of loading events, and the contribution of the neuronal signaling to functional adaptation of bone.Mechanical signals play an integral role in the regulation of bone mass and functional adaptation to bone loading. The osteocyte has long been considered the principle mechanosensory cell type in bone, although recent evidence suggests the sensory nervous system may play a role in mechanosensing. The specific signaling pathways responsible for functional adaptation of the skeleton through modeling and remodeling are not clearly defined.  Supernatant protein concentration of each sample was determined by the DC protein assay , and equivalent amounts of protein from each sample were analyzed on a Western blot as previously described  signaling did not appear to be altered by the loading protocol employed in this study. Similarly, no significant changes were identified in our panel of candidate genes associated with neuronal signaling, or in correlations with bone kinase signaling.We demonstrate the ability to detect signaling events in bone tissue in vitro, including the MAPK’s, WNT/β-catenin, calcium ions, integrins, nitric oxide and prostaglandins [in vitro models studying manipulation of the mechanical environment of osteoblasts and osteocytes in cell culture, or immunostaining of bone tissue after mechanical stimulation [in vivo model to investigate signaling responses in bone tissue in response to loading is likely to be important to future work. Loaded and non-loaded bones were analyzed at various times after a mechanical stimulus that has been shown to induce functional adaptation in multiple bones and was based on previous work in our laboratory [in vivo is very limited. We expected that some signaling events would be very rapid. Investigation of additional time points, particularly later times, may be informative with regards to signaling responses in non-loaded bones. Alternative loading regimens, including different levels of peak strain, different numbers of load cycles, periods of rest [Bone has a remarkable ability to alter its mass and architecture in response to changes in its local environment. Various mechanical stimuli have been shown to influence bone cell metabolism and activate signal transduction in bone cells glandins . Limitedmulation ,48. Use boratory . Bone ti of rest , or repe of rest ,50, and In the present study, the most consistent and notable impact of mechanical stimulation was increased signaling through JNK and ERK shortly after loading. ERK, JNK, and p38 are the best studied members of the MAPK family and have a central role in regulation of bone mass. MAPK’s mediate bone formation through differentiation of both osteoblasts  and osteIn vitro work suggests that Akt signaling is one of several key pathways important for bone cell differentiation, function, and survival [The lack of a load effect on signaling through the PI3K/Akt/mTOR pathway was unexpected. survival . A trendin situ hybridization may provide support in correlation of a molecular response to a tissue level response.Identification of significant loading effects was influenced by data variation within individual time points and signaling pathways. Mechanical signaling machinery can be exquisitely sensitive to any iatrogenic stimulus. Although mechanical loading, tissue collection and processing was efficient and meticulous, variation in bone collection technique or time may have contributed to data variance as some variation in the concentration of protein extracted from the ulna of similar sized rats found. Load-induced bone formation in the rat ulna after end-loading is greatest in the mid-diaphysis where strain is concentrated ,56. PeakEarly signaling events were only detected in loaded bones. Similarly, the pattern of bone signaling and DRG neuron gene expression was similar between right and left limbs of individual animals for all pathways and genes studied. A neuronal mechanism is one possibility that may be responsible for such effects ,34. To bIn a pilot study, the yield of protein for Western blot analysis from the cervical intumescence DRG in rats was limited. Consequently, we chose to pursue analysis of gene expression from neuronal tissue using qRT-PCR to examine downstream products from relevant signaling pathways. Use of more sophisticated and comprehensive methods for detecting RNA products such as microarrays or RNA-Seq may be useful to analyze the transcriptome in DRG, periosteum, and bone from loaded and contralateral limbs. Upregulation of Wnt signaling among other genes at 3 hours and at 24 hours in cortical bone has been demonstrated using this approach . Ultimatin vitro work. Other pathways, such as Sost/Sclerostin, Wnt/ β catenin, or NGF/TrkA signaling [in vivo using our experimental approach.Several limitations of this study exist. The time course experiment was chosen to identify time points with increased signaling activity in various pathways. Activation of signaling can be transient. Consequently, peaks of signaling activity may occur in between or beyond the selected time points used in the study. A trend towards increased activity was noted at several time points. However, sample sizes precluded identification of additional significant findings. Post hoc power analysis revealed a group sizes of >20 animals per time point would be needed to achieve significance for the effect of loading on these trends. Selected pathways were studied based on previous ignaling ,39,58, ain vitro models. It is clear the skeletal responses to mechanical loading are complex, particularly when considering the whole animal model in which neurovascular responses to bone loading are intact. Signaling by the MAPK pathway appears to be an important early component to bone mechanotransduction, but the role of other signaling pathways and sensory innervation needs further investigation. Further characterization of the molecular events involved in functional adaptation using this model is needed in future work.In conclusion, our findings from this study extend knowledge of bone mechanotransduction beyond results of S1 File(XLSX)Click here for additional data file.


