Pos satellite cell-derived myogenic progenitors obtained from healthy individuals to study the role of Wnt-β-catenin signalling in myogenic differentiation. We show that dephosphorylated β-catenin (active-β-catenin), the central effector of the canonical Wnt cascade, is strongly upregulated at the onset of differentiation and undergoes nuclear translocation as differentiation progresses. To establish the role of Wnt signalling in regulating the differentiation process we manipulated key nodes of this pathway through a series of β-catenin gain-of-function (GSK3 inhibition and β-catenin overexpression) or loss-of-function experiments (dominant negative TCF4). Our data showed that manipulation of these critical pathway components led to varying degrees of disruption to the normal differentiation phenotype indicating the importance of Wnt signalling in regulating this process. We reveal an independent necessity for active-β-catenin in the fusion and differentiation of human myogenic progenitors and that dominant negative inhibition of TCF4 prevents differentiation completely. Together these data add new mechanistic insights into both Wnt signalling and adult human myogenic progenitor differentiation.Wnt-β-catenin signalling is essential for skeletal muscle myogenesis during development, but its role in adult human skeletal muscle remains unknown. Here we have used human primary CD56 In the absence of Wnt ligands, a cytoplasmic ‘destruction complex’ maintains the cellular pool of β-catenin at a low level. A key action of this complex is to immobilise β-catenin providing glycogen synthase kinase 3β (GSK3β) the opportunity to phosphorylate it on key N-terminal serine and threonine residues, thereby marking β-catenin for degradation via the ubiquitin-proteasome pathway. Conversely, when Wnt ligands are present, this complex is inhibited and β-catenin remains in its active, unphosphorylated, form, which can translocate to the nucleus and regulate Wnt responsive genes via its partnership with TCF/LEF transcription factors4.Canonical Wnt signalling acting via the transcriptional co-activator β-catenin, is known to be critical for skeletal muscle myogenesis during embryonic development5. In resting muscle, undifferentiated satellite cells exist in a quiescent state beneath the basal lamina and directly adjacent to the terminally differentiated syncytial myofibres. In many species these dormant satellite cells are most commonly distinguished by their characteristic expression of the paired homeobox transcription factor 7 (Pax7); however, unlike in mice, human satellite cells can also be identified by their cell surface expression of CD56 (N-CAM)6. When muscle is damaged, satellite cells exit quiescence and sequentially express a well characterised cascade of myogenic regulatory factors, which in turn drive the expression of muscle specific genes. Two of these factors Myf5 and MyoD, are expressed immediately post-activation as the cells undergo proliferative expansion as myogenic progenitors, whilst myogenin and myogenic regulatory factor (MRF) govern the differentiation of these cells as they work to repair or replace damaged myofibres7.Little is known about the role of β-catenin in adult skeletal muscle, where it is assumed that many aspects of muscle development are recapitulated by the resident progenitor cell population, known as satellite cells, upon their activation in response to damage10 and promote15 myogenic differentiation. Studies focusing on primary cells from adult mouse muscle and also mature mouse muscle in vivo, indicate roles for Wnt-β-catenin signalling in the regulation of both myotube16 and myofibre hypertrophy17. Although Wnt signalling is believed to be conserved between species, differences exist in the way satellite cells are identified in mice and humans and there are also emerging differences in the behaviour of isolated satellite cell-derived myogenic progenitors18. These differences mandate the need for species-specific studies to identify the role of Wnt-β-catenin signalling in human muscle adaptation and repair. This is particularly important as dysregulation of Wnt signalling has been implicated in sarcopenia, the age-regulated loss of skeletal muscle mass19.The overwhelming majority of studies investigating the role of canonical Wnt-β-catenin signalling in skeletal muscle have been performed using cultured mouse cell lines. Manipulation of β-catenin levels in these lines has been reported to both inhibit20 in combination with small molecule inhibitors and high-efficiency lentiviral expression constructs, we manipulated three key nodes in the Wnt pathway: GSK3, β-catenin, and T cell factor (TCF4/TCF7L2)21, in bulk populations of purified CD56Pos myogenic progenitors obtained from numerous healthy individuals at early passage, therefore mitigating the potential impact of extended tissue culture artifacts22.We have investigated the role of canonical Wnt signalling in early passage adult human myogenic progenitors and, in particular, whether it acts as a facilitator or inhibitor of differentiation. Using an immunomagentic isolation procedurein vitro differentiation.Manipulation of the core canonical Wnt components we selected disrupted the normal differentiation of human myogenic progenitors indicating the importance of this pathway in the regulation of these cells. We found that active GSK3 is required for the fusion and differentiation of human myogenic progenitors and that its inhibition represses the normal onset of myogenin expression. We show that while differentiation is not enhanced by overexpression of β-catenin, prevention of canonical Wnt signalling during differentiation via disruption of the nuclear β-catenin-TCF transcriptional complex completely abolishes differentiation. Together these data show that in adult human primary myogenic progenitor cells Wnt signalling is a central regulator of myogenesis and is essential for Pos, satellite cell-derived myogenic progenitor cells 6. In proliferating CD56Pos/desminPos/Ki67Pos myogenic progenitors, 8–9 days after isolation from human muscle at passage 1, β-catenin was detectable only at very low levels in the cytoplasm and there was no clear nuclear accumulation  with or without Wnt3a or BIO supplementation. By late differentiation, control populations differentiated into large, multinucleated myotubes and the nuclei within these MHCnin Fig. . Quantifnin Fig. . This lalls Fig. ; nor canlls Fig. . Contraslls Fig. ; Fig. S5lls Fig.  and MHC lls Fig. .Neg), which are comprised of myogenic cells that have failed to differentiate (including reserve cells)  Fig. ; Fig. S5ls) Fig. ; Fig. S5res Fig. ; Fig. S5Pos cells in proliferation media (24 h), differentiation media and differentiation media supplemented with BIO (72 h). Immunostaining revealed that as expected myogenic cells maintained in proliferation media were almost exclusively Ki67Pos/myogeninNeg while cells maintained in differentiation media contained a high percentage of myogeninPos cells and lacked discernible expression of Ki67  which cannot be targeted for degradation by