WIREs Dev Biol 2017, 6:e271. doi: 10.1002/wdev.271Tissue patterning during animal development is orchestrated by a handful of inductive signals. Most of these developmental cues act as morphogens, meaning they are locally produced secreted molecules that act at a distance to govern tissue patterning. The iterative use of the same signaling molecules in different developmental contexts demands that signal interpretation occurs in a highly context‐dependent manner. Hence the interpretation of signal depends on the specific competence of the receiving cells. Moreover, it has become clear that the differential interpretation of morphogens depends not only on the level of signaling but also the signaling dynamics, particularly the duration of signaling. In this review, we outline molecular mechanisms proposed in recent studies that explain how the response to morphogens is determined by differential competence, pathway intrinsic feedback, and the interpretation of signaling dynamics by gene regulatory networks. WIREs website.For further resources related to this article, please visit the  Embryonic development is a progressive program in which a single totipotent cell, the fertilized egg, gives rise to hundreds of distinct differentiated cell types. For this to result in the successful completion of embryogenesis, and the well‐organized assembly of functioning organs, the appropriate cell types must be produced at the right time, at the right place and in the correct number. Fundamental therefore, is the elaboration of cell lineages in which multipotent progenitors are transformed to specific cell types in spatially stereotypic arrangements. Strikingly, a handful of inductive signals, iteratively used during development, coordinate this process. The repeated use of a limited set of signals means that the identity of a signal is not itself sufficient to confer specificity.A key finding is that many inductive signals act as morphogens: locally produced, secreted signaling molecules that act over long distances and control growth and patterning throughout a region of tissue.Recent findings from a number of systems have led to the realization that interpretation of developmental cues depends not only on the level but also the duration of signaling.In this review, we first discuss how differential competence underlies the specific response of cells to inductive signals. We then outline molecular mechanisms introducing dynamics into morphogen distribution and signaling and discuss how these dynamics affect signal interpretation. Finally, we highlight the role of transcriptional networks for integrating both levels and dynamics of morphogen signaling to control tissue patterning.As outlined above, the iterative deployment of the same inductive signal in different developmental contexts suggests that interpretation of a signal is highly context‐dependent. This behavior was originally recognized in classic grafting experiments,cubitus interruptus (ci), the transcriptional effector of the Hh pathway,ciHow is competence mediated at the molecular level? In its simplest instance, competence is imposed by the expression of all the necessary components for receiving a signal. Loss of competence results if parts of a signal transduction cascade are missing or blocked. This is for example the case for cells in the Drosophila wing disc. Cells in the anterior compartment of the wing disc express CIP1 and induces neuronal differentiation.This mechanism, however, only allows the discrimination between ON and OFF states of the signaling pathway and does not explain the induction of a distinct set of target genes in different tissues in response to the same signaling molecule. This phenomenon, in principle, could be explained by signals acting through different context‐dependent receptors or transcriptional effector proteins. Such a mode of action has been for example demonstrated for bone morphogenetic protein (BMP) signaling in the dorsal neural tube.In addition to the role of sequence specific TFs, epigenetic changes, such as alterations in chromatin modifications, can modify the accessibility or operability of genomic regulatory elements. One of the best‐described mediators for such epigenetic changes during development is the TF REST , which orchestrates the repression of neuronal genes in non‐neuronal tissues.in vitro work revealed that the timescales of epigenetic memory depend on the type of modification and the duration of recruitment of modifying enzymes to genomic loci and can easily exceed several weeks.In many cases such epigenetic mechanisms also confer memory to cells and are critically required for the maintenance of cell states.In most cases, competence to a signal defines specific developmental stages. Such ‘competency windows’ can arise in response to pathway‐intrinsic feedback, resulting in self‐limiting competency. An example of how this provides a temporal window for competence is the delayed expression of a pathway specific inhibitor. This appears to be the case for mesendoderm specification in response to Nodal signaling in the zebrafish embryoWindows of competence can also arise from the temporal sequence of different signals or by continuous exposure to the same signal. For example, cells in the neural tube can differentiate into FP in response to Shh or roof plate (RP) in response to BMP signaling.Equally, longer exposures to signals can lead to cascades of gene activation, where the competence to induce target genes of the next step, depends on target genes activated in the previous—a mechanism dubbed ‘sequential cell context.’In the previous section, we outlined how differences in cell competence can produce different cell fates in response to the same inducing signals. In the following sections, we will focus on how signaling dynamics affect cell fate decisions, and thus allow further diversification of the repertoire of cell types that can form in a given tissue.Dynamics in morphogen signaling can arise in three main ways: (1) the inductive signal itself can have a dynamic pattern of production or movement through the tissue; (2) positive and negative feedback in the signaling pathway can modulate the response of cells to the ligand; or (3) the underlying GRN can interpret and influence the dynamics of the signal.In the conventional view of morphogen activity all information for differentially inducing target genes is provided by the concentration of the ligand producing different levels of signal transduction. In addition to the level, however, dynamical properties of a signal can be used to carry information—the fold change in signal level, the duration, the amount of signal accumulated over time  and the rate of change of signaling (the speed at which the level increases or decreases). Additionally, if a signal oscillates, the number and frequency of the oscillations can also communicate information. Obviously, these combinations are not mutually exclusive. For example, a signal might need to exceed a certain threshold within a certain time period to induce a specific response, and exceeding this threshold at later stages does not result in the same response.Inductive signals are not only gradients in space, but also in time. How a morphogen is produced and disseminated can lead to dynamics in signaling. The rate of production, rate of clearance in receiving cells, and the speed of movement/diffusion determine the distribution of a morphogen in a tissue.The extent to which morphogen gradients are pre‐steady directly depends on the persistence time of the morphogen in the tissue and the speed with which patterning is read out from the gradient. The longer the persistence time and the faster the readout speed by the patterning system, the more likely it is that pre‐steady state dynamics of the morphogen govern patterning. Pre‐steady state decoding has been proposed for the Bicoid gradient in the Drosophila embryo.wg is transcribed throughout the entire prospective wing field and its expression only later recedes to the DV boundary  in the Drosophila wing disc.y Figure . These efgf8 mRNA is restricted to the posterior tip of the embryo  and Goosecoid (Gsc) in de novo production of the effector proteins and the degradation of the previously formed repressors. This effectively slows the transduction of the signal and imparts some resistance to rapid fluctuations in the level of ligand. Similarly, ligands can stay bound to and activate receptors over a longer timescale, thereby allowing cells to ‘remember’ a signal over a certain timescale. Such a mechanism has been proposed for Activin signaling in Xenopus blastula cells. Here cells can remember hours later Activin exposures that were as short as 10 min.The mechanisms employed within the signaling pathway can introduce dynamics. For example, both the Wnt and Hh signaling pathways, which are used in several developing tissues, exploit the irreversible degradation of the transcriptional effectors of the pathway, either completely degrading them or proteolyzing them into repressors in the absence of signal.Analyses of how much information can be communicated at any one moment by signaling pathways emphasize the limited capacity to control multiple decisions.Several mechanisms have been described that introduce specific nonlinearities into a signal. Ultrasensitivity and positive feedback result in the conversion of a continuous graded signal into a step‐like signal, whereas negative feedback allows cells to adapt to a signal. Besides affecting the output of a signaling pathway, these mechanisms have been shown to control the shape of morphogen gradients and buffer them against fluctuations. Consequently, disruption of these feedback mechanisms usually results in abnormal patterning. Thus, these feedback mechanisms are a vital part of their patterning systems and their function is indispensible for inducing expression domains at the right place, size, and time.Ultrasensitivity can arise when two enzymes with opposing activities (e.g. a kinase/phosphatase pair) are saturated by their substrates.Another way in which graded signals can be converted into step‐like cellular responses is via positive feedback of the signaling pathway, where high levels of signaling enhance further signaling, for example by promoting ligand–receptor interactions, and low levels of signaling result in a downregulation of signaling capacity. Such a mechanism has been proposed to explain signaling activities of the BMP ligands Dpp and Screw during dorsoventral patterning in Drosophila.One of the most commonly used mechanisms for temporal signal integration is negative feedback, in which a signaling pathway actively promotes expression of an inhibitor of the signal transduction cascade. In many cases these inhibitors are considered to be canonical target genes of the respective signaling pathways, and their expression levels are proxies for levels of pathway activity . Such negative feedback systems confer several properties, namely they can increase the dynamic range of ligand a cell can sense, lead to pathway adaptation and provide a means for cells to measure duration of signaling. Another form of negative feedback is ligand‐mediated receptor degradation, which has been described for EGF and TGFβ signaling.A negative feedback inhibitor that directly interacts with the ligand or the receptor can allow the cell to increase the range of ligand concentrations it can measure with the same number of receptors, as high levels of ligand promote strong production of inhibitors, which will sequester the number of available ligands.In the vertebrate spinal cord neuronal progenitor domains are specified along the DV axis by the distinct activities of different morphogen systems. Subdivision of the ventral neural tube is mediated by the regulation of a set of TFs in response to graded Shh signaling  and does not reflect levels or duration of pathway stimulation. Instead cytoplasmic relocalization depends on de novo protein synthesis, but not ubiquitin‐mediated protein degradation. The molecular details of this mechanism remain to be determined and it seems unlikely that this is the only mechanism at work, as mice expressing a constitutively nuclear version of Smad4 are viable.In the case of TGFβ, signaling leads to receptor mediated phosphorylation of Smad2/3 (R‐Smads) that then form a complex with Smad4, translocate to the nucleus and activate target genes , another Dpp target gene. Thus, these genes are linked together in a feed‐forward loop where Dpp must first induce zen and then the combination of zen and continued Dpp signaling induces expression of Race and C15.One way to integrate dynamics of signaling is via feed‐forward loops in which a signal elaborates a cascade of target gene expressionAnother commonly used GRN motif is mutual cross repression, where a signal induces two TFs, which repress each other  GRNs allow the conversion of a graded morphogen input into discrete domains of gene expression. Furthermore cross‐repressive interactions between genes induced in adjacent domains can refine patterning and buffer the patterning system against fluctuations. (2) Positive autoregulation and mutual cross‐repression can confer memory to patterning systems and thereby transform a transient input into a developmentally stable output. (3) Transcriptional feed‐forward cascades and more complex GRNs provide mechanisms for interpreting levels, duration and dynamics of morphogen signaling.We have outlined in this review how cells diversify their response to morphogens by changing their competence to the signal over time, using the dynamics of signaling and employing transcriptional networks to convert signals into stable patterns of gene expression. Thus to understand development we need to be able to assay with increasing precision the dynamics of signaling and measure the regulation of multiple genes simultaneously. This is necessitating sophisticated experimental approaches. To this end, a wide range of exciting new techniques have emerged that allow the quantification of various aspects of gene regulation in single cells. These range from quantitative imaging approaches to sequencing methods that allow assays of transcriptomes, genome accessibility, and epigenomic modifications. Complementing these, mathematical tools are needed. Current models are largely restricted to relatively simple GRNs. In contrast, the predictive value of models of more complex GRNs is usually limited. Combining theoretical and experimental approaches will likely allow us to construct more detailed and quantitative models and thereby provide a deeper insight into the regulatory logic of the genome.