Specifically, Cx40-formed channels at high cell density could increase PDT photocytotoxicity. This action was substantially restricted when Cx40 expression was not induced or Cx40 channels were restrained. Additionally, the presence of Cx40-composed channels enhanced the phototoxicity of PDT in the tumor xenografts. The above results indicate that enhancing the function of Cx40-formed channels increases PDT efficacy. The enhancement of PDT efficacy mediated by Cx40 channels was related with intracellular pathways mediated by ROS and calcium pathways, but not the lipid peroxide-mediated pathway. This work demonstrates the capacity of Cx40-mediated channels to increase PDT efficacy and suggests that therapeutic strategies designed to maintain or enhance Cx40 expression and/or channels composed by Cx40 may increase the therapeutic efficacy of PDT.Despite responses to initial treatment of photodynamic therapy (PDT) being promising, a recurrence rate exists. Thus, finding novel therapeutic targets to enhance PDT efficacy is an urgent need. Reports indicate that connexin (Cx) 40 plays an important role in tumor angiogenesis and growth. However, it is unknown whether Cx40-composed channels have effects on PDT efficacy. The study uniquely demonstrated that Cx40-formed channels could enhance the phototoxicity of PDT to malignant cells  Photodynamic therapy (PDT) is a novel cancer treatment and acts primarily via the generation of reactive oxygen species (ROS) through the irradiation of photosensitized cells, resulting in the destruction of subcellular sites in which the photosensitizer has localized . PDT hasConnexin (Cx) channels, which are composed of connexins (Cxs), mediate direct intercellular transfer of molecules and/or electrical charge . Six Cxs2+, which are responsible for the photodamage of malignant cells, can spread via Cx channels to untreated neighboring cells  was obtained from Union Med. Group Limited Company, Hong Kong, China. Doxycycline (Dox) was supplied by Abcam. Puromycin was provided by Abcam. Cell culture reagents were obtained from Life Technology. Other reagents were all supplied by Sigma-Aldrich unless otherwise stated.2 for 2 J/cm2) and then cultured in complete DMEM medium without photosensitizer for 24 h in the dark until Photofrin phototoxicity was measured using CCK-8 assay. 18α-GA  was exposed to the cells 2 h before Photofrin treatment.The HeLa cell line used in the study was transfected with tetracycline operon (Tet-on) inducible gene expression system as described in our previous study . The exp4 cells/cm2 cells were seeded with 70%-100% confluence. Under such circumstances, a single cell was allowed to contact 3–5 others and provided substantial opportunity for channel formation when treated with Photofrin. For the low-cell density, 3 × 103 cells/cm2 cells were seeded into 96-well plates. Under such conditions, cells did not have the opportunity to contact each other and channel formation was not allowed. After PDT, CCK-8 solution  was added and incubated with cells for 2 h. The OD values were measured to determine cell survival using an Enzyme-labeling instrument.For investigating the role of Cx40-formed channels in PDT photosensitivity, after cells were treated with Dox in one culture flask to induce Cx40 expression and channel formation, they were seeded in 96-well plates under low and high cell density conditions. At high cell density, 3 × 10in vivo, BALB/c-nude mice were transplanted with HeLa cells expressing Cx40 according to our previous study  was intravenously administrated; the mice were kept in darkness for 24 h and then the tumors were irradiated . After PDT, the xenograft volume (V) was determined based on the equation V = (L × W2) ×0.5, where L is the longest axis of xenograft and W is the axis perpendicular to L. The relative tumor volume (RTV) of each xenograft was calculated according to the equation RTV = volume of each time point/volume prior to