GSK3-mediated phosphorylation. In addition, to examine the necessity of the transactional co-activator role of β-catenin in human myogenic cell differentiation we also transduced purified primary cell lines with a construct overexpressing a dominant negative form of TCF4 (EdTC). This dominant negative mutant form of TCF4 harbours an N-terminal deletion, such that it binds Wnt target genes, but cannot transactivate them due to its inability to bind β-catenin27. In both cases successful integration was identified by mCherry expression  compared to control cultures (74% ± 4%)  Fig. . In aggr4%) Fig. , suggestAXIN-2 expression relative to mock control, however, a variable response was noted depending on the individual, ranging from a moderate, 2–7 fold, increase to a very high, 17–19 fold, increase. EβC-transduced cells cultured in growth medium underwent active suppression of myogenic regulatory genes, with the exception of Myf5 , MYF5, MYOD, and MHC transcripts when compared to mock transduced controls  using three structurally diverse molecules , all of which have been shown to activate canonical Wnt signalling in a variety of different systems. We confirmed the ability of these compounds to bypass AXIN-mediated negative feedback to stabilise the active form of β-catenin in our human myogenic progenitors . Overexpression of active-β-catenin produced a phenotype of reduced fusion and myotube size, which was similar but less severe than that observed with GSKi Fig. . This waet al., also observed a reduction in myogenin following β-catenin overexpression from a retroviral vector in growth medium8. We did however uncover a reduction in the proportion of proliferating progenitors with EβC overexpression . EdTC is able to bind DNA at Wnt target genes, but cannot transactivate them due to its inability to bind β-cateninion Fig. . Thus, w56. This was also seen in vivo with β-catenin overexpression in mouse muscle34. Another study, which knocked out APC in mouse satellite cells leading to unrestrained β-catenin activation produced similar findings57. Recently, Rudolf et al. showed that loss of beta-catenin lead to protracted and incomplete regeneration but that over-expression of increased active beta-catenin resulted in precocious differentiation in mouse myogenic cells. These authors argued that both increased or loss of Wnt-β-catenin signalling leads to impaired in vivo muscle regeneration with decreases in satellite cell self-renewal, decreased fibre size and excess collagen deposition10. Collectively, these studies and ours suggest that the manipulation of β-catenin above or dramatically below physiological levels is broadly disruptive to myogenesis. In contrast, Murphy et al. utilised a Pax7-Cre driver and did not observe any phenotype from loss of β-catenin suggesting that it is dispensable for regenerative myogenesis in the adult34. However, because Cre-based recombination is imperfect, not all satellite cells will have deleted the gene entirely. In addition satellite cells are abundant in muscle with estimates of 5–7 per fibre; once activated they are highly proliferative and only a few cells are needed to regenerate and repopulate a large area of muscle58. If a knockout and wild type cell were able to fuse, β-catenin from the wild type cell could be shared rendering the knockout obsolete. Thus a very small number of ‘escaper cells’ with functional β-catenin could potentially compensate. As the level and localisation of β-catenin in this study was not quantified over time, the extent of β-catenin expression following the experimental manipulations remains unclear34.Recent work in mice whereby either the Wnt agonist BIO was administered or β-catenin was overexpressed specifically in satellite cells following muscle injury, demonstrate a striking phenotype of vastly protracted regeneration and the appearance of many small fibresin situ and active in CD56Pos myogenic progenitors in vitro. The present study provides evidence that β-catenin is dynamically regulated during the different phases of myogenesis increasing as differentiation progresses. Manipulation of key Wnt signalling components during differentiation has unique outcomes. GSKi leads to inhibition of myogenesis whilst Wnt proteins, which activate the signalling pathway  who gave written informed consent to participate in this study (n = 22). Muscle-derived cells were isolated, cultured for 5–7 days to expand numbers and immunomagnetically purified using anti-CD56 microbeads as described previously20. In all studies reported here primary human muscle-derived cells at passage 1 or 2 were used and at least 2–3 replicate wells were plated and analysed per condition.All studies were performed with UK National Health Service Ethics Committee approval and in accordance with the Human Tissue Act and Declaration of Helsinki. Using the Bergström needle biopsy techniquePos purification, cells were plated on collagen-coated  borosilicate glass coverslips  in 24 well plates . For western blotting and qRT-PCR experiments, cells were plated at 40,000 cells/35 mm dish. For proliferation cells were plated at 10,000 cells/well in 24 well plates and for differentiation cells were plated at 25–50,000 cells/well. For all studies investigating differentiation the cell monolayer was rinsed three times with PBS prior to the switch from proliferation to differentiation medium to remove all traces of serum containing medium60. Full details of media conditions as described previously6.After CD56in situ, human skeletal muscle biopsies obtained from healthy individuals were snap frozen in dry ice-cooled isopentane and cryosectioned at 10 μm thickness (n = 2). Tissue sections were stained as detailed below.To characterize the expression of CD56 and β-catenin 61, lithium chloride (LiCl)  was diluted to a final working concentration of 20 mM/L, CHIR99021(CHIR)  was diluted to a final working concentration of 5 μM. Each was administered to cells at their working concentrations in an appropriate volume of serum-free differentiation medium. In all cases the final concentration of DMSO was <0.2% v/v. Recombinant proteins Wnt3a and Dkk-1  were reconstituted in sterile PBS containing 0.2% BSA. Proteins were administered at 200–400 mg/ml (Wnt3a) and 600–1000ng/ml (Dkk-1) in serum-free skeletal muscle differentiation medium containing 0.3% (w/v) BSA as a carrier protein.6-bromoindirubin-3′-oxime (BIO) or its kinase inactive form 6-Bromo-1-methylindirubin-3′-oxime (meBIO)  were diluted to a final working concentration of 5 μM4 cells/well were seeded per well into a 24-well plate. After overnight incubation, the cells (90% confluence) were transiently co-transfected with 0.4 mg DNA of reporter constructs (TOP/FOP Flash) and control plasmid (pCMV-GLuc) using 1 mL Lipofectamine 2000TM (Invitrogen) in 50 mL OptiMEM I reduced serum media . Thereafter, the test cells were stimulated with control unconditioned media or conditioned media for 24 hours. Cells from the treated and control conditions were collected for dual luciferase assay  according to manufacturer’s instructions. Each assay was performed in