As a result, infected cells survive, proliferate and infiltrate into the tissues, which is critical for transmission of the virus. Thus, the strategy of this virus is indivisibly linked with its pathogenesis, providing a clue for prevention and treatment of HTLV-1-induced diseases.Human T-cell leukaemia virus type 1 (HTLV-1) causes not only adult T-cell leukaemia-lymphoma (ATL), but also inflammatory diseases including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 transmits primarily through cell-to-cell contact, and generates abundant infected cells in the host in order to survive and transmit to a new host. The resulting high proviral load is closely associated with the development of ATL and inflammatory diseases. To increase the number of infected cells, HTLV-1 changes the immunophenotype of infected cells, induces proliferation and inhibits apoptosis through the cooperative actions of two viral genes, This article is part of the themed issue ‘Human oncogenic viruses’. HTLV-1 has a simian origin called simian T-cell leukaemia virus type 1 (STLV-1). STLV-1 is endemic in many species of Old-World monkeys and apes. The precise origins of the ancestors of HTLV-1 now present in humans remain uncertain, i.e. the species of monkey and the time and place of the putative interspecies transmission. However, it is thought to be an ancient interspecies transfer . In this2.in vitro. Thus, transmission of this virus needs living infected cells. Infected cells transmit to new hosts through three routes: breast-feeding, sexual intercourse, and the parenteral route [in vivo. For this purpose, HTLV-1-infected cells need to evade host immune surveillance, promote proliferation and inhibit apoptosis. Another important issue for transmission is that infected cells enter into breast milk and semen for transmission. HTLV-1 has therefore evolved mechanisms to increase the migratory capacity of infected T cells. These two broad viral strategies—host cell proliferation and cellular motility—are closely linked to the pathogenesis of the diseases caused by this virus [One of the most striking characteristics of HTLV-1 is that this virus can transmit only through cell-to-cell contact . Free vial route . Therefois virus ,6.3.gag, pol and env), it encodes regulatory genes (tax and rex) and accessory genes ) [HBZ is encoded on the minus strand of the provirus and transcribed from the 3′ long terminal repeat (LTR). Other viral genes are transcribed as sense transcripts from the 5′LTR. Transcription from the 5′LTR is highly inducible by Tax, in which CREB and p300/CBP are involved [tax is intermittently transcribed in vivo, HBZ undergoes constant expression, in which SP1 is critically involved [HBZ transcription by cooperating with Sp1 [+ T cells in vitro, suggesting that cytotoxic T lymphocytes (CTLs) suppress Tax expression in vivo [HTLV-1 has a similar structure to other complex retroviruses. In addition to structural genes (r (HBZ)) . As showinvolved . Whereasinvolved . JunD auwith Sp1 . Tax exp in vivo . These d in vivo . Therefotax and HBZ genes are encoded respectively by the plus and minus strands of the provirus. Transcription of these genes appears to be reciprocally controlled. In valproate-treated infected cells with high Tax expression, the HBZ transcript was suppressed [The ppressed . However4.de novo infection [+ T cells, indicating that this subpopulation is infected with HTLV-1 [HBZ transgenic mice possessed the immunophenotype of effector/memory T cells, whereas effector/memory T cells were not increased in tax-transgenic mice [As noted above, the infectivity of free HTLV-1 virions is very poor, and HTLV-1 can transmit efficiently only through cell-to-cell infection . Infectenfection . Therefonfection . In all nfection ,19. The h HTLV-1 . Most T h HTLV-1 . Most HBnic mice . Thus, H5.Because primary infection with HTLV-1 is asymptomatic, there are few data on the rate of propagation of the virus during the establishment of the proviral load. In three recipients of organ transplants from an infected donor, the proviral load in the circulation doubled approximately every 1.4 days during the first few weeks of infection . It is nLike other replication-competent exogenous retroviruses, HTLV-1 can propagate by two routes . First, 5-fold lower than that of reverse transcriptase. Mitotic replication therefore generates much less sequence diversity than infectious replication.Second, mitosis of an HTLV-1-infected cell produces two daughter cells that carry the provirus at the same genomic site. In contrast to the infectious route of spread described above, this ‘mitotic’ route involves replication of the provirus by DNA Pol2, whose nucleotide misincorporation rate is about 10Integration of the HTLV-1 provirus in the host genome is not random, but is determined by factors at four successive physical scales . First, tax and HBZ (see below), and certain HTLV-1-infected T-cell clones reach a very high abundance in the circulation. Until recently, it was believed that a typical HTLV-1-infected host carried about 100 infected T-cell clones [in vivo and contributed to the pathogenesis of the inflammatory and malignant diseases associated with HTLV-1. Recent evidence from high-throughput sequencing and quantification of HTLV-1 integration sites has changed this view: the number of HTLV-1-positive T-cell clones in the circulation in each host is of the order of 104–105 [HTLV-1 infection drives proliferation of the infected cell, through the products of the HTLV-1 genes l clones , and tha 104–105 , and it  104–105 . These o+ and CD8+ T cells are infected by HTLV-1: about 95% of the proviral load is present in CD4+ T cells, and 5% in CD8+ T cells [+ and CD4+ T cells are preferentially infected with the virus if they are HTLV-1-antigen-specific [in vitro; small numbers of dendritic cells, monocyte/macrophages and epithelial cells are infected in vivo. It has been reported that infected dendritic cells and macrophages play important roles in propagation of HTLV-1 [Both CD4 T cells . Both CDspecific ,39. Mostf HTLV-1 .6.+ T cells in vivo [+ T cells. However, the precise mechanisms of cell specificity of HTLV-1 and HTLV-2 remain to be elucidated.Human T-cell leukaemia virus type 2 (HTLV-2) was discovered in a T-cell line established from a patient with T-cell variant hairy cell leukaemia . However in vivo , whereas7.The immune response to HTLV-1 has been extensively reviewed elsewhere –46, and (a)in vivo, but since it targets the minus strand during reverse transcription, it spares HBZ [HBZ expression may favour viral persistence. Like HIV-1, HTLV-1 incorporates APOBEC3G into the virion, but this incorporation is limited by a peptide motif in the nucleocapsid [Efficient spread of HTLV-1 virions across the intimate cell–cell contact in the virological synapse minimizes the ability of tetherin to inhibit HTLV-1 propagation . The deaares HBZ ; the coneocapsid .SAMHD1, which limits the supply of nucleoside triphosphates for virion replication, inhibits HTLV-1 infection of macrophages . Type 1 (b)+ CTL plays a dominant role in host protection in most viral infections, and the CTL response is an important determinant of the outcome of HTLV-1 infection. HTLV-1-infected individuals typically have a high frequency of persistently activated, HTLV-1-specific CTLs in the circulation; the activated state indicates recent exposure to newly synthesized viral antigen, and therefore demonstrates that the virus is persistently expressed in vivo. Although the Tax protein is highly immunodominant in the anti-HTLV-1 CTL response, it is the efficacy or ‘quality’ of the CTL response to the HBZ protein that is associated with control of the virus in vivo [The frequency and activity of NK and NKT-like cells are abnormally low in HAM/TSP –57, but  in vivo . The cla in vivo ; in sout in vivo . For rea in vivo .+ T cells is significantly higher in patients with HAM/TSP than in asymptomatic carriers of the virus. CD4+ T cells predominate in the early, active lesions in the central nervous system in HAM/TSP [The frequency of HTLV-1-specific CD4 HAM/TSP , and the HAM/TSP ,61.+ T cell to secrete the chemokine CCL22, which maintains a high frequency of CD4+ Foxp3+ cells in the circulation because these cells characteristically express the CCL22 receptor, CCR4 [+ cells may diminish the protective effect of the anti-HTLV-1 CTL response [HTLV-1 Tax protein induces the infected CD4or, CCR4 . The regresponse . Althougresponse ,65.8.(a)de novo infection does not contribute significantly to the proviral load in the chronic phase [After infection, HTLV-1 increases the proviral load mainly by driving proliferation of infected cells (mitotic division) ,67. Indeic phase . HBZ andic phase . HBZ alsic phase . Furtheric phase ,72, whicHBZ RNA possesses functions that are distinct from those of its protein. HBZ RNA is more strongly retained in the nucleus than the other viral gene transcripts. HBZ RNA inhibits apoptosis, and promotes proliferation of expressing cells [In addition to these functions of HBZ protein, ng cells ,74. Thesc-FLIP [in vivo.Tax strongly activates NFκB, which leads to expression of the anti-apoptotic gene c-FLIP ,76 and gc-FLIP . In addic-FLIP . Thus, Tc-FLIP . However(b)HBZ gene is persistently expressed at a low level in most—if not all—infected cells in vivo [in vivo. It is unknown what regulates this differential expression of plus and minus strands and, in particular, the frequency, intensity and duration of what are presumably bursts of plus-strand expression.The  in vivo . The pluet al. [in vivo, but they also observed an unexplained border in the methylation in the pX region of the provirus. It has now been shown [HBZ) expression. Another major function of CTCF is to form chromatin loops, both to organize the chromatin structure and to bring enhancers near their cognate promoters. The abnormal CTCF-binding site present in the HTLV-1 provirus can indeed form loops with flanking host chromatin [Taniguchi et al.  showed ten shown  that CTChromatin ; this loThe genomic integration site influences the expression of the provirus, and consequently each infected T-cell clone will have its own characteristics of proviral expression. Two chief specific features of the integration site are associated with the regulation of plus-strand expression . First, ex vivo? The answer to this question will illuminate the mechanisms that maintain plus-strand