treatment. After euthanasia, the xenografts were excised and weighted. The weight inhibition rate was defined by the following equation: Tumor weight inhibition rate = (1-mean tumor weight of treated groups/mean tumor weight of control group) ×100%.For determining the role of Cx40 channels in photosensitivity us study . BrieflyThe western blot assay was utilized as described in previous studies , 18. BriThe assay was utilized for measuring the function of Cx40 channels as previously described , 12. In 2+ concentrations was performed according to our previous study  or Ca2+ free BBS  was added and illumined. After PDT, cell suspension was prepared after cells were trypsinized and collected. The intracellular concentrations of Ca2+ and ROS of Dox-treated and -untreated cells were determined by flow cytometer and normalized to Dox-treated and -untreated cells in control group, respectively.The determination of intracellular ROS and Caus study . In brie4-HNE and ceramide were measured using ELISA according to our previous study . Briefly4cells/cm2), the formation of the Cx channel was possible, while at low density (3 × 103cells/cm2), cells did not directly contact each other, and no channel was formed. As illustrated in For exploring the role of Cx40 channels in cellular survival after Photofrin-mediated PDT, Cx40-expression cells were seeded at high and low cellular density. Under the condition of high density  and without Dox (no channel formation) at high cellular density was determined in The above data indicate that at medium Photofrin concentration, under high-density conditions, Cx40-formed channel increases the phototoxic effect of Photofrin-PDT as the phototoxic action at high density significantly decreased when Cx40 was not expressed or Cx40-formed channels were suppressed. In short, these results demonstrate that Cx40-formed channels have an ability to increase PDT phototoxicity under high-density conditions.in vivo, a xenograft model using nude mice subcutaneously transplanted with HeLa cells transfected with Cx40 was employed. For controlling the expression of Cx40 in tumor xenografts, drinking water with or without Dox was supplied. Before PDT treatment, tumor xenografts in Dox-treated and Dox-untreated groups were randomly selected for detecting Cx40 expression. For exploring the role of Cx40-formed channels in PDT phototoxicity 3, Photofrin or 0.5% sterile dextrose were injected through the tail vein. Twenty-four hours after the injection, the xenografts were irradiated. After PDT, tumor volume was measured and the mean RTV of each group was calculated for assessing tumor growth. The results showed that the tumor growth in xenografts treated with and without Dox was significantly inhibited after Photofrin treatment  due to the release from intracellular Ca2+ store and Ca2+ influx from the extracellular medium, thereby inducing apoptosis and cell death  cells significantly increased compared to Dox-untreated (channel-unformed) cells, indicating that Cx40-formed channels stimulate the influx of Ca2+ from the extracellular medium after PDT. For exploring the role of Cx40-composed channels in Ca2+ release after PDT, cells in Ca2+-free balanced salt solution were irradiated. As illustrated in 2+]i of cells treated with Dox (channel formation) were significantly enhanced compared to cells treated without Dox (no channel formation), suggesting that Cx40-formed channels promote intracellular Ca2+ release after PDT.Studies have demonstrated that PDT causes an increment of intracellular Call death , 20. Sinchannels , the prePDT induces the accumulation of intracellular lipid peroxides, among which 4-HNE and ceramide might diffuse via Cx channels due to their small molecular weights , 21. ThuAlthough lack of Cxs or Cx channels in many tumors is generally associated with tumorigenic phenotype, the expression of Cxs can be maintained or up-regulated in some cases. Reports have shown that in testicular