triplicate and the reporter activity expressed as mean ± SD.Dual Luciferase assay kit (Promega) based on TOPflash/FOPflash reporter plasmid system was used for the detection of β-catenin driven Wnt-transcriptional activity. The TOP Flash reporter construct contains three optimal copies of TCF/LEF sites upstream of a thymidine kinase minimal promoter that, when bound by β -catenin induces transcription of luciferase reporter gene. FOP Flash contains mutated copies of the TCF/LEF that cannot be activated by β -catenin and was used as negative control. Luciferase reporter gene analysis was performed using the SaOS cell line treated with either control unconditioned media or media conditioned by differentiating myogenic cells for 24 h. One day before transfection, 1 × 10To generate lentiviral supernatants, HEK293T cells were cultured overnight in dishes pre-coated with 0.1 mg/ml collagen solution  in DMEM, 10% FCS, 2% glutamax and 1% pen/strep until 70% confluent. A solution containing 6.5 µg pCMV Δ8.9 packaging plasmid, 3.5 µg VSV-g envelope plasmid and 10 µg target construct were diluted in OptiMEM-1, combined with 30 µl Lipofectamine 2000  and incubated at room temperature for 20 min to allow complexes to form. The plasmid/lipofectamine mixture was added dropwise to the HEK293 cells and incubated at 37 °C for 4 h before an equal volume of OptiMEM-1 was added. The lentiviral supernatant was collected 48 h post-transfection and concentrated using a lenti-X-concentrator kit  according to manufacturer’s specification and viral titre measured using GoStix . The pseudovirus was stored at −80 °C until ready to use. Fresh primary human muscle-derived cells at passage 1 were seeded in 6 or 24-well plates and maintained at 37 °C for 24 h in growth media prior to transduction. Human myogenic cells were transduced at a supernatant to media ratio ranging from 1:15 to 1:40 with 8 µg/ml polybrene  in growth media devoid of antibiotics. A mock control, consisting of the same transduction mixture without the addition of lentivirus was performed for each subject. The infected target cells were monitored for mCherry expression using fluorescent microscopy and after 48 h media was changed to growth media or differentiation media. In both cases the mouse ORF for β-catenin and TCF4/were expressed as β-catenin  binding domains are 100% conserved between humans and mice. Primers were designed, which allowed us to measure endogenous transcripts to study activity at the host locus.3 (0.01%) for 10 minutes, then blocked and incubated with primary antibodies. This was followed by incubation with species-specific fluorescently-labelled secondary antibodies, and 10 minute incubation in Hoechst 33342 dye. Stained tissue sections and cells were mounted using ‘anti-fade gold’ medium (DAKO USA). Details of all antibodies used are shown in Supplementary Table Cryosections and cultured cells were fixed by the addition of 4% paraformaldehyde in ice-cold PBS for 10 minutes. Cells were permeabilised after fixation by addition of 0.2% Triton-X100 in PBS with 1% BSA and NaNet al.6. For all analysis 6–10 independent fields of view were analysed from two separate coverslips on cells derived from at least three representative myogenic cell populations. Only raw, greyscale images were used for quantitative analysis. Local background intensity values were subtracted from measurements to account for potential field to field variation in non-specific background fluorescence62.Immunofluorescent probes were illuminated by epifluorescence and signals were visualised through red (filter set 45 HQ Texas red shift free), green  and blue (filter set 49 DAPI shift free) band pass filters (Zeiss) on an AxioPlan microscope . Greyscale images were captured using an AxioCam MRm digital camera (Zeiss). To measure the expression of myogenic transcription factors and transcriptional co-activators in individual nuclei, image analysis was performed using techniques described in Agley Cell lysates were prepared using ice cold lysis buffer containing phosphatase and protease inhibitors . Protein content of the samples was quantified using the Bradford assay . Protein samples to be analysed were mixed at a 1:1 ratio with 2x Laemmli sample buffer and denaturing discontinuous SDS-PAGE was performed using 4% and 10% acrylamide: bis-acrylamide (37.5:1) stacking and resolving gels respectively. After SDS-PAGE had been performed, electro-blotting was performed using standard methods. Immobilised proteins were then probed overnight with primary antibodies. After washing, blots were incubated with the appropriate species-specific horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000 dilution) for one hour at room temperature. Immuno-labelled protein bands were then visualised using ECL Plus Western Blotting Detection Reagents  and ECL Hyperfilm . Band density was quantified using Scion Image analysis software . β-tubulin or GAPDH expression were used for the normalisation of protein loading. Details of all antibodies used are shown in Supplementary Table et al.63 and normalised to 4 stable reference genes or as performed by Lewis et al.64. Primer sequences can be found in Supplementary Table Cell monolayers were rinsed with PBS and cells lysed directly in their culture dishes by the addition of TRIzol reagent . Isolation of RNA from TRIzol was performed as per the manufacturer’s instructions. RNA was reverse transcribed using the QuantiTect Reverse Transcription kit  and RotorGene 6000  qPCR machine following the manufacturer’s instructions. qPCR was performed as described by Bailey n numbers are given for specific experiments and an alpha value of P < 0.05 was accepted as statistically significant. For quantification of immunofluorescent staining 6–10 independent fields of view were analysed per condition (pooled from two separate coverslips) on cells derived from at least 2–3 representative myogenic populations per experiment. In all other cases n numbers are given for specific experiments and an alpha value of P < 0.05 was accepted as statistically significant.For experiments comparing only two groups, data were compared by unpaired t-test with Welch’s correction, which does not assume equal variances between groups; where data did not satisfy all parametric assumptions Mann-Whitney U tests were used to compare differences between groups. For all experiments comparing more than two groups a one or two way (if repeated measures or time as a factor) analysis of variance (ANOVA) was performed and specific time points were analysed via post-hoc Bonferroni’s multiple comparison test. In all cases Supplementary Information