latency in vivo. Second, what are the molecular mechanisms that give rise to cell-to-cell heterogeneity in proviral expression, and what is the importance of this heterogeneity in viral persistence?Two central questions remain in the regulation of HTLV-1 proviral latency. First, what causes  the rapid spontaneous transcriptional activation of the provirus when PBMCs are taken (c)4 and 105 (ranging from 103 to 106). It is the large number of low-abundance clones that constitute the high proviral load [in vivo.It was formerly believed that a typical host possessed about 100 clones of HTLV-1-infected T cells , and tharal load : these cThe current picture of HTLV-1 clonality is depicted schematically in 9.in vitro [in vivo. However, the cell type of the cancer caused by Tax depended on the promoter. Tax expression by the granzyme B promoter induced a tumour of NK cells [For many years, the Tax protein was believed to be necessary and sufficient to cause malignant transformation of HTLV-1-infected cells. Transduction of Tax-expressing vectors can immortalize T cells in vitro ,92, and NK cells , whereasNK cells . Thus, ttax gene, (ii) DNA methylation of the 5′LTR, and (iii) deletion of the 5′LTR. However, HBZ is expressed in all ATL cases, suggesting that HBZ is indispensable. DNA methylation of the 5′LTR accumulates during the natural course of infection, which finally silences transcription of the sense strand from the 5′LTR [HBZ transcription. Recently, a CTCF-binding region was found in the pX region, which may account for the arrest of DNA methylation before pX and 3′LTR, and ensure continued HBZ expression [The most direct evidence of HTLV-1 infection in ATL cells is the presence of the monoclonally integrated provirus. Therefore, analysis of the structure and genomic integration site of HTLV-1 proviruses in ATL cells can provide critical clues on leukaemogenesis by HTLV-1. Studies of HTLV-1 proviruses and transcripts of viral genes showed that ATL cells do not express Tax in approximately half the cases of ATL. There are three known mechanisms to inactivate Tax expression: (i) nonsense mutations of the he 5′LTR . Howeverhe 5′LTR , which ipression . Furtherpression . The lactax gene were found in approximately 10% of ATL cases. There is a hotspot of nonsense mutations in the HTLV-1 provirus in a target sequence of APOBEC3G [tax gene was found in some asymptomatic carriers [tax gene can transform to ATL cells, suggesting that HBZ plays critical roles in oncogenesis.Nonsense mutations of the APOBEC3G , suggestcarriers . These fin vivo through the actions of HBZ and Tax during asymptomatic carriage of the virus. Thereafter, genetic and epigenetic alterations fix or potentiate these changes. For example, HBZ induces CCR4 expression [A long latent period is necessary before onset of ATL, suggesting that multiple genetic and epigenetic alterations are needed for ATL . Recentlpression . Gain-ofpression ,85. Furtpression . These t10.(a)There are four subtypes of ATL according to clinical criteria: acute, lymphoma-type, chronic and smouldering types . In geneIt has been reported that sustained treatment with azidothymidine (AZT) plus interferon-α can be effective in cases of ATL of the leukaemic types, but is less effective in the lymphoma type . Althougin vivo [in vivo, which may account for the prolonged suppressive effect of this antibody on ATL cells.Most HTLV-1 infected cells and ATL cells express the chemokine receptor CCR4 on their surfaces . CCR4 exin vivo . CCR4 isin vivo . After ain vivo . Thus, t(b)in vivo during chronic infection mainly by sustained proliferation of infected cells. The high resulting proviral load is also critical in the transmission of the virus, which requires transfer of infected cells to the new host. Therefore, a logical strategy to prevent ATL is to reduce the number of infected cells. Immune responses, primarily CTLs, strongly suppress proliferation of ATL cells and HTLV-1-infected cells. Thus, vaccines against Tax and HBZ might be effective in preventing HTLV-1-associated diseases. In addition, the recent genetic and epigenetic analyses of ATL cells reveal critical pathways and molecules for ATL. Therapy targeted to these pathways might improve the prognosis of this intractable disease.As discussed above, HTLV-1 persists


In this review, we discuss several studies that determine specific molecular pathways affected by autism-associated mutations, and then discuss more in-depth into the biological roles of a few of these pathways, and how they may be involved in the development of ASD. Considering that these pathways may be targeted by specific pharmacological intervention, they may prove to be important therapeutic targets for the treatment of ASD.In recent decades it has become clear that Autism Spectrum Disorder (ASD) possesses a diverse and heterogeneous genetic etiology. Aberrations in hundreds of genes have been associated with ASD so far, which include both rare and common variations. While one may expect that these genes converge on specific common molecular pathways, which drive the development of the core ASD characteristics, the task of elucidating these common molecular pathways has been proven to be challenging. Several studies have combined genetic analysis with bioinformatical techniques to uncover molecular mechanisms that are specifically targeted by autism-associated genetic aberrations. Recently, several analysis have suggested that particular signaling mechanisms, including the Wnt and Ca Autism Spectrum Disorder (ASD) is a developmental disorder characterized by persistent deficits in social communication, as well as restricted and repetitive patterns of behavior. It has been well characterized that genetic aberrations have a prominent role in the etiology of ASD . Among tFMR1) gene, up to 30% of these male patients also develop ASD [CNTNAP2), where a subgroup of patients express autistic behavior [TSC1/TCS2), where up to 61% of patients express autistic behaviors [MECP2) [SHANK3/PROSAP2, where mutations in this gene alone have been shown to be associated with ASD [A heterogeneous genetic etiology for ASD has been firmly established. Historically, some of the first genes that were successfully associated to autism were those responsible for syndromic forms of autism. Thus far, approximately 35 such syndromes have been documented . A sub-gelop ASD . Corticabehavior . Tuberouehaviors . Rett sy [MECP2) . Another [MECP2) , which c [MECP2) . The moswith ASD ,17. Addiwith ASD , the chrwith ASD ,20 and twith ASD .FOXP1, GRIN2B, SCN1A and LAMC3 [SCN2A, CHD8 and NTNG1 [STXBP1, UBE3A, KATNAL2, THRA, KCNQ4, MYH14, GJB6 and COL11A1, some of which have been previously associated with ASD and some with overlapping conditions such as hearing loss [A wider search for ASD’s genetic etiology started with linkage studies, which uncovered several candidate loci, including 2q, 3q 7q and 20p. However, it was primarily through Next Generation Sequencing (NGS) technologies that majority of genes were discovered, and by 2012, 10–20% of ASD cases had a known genetic link . One appnd LAMC3 . In 2012nd NTNG1 ,23,24. Bing loss . In addiing loss . To this day, over 800 genes have been identified and associated with ASD development . Given tOne approach to discovering molecular pathways that are commonly targeted by autism-associated mutations is to perform Protein-Protein Interaction (PPI) network analysis to identify groups of proteins within a given list of proteins that physically interact with each other. This analysis uses different databases that curate experimentally validated or predicted protein-protein interactions . PPI alsNature, a subset of these de-novo mutations were enriched for genes involved in chromatin-binding [As part of the effort conducted by the SSC in 2012, O’Roak et al. sequenced the exome of 209 families with sporadic cases of ASD, revealing 126 genes which had truncation or missense mutations, followed by PPI analysis, to determine if these genes form a biologically-relevant network . They di-binding . A separ-binding . An addi-binding . 2+/Calmodulin-signaling [A different strategy to identify molecular pathways that are dysregulated by autism-associated mutations is to use publicly available data of gene expression patterns in the human brain to build gene expression networks and PPI networks that are found in specific brain regions, and then to probe if any of these networks are enriched for autism-associated genes, and are therefore likely to be perturbed by autism-related mutations. Lin et al. used such an approach to identify PPI networks in the brain that are effected by the autism-associated CNV at chromosome 16p11.2 . Using pignaling ,38,39,40Wnt-signaling pathways orchestrate cellular proliferation, polarity and differentiation; processes that are crucial for healthy tissue morphogenesis, especially in the embryonic stage . DysreguCanonical Wnt-signaling has a pivotal role both in the developing and mature brain. Much evidence has indicated that during development the Wnt pathway regulates the balance between proliferation and differentiation of neuronal progenitor and precursor cells, as was recently reviewed by Noelanders and Vleminckx . In tranIn addition to its roles in development, Wnt-signaling also affects Neuronal Stem Cell (NSC) proliferation and differentiation in the mature brain. One study of Wnt-signaling in NSCs used mice which express lacZ under the control of a TCF transcription response . These mN-cadherin complex acts as a scaffold between intracellular actin and extracellular cadherin-dependent interactions [N-cadherin individually, and co-expression of β-catenin and N-cadherin in vitro, both induce increased dendritic branching [N-cadherin complex. By inducing a conditional knockout of β-catenin in the cerebral cortex and hippocampus of mice, spine density is increased and is comprised mostly from immature spines [Apart from its role in NSCs, Wnt-signaling has a positive developmental role in the maturation of dendrites and spines. The α-catenin/β-catenin/ractions ,51. Thisranching . In addie spines . This suN-cadherin complex, and the complex’s importance in inducing cell-cell contacts [In addition to the roles in neuronal differentiation and morphology, β-catenin has been suggested to have an additional role in neurotransmission through two distinct mechanisms. First, translocation of β-catenin to spines