tumors, Cx40 expression is increased when compared with the testis , and in Our previous studies demonstrated that a substantial increase of PDT efficacy depended on Cx channels composed of Cx32 and Cx26 among the target cells , 12. TheIt should be noted that several analgesics, such as tramadol and flurbiprofen, have been shown to inhibit the function of Cx channels . Thus, iIt is worth noting that the effects dependent on Cx40 channels on phototoxicity were absent at high (such as 5 and 10 μg/mL) and low (such as 1 μg/mL) Photofrin concentrations in the study . This inIt is generally believed that the “bystander effect” of Cx channels depends on the diffusion of toxic signals from one cell to neighboring cells through Cx channels . Reports2+ release from calcium stores and extracellular Ca2+ influx, causing the enhancement of [Ca2+]i, ultimately resulting in cellular death and apoptosis (2+ was allowed to propagate via Cx channels to unexposed neighbors (2+ through Cx channels may have impacts on PDT photocytoxicity. Nevertheless, reports have indicated that the enhancement of [Ca2+]i may result in Cx coupling closure (2+]i impacting Cx channels is dependent on the species of cell and Cx expressed (2+ concentration was mediated by Cx40-formed channels suggested that the increase in intracellular Ca2+ concentration induced by PDT has no impact on Cx40-formed channels (2+ influx from the extracellular medium and Ca2+ release from intracellular Ca2+ store by PDT (2+ pathway. Nevertheless, whether the intracellular Ca2+ pathway is required for the increment of phototoxicity mediated by Cx40 channels needs to be investigated in further studies.It has been documented that PDT can induce intracellular Capoptosis . Studieseighbors . Thus, t closure . It is wxpressed . The reschannels . More ime by PDT . Taken t2+ is allowed to propagate through Cx40-formed hemichannels, contributing to ATP release in juxtaglomerular endotheliocytes (2+ concentration may be partially attributed to Ca2+ influx via hemichannels composed by Cx40, which may contribute to Cx40-mediated increment of PDT phototoxicity. However, the role of hemichannels in oncotherapy including PDT has been less reported. It would be interesting to uncover the role of hemichannel in cancer treatment and its underlying mechanisms in further study.It has been established that Caliocytes . Thus, CIt has been established that PDT can cause an increment of lipid peroxide production, which may be responsible for PDT-induced apoptosis and cell death . Among tCollectively, the study demonstrates the increased effects of Cx40-formed channels on PDT-mediated oncotherapy and suggests a key insight in considering bystander effects and intercellular signaling via Cx40-formed channels in response to PDT-mediated cancer treatment. The function of Cx40-formed channels in human malignant tumors may be an important determining factor of the response to PDT-mediated oncotherapy in clinic, which brings about a few therapeutic considerations. First of all, maintenance or even transitory enhancement of Cx40 expression and Cx40-formed channels is a beneficial strategy for increasing the therapeutic effect of PDT. Oppositely, factors inhibiting the function of Cx40-composed channels may cause a decrease in the sensitivity of malignancies to PDT, resulting in a significant decline in the therapeutic effect of PDT.This study was carried out according to the principles of the Basel Declaration and recommendations of Guidance Suggestions for Caring for Laboratory Animals, Animal ethics committee of Xuzhou Medical University. The protocol was approved by the Animal Ethics Committee of Xuzhou Medical University.yinxx@xzhmu.edu.cn.The datasets for this manuscript are not publicly available because of data confidentiality. Requests to access the datasets should be directed to Professor Xiao-Xing Yin, X-XY and D-PW designed the experiments. D-PW, L-RB, Y-FL, YZ, C-HD, S-MY, FZ, J-LH, and Y-YW performed the experiments. D-PW, L-RB, and Y-FL analyzed the data. D-PW wrote the paper. D-PW, L-RB, Y-FL, and YZ contributed analysis tools, reagents, and materials.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