The chemokine fractalkine (CX3CL1) and its receptor CX3CR1 play a fundamental role in the pathophysiology of stroke. Previous studies have focused on a paracrine interaction between neurons that produce fractalkine and microglia that express CX3CR1 receptors in the central nervous system. Recent findings have demonstrated the functional expression of CX3CR1 receptors by hippocampal neurons, suggesting their involvement in neuroprotective and neurodegenerative actions. To elucidate the roles of neuronal CX3CR1 in neurodegeneration induced by ischemic stroke, a mouse model of permanent middle cerebral artery occlusion (pMCAO) was employed. In the pMCAO mice, increased CX3CR1 levels, apoptosis-associated morphological changes, and Caspase 3-positive neuronal cells were observed in the striatum and in the hippocampus 24 hours after occlusion. Upregulation of CX3CR1 in ischemic neurons is associated with neuronal apoptotic cell death. In contrast, ischemia-induced apoptotic neuronal cell death was decreased in CX3CR1 deficient mice. Cultured primary hippocampal neurons obtained from CX3CR1 deficient mice were more resistant to glutamate-induced excitotoxicity by blocking calcium influx than those from wild-type mice. For the first time, we reported that neuronal CXCR1 mediates neuronal apoptotic cell death in ischemia. Our results suggest that modulating CXCR1 activity offers a novel therapeutic strategy for stroke. During an ischemic event, impaired blood flow does not allow the delivery of oxygen and glucose, leading to energy depletion, over-activation of glutamate receptors and release of excess glutamate, increase of intracellular calcium, loss of membrane potential and cell depolarization, and, eventually, cell death3. The need for better stroke therapies has encouraged research in the cellular and molecular mechanisms of ischemic brain damage, with a major focus on the neuronal response under ischemic conditions.Ischemic stroke is the fourth leading cause of death in the United States and Europe, and it is a major cause of adult disability6. It has been previously reported that CX3CR1 deficiency is associated with decreased neuronal apoptosis and improved outcomes following ischemic brain injury8. The underlying mechanism has focused on the signaling from neurons to microglia specific to microglia activation, survival, proliferation, and neurotoxicity, since the expression of fractalkine in the brain has been found to be widespread and localized mainly to neurons9, whereas the expression of its G protein-coupled receptor CX3CR1 is thought to be restricted to microglia and astrocytes10. Meucci et al. recently provided direct evidence for the functional expression of CX3CR1 receptors by hippocampal neurons and demonstrated their involvement in the neuroprotective action of fractalkine11. CX3CR1 immunoreactivity was also transiently detected in neurons, particularly in CA1 pyramidal cells, in a rat model of pilocarpine-induced status epilepticus12.The chemokine fractalkine (CX3CL1)/CX3C chemokine receptor 1 (CX3CR1) system has been shown to play an important role in modulating the communication between neurons and resident microglia in the central nervous system (CNS)We hypothesized that CX3CR1 directly mediates brain ischemia-induced neuronal apoptosis. We now report that CX3CR1 is upregulated in ischemic neurons in mice subjected to focal cerebral ischemia. Higher levels of CX3CR1 are associated with apoptotic neuronal death, and calcium influx plays a crucial role in this process of cell death.16 and has not yet been investigated in our system; we first assayed mature microglia-depleted hippocampal cultures from postnatal C56BL/6 mice for CX3CR1 expression. Microglial depletion was attained using a rigorous densitometric segregating centrifugation protocol, and it was ensured for the lifespan of the culture by treatment with 2.5 μM cytosine arabinoside (AraC) on days in vitro (DIV) 3. We found that CX3CR1 expression significantly co-localized with microtubule-associated protein (MAP) 2-positive cell bodies in hippocampal cultures , CX3CR1, and NeuN (neuron marker) in the infarct. Triple immunofluorescence staining demonstrated that CX3CR1 is co-localized in most apoptotic neurons in ischemia Fig. . Comparein vivo, we used CX3CR1−/− mice. Figure +/+) and CX3CR1−/− mice. There was no significant difference between the WT and CX3CR1−/− mice in the decrease of regional cerebral blood flow (CBF)  Fig. . The appice Fig. . The infice Fig. . Given t−/− pMCAO mice by immunostaining the cleaved Caspase-3. Cells positive for Caspase-3 were observed in these three locations of the ischemic brain of CX3CR1+/+ mice  neuronal CX3CR1 elevation is associated with apoptotic neuronal death; (3) CX3CR1 deficiency protects mice from ischemic brain damage in vivo; (4) CX3CR1 deficiency reduces glutamate-mediated excitotoxicity in hippocampal neurons cultured in vitro with a mechanism dependent on intracellular calcium. Taken together, our data suggest that CX3CR1 promotes acute brain ischemic pathology by exacerbating excitotoxicity and neuronal apoptosis.In this paper, we describe for the first time that neuronal CXCR1 mediates neuronal apoptotic cell death in ischemia. Our principal findings are: (1) CX3CR1 is unregulated in ischemic neurons 18. Consequently, the tissue in the penumbra is the most clinically relevant target for interventional treatments in acute stroke and the damage may be reversed if treatment is implemented at an early stage. Evidence suggests that excitotoxicity and apoptosis comprise a major route of ischemic neuronal death in the penumbra, while tissue death in the core zone is mainly necrotic2. A deep understanding of the mechanisms that promote or prevent recovery is crucial in identifying the means of preventing neuronal death and designing therapies for stroke. Previously, we had found that CX3CR1 deficiency is associated with an improved outcome following transient ischemic brain injury7. Our results are consistent with others’ findings in acute brain and spinal cord injury models in that the absence of CX3CR1 significantly reduces ischemic damage and inflammation20. Based on the previous observation that CX3CR1 receptors are exclusively found on microglia and astrocytes10, we and others had focused on the microglia-neuron communication via CX3CL1/CX3CR1 signaling. In particular, we demonstrated that CX3CR1-mediated microglia activation was associated with neuronal apoptosis in the peri-infarct area at 72 hours after transient middle cerebral artery occlusion (tMCAO) in mice7. However, recent findings of the expression of CX3CR1 by hippocampal neurons indicate that these receptors may also be a target for fractalkine produced by microglia and astroglia or adjacent neurons11. In this study, we found that CX3CR1 was expressed at low levels in the intact brain and was transiently upregulated in ischemic neurons post-pMCAO. Furthermore, we demonstrated that the upregulated CX3CR1 levels were associated with ischemic neuronal apoptosis in hippocampus at 24 hours after permanent middle cerebral artery occlusion. Our results shed more light on the neurotoxic role of CX3CR1 in ischemia and support the hypothesis that fractalkine and its receptor are involved in a complex network of both paracrine and autocrine interactions between neurons and glia21.In ischemic stroke, the concept of a penumbra has been proven by studies in patients in whom thrombolytic treatment had reversed ischemia and had decreased the infarct volume16. The neurodegenerative role of CX3CR1 