may increase the size of the post synaptic density (PSD) which leads to increased synaptic transmission . This wacontacts , the autcontacts . Veli hacontacts ,58. It hcontacts . In thatcontacts . An addicontacts . These sFrom the known roles of Wnt-signaling in neuronal differentiation, morphology, and neurotransmission, we can propose possible roles for dysregulated Wnt-signaling in the processes that are dysregulated in the brain of individuals diagnosed with ASD. Numerous studies have witnessed differences in cortical patterning or spine morphology in the brain of individuals with ASD. In some cases, it has been shown that cortical dendritic spine density is increased in the brain of individuals with autism . This maExperimentation in mouse models have also given more insight into the possible roles of Wnt in the development of ASD. Interestingly, a conditional knockdown of β-catenin in Parvalbumin (PV) neurons in mice induced increased repetitive behaviors and anxiety, decreased social interaction, and an increase in PV neuron density in the prefrontal cortex . An addiCHD8, a gene that was recently found to be strongly associated to ASD and encodes for a chromatin-binding protein. One of the first studies on CHD8 in the brain shows that it acts as a positive regulator of the Wnt pathway in the brain [In addition, recent studies in mouse models have determined how autism-related mutations may induce autism-related behaviors through the dysregulation of Wnt-signaling. One such example is he brain . CHD8 bihe brain . Surprishe brain ,75. By k2+) concentrations have been shown to affect the function of several tissues and organs such as the heart, pancreas and components of the Central Nervous System (CNS). Therefore, it is of no surprise that disequilibrium in signaling leads to pathology [2+ performs specific functions in the presynapse and postsynapse. In the presynapse, arrival of action potentials induce an increase of presynaptic Ca2+ levels through activation of the N and P/Q-voltage-gated channel. The influx of Ca2+ leads directly to the release of neurotransmitters [2+ in postsynaptic signal transduction pathways has been found to be dysregulated in the autism genetic studies discussed previously. Shifts in Calcium , which induces signaling to additional downstream CaM Kinases [2+ influx at the postsynapse by binding to N-methyl-d-aspartate receptors (NMDARs) and metabotropic glutamate receptors such as mGlur1, which initiates the release of internal Ca2+ stores [2+, CaM may induce the activation of CaMKII or CaMKK2. CaMKII is a dodecamer holoenzyme which has 28 isoforms composed of four different subunits (CaMKIIα through δ), and resides mostly in spines and dendritic shafts of excitatory neurons [CAMKIIA, or inhibiting the binding of CaM and Ca2+ to CaMKII, inhibits LTP induction [A β, γ2 and α1 subunits, and regulates GABAA trafficking to the synaptic membrane [Ca Kinases ,78. Glut+ stores . After b neurons ,80,81. T neurons . Active nduction . CaMKIIβnduction . Furthermembrane . Since GCAMKIG and CAMKIV are among the genes which were enriched in the Calmodulin-binding pathway in the Ben-David et al. publication, which looked for common molecular pathways affected by rare and common variations in ASD [2+ signaling and the CaM pathway are involved in neurological functions by affecting a variety of synaptic characteristics, neurotransmission via excitatory and inhibitory receptor regulation and important biological functions such as LTP and LTM. Deficits in the CaM pathway and its branching cascades have the potential to be involved in many neuropsychiatric conditions due to their broad influence on many biological systems, and more specifically by the way it regulates neurotransmission and synaptic characteristics.CaM also activates CaMKK2, whose activity is crucial for spatial memory formation, and the downstream activation of additional kinases: CaMKI is a positive transducer of growth cone motility which is essential for neurite elongation and arborization ; phosphos in ASD . Overall2+ and CaM signaling in synaptic function and neuronal connectivity, it is reasonable to presume that dysregulation of this pathway could lead to autism-related symptoms. However, it is technically challenging to decipher if there are any dysregulation in synaptic functions such as LTP in humans diagnosed with ASD, while evidence for such dysregulation have been frequently observed in several ASD mouse models [2+ signaling in autism is still at its infancy, compared to more established role of the Wnt pathway. Of great interest, one human study has used Transcranial Magnetic Stimulation (TMS) to study changes in long term potentiation-like synaptic plasticity in humans diagnosed with ASD [Considering the central role of Cae models ,89. Therwith ASD . In thiswith ASD . These s2+ signaling in the human brain remains challenging, recent studies have determined disturbances in Ca2+ signaling in cells derived from individuals with ASD. Agonist-evoked Ca2+ signaling has been shown to be dysfunctional in skin fibroblasts derived from individuals diagnosed with autism [2+ signaling and changes in activity-dependent gene transcription. While these studies suggest that Ca2+ signaling and CaM may be involved in the biology of ASD, technological improvements of Ca2+ imaging in the human brain, and more high-throughput studies in individuals diagnosed with ASD, are necessary to understand the role of Ca2+ in the specific behaviors and brain regions that are particularly relevant to ASD.While studies of Cah autism . An elegh autism , a syndr2+ and Calmodulin-binding may be involved in abnormal neurodevelopment. For example, CaMKIV positively regulates the transcription of FMRP , the causative gene of FXS [FMR1 knockout mice improved learning and memory, which highlighted this therapy as a promising pharmaceutical treatment [Animal models and in vitro studies have given some additional insights into how dysregulation of Cae of FXS . A folloe of FXS . In addie of FXS . It is se of FXS . Inhibitreatment . Howeverreatment . In one reatment . In anotreatment . 2+ signaling dysregulation is Angelman Syndrome (AS), which is characterized by the deletion of the maternal allele of 15q13-11, including the gene UBE3A. In the UBE3A maternal allele null mouse model, disruptions in the autophosphorylation of CaMKIIα, which is essential for its kinase activity, is responsible for LTP deficits of the hippocampus [2+ signaling enzymes that are most likely to be involved in ASD. On one hand the Ca2+ and Calmodulin-binding pathways may be interesting targets for therapeutic interventions. However, future studies in mouse models, and electrophysiology studies in humans, are necessary to understand how and why the Ca2+ signaling pathway is involved in ASD. Further studies are particularly needed to clarify the potential roles of Ca2+ signaling in social behaviors or repetitive behaviors, which form the core features of ASD. An additional syndromic autism linked with Capocampus . CaMKIIαpocampus . In summThus far, we have discussed the Wnt-signaling and Calmodulin-binding pathways as potential molecular mechanisms that are involved in ASD, due to the fact that these pathways have been found to be enriched for ASD-associated genes in multiple network-based analysis. In addition to these pathways, the mTOR-signaling and NGF-signaling pathways have also appeared in some of the discussed bioinformatic publications, although not quite as often as the Wnt and Calmodulin pathways. The mTOR pathway has already gained significant attention in connection to autism due to its significant involvement in syndromic autisms, as explained below. There is yet little known connection between NGF and ASD, however it is worth shortly considering the possible connections, considering its roles in brain development and function.Mammalian Target of Rapamycin (mTOR) is a serine/threonine kinase, which is considered a central kinase in organism development. Therefore it is of no surprise that ablation of mTOR results in in-utero death a short time after the implantation stage of the embryo in the uterus endometrium . The mTOPTEN-related syndrome, Neurofibromatosis Type 1 (NF-1) and FXS [PTEN mutations in a subset of ASD cases are co-morbid with overgrowth and macrocephaly [PTEN in mature neurons of the cerebral cortex and hippocampus display axonal overgrowth, ectopic axonal projections, and abnormal synapses, as well as reduced social interaction, increased anxiety, and hyperexcitability [There is an abundance of publications linking mTOR-signaling to ASD, and it is considered to be one of the most promising converging signaling pathway candidates for ASD development. The involvement of mTOR-signaling in ASD and other neuropsychiatric conditions has been thoroughly reviewed previously . However and FXS ,108,109. and FXS . Interes and FXS . PTEN isocephaly . Mouse mtability . In a retability . Neurofitability . It is tIn all of the examples given above, inability to downregulate mTOR-signaling is associated with the autism phenotype. In fact, studies determined that blocking mTOR through the use of Rapamycin improves social deficits in the TS and BTBR mouse models ,116 whicNerve Growth Factor (NGF) is a primary neurotrophin for peripheral organ innervation and sensory neuron development. As the nervous system develops, target-organs secret NGF that is detected by elongating axons, and through receptor-mediated endocytosis it enhances survival, neurite outgrowth and synaptic plasticity and connectivity . A role Given its important role in nervous system development, it is not surprising that dysregulation in NGF-signaling has been implicated in psychiatric disorders such as depression, schizophrenia and Alzheimer’s disease . Neverth2+ signaling pathways that we discussed in-depth in this review are potential targets for pharmacological intervention. However, while these pathways have been well characterized, there is still a great need to understand exactly how dysregulation of these pathways are involved in the core characteristics of ASD, including social and repetitive behaviors. A more clear understanding of the specific roles of these pathways in specific brain region is also likely to shed some light into this issue.ASD’s elusive genetic etiology imposes a great challenge to our understanding of the disorder’s pathology. The growing number of genes associated with ASD, both rare and common variants, and the fact that these genes are involved in a variety of biological processes, makes it difficult for the research community to find a specific target for therapy. Taking into account that there are currently no pharmacological agents that treat the core symptoms of ASD, there is great need to understand the biological pathways that are targeted by ASD-mutations, which can be novel pharmacological targets. Both Wnt and the CaUp until now, the only way to find common pathways affected by ASD-associated genes was by in-silico network analysis. However, with the creation of multiple mouse models based on these genetic aberrations, a current method can be to decipher common molecular dysregulations found in these multiple autism mouse models, and to correlate these dysregulations with the animal’s behavioral and neurodevelopmental phenotypes. For example, Ellegood et al. revealed that autism mouse models can be clustered according to neuroanatomical differences such as changes in the volume of different brain regions . In a re