EphrinB2, a membrane-tethered ligand preferentially binding to its receptor EphB4, is ubiquitously expressed in all mammals. Through the particular bidirectional signaling, EphrinB2 plays a critical role during the development of cardiovascular system, postnatal angiogenesis physiologically and pathologically, and cardiac remodeling after injuries as an emerging role. This review highlights the pivotal involvement of EphrinB2 in heart, from developmental cardiogenesis to pathological cardiac remodeling process. Further potential translational therapies will be discussed in targeting EphrinB2 signaling, to better understand the prevention and treatment of cardiovascular diseases. Noteworthy, Ephrin reverse signaling, in certain circumstances, could be activated independently of the associated Eph receptors [Ephrins (erythropoietin-producing hepatocellular receptor interacting proteins) are membrane-tethered ligands for Eph tyrosine kinase receptors (erythropoietin-producing hatocellular receptors), ubiquitously expressed in mammal. According to the ways of anchoring cellular membrane, Ephrins were divided into type A and type B subfamilies. All five members of EphrinAs connected the extracellular Eph-binding domain to the transmembrane segment by utilizing a glycosyl phosphatidylinositol (GPI) anchor. Whereas, all three members of EphrinBs employed a single-pass transmembrane region to fuse the Eph-binding globular domain with a cytoplasmic tail followed by several phosphorylation sites and PDZ domains . Classiceceptors –4.The interaction between Ephrin and Eph was critical in development and a variety of diseases. EphrinBs have been evidenced essential in a serious of developmental processes including vasculogenesis, neural and cardiac development. Meanwhile, EphrinBs were physiologically involved in regulating synaptic plasticity, axonal guidance and maintaining vascular homeostasis , 6. PathEphrinB2 was a highly conserved protein in mammal. Although human EphrinB2 was constituted 333 amino acids, while murine EphrinB2 had three more amino acids. They shared over 98% homology of amino acid sequence and roughly same structure. The domain structure of EphrinB2 consisted of ectodomain , transmembrane domain  and cytoplasmic domain , while the last three amino acids consisted a PDZ-binding motif in both human and murine . The revΔV-βgal) allowed full embryonic development and birth of neonatal mice, in which EphrinB2-mutant protein preserved the surface binding activity and functioned as a proper ligand to stimulate EphB forward signaling [ΔV mice, which was lacking the C-terminal PDZ interaction residue to disrupt PDZ-dependent EphrinB2 signaling and (2) EphrinB25F mice, which was lacking phosphotyrosine-dependent EphrinB2 signaling with inactive mutation in 5 conserved tyrosine sites. Both of the mice survived the requirement of EphrinB2 in embryonic angiogenesis, while EphrinB25F mice survived to adulthood but EphrinB2ΔV mice died from failure of lymphatic vasculature maturation [EphrinB2 was of great importance in heart development. Conventional knockout of EphrinB2 was embryonic lethal in mice, which attributed to the defects of angiogenesis in yolk sac and myocardial trabeculation , 20. Meaignaling . Thus, tturation . These dΔV-βgal mice eventually died within first day after birth, exhibiting extremely thickened aortic and pulmonary valves [Additional data revealed that EphrinB2 reverse signaling is indispensable for cardiac valve maturation and the following neonatal survival. The EphrinB2y valves . A massiThe genetic evidences in mice have identified that the cellular location of EphrinB2 was not restricted in endothelial cells, but was also in mesenchymal