was aslo observed in a rat model of epilepsy12, although neuronal CX3CR1 receptors were reported to mediate the neurotrophic effects of fractalkine by activating Akt, a major component of prosurvival signaling pathways, and nuclear translocation of NF-κB, a downstream effector of Akt. This prosurvival signaling pathway protects hippocampal neurons from the neurotoxicity induced by the HIV-1 envelope protein gp120IIIB11. Discrepancies in various reports on the neuroprotective versus neurodegenerative functions of neuronal CX3CR1 might be attributed to the regional specificity of its expression in different neurological disorders. In our experiments, we found that both striatal and hippocampal neurons expressed CX3CR1 in accordance with previous reports of selective CX3CR1 expression22. Since both neurons and microglia appear to express CXCR1, in some contexts particular to the CX3CR1+/+ animal model, the result will reflect the complexity of the CX3CL1/CX3CR1 system involving both microglia-neuron and neuron-neuron communication16.Our results suggest that neuronal CX3CR1 deficiency may play a neuroprotective role in ischemia, similar to previous reports that CX3CR1 knockout protects against amyloid-β induced neurotoxicity23. In a previous study, we have shown ischemic neuronal excitability and synaptic excitatory transmission using somatic whole-cell current-clamp recording24. In this report, we have expanded upon previous researches by showing that CX3CR1 deficiency prevents glutamate-induced excitotoxicity by blocking calcium influx. Neutralizing the CX3CR1 with anti-CX3CR1 antibody can also attenuate glutamate-induced damage. It has been shown that the protective effect of CX3CR1 on gp120-mediated neurotoxicity is through prosurvival signaling of Akt and NF-κB11 and that CX3CL1 reduces glutamate-mediated neuronal apoptosis with a mechanism that involves the activation of extracellular-signal-regulated kinases (ERK) and Akt pathways25. Additionally, the protective properties of CX3CR1 may be related to the reduction of glutamate-activated currents or CX3CR1-mediated activation of cytosolic pathways. Considerable crosstalk may exist between the proapoptotic and prosurvival signaling pathways mediated by neuronal CX3CR1 and is worthy of further study in the field of ischemic neuronal damage.Excitotoxicity is a primary mechanism of neuronal injury following stroke. It is a form of cell death induced by prolonged neuronal stimulation with high glutamate concentrations, a consequence of impairment of the local energy supply. Glutamate activates glutamate-ionotropic receptors at synaptic and extra-synaptic sites, causing prolonged neuronal depolarization and triggering deregulation of cellular ion homeostasis, mainly intracellular calcium and sodiumIn conclusion, we provide valuable insight into the specific mechanisms of ischemic brain injury by suggesting a role for neuron-expressing CX3CR1 in the evolution of ischemic neurons Fig. . Targeti−/− mice were obtained from The Jackson Laboratory . Knockouts were generated by replacing the second exon of the CX3CR1 gene with the enhanced green fluorescent protein (GFP) reporter gene, and the resulting mice were backcrossed for more than 10 generations to C57BL/6 mice26. Cells under the control of the endogenous CX3CR1 locus  in homozygous CX3CR1−/− mice were labeled with GFP and lacked CX3CR1 receptor function. All animals were housed in pathogen-free conditions at the animal facilities of the Barrow Neurological Institute, Phoenix, AZ, USA. All experimental procedures were approved by the Institutional Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals .Wild-type (WT) C57BL/6 mice and breeding pairs of CX3CR127. Mice were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). In anesthetized mice, the right side of the common carotid artery was exposed and isolated. The middle cerebral artery (MCA) was occluded by inserting a 6–0 surgical monofilament nylon suture with a heat-rounded tip into the internal carotid artery, which was advanced further until it closed the origin of the MCA. During the experiments, MCA blood flow was monitored using a single fiber optic probe  adhered onto the skull surface of the core area supplied by the MCA  before and after clamping the right MCA. Mice that showed a decrease in MCA blood flow of less than 70–80% of the initial value were excluded. Rectal temperature was maintained at 37 ± 0.5 °C by means of a heating blanket and heating lamp throughout the surgery. Sham-operated control mice received the same surgical procedure without inserting a filament.Adult male mice  were exposed to permanent middle cerebral artery occlusion (pMCAO) with an intraluminal filament2 along each direction. To assess whether the ischemia was induced successfully, single slice cerebral blood flow (CBF) images were acquired 1 hour after pMCAO using a Continuous Arterial Spin Labeling (CASL) sequence with parameters: TR = 3000 ms, TE = 6.95 ms, segments = 4, slice thickness = 1.5 mm, field of view (FOV) 2.0 cm × 2.0 cm, matrix: 64 × 64, total scan time of 20 min. T2-weighted images were acquired 24 hours after pMCAO to assess the infarction volume using a Rapid Acquisition with Refocused Echoes (RARE) sequence with parameters: TR = 3000 ms, effective TE = 60 ms, RARE factor = 8, 20 slices were acquired with thickness = 0.5 mm, field of view (FOV) 2.56 cm × 2.56 cm, matrix: 128 × 128, total scan time of 3 min 12 seconds. The MRI data were analyzed using the MED × 3.4.3 software package  on a LINUX workstation.MRI experiments were carried out on a 7 T small animal system, 30-cm horizontal-bore magnet, and BioSpec Avance III spectrometer  with a 116-mm high power gradient set (600 mT/m) and a 72-mm whole-body mouse transmit/surface receiving coil configuration. Anesthesia was induced with 3% isoflurane and maintained with 1.5% isoflurane carried by 1.5 L/min medical air. During MRI scanning, the animal’s respiration was continually monitored by a small animal monitoring and gating system  via a pillow sensor positioned under the abdomen. Mice were placed on a heated circulating water blanket  to maintain normal body temperature (36–37 °C). Segments of echo-planar imaging (EPI) sequences were used to acquire the apparent diffusion coefficient (ADC) reduction 30 min after pMCAO to assess the damage volume with the following parameters: TR = 5000 ms, TE = 30 ms, 0.5 mm slice thickness without slice gap, field of view (FOV) 2.56 cm × 2.56 cm, matrix: 128 × 128, 18 slices, total scan time of 2 min 20 seconds, and 6 directions of B-field were applied, with b = 100, 200, 400, 600, 800, and 1000 s/mm28 was performed immediately after MRI scanning. The animals were quickly anesthetized with isoflurane, and the brains were removed rapidly and frozen at −20 °C for 5 min. Coronal slices were made at 1–2 mm from the frontal tips, and sections were immersed in 2% TTC  at 37 °C for 20 min. The presence of infarction was determined by the area that was stained negative with TTC.To confirm the infarction volume determined by the T2-weighted MRI images, TTC staining29. The