Since the initial discovery of the oncogenic activity of WNT ligands our understanding of the complex roles for WNT signaling pathways in lung cancers has increased substantially. In the current review, the various effects of activation and inhibition of the WNT signaling pathways are summarized in the context of lung carcinogenesis. Recent evidence regarding WNT ligand transport mechanisms, the role of WNT signaling in lung cancer angiogenesis and drug transporter regulation and the importance of microRNA and posttranscriptional regulation of WNT signaling are also reviewed. Lung cancer (LC) is one of the deadliest forms of cancer worldwide , 2 affecAn increasing amount of evidence has shown that the WNT pathway is one of the main signaling pathways involved in maintaining lung homeostasis and that aberrant activation of this pathway may underlie several debilitating lung diseases. Similarly, to other human cancers, WNT signaling plays an important part in lung carcinogenesis. Interestingly, however, while some epigenetic changes that affect WNT pathway inhibitors are similar to those seen in other malignancies, genetic mutations of the WNT pathway are uncommon in NSCLCs .This review will summarize some novel aspects of WNT signaling, what is currently known about WNT associated LC pathogenesis as well as some important features of WNT mediated events in LC therapies.WNT proteins are secreted glyco-lipoprotein morphogens that are required during lung development for cell-fate specification, cell proliferation and the control of asymmetric cell division. In adults, WNT signaling is essential for stem cell maintenance for regulation of tissue homeostasis . Most ofIn the lung, the role of WNT signaling has been examined in detail by multiple studies which mostly focus on beta-catenin-dependent signaling. In the canonical pathway during the absence of WNT, a beta-catenin destruction complex is assembled, consisting of: Axis inhibition protein (AXIN), adenomatous polyposis coli (APC), and glycogen synthase kinase 3-beta (GSK-3-beta) whereby beta-catenin is phosphorylated at serine and threonine sites and then proteolytically degraded , 12. If . This, in turn, activates the stress kinases JNK  and ROCK (Rho-associated coiled-coil-containing protein kinase 1) that initiates remodeling of the cytoskeleton thus leading to changes in cell adhesion and motility [The two non-canonical WNT pathways are activated by several WNT ligands including WNT4, WNT5a, WNT7a, WNT11 and WNT16 . Activatmotility –21.The best known activator of Ca2 + −dependent WNT signaling is WNT5a. It triggers signal transduction via DVL-3, heterotrimeric G proteins and phospholipases . ActivatAlthough WNT signaling pathways seem distinct, WNT proteins are promiscuous and can share receptors and regulate the expression of WNT signaling molecules, as well as modify WNT signaling pathway activity. Non-canonical WNT signaling, for example, represses canonical WNT activity via various mechanisms involving PKC-alpha, CaMKII-Transforming growth factor beta-Activated Kinase (TAK)1, Nemo-like Kinase (NLK), Siah2 E3 ubiquitin ligase or calcineurin-NFAT , 25–28. Functional analyses of the canonical and non-canonical WNT pathways revealed that the canonical, PCP and Ca2+ pathways regulate multiple cellular activities in the lung that are dependent on the specific cellular context. In most cell types, non-canonical WNT signaling regulates canonical WNT activity, which is also critical for many aspects of lung biology. In response to canonical WNT signaling for example, beta-catenin/TCF/LEF signaling is activated in different lung cell types including the primordial epithelium (PE), alveolar epithelium (AE), and adjacent mesenchyme . Human tConsequently, constitutive activation of either the canonical or the non-canonical WNT pathways in the developing lung can result in non-differentiated, dysfunctional lung phenotypes that resemble certain subtypes of LCs . In suppVarious LC subtypes are believed to originate from stem cells in different histological parts of the lung. Adenocarcinoma, one of the NSCLC subtypes, has been reported to develop from various progenitor cells including alveolar type (AT) II cells, Clara cells, and bronchioalveolar stem cells (BASCs) –41. The In murine models, activation of WNT signaling is associated with increased carcinogenic potential  especialThe most studied WNT pathway mutations in cancers include inherited and sporadic mutations in APC and beta-catenin genes. Since APC is part of the degradation scaffold for beta-catenin, mutations of APC result in reduced degradation and increased nuclear accumulation of beta-catenin, leading to activation of target oncogenes including cyclin D1 and c-myc . Such muA shift from canonical to non-canonical WNT signaling, or vice versa, has also been reported in certain NSCLS subtypes. Up-regulation of the canonical WNT7b was detected in adenocarcinomas, while increased expression of WNT5a was found in primary squamous cell carcinomas . AdditioIt is not only the WNT ligands, but also various signaling molecules that are dysregulated in LCs. For example, overexpression of DVL-3, a signal transducer molecule and positive regulator of WNT signaling pathways, was reported in 75% of primary NSCLCs compared to autologous matched normal tissue controls . Down-reGenome-wide association studies recently identified three LC susceptibility loci in chromosome regions 15q25, 5p15 and 6p21 . ImportaGradients of WNT proteins are essential for tissue maintenance. Importantly, WNT gradients lead to different gene expressions at certain points of the gradient in the tissue. Such concentration differences generated along the gradient might even explain how designated “canonical” and “non-canonical” WNT proteins can alternate between signaling pathways. How the gradient is maintained is not yet entirely clear. Due to their lipid residues, WNT proteins are highly hydrophobic; they attach tightly to cell membranes  and then, sFRPs 1–5, WIF-1, DKK-1, DKK-3, human homolog of Dapper (HDPR)1, runt-related transcription factor (RUNX)3, APC, caudal type homeobox (CDX)2, Dapper homolog (DACT)2, transmembrane protein (TMEM)88, Chibby, naked cuticle homolog (NKD1), empty spiracles homeobox (EMX)2, inhibitor of growth family (ING)4, and miR-487b. Although the mechanisms are not yet entirely clear, methylation and hypermethylation are the likely causes of reduced inhibitor levels [Secreted inhibitors and activators that regulate complex interactions within the complicated molecular network of WNT signaling  are under levels , 95. DACr levels . Similarr levels . Restorar levels .Epigenetic modulation of the “anti-aging” Klotho  is also Methylation, however, is not the only posttranslational modification that modulates WNT signaling. AXIN, for example, can be destabilized by tankyrases  that regThe non-coding microRNAs or miRNAs suppress gene expressions by inhibiting translation or increasing degradation of target gene mRNAs . miRNAs,Several miRNAs have been identified in association with various types of LCs in recent years Table . For exaIn contrast, miR-410 can accelerate tumor growth by suppressing the expression of SLC34A2, a type 2b sodium-dependent phosphate transporter (NaPi-IIb) that is located in the apical membrane of ATII cells. Decreased NaPi-IIb levels have been shown to activate the WNT/beta-catenin pathway leading to enhanced tumor growth and invasion . PerhapsAlthough some miRNAs have not been directly associated with WNT signaling in NSCLCs, in other tumor types they have been demonstrated to play important WNT pathway regulatory function. For example miR-148b suppresses tumor growth via inhibiting canonical WNT signaling in hepatocellular carcinoma , while mWNT signaling has a fundamental role in both normal and tumor angiogenesis . The canCancer cells can induce neovascularization when the solid tumor reaches more than 2 mm in diameter  and hypoThe canonical WNT pathway mediated EMT , which cAs the angiogenic process is a therapeutic target in solid tumors, anti-angiogenic agents have become important in therapy. The anti-VEGF-A monoclonal antibody, bevacizumab is the first approved antiangiogenic agent to be applied in NSCLSs . DespiteThe accumulation of highly chemotherapy resistant cancer stem cells (CSC) are thought to play an important role in the incurability of LCs . In CSCsAlthough increased expression of drug transporters in chemoresistant lung tumors suggests that the drug transporters might be useful therapeutic targets, sadly, clinical trials using ABC transporter inhibitors have not been successful. For example, the ABCB1 (MDR1) inhibitors tariquidar and CBT-1(R) –177 wereComplex deregulation of WNT signaling is an important element of lung carcinogenesis, controlling not just the carcinogenic process, but also tumor vascularization, drug response and disease progression. While current cancer research frequently cites the importance of precision medicine, therapeutic approaches and even drug development are still strongly focused on genetic mutations . Individ