cells, vascular smooth muscle cells and pericytes surrounding the angiogenic vessles , 24, 25.Similarly, Sonia et al. reported that in avian embryos, both EphrinB2 and B1 were expressed in epicardium and then restricted in perivascular fibroblasts of the atrioventricular sulcus during the development. These EphrinBs regulated the migration of epicardial cells in the early stage and later participated in the coronary vascular development via perivascular fibroblasts .Recently, accumulating researches uncovered the role of EphrinB2 in cardiomyocyte differentiation. In-vivo genetic data showed that the EphrinB2, directly induced by Notch1/RBPJK signaling in primitive myocardial epithelium during embryonic cardiogenesis, guided the endocardial cells towards the cardiomyocyte differentiation, leading to the transition of primitive myocardial epithelium into myocardial trabeculation and ventricular chamber formation . ConsistIn adult mouse heart ventricles, the EphrinB ligands are preferentially expressed in the vasculature and EphB receptors in cardiomyocytes . CardiomRevascularization could therapeutically recover the injured myocardium in ischemic cardiovascular diseases such as myocardial infarction (MI) and ischemia/reperfusion (I/R) injury. Both the expression of EphrinB2 and EphB4 dramatically increased in MI, Brain IR injury and limb ischemic models in murine –41, suggAs expanded above, EphrinB2 was also expressed in mesenchymal cells, especially high in fibroblast. A decade before, pericyte-EphrinB2 knockout mice were observed with massive fibrosis surrounding aberrant vessels in skin , implyinUp to now, the significant roles of EphrinB2 in human carcinogenesis have been clearly identified. High-level expression of EphrinB2 was associated with low-stage, progressive and metastable human solid tumors , 59, sugSeveral proteases can cleave EphrinBs from their extracellular, transmembrane and intracellular domains in a manner that can be dispensable of Eph interaction. Protease of ADAM family, especially ADAM10, has been confirmed to cleave EphrinB2 extracellularly , while ARecombinant extracellular domain (ECD) of Eph/ephrins were widely used in research, serving as soluble surrogates to activate as well as inhibit the bidirectional signaling . The ECDAlthough EphrinB2 ECD displayed as a soluble ligand activating EphBs, the soluble EphB4 ECD (sEphB4) , as wellAdditionally, inhibitory antibodies specifically targeting EphrinB2 and EphB4 have been applied in cancer research, showing high efficacy in inhibiting angiogenesis, lymphangiogenesis and tumor growth –86.To date, small molecule targeting EphrinB-EphB interaction has been synthesized though with limited binding affinities. The low affinity could be blamed by the flexibility and large size of Ephrin-binding domain in EphBs . Hence, EphrinB2 was a highly conserved protein ubiquitously expressed in all mammals, transducing signaling through a particular bidirectional pathway. EphrinB2-regulated bidirectional signaling was of great importance in cardiogenesis, guiding the completion of cardiac looping, expansion of endocardium, formation of myocardial trabeculation and cardiac valve and development of cardiac lineage differentiation as an emerging role. Lately, beyond cardiogenesis, EphrinB2 was identified critically involved in pathological cardiac remodeling process, such as angiogenesis after cardiac injury and cardiac fibrosis, which uncovered the tip of the iceberg regarding the roles of EphrinB2 under pathological conditions. Several proteases which were naturally existed in mammals, such as MMPs, ADAMs, γ-secretase, were capable of cleaving EphrinBs, leading to (de) activation of EphrinBs. Besides, synthesized recombinant ECD of Ephrin or Eph, inhibitory antibodies and small molecules were also able to (de) activate EphrinB2 signaling, providing a strongly potential target for further translational application in cardiovascular diseases.