following rating scale was used: 0 = no deficit, 1 = failure to extend left forepaw, 2 = decreased grip strength of left forepaw, 3 = circling to left by pulling the tail, and 4 = spontaneous circling.Neurological deficit assessments were performed by investigators who were blinded to the experimental groups, as described previously−/− sections were incubated with 10% methanol in PBS to quench endogenous GFP. For double immunofluorescent staining, antibodies against NeuN  and cleaved Caspase-3  were used to identify apoptotic neurons. For triple immunofluorescent staining, NeuN , CX3CR1 , and cleaved Caspase-3  were used as primary antibodies. Anti-CX3CR1 (sc-30030) specificity has been confirmed by histochemistry staining of brain tissues from the CX3CR1 mutant mice .The mice were anesthetized with isoflurane 24 hours after pMCAO. Terminally anesthetized mice were perfused intracardially with saline followed by 4% paraformaldehyde. The fixed brains were embedded in paraffin and cut into 6 µm thick serial coronal slides. Immunohistochemistry was performed with antibodies against mouse CX3CR1 . Immunolabeling was detected by applying the peroxidase-antiperoxidase procedure with 3, 3′-diaminobenzidine (DAB) as a co-substrate. Prior to immunofluorescence staining, CX3CR1Ipsilateral and contralateral hemispheres of pMCAO mice were homogenized in 150 mM NaCl containing 50 mM Tris-HCl (pH 7.2), 1% Triton X-100, and complete mini protease inhibitor mixture tablets . After centrifugation, the supernatants were used for immunoblot analysis. Anti-CX3CR1  and anti-beta Actin  were used as the primary antibodies. The specificity of anti-CX3CR1 (ab8021) was verified by using CX3CR1 deficient mice  at 37 °C for 20 min. Cells were mechanically dissociated and plated at a density of 2.5 × 105 in poly-L-lysine coated plastic 24-well dishes in serum-free Neurobasal medium supplemented with B27, 0.5 mM L-glutamine and 100 μg/ml gentamicin. Two days after plating, the cells were treated with 2.5 μΜ cytosine arabinoside, an inhibitor of DNA replication  for 3 days to prevent mitotic, non-neuronal cell growth30. Under these conditions, an average neuronal purity of 92% can be achieved. Neuronal cultures were kept at 37 °C in 5% CO2 for 10–11 days in vitro with a biweekly medium replacement by removing half of the old media and replacing it with the same volume of fresh media31.Primary hippocampal cultures were prepared from the brains of WT and CX3CR1+/+ (WT) and CX3CR1−/− hippocampal cultures, the conditioned medium was removed and stored for later use; neurons were washed and treated with glutamate  in modified Locke’s buffer (without MgCl2 plus 1 μM glycine) to stimulate all types of glutamate receptors. When necessary, cells were pre-treated with polyclonal CX3CR1 Ab  or rabbit IgG  in culture medium; drugs were present during and after the glutamate challenge. After treatment, cells were re-incubated in the original conditioned medium for 18–20 hours.In primary CX3CR1Glutamate excitotoxicity towards neurons was measured using the lactate dehydrogenase (LDH)-based Cyto Tox 96 non-radioactive cytotoxicity assay kit  in accordance with the manufacturer’s protocol. Glutamate-treated cultured neurons were pelleted by centrifugation at 250 × g for 4 min. The supernatant was transferred to a new 96-well plate, and the reconstituted substrate mix was added. The absorbance was measured at 490 nm. Each assay was tested in triplicate. The percentage of specific lysis was determined as follows: / (target maximum release-target spontaneous release) × 100. The maximum release was determined by detecting the absorbance of the target cells lysed with LDH assay lysis solution.The viability of glutamate-treated cultured neurons was tested by an MTT assay. Specifically, 5 mg/ml MTT was added 1:10 to the cultures and incubated for 2 hours; the medium was aspired, then the cells were treated with DMSO and incubated at 37 °C for 10 min. Samples were then analyzed with a microplate reader at 490 and 630 nm to subtract the background. The results were expressed as a percentage (%) of viable cells in the control cultures.After being cultured for 10 days, primary neuronal cells on coverslips were washed in warmed HBSS. They were then incubated in 3 μM Fluo-3-acetoxymethyl ester (Fluo-3/AM) in HBSS at 37 °C for 20 min. Coverslips were washed again with HBSS and placed on the stage of a laser scanning confocal microscope .32.For live cell calcium imaging, cells were excited with 488 nm light from an argon laser, and an emission of 530 nm was detected. The cells were scanned once every second for 240 seconds. After 60 seconds of baseline recording in HBSS, cells were exposed to glutamate (50 μM glutamate + 10 μM glycine) in HBSS for 180 secondsThe intensity of the fluorescence in each cell, which represented the concentration of intracellular calcium, was recorded. The baseline fluorescence was normalized to 1, and the data were presented as the fold increase over the baseline.t-test. Multiple comparisons were performed with two-way ANOVA accompanied by Bonferroni’s post hoc test. Values of p < 0.05 were considered significant.The results are presented as the means ± SEM. Statistical differences between two groups were evaluated by the two-tailed unpaired Student’s All data generated or analyzed during this study are included in this article.Supplementary Information


Increasing evidence showed that the endocannabinoids control tumor growth and progression, both in vitro and in vivo. We evaluated the effect of Rimonabant, a Cannabinoid Receptor 1 (CB1) inverse agonist, on the Wnt/β-Catenin pathway in HCT116 and SW48 cell lines carrying the genetic profile of metastatic CRC poorly responsive to chemotherapies. In these models, Rimonabant inhibited the Wnt/β-Catenin canonical pathway and increased β-Catenin phosphorylation; in HCT116 cells, but not in SW48, the compound also triggered the Wnt/β-Catenin non canonical pathway activation through induction of Wnt5A and activation of CaMKII. The Rimonabant-induced downregulation of Wnt/β-Catenin target genes was partially ascribable to a direct inhibition of p300/KAT3B histone acetyltransferase, a coactivator of β-Catenin dependent gene regulation. Finally, in HCT116 xenografts, Rimonabant significantly reduced tumor growth and destabilized the nuclear localization of β-Catenin. Obtained data heavily supported the rationale for the use of cannabinoids in combined therapies for metastatic CRC harbouring activating mutations of β-Catenin.In a high percentage (≥85%) of both sporadic and familial adenomatous polyposis forms of colorectal cancer (CRC), the inactivation of the APC tumor suppressor gene initiates tumor formation and modulates the Wnt/ WNTs are able to modulate both the ‘canonical’ β-Catenin-dependent and the ‘non-canonical’ β-Catenin-independent Wnt signalling pathways1. In the canonical pathway, in the absence of Wnt ligands, GSK3 (Glycogen Synthase Kinase 3), casein kinase 1 α (CK1 α), axin and APC promote the phosphorylation of β-Catenin, its ubiquitylation and degradation. Interaction of Wnt