Raw materials used were yttrium nitrate and yttrium chloride, in methanol. In order to promote oxygen vacancies, P-123 poloxamer was incorporated. Synthesized systems were heat-treated at temperatures from 700 °C to 900 °C. Systems at 900 °C were prepared in the presence and absence of P-123 using different molar ratios (P-123:Y = 1:1 and 2:1). Fourier transform infrared spectroscopy (FTIR) results revealed a characteristic absorption band of Y–O vibrations typical of Y2O3 matrix. The structural phase was analyzed by X-ray diffraction (XRD), showing the characteristic cubic phase in all systems. The diffraction peak that presented the major intensity corresponded to the sample prepared from yttrium chloride incorporating P-123 in a molar ratio of P-123:Y = 2:1 at 900 °C. Crystallites sizes were determined by Scherrer equation as between 21 nm and 32 nm. Antioxidant properties were estimated by 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assays; the results are discussed.Yttrium oxide (Y The results demonstrate that Y10 reduces DPPH• to an extent and antioxidant activity increases with time reaching it highest value after one hour. As has been already observed in other materials, such iron oxide nanoparticles , the inc2O3 was prepared by sol-gel method according to the following procedure. All experiments were carried out at room temperature. Experimental strategies for Y2O3 powder preparation used two raw materials: yttrium nitrate and yttrium chloride. For Y2O3 synthesis hexa-hydrate yttrium nitrate (Y(NO3)3·6H2O, 99.8%, Sigma-Aldrich, St. Louis, MO, USA), hexa-hydrate yttrium chloride , methanol (CH3OH (MeOH), 99.8%, Sigma-Aldrich), acetylacetone (CH3COCH2COCH3 (AcAc), 99.9%, Sigma-Aldrich), and for embedded system P-123 poloxamer ((C3H6O·C2H4O)x, molecular weight (MW): ~5800, Sigma-Aldrich) were used. 0.0026 mol of Y(NO3)3·6H2O or 0.0051 mol of YCl3·xH2O were dissolved in MeOH in a molar ratio of MeOH/Y = 123 under vigorous magnetic stirring for 15 min. Thereafter, AcAc was added in order to obtain a stable sol of Y2O3. In selected systems, P-123 poloxamer was incorporated in a molar ratio described in 2O3 xerogel was formed at 90 °C for 24 h. Finally, different heat-treatments were carried out in order to produce dense materials and to promote the crystallization of the Y2O3 powder at 270 °C (2 h), 500 °C, 700 °C, 800 °C and 900 °C for 1 h.Y2O3 powders heat-treated at different temperatures using a Bruker Tensor 27 Model  in the range of 4000–400 cm−1. Samples were analyzed using the KBr pelleting technique . The structure was determined by a Bruker Diffractometer D8 Advance using a copper anticathode at 35 kV and 25 mA in a range 2θ from 15° to 80°. The crystallite size was determined according to the Scherrer equation, considering the line broadening of the diffraction peak due to the effect of crystal size [FWHM is the full-width at half-maximum of the peak, and θ is the Bragg angle of XRD peak. The microstructure of powders was determined by a SEM and TEM by means of JEOL JSM-6510LV and JEM 1400 Equipment , respectively.The FTIR transmittance spectra were recorded from Y2O3 nanocrystallites were successfully synthesized by sol-gel method from yttrium nitrate and yttrium chloride as precursors. Y2O3 nanostructured powders prepared from yttrium chloride and in the presence of P-123 poloxamer in a molar ratio of P-123:Y = 2:1 presented better physicochemical properties  than systems prepared from yttrium nitrate precursor. Y2O3 powders presented a crystallite size range of 21 nm to 32 nm in agglomerate arrays. The DPPH• studies are reported for the first time for Y2O3 synthesized by sol-gel method, thus, there is no reference to compare. Y2O3 nanocrystallites in presence of P-123 compose of porous morphologies integrated with nanosized particles showed enhanced antioxidant properties, therefore they can be considered as new promising materials in biological systems.Y


SZ-123, a murine monoclonal antibody that targets the human von Willebrand factor (VWF) A3 domain and blocks the binding of collagen, is a powerful antithrombotic. In a Rhesus monkey model of thrombosis, SZ-123 had no side effects, such as bleeding or thrombocytopenia.The mouse/human chimeric version of SZ-123, MHCSZ-123, was developed and maintained inhibitory capacities in vitro and ex vivo after injection into monkeys. CHO-S cells were selected for stable expression of MHCSZ-123. Cell clones with high levels of MHCSZ-123 expression were screened with G418 then adapted to serum-free suspension culture. The antithrombotic effect of MHCSZ-123 on acute platelet-mediated thrombosis was studied in monkeys where thrombus formation was induced by injury and stenosis of the femoral artery, which allowed for cyclic flow reductions (CFRs). CFRs were measured in the femoral artery of anesthetized Rhesus monkeys before and after intravenous administration of MHCSZ-123. Ex vivo VWF binding to collagen, platelet aggregation, platelet counts, and template bleeding time were used as measurements of antithrombotic activity. In addition, plasma VWF and VWF occupancy were measured by ELISA.Injection of 0.1, 0.3, and 0.6 mg/kg MHCSZ-123 significantly reduced CFRs by 29.4%, 57.9%, and 73.1%, respectively. When 0.3 and 0.6 mg/kg MHCSZ-123 were administered, 46.6%–65.8% inhibition of ristocetin-induced platelet aggregation was observed between 15 and 30 min after injection. We observed minimal effects on bleeding time, minimal blood loss, and no spontaneous bleeding or thrombocytopenia.The VWF-A3 inhibitor MHCSZ-123 significantly reduced thrombosis in Rhesus monkeys and appeared to be safe and well tolerated. Platelets are the key players in primary hemostasis. The interaction between von Willebrand factor (VWF), and platelet glycoprotein (GP) Ibα is the key for initiating the response to vascular injury that leads to hemostasis or, under pathological conditions, thrombosis –3. VWF bMany different components have been identified that contribute to thrombosis, and several therapeutic approaches have been developed for this severe clinical complication. While these treatments have been effective in the clinical setting, they have a limited therapeutic window and are associated with clinically significant bleeding, particularly when high doses of the drugs are used , 5. In tPreviously, we prepared and characterized a murine monoclonal antibody (mAb) against the human VWF A3 domain, designated SZ-123. SZ-123 specifically blocked binding of purified human VWF to collagen type III and prevented the VWF-dependent adhesion of human platelets to collagen under high shear stress . We demoAs a further step in the development of SZ-123, we constructed a mouse/human chimeric antibody derived from hybridomas producing murine antibody against the human VWF A3 domain and induced its expression in Chinese hamster ovary (CHO-S) cells. The chimeric antibodies consisted of variable regions from the mouse antibody and constant regions from the human antibody. This type of antibody eliminates the highly immunogenic murine constant region while maintaining its antigen binding affinity . The mouBlood donations were from healthy volunteers who provided written informed consent. The pMH3 vector and CHO-S cells were purchased from AmProtein . Restriction enzymes, T4 DNA ligase, and Taq DNA polymerase were obtained from New England BioLabs . Rabbit anti-human VWF polyclonal antibody and rabbit anti-human VWF-HRP antibody were purchased from Dako Cytomation . Ristocetin, tetramethylbenizidine (TMB), and collagen type III from human placenta were purchased from Sigma . Goat anti-mouse IgG and goat anti-mouse IgG labeled with horseradish peroxidase (HRP) were purchased from Beckman-Coulter . SZ-123, a murine anti-human VWF A3 domain mAb (IgG1) was produced by standard hybridoma technology in our laboratory as described previously . SZ-34, Complementary DNAs (cDNAs) for the variable gene of the heavy and light chains of SZ-123 were amplified using the 5’ rapid amplification of cDNA end (5’RACE) method. Briefly, total RNA was isolated from an SZ-123 monoclonal antibody-expressing hybridoma with TRIzol reagent according to the manufacturer’s protocol. The 5’RACE method was performed according to the protocol provided by the manufacturer . The gene-specific primers HGSP1 and KGSP1 used for reverse transcription are listed in Table The pMH3 vector , a eukaryotic expression vector that includes the chicken beta actin gene intron-1 promoter , was selected for high expression of the chimeric antibody . pMH3H aTo construct the pMH3-MHC-SZ-123VH and pMH3-MHC-SZ-123VL expression vectors, two sets of primer pairs, SZ-123HR, SZ-123HF for VH and SZ-123LR, SZ-123LF for VL, were designed and synthesized based on the sequences of 5’RACE results. Primer sequences are listed in Table 7 cells per milliliter on the day of transfection. pMH3-MHC-SZ-123VH and pMH3-MHC-SZ-123VL expression vectors were transfected into CHO-S cells using electroporation. Briefly, 3 × 106 CHO-S cells in 200 μL DMEM/F12 medium (Gibco) with 10% FBS, 20 μg of expression vector, and 10 μg salmon sperm DNA were mixed together. Electroporation was performed four times at 500 V, 500 μs using a Gene Pulser . The transfected CHO-S cells were then cultured under selection in DMEM/F12 medium supplemented with 10% FBS containing 2.4 mg/ml G418  in 10 cm dishes for 1 week at 37 °C. Surviving cell clones were picked and cultured in 96-well plates for another 7 days. Stably transfected cells were screened under G418 for gene copy-number amplification. Twenty-four hours after replacing the G418-containing medium with serum-free medium, the amount of MHCSZ-123 secreted into the medium was quantified using a sandwich ELISA kit . The high-expression cell clones were picked and used for the second round of selection where 100 cells of each high-expression clone were seeded into 24-well plates and selected again according to protocol described above. Finally, the surviving clones were expanded for suspension cultures and freezing. The cells were then adapted to serum-free suspension medium B001 (AmProtein) containing 2.5 mM L-glutamine (Gibco) on a shaker at 120 rpm in a humidified atmosphere at 37 °C and 8% CO2.CHO-S cells were cultured in DMEM/F12 medium  supplemented with 10% FBS (Gibco) to obtain 1.5 × 102SO4 upon incubation for 15 min at 37 °C. The plate was read at 450 nm in a microtiter plate reader .MHCSZ-123 concentrations were determined in an ELISA assay as described previously (23). Briefly, MHCSZ-123 was captured by goat anti-human IgG1 polyclonal antibody (10 μg/ml) precoated on an ELISA plate. Supernatants from growing colonies and human IgG1 as standard