Membrane-associated estrogen receptors (ER)-α36 and G protein-coupled estrogen receptor (GPER) play important roles in the estrogen’s rapid non-genomic actions including stimulation of cell proliferation. Estrogen via these receptors induces rapid activation of transcription factor nuclear factor-E2-related factor 2 (Nrf2), a master regulator of detoxification and antioxidant systems, playing a key role in the metabolic reprogramming to support cell proliferation. This review highlights the possible mechanism underlying rapid Nrf2 activation via membrane-associated estrogen receptors by estrogen and phytoestrogens. Stimulation of ER-α36-GPER signaling complex rapidly induces Src-mediated transactivation of epidermal growth factor receptor (EGFR) leading to a kinase-mediated signaling cascade. We propose a novel hypothesis that ER-α36-GPER signaling initially induces rapid and temporal activation of NADPH oxidase 1 to generate superoxide, which subsequently activates redox-sensitive neutral sphingomyelinase 2 generating the lipid signaling mediator ceramide. Generation of ceramide is required for Ras activation and ceramide-protein kinase C ζ-casein kinase 2 (CK2) signaling. Notably, CK2 enhances chaperone activity of the Cdc37-Hsp90 complex supporting activation of various signaling kinases including Src, Raf and Akt (protein kinase B). Activation of Nrf2 may be induced by cooperation of two signaling pathways, (i) Nrf2 stabilization by direct phosphorylation by CK2 and (ii) EGFR-Ras-PI 3 kinase (PI3K)-Akt axis which inhibits glycogen synthase kinase 3β leading to enhanced nuclear transport and stability of Nrf2. Estrogens have long been known as important regulators of the female reproductive functions. However, it is now commonly accepted that estrogens are locally synthesized and play important roles in the cardiovascular protection, bone preservation, and neuroprotection in both female and male . EsOne of the biological functions of estrogen is stimulation of cell proliferation. ER-α is the major driver of breast cancer and multiple ER targeting drugs are routinely used clinically to treat patients with ER positive breast cancer ,16,17. ARapid Nrf2 activation can be induced by both stabilization of Nrf2 and enhanced nuclear import of Nrf2 protein. Nrf2 protein is unstable under normal conditions because the cytosolic Nrf2 binding protein Kelch-like ECH-associated protein 1 (Keap1) facilitates degradation of Nrf2 by proteasome ,28,29. INTR (neurotrophin receptor) [NTR-depndent manner [NTR induces generation of the lipid signal mediator ceramide via activation of neutral sphingomyelinase (nSMase) [NTR-ceramide-PKCζ-CK2 pathway [NTR is currently not known. We here propose novel signaling pathways downstream of membrane-associated estrogen receptors leading to Nrf2 activation with a special interest in the regulation of proliferation of estrogen-dependent breast cancer cell MCF-7.This review highlights the signaling pathways leading to rapid activation of Nrf2 by 17β-estradiol and some estrogen mimetics via membrane-associated estrogen receptors. Nrf2 activation via hormone-receptor signaling cascades differs from that induced by toxic electrophiles which directly target Keap1. We recently proposed that neurotrophins can activate Nrf2 by a mechanism dependent on the common neurotrophin receptor p75eceptor) ,40,41. Tt manner  and thatt manner ,37. Nota(nSMase) . As cera(nSMase)  and PKCζ(nSMase) , we hypo pathway , althougS-palmitoylation, which is a reversible post-translational lipid modification important for membrane anchoring [ER-α36 is widely expressed in estrogen sensitive cancer cells, and high expression levels correlates with a poor survival prognosis for breast cancer patients . MCF-7 cnchoring .ER-α36 localizes in the cytoplasm and at the plasma membrane, mediating rapid estrogen signaling in human breast cancer cells . Post-tr2+ elevation is required for the activation of tyrosine kinase Src which lead to activation of ADAM17 [EGFR is expressed in various cell types and plays a key role in the regulation of cell proliferation, survival and differentiation. In pathological settings, mostly in lung and breast cancer and in glioblastoma, EGFR is overexpressed or mutated ,52,53. Af ADAM17 , precisef ADAM17 ,56. Notaf ADAM17 , and actf ADAM17 . As GSK3f ADAM17 ,31,32,33f ADAM17  showed tf ADAM17 . These sf ADAM17  also shof ADAM17 .ER-α36 is expressed in rat and human brain especially high levels in cerebral cortex and