ligands, such as Wnt3a, with Frizzled (Fzd) receptors and the Wnt co-receptor low density Lipoprotein Receptor-related Protein 5 (LRP5) or LRP6 activates the Dishevelled (Dvl) cytoplasmic phospho proteins, which inhibit β-Catenin phosphorylation and block its degradation. Then β-Catenin accumulates in the nucleus, binds to Lymphoid Enhancer-binding Factor (LEF) and T Cell Factor (TCF) proteins and acts as a transcriptional co-activator to modulate the expression of target genes2. Some WNTs, such as Wnt5A and Wnt11, fail to stabilize β-Catenin and can also induce a calcium flux and the activation of various pathways, such as PKC (Protein kinase C), CaMKII  and JNK 3. Moreover, Wnt5A inhibits Wnt3A-induced canonical pathway in a dose-dependent manner4. Wnt5A is frequently silenced in human CRC cell lines and in human primary tumors due to its promoter methylation, resulting thus as a potential epigenetic biomarker or therapeutic target for CRC5. Wnt/β-Catenin signalling regulates different cellular processes in both embryonic and adult stages and increasing evidence suggests that Wnt target genes are mainly cell and context specific. The multifaceted role of Wnt/β-Catenin pathway is a topic still debated and the analysis of the Wnt targetome6 and of β-Catenin target genes7 identified new players associated with CRC. Intriguingly, anandamide (AEA), a cannabinoid receptor 1 (CB1) agonist, inhibits cholangiocarcinoma growth through activation of the non-canonical Wnt pathway mediated by Wnt5A8 and, in human breast cancer cells, methyl-F-AEA reduces β-Catenin levels, inhibits the transcriptional activation of TCF responsive elements and decreases the expression of mesenchymal markers9. These data strongly suggest our hypothesis of a potential effect of cannabinoids on the Wnt/β-Catenin in CRC, but to date no data dissected these interactions. The endocannabinoid (EC) system possesses antitumor effects in vitro and in vivo12. Δ9-tetrahydrocannabinol induces apoptosis in CRC by CB1-mediated inhibition of both RAS-MAPK/ERK and PI3K-Akt signalling and activation of BAD (BCL2 Associated Agonist of cell Death)10. In DLD1 and HT29 CRC cell lines, both CB1 and CB2 (cannabinoid receptor 2) receptor agonists induce apoptosis13. In the azoxymethane (AOM) induced aberrant crypt foci (ACF) model, the inhibition of EC hydrolysis decreases the development of precancerous lesions in the mouse colon14. Moreover, cannabinoids significantly reduce the proliferation of CRC cell lines15 and raise the expression of CB1 and estrogen receptors (ER)15, whose loss might promote and accelerate colorectal carcinogenesis in APCMin/+ mice16. Last but not least, cannabinoids improve the efficacy of chemotherapic drugs used in the clinical practice18. A priority in the treatment of human cancer is the finding of strategies able to reduce the occurrence of resistance to chemotherapies or, alternatively, to identify new pathways as targets of drugs enabling to bypass these events. In this issue the ECs could represent good therapeutic chances acting at several levels. We previously found that among other cannabinoid compounds, Rimonabant (SR141716), an inverse agonist at the CB1 receptor, shows a powerful antitumor effect both in precancerous lesions and in CRC cell lines18. Here we dissected the role of Rimonabant in CRC analyzing, in vitro and in vivo, its effects on the Wnt/β-Catenin mediated signalling.The majority of both sporadic and familial forms of adenomatous polyposis (FAP) in colorectal cancer (CRC) originates from inactivation of APC (Adenomatous Polyposis Coli) tumor suppressor gene. APC negatively regulates the levels of 11. Here we found that SR141716 inhibited HCT116 cell growth  marks β-Catenin for ubiquitin-mediated proteolysis20. In HCT116 and SW48 cell lines, SR141716 increased p-β-Catenin starting from 8 hours of treatment  tyrosine kinase receptor activating actin-binding protein, filamin A, and the JNK signalling pathway26. However, the non-canonical pathway triggers intracellular calcium flux, associated with CaMKII activation and canonical signalling inhibition. In HCT116 cells SR141716 increased protein levels of both Wnt5A and ROR2 at 8 hours of treatment and induced activation of CaMKII  and COX-2 (Cyclooxygenase-2), involved in CRC progression  HCT116 xenograft model. Tumor cell suspension was injected s.c. into 20 female SCID mice and when the tumor reached approximately the size of 50–70 mm3, 10 mice in the treated group received the peri-tumoral injection of SR141716, while 10 mice in the control group received vehicle alone, three times a week for 6 weeks. The tumor sizes have been recorded on the first day of SR141716 treatment (day 0) and bi- or three-weekly at the indicated time points. Mice in the control group developed tumors beyond 2,0 cm3 on average by day 42. In contrast, the mice in SR141716 group developed much smaller tumors   is a component of the Adenosine receptor family comprising four G-protein coupled receptors  linked to Ca2+ mobilization and Cyclic AMP increase. Low amount of A1, A2A, and A2B receptors have been detected in colon cancer cell lines cultured in normoxic condition, as in our experimental procedures33, whereas both HCT116 and DLD1 cells expressed high levels of the A3 receptor subtype34. ErbB4/Her4 (Erb-B2 Receptor Tyrosine Kinase 4) is a member of the ErbB protein tyrosine kinase family, which also includes EGFR/ErbB1/Her1. Despite recently an over-expression of ErbB4 was found in human CRC tissues, in cultured colon cancer cell lines ErbB4 protein expression is difficult to detect and mainly unmistakable in poorly differentiated CRC cells such as HCT116 in our panel36. Therefore, among the obtained results, we were intrigued by p300/KAT3B target at the 3rd position in the final ranking of predicted most affine proteins of SR14171637. Specifically, the careful analysis of the sampled docking poses enforced this result, showing a good accommodation of SR141716 in the p300/KAT3B binding site and supporting the potential inhibition of the histone acetyltransferase (HAT) activity exerted by the investigated compound. We found two interesting binding modes in which SR141716 is placed in p300/KAT3B occupying the ligand binding site (LBD) and exerting both polar and hydrophobic interactions. The analysis of the first pose, associated to the best docking score , disclosed the arrangement of SR141716 in the deep part of the LBD supported by an edge-to-face π-π interaction between the pyrazole core and the indole moiety in the side chain of Trp1466, and an H-bond with the carbonyl oxygen in the backbone of Leu1398  showed the placement of the molecule in a more external part of the binding site, supported by halogen bonds between the dichloro-phenyl part of SR141716 and Arg1410   Fig. . MoreoveμM) Fig. . The decnes Fig. .Figure 7β-Catenin signalling resulting in increased transcriptional activity of the β-Catenin. Despite the difficulties in to dissect the engagement of Wnt/β-Catenin pathway in the onset and progression of CRC, the study of these mechanisms is now emerging as a promising