were added to the wells. After 1 h incubation at 37 °C, bound MHCSZ-123 was detected with horseradish peroxide (HRP)-conjugated goat anti-human IgG kappa antibody (1 μg/ml), followed by color development after addition of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution. The reaction was stopped with 1 N HThe chimeric antibody against the VWF A3 domain was purified using affinity chromatography with Protein A-Sepharose resin. Briefly, supernatant was harvested then applied to a Protein A-Sepharose 4B column . The bound MHCSZ-123 was eluted with 0.1 M glycine-HCL (pH 3.0). After dialysis against phosphate-buffered saline (PBS) overnight at 4 °C, the antibody was clarified through a 0.22 μm filter. The final purified MHCSZ-123 was stored in PBS at −80 °C. The protein concentration was estimated using a BCA protein assay reagent (Pierce), with BSA as standard.In a previous study, we verified that SZ-123 cross-reacted with Rhesus monkey VWF and efficiently inhibited binding of plasma VWF from Rhesus monkey to human collagen type III [Collagen binding assays were performed by ELISA as described , 23. Bri5 per microliter by mixing platelet-rich plasma (PRP) and autologous platelet-poor plasma (PPP) prepared by differential centrifugation from 0.38% citrated blood. After incubation with 10 μg/mL of mAb SZ-123, human IgG, or different concentrations of MHCSZ-123 at 37 °C for 10 min, platelet aggregation was induced by 1.25 mg/mL ristocetin (Sigma) under constant stirring (1000 rpm). Platelet aggregation was measured according to the change in light transmission using a lumiaggregometer .Platelet aggregation assay was performed as described previously , 23. Hum−1 and 300 s−1 in a parallel-plate flow chamber . Acid-soluble collagen type III (200 μg/ml) from human placenta (Sigma) was coated onto dishes  at 4 °C overnight. Non-specific binding sites were blocked with 2% BSA-PBS. Whole blood from healthy volunteers was collected , and platelets were labeled with calcein acetoxymethyl ester . After incubation with 10 μg/mL mAb SZ-123, SZ-34, or MHCSZ-123 for 10 min at 37 °C, perfusions were carried out at 37 °C in a water bath. During the 5-min perfusion, images were captured using a fluorescence microscope  at 0, 1, 3, and 5 min, respectively.To examine whether MHCSZ-123 could inhibit platelet adhesion to collagen under physiological flow conditions, a flow chamber assay was performed. The effects of the antibody were tested under different shear rates of 1200 sHousing, treatment, surgery, and veterinary care for the animals used in the study were approved by the Animal Ethics Committee of Soochow University. Twenty-four Rhesus monkeys obtained from Xishan Zhongke Laboratory Animal Co.,  were used in this study. The monkeys weighed between 4.0 and 6.5 kg and were aged 3–5 years old. Monkeys were distributed into four treatment groups according sex and weight. Groups 1–4 were administered saline, 0.1, 0.3, and 0.6 mg/kg MHCSZ-123, respectively. Each group contained six animals, half male and half female. Each animal underwent the experiment one time.Arterial thrombosis was established in the monkeys as described previously , 27. Brit = −60–0 min), 5 mL of the test agents  were administrated by intravenous injection within 1 min (t = 0 min), and monkeys were monitored up to 1 h after administration (t = +60 min). The antithrombotic effect of MHCSZ-123 was quantified by comparing the number of CFRs per hour before and after administration of MHCSZ-123. Blood samples were drawn at different time points for ex vivo analysis.Baseline CFRs were recorded for 60 min  trisodium citrate, and the platelet count was measured immediately using an automatic blood cell analyzer . The template bleeding time was measured using bleeding time devices . The surface of the foreleg was shaved, and a pressure cuff was applied with constant inflation to 20 mmHg during measurements. A wound incision approximately 5 mm long and 1 mm deep was then carefully dabbed every 15 s with sterile filter paper. All measurements were performed once at each time point. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured at 37 °C using an automatic coagulometer .Plasma VWF levels were measured by ELISA. Briefly, 96-well microtiter plates were coated with monoclonal antibody anti-VWF SZ-29-IgG (5 mg/mL) overnight at 4 °C  and blocVWF occupied by MHCSZ-123 was estimated using a competition ELISA assay with a fixed concentration of biotinylated SZ-123 (Bio-SZ-123) (made by our lab) in combination with streptavidin-HRP (Thermo). Briefly, 96-well microtiter plates were coated with a polyclonal anti-VWF-Ig solution (10 μg/well) overnight at 4 °C and blocked with 2% BSA-PBS at 37 °C for 2 h. A dilution series (1:2 in PBS) of the plasma samples (pre-warmed for 5 min at 37 °C) was added for 2 h at 37 °C. Samples containing 100% occupied VWF were obtained by adding a saturating amount of MHCSZ-123 (6 μg/mL) to each corresponding Rhesus monkey plasma sample. Bound MHCSZ-123 was detected by addition of a fixed concentration of biotinylated SZ-123 (Bio-SZ-123) for another 1 h. After washing, bound Bio-SZ-123 was combined with streptavidin-HRP for 1 h at 37 °C. Visualization and wash steps were performed as described above. The VWF occupancy of each sample was calculated as follows: (A450 nm sample saturated with MHCSZ-123/A450 nm sample) × 100%.t = 0 min), which was set as 100%.VWF-collagen binding assays were performed by ELISA. Briefly, 96-well microtiter plates were coated overnight at 4 °C with type III collagen from human placenta and blocked with 2% BSA-PBS. Rhesus monkey plasma was then added to the wells and bound VWF detected using rabbit anti-human VWF-HRP antibodies. Binding of VWF to collagen in plasma samples at each time point was compared to the binding of VWF in each blood sample taken at time zero  and one-factor ANOVA followed by Fisher’s exact test. P < 0.05 was considered significant.Data were expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 5  for Student’s We used 5’ RACE to clone the SZ-123 variable gene and the signal peptide sequence. The PCR products were 663 bp for VH and 543 bp for VL , transfected into CHO-S cells as an expression system . DetaileMore than 50 clones secreted MHCSZ-123 at higher levels than other clones, which were >12.5 mg/L as determined by ELISA. These clones were selected as high-expression cell clones and picked for the second round of selection. After two rounds of selection, five clones with the highest titers were selected under G418 for gene copy-number amplification. To select the cell clone that produced the highest level of antibody, the five chosen cell clones were adapted to suspension culture in serum-free medium and assays were performed on a small scale of 50 ml. The highest antibody-producing clone (23 μg/ml) was designated MHCSZ-123-CHO and chosen for further studies. We scaled the cultures of the highest producer clone up to 10 L for a fed-batch culture. MHCSZ-123 was collected and purified for further studies. As estimated using a Coomassie blue-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, MHCSZ-123 protein of >95% purity was obtained after affinity chromatography. The recovery rate of MHCSZ-123 reached 78.8% (63 mg/80 mg).50) of MHCSZ-123 was 14.06 ± 1.34 ng/ml, which was slightly lower than that of the parent antibody SZ-123 (16.91 ± 2.07 ng/ml). However, the difference was not significant. We concluded that MHCSZ-123 cross-reacted efficiently with plasma VWF from Rhesus monkeys and specifically inhibited the binding of plasma VWF from Rhesus monkeys to human collagen type III. Thus, Rhesus monkeys are an appropriate model for investigating MHCSZ-123 as a potential therapeutic agent for arterial thrombosis.As shown in Fig. The activity of MHCSZ-123 was further tested for its effect on ristocetin-induced platelet aggregation in vitro. Similar to murine SZ-123, purified MHCSZ-123 at a concentration of 8 μg/mL completely inhibited ristocetin-induced human platelet aggregation Fig. .Fig. 4MH−1 shear stress  by administration of MHCSZ-123 at 0.1, 0.3, and 0.6 mg/kg  of MHCSZ-123 to Rhesus monkeys in a modified arterial platelet thrombosis model. CFRs were significantly inhibited  observed in any of the groups (data not shown). All animals survived after the procedures, and none exhibited bleeding complications.VWF initiates thrombus formation by mediating platelet adhesion to exposed subendothelial collagen on an injured vessel wall. It is known that bridge formation between platelets and collagen by VWF allows platelets to withstand the elevated shear forces, whereas the role of VWF at lower shear forces is redundant at best. Thus, interfering with this adhesion pathway might allow specific targeting of the antithrombotic action to stenotic arteries, leaving the rest of the circulation intact, with presumably less risk for spontaneous bleeding .We showed that the chimeric monoclonal anti-VWF-A3 antibody, MHCSZ-123, inhibited platelet adhesion to human collagen type III in a perfusion flow chamber; this effect was more efficient with increasing shear rate as observed with the parent mAb SZ-123 . The antThe stable expression system we used allowed high yield and quality of recombinant protein in different batches for further research. The HEK293 and CHO cell lines are the most frequently used mammalian cells for protein expression because they produce proteins with correct folding and post-translational modifications . The CHOIn the present study, we demonstrated again that MHCSZ-123 inhibited VWF binding to collagen ex vivo in a dose- and time-dependent manner. As we previously observed with the murine SZ-123, 60% of VWF occupied by SZ-123 is enough to totally inhibit the thrombus formation but also to inhibit the ex vivo platelet aggregation induced by 1.25 mg/ml ristocetin . KageyamIn conclusion, the present study demonstrated that the chimeric monoclonal antibody MHCSZ-123 was a powerful inhibitor of the GPIbα-VWF-collagen axis. In addition, we confirmed that inhibiting the platelet VWF-collagen interaction is an efficient way to prevent thrombus formation in the injured and stenosed Rhesus monkey femoral artery without causing prolongation of the bleeding time or increased blood loss. Because of the lack of effect on the bleeding time, it will be worthwhile to validate this approach in clinical trials aimed to prevent acute arterial thrombotic syndromes.