plays a role in protection of neurons under oxygen and glucose deprivation . EstrogeCloned GPER cDNA predicts GPER is a 375 amino acid protein with the structural signature of the Class A Rhodopsin family 7-transmembrane receptor with about 30% amino acid identity to angiotensin II (AngII) and interleukin 8 receptors . Compares protein and activates phospholipase C (PLC) to catalyze the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3)and diacylglycerol (DAG) [2+ signaling via interaction with plasma membrane Ca2+-ATPase (PMCA) [2+ and for shaping the time courses of Ca2+-dependent activity. The authors show that GPER physically associates with PMCA and suppresses PMCA activity. GPER agonist G1 further inhibits PMCA activity through tyrosine phosphorylation of the pump resulting increase in intracellular Ca2+ levels. Second, GPER enhances expression of NADPH oxidase 1 (NOX1) [2O2) by superoxide dismutase. Meyer et al. [GPER associates with Gαol (DAG) . DAG actol (DAG) . Additioe (PMCA) . PMCA is1 (NOX1) ,65. NOX1r et al.  observedr et al. ,65.2O2 serve as co-stimulatory signals for cell proliferation in many normal and cancer cells [In addition to vascular cells ,67, NOX1er cells ,71,72,73er cells . As impoer cells , GPER ma1R), NOX1, NOXO1 and NOXA1 using virus vectors into HEK293 and CHO cells and characterized mechanism and kinetics of NOX1 activation by AngII [1R signaling is Gαq-mediated activation of phospholipase-β, which is followed by Ca2+ mobilization and activation of PKC, and G protein-dependent and -independent activation of Src family kinases [1R-NOX1 under the experimental conditions [Activation of NOX1 requires association with its regulatory components p22phox, NOX organizer 1 (NOXO1), NOX activator 1 (NOXA1) and Rho family small GTPase Rac1 . Choi etby AngII . The act kinases . The autnditions . This renditions . Notablynditions .2+ mobilization may activate PKC and contribute in NOX1 activation like the phorbol ester , which hydrolyzes sphingomyelin to generate ceramide. Ceramide is a pleiotropic signal mediator of cellular responses including differentiation, proliferation, cell cycle arrest and apoptosis .A notable characteristic of nSMase2 is that enzyme activity can be reversibly inhibited by GSH. Liu and Hannun  partiall2O2 rapidly and effectively oxidizes GSH to generate disulfide GSSG [Taken together, nSMase2 activity seems to be suppressed in cells under normal conditions with high GSH and that the enzyme activation requires downregulation of cellular GSH. Notably, superoxide but not Hide GSSG , which cide GSSG . ImportaThe activation of nSMase2 is also modulated by protein kinases. PKCδ mediates translocation of nSMase2 from the Golgi to the plasma membrane . Phospho2O2. In case of amyloid-β-induced neuronal death, nSMase activation occurred by a NADPH oxidase-superoxide-H2O2 dependent manner [In fact, it was shown that superoxide can activate nSMase in cultured cells ,110. Sawt manner . TherefoCeramide generation by nSMase2 seems to be required for the EGFR mediated signaling cascade. This idea comes from the two facts; (i) Ras scaffolding protein KSR1 is a ceramide-binding protein requiring low levels of ceramide for membrane translocation to trigger the Ras signaling via EGFR ,112 and Gardenia fruit, is an agonist for G-protein coupled glucagon-like peptide-1 (GLP-1) receptor [Geniposide, a major iridoid component of receptor  and GLP-receptor . Thereforeceptor . Geniposreceptor ,119. Penreceptor  showed treceptor  leading CAPE is an active ingredient of coffee, vegetables and beehive propolis and shown to induce Nrf2 activation in renal epithelial cells  and in mIn addition to ceramide-KSR1 signaling, ceramide-PKCζ-CK2 signaling also plays a key role in enhancing kinase signaling via interaction of CK2 with molecular chaperone Cdc37 . ActivatWe propose that the stimulation of membrane-associated estrogen receptors initially activates NOX1. NOX1-derived superoxide activates nSMase2 to generate ceramide which subsequently activates Ras and PKCζ-CK2 axis. The CK2-Cdc37-Hsp90 axis supports EGFR-mediated signaling kinase cascade. Activation of Nrf2 depends on both PI3K-Akt-GSK3β and PKCζ-CK2 signaling pathways . As PI3KThe importance of superoxide in Nrf2 activation has been demonstrated in vascular endothelial cells exposed to vasoprotective unidirectional laminar shear stress . Fluid sRapid activation of Nrf2 by low levels of 17β-estradiol will favor proliferation of MCF-7 cancer cells  by facil