ground to identify potential targets of intervention for CRC treatment. The porcupine inhibitor LGK974, which blocks the palmitoylation and secretion of Wnt ligands40, has been recently inserted in a Phase II multi-centric clinical trial to assess the safety and antitumor efficacy of the triple combination of LGK974, LGX818 (a highly selective BRAFV600 inhibitor) and Cetuximab in BRAFV600-mutant metastatic CRC .The heterogeneous molecular profiles of CRC and the need to identify patients which could effectively take clinical advantage from combined chemotherapies, triggered the dissection of the mechanisms responsible for sensitivity and resistance to treatments. The majority of sporadic forms of CRC are characterized by deregulation of Wnt/11. The cannabinoid compounds are able to reduce the progression of CRC in vivo in the AOM-induced ACF model in mice14 and improve the efficacy of chemotherapic drugs18.Rimonabant, an inverse agonist at CB1 receptor, produced mitotic catastrophe and modulated the expression of Cyclin B1, PARP-1, Aurora B and phosphorylated p38/MAPK and Chk1, in CRC cell lineβ-Catenin pathway. We tested the antitumor efficacy of Rimonabant in HCT116 and SW48 CRC cell lines, expressing APC and BRAF wild type and harbouring β-Catenin mutation  and PIK3CA activating mutations. Despite this genetic profile represents a small percentage of the human primary CRCs, the constitutive and simultaneous activation of KRAS and PIK3CA pathways, associated with β-Catenin stabilization, confers maximal resistance to Cetuximab41 and the mesenchymal phenotype reduces the sensitivity to Erlotinib42, features accounting for a significant number of metastatic cancers unresponsive to therapies.In this study, we hypothesized that cannabinoids could directly interact with the Wnt/11. Unlike our previous results, here we found that Rimonabant also inhibits proliferation in TP53 wild-type highly invasive HCT116 and SW48 cell lines through induction of apoptosis clearly highlighted by an increase of Caspase 3 and PARP cleaved forms in treated cells.In DLD1 and SW620 cells, carrying mutated form of tumor protein p53 (TP53), Rimonabant exerted cytotoxic effects without induction of apoptosis and through activation of mitotic catastropheβ-Catenin43. Finally, alterations in several tyrosine kinase genes dramatically establish the sensitivity of CRC cells and primary tumors to therapies43. These molecular profiles could explain the different Rimonabant-mediated effects observed in to Wnt/β-Catenin pathway.Beside TP53 mutations, CRC cell lines used in this study have heterogeneous genetic features partially representatives of the broad diversity observed in CRC patients. All cells in the study, except SW48, express mutated KRAS. Allelic loss of APC has been found in SW620 and DLD1  but not in APC wild-type HCT116 and SW48 cell lines which express stabilizing mutations of β-Catenin phosphorylation whereas this evidence seems provisional in other cell lines. These results strongly suggest the engagement of mechanisms able to antagonize β-Catenin phosphorylation and to induce its dephosphorylation.In HCT116 and SW48 cells, Rimonabant induced a significant and stable increase of β-Catenin degradation complex includes protein phosphatase 2A (PP2A) and heat shock protein 105 (HSP105), two components recently identified as fine regulators of the balance between degradation and stabilization of β-Catenin. Although HSP105 does not possess intrinsic phosphatase activity, this protein seems essential to anchor PP2A into the degradation complex and to induce PP2A-mediated β-Catenin dephosphorylation44. In primary CRCs increased expression of HSP105 correlates with nuclear localization of β-Catenin and poor prognosis. Moreover, in SW480 CRC cells, characterized by stable accumulation of β-Catenin and truncated APC, the depletion of HSP105 decreased β-Catenin levels, reduced Wnt target genes expression and impaired cell proliferation through cleavage of PARP and Caspase 3, then substantially triggering apoptosis44. In our study we did not evaluate the HSP105 expression but we speculate that in SW620, a metastatic CRC cell line derived from the same patient originating SW480 cells, and presumably in DLD1, the transient phosphorylation of β-Catenin induced by Rimonabant could be ascribable to a prevailing phosphatase activity in the degradation complex.The β-Catenin pathway thanks to which the stabilization of the phosphorylated form of β-Catenin localized the protein in the cytoplasm and inhibited its transcriptional effects. As expected, in DLD1 and, mainly, in SW620 cell lines, the Rimonabant-induced increase of p-β-Catenin did not seem associated to a stable inhibition of the canonical Wnt/β-Catenin signal, evaluated by luciferase reporter gene assay. These results originate from the different genetic profile of the used CRC cell lines and it is in agreement with the finding that APC truncations, in the SW620 and DLD1 cell lines, did not prevent β-Catenin phosphorylation but inhibited β-Catenin ubiquitination and degradation45. Moreover, despite all cell lines analyzed in this study expressed ROR2 receptor, LRP5 typically associated with prevalent function of the canonical Wnt/β-Catenin signal, was detectable in DLD1 and SW620 but not in HCT116 cells  and members, other than EGFR/ErbB1/Her1, belonging to the ErbB/Her family of protein-tyrosine kinases, have recently received renewed interest from the scientific community56. Cetuximab, a chimeric monoclonal antibody approved for the treatment of metastatic CRCs expressing EGFR  and wild type KRAS, inhibits several transcription pathways downstream EGFR. Despite Cetuximab, used as single agent or in combination with chemotherapies, significantly improved prognosis and median survival, several patients are resistant to the compound and accumulating evidence suggests that EGFR expression, routinely tested in tumor samples with immunohistochemical assays, failed to predict the responsiveness to EGFR-targeting treatment. In a recent paper Mitsui and co-workers36 showed that the expression of phosphorilated ErbB family members, but not of their total forms, correlated with worse overall survival in CRC patients. Although ErbB4 protein expression is solely detectable in more aggressive CRC cell lines36, high levels of ErbB4 were found in about 43% of primary CRC tissue microarrays35, and the nuclear staining of its 80kDa cleaved fragment, able to participate in transcriptional events, correlated with poor prognosis36. In a recent work, tumor tissue specimens from patients with metastatic CRC unresponsive to cetuximab coexpressed ErbB4 and kal1 C-terminal interacting tetrasponin (KITENIN)55. The Authors suggested an interesting EGFR-independent interaction KITENIN/ErbB4 able to stabilize c-jun and the transcription triggered by a c-jun-TCF4-β-Catenin complex55. According to previous reports, in our study only HCT116 express both ErbB4 and KITENIN but the inhibition of Wnt/β-Catenin mediated transcription seems a common event in Rimonabant-treated cells probably acting downstream ErbB4 containing