Central nervous system tumors are the most common cancer type in children and the leading cause of cancer related deaths. There is therefore a need to develop novel treatments. Large scale profiling studies have begun to identify alterations that could be targeted therapeutically, including the phosphoinositide 3-kinase (PI3K) signaling pathway, which is one of the most commonly activated pathways in cancer with many inhibitors under clinical development. PI3K signaling has been shown to be aberrantly activated in many pediatric CNS neoplasms. Pre-clinical analysis supports a role for PI3K signaling in the control of tumor growth, survival and migration as well as enhancing the cytotoxic effects of current treatments. Based on this evidence agents targeting PI3K signaling have begun to be tested in clinical trials of pediatric cancer patients. Overall, targeting the PI3K pathway presents as a promising strategy for the treatment of pediatric CNS tumors. In this review we examine the genetic alterations found in the PI3K pathway in pediatric CNS tumors and the pathological role it plays, as well as summarizing the current pre-clinical and clinical data supporting the use of PI3K pathway inhibitors for the treatment of these tumors. Neoplasms of the CNS are the most common type of solid tumor that occur in children and the leading cause of cancer related deaths . CurrentThe PI3K pathway is one of the most commonly activated pathways in cancer. PI3Ks are lipid kinases that activate a signaling cascade which controls diverse biological functions including cellular proliferation, survival and motility. PI3Ks can be broadly divided into three structural classes; Class I, II and III.Class I PI3Ks can be further divided into two subtypes, Class IA and IB, dependent on their method of activation. Class IA PI3Ks are activated by receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs) and oncogenes, whereas Class IB PI3Ks are activated by GPRCs only . Class I2) to produce phosphatidylinositol-3,4,5-bisphosphate (PIP3). This reaction is negatively regulated by phosphate and tensin homolog (PTEN) which reduces levels of PIP3 by converting it back to PIP2. PIP3 forms a docking site for the recruitment of many proteins to the plasma membrane including the serine threonine kinase V-Akt murine thymoma viral oncogene homolog (AKT), where it is activated by phosphorylation by phosphoinositide-dependent protein kinase 1 (PDK1) and mammalian target of rapamycin complex 2 (mTORC2). Once activated, AKT regulates key cellular activities downstream, including glycogen synthesis by the forkhead family of transcription factors (FOXOs) and apoptosis through p53, BAD and NfκB . Signaling through mTOR regulates key cellular activities including cell growth and protein synthesis . mTOR foctivator .Class II PI3Ks consist of a single catalytic subunit of which there are three isoforms PI3KC2α, PI3KC2β and PI3KC2γ. They can be activated by RTKs, cytokine receptors and integrins. Their specific cellular functions remain poorly understood. Recent evidence suggests signaling may play a role in angiogenesis, growth and survival . Class I3 levels and un-restrained PI3K signaling.Signaling through class I PI3Ks has commonly been implicated in cancer. A number of genetic alterations resulting in pathway activation have been identified. PTEN is one of the most frequently inactivated tumor suppressor genes, with loss commonly occurring through deletions and inactivating mutations . Loss ofPI3KCA, which encodes the p110α catalytic subunit of class IA PI3K, is frequently mutated in some of the most common human cancers , 20. 80%Activating mutations or amplifications of AKT1/2 have also been identified . MutatioClass II PI3K isoforms are less well understood. However, they have been implicated in cancer. Amplification and somatic mutations have been identified in PIK3C2B . HoweverMutations have also been identified in the negative regulators of mTOR signaling TSC1 and TSC2. Mutations in these genes occur in the familial syndrome tuberous sclerosis complex (TSC), a rare autosomal dominant disorder. Patients with TSC develop benign tumors in multiple organs including brain, heart, kidney, lungs and skin . Loss ofThe common occurrence of PI3K pathway deregulation in cancer has sparked interest in the development of inhibitors targeting PI3K signaling, with over 30 inhibitors being trialed clinically Figure . The firAgents targeting mTOR signaling are more clinically advanced than other nodes of the pathway. The first inhibitor to be discovered was rapamycin, a bacterially derived product which was originally developed as a fungicidal and immunosuppressive drug , 33. AnaRecent evidence also suggests that PI3K inhibition can be used not only to target tumor cells directly but also the surrounding stroma, including tumor vasculature and infiltrating immune cells , 40. ThepHGG account for 5-10% of all pediatric CNS tumors. Prognosis for children with pHGG is dismal with a 5 year survival rate of less than 20% [Activation of PI3K signaling has been found in a high percentage of pHGG, which in some studies was linked to a poorer prognosis , 43, 44.However, the mechanism of aberrant pathway activation differs between adult and pediatric tumors. In adult tumors inactivating PTEN mutations are commonly seen. In contrast, in pediatric cases they are infrequent . HoweverPathway activation through amplification or overexpression of growth factor receptors is also common. Amplification or overexpression of EGFR was commonly found in adult HGGs but was rarely seen in pHGG . HoweverA recent whole genome sequencing study identified frequent activation of PI3K signaling through mutations in 67% of non-brainstem HGGs and 69% of DIPGs, including fusions involving the kinase domain of neurotrophin receptor (NTRK) genes, highlighting the importance of this signaling pathway in these tumors . HoweverpLGG are the most common type of pediatric brain tumor and account for approximately 40% of cases . pHGG enPI3K signaling has been implicated in pLGG. mTOR activation has been associated with subependymal giant cell astrocytoma (SEGA) which occurs in up to 15% of TSC patients . EvidencPre-clinical analysis has produced evidence supporting the potential for therapeutic targeting using mTOR inhibitors in pLGG. Decreased growth was seen when two pLGG cell lines were treated with an mTORC1 inhibitor . Kaul etEpendymoma is the second most common malignant pediatric CNS tumor, accounting for approximately 10% of brain tumors . PrognosAn immunohistochemical analysis of phosphorylated AKT demonstrated positive staining, and inferred pathway activation, in a high percentage of ependymomas (72% of primary tumors), which was significantly linked to a poorer progression free survival. Cyclin D1 was significantly associated with active PI3K signaling suggesting control of cell proliferation . Active A definitive mechanism for activation of PI3K signaling has not been determined for ependymoma. Sequencing studies have not identified PIK3CA mutations , 64. CopA decrease in cell viability has been demonstrated in ependymoma cell lines following treatment with PI3K pathway inhibitors suggesting the potential for therapeutic targeting , 65. A hMedulloblastoma is an embryonal tumor and is the most common malignant brain tumor in children . Recent Active PI3K signaling has been detected in a high percentage of medulloblastomas –73, and Inhibition of PI3K signaling has demonstrated a role for the pathway in control of medulloblastoma cell growth and migration. Treatment with inhibitors at various nodes of the signaling pathway, including PI3K, AKT and mTOR, resulted in impaired growth and migration of medulloblastoma cells using in vitro and in vivo models , 84. TrePI3K signaling has also been linked to tumor resistance in medulloblastoma. Inhibition of PI3K signaling in a sonic hedgehog pathway induced medulloblastoma mouse model delayed the development of smo-induced resistance .ATRT is a highly malignant embryonal tumor, distinguished by alterations in the tumor suppressor gene INI1 . It is cStudies have implicated PI3K signaling in the control of ATRT cell growth. ATRT cell lines were found to overexpress RTKs, including insulin receptor and insulin-like growth factor-I receptor, as well as PI3K signaling components, compared to normal brain. PI3K signaling was also shown to play a crucial role in ATRT cell proliferation induced by insulin signaling with evidence that induced signaling was mediated specifically through the Class IA PI3K isoform p110α . PharmacEmbryonal tumors previously classified as central nervous system primitive neuroectodermal tumors (CNS-PNET) have undergone a major re-organization in the recent WHO classification, to reflect their molecular basis . They enAmplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to an aggressive group of embryonal tumors which span histological subtypes .The high frequency of alterations in PI3K signaling in pediatric brain tumors has led to the initiation of clinical trials testing these agents Table . The mosEverolimus has recently been approved for treatment of TSC patients with SEGA . EveroliTemsirolimus was the first mTOR inhibitor approved for treatment in oncology. In phase I trials of pediatric patients with recurrent/refractory solid tumors temsirolimus was shown to be well tolerated . In a phA study investigating the efficacy of rapamycin in combination with the EGFR inhibitor erlotinib in children with recurrent LGG was well tolerated but had questionable to no activity . A seconA phase I study of ridaforolimus in pediatric patients with refractory solid tumors demonstrated the drug to be well tolerated . In a seA recently opened proof of concept therapeutic stratification trial in children, adolescents and young adults with relapsed or refractory tumors aims to use molecular analysis to identify treatment targets. The trial includes use of everolimus and the dual mTORC1 and 2 inhibitor AZD2014 (NCT02813135).Trials investigating inhibitors of upstream PI3K signaling are less advanced. The AKT inhibitor MK2206 was investigated in a phase I trial of children with refractory malignancies where the drug was well tolerated . The AKTFurther PI3K inhibitors have been investigated pre-clinically as part of the pediatric preclinical testing program. Studies investigating the PI3K inhibitor XL146, the AKT inhibitor GSK690693 and the mTOR inhibitor MLN0128 all demonstrated modest anti-tumor activity in preclinical models –106.The PI3K signaling pathway has been implicated in a high proportion of pediatric CNS tumors suggesting it represents a promising target for therapy. Pathway activation has been identified in primary tumors and pre-clinical analysis has demonstrated PI3K signaling plays a role in the control of tumor growth, survival and migration.Pre-clinical analysis of pathway inhibitors also strongly supports the potential therapeutic use of agents targeting PI3K signaling in pediatric CNS malignancies, with inhibitors able to impair the growth and migration of tumor models as well as enhancing the cytotoxic effects of chemotherapeutic agents. Initial clinical trials have been promising with agents generally well tolerated. The evidence is particularly strong for the use of mTOR inhibitors, with approval of everolimus for the treatment of SEGA patients.It will however be important to take into consideration problems encountered in other cancers, including adult glioma, where effects of inhibitors so far have, in general, been modest . This maA further issue specific to brain tumors is the ability of a drug to cross the blood brain barrier and achieve high enough concentrations within the brain without systemic toxicity. Caution must also be undertaken when targeting key cellular signaling pathways that are fundamental to normal development in childhood. Evaluation of toxicity to the normal brain therefore needs to be taken into consideration.To overcome these challenges it will firstly be important to develop sophisticated patient selection strategies, using predictive biomarkers, to ensure patients entered into a trial have a responsive tumor. Secondly, a combination approach will be needed using PI3K inhibitors alongside conventional treatments as well as other novel targeted agents to increase effectiveness and avoid development of resistance. The best and most rational combinations will need to be identified. This can only be achieved if we increase our understanding of the underlying biology of these tumors and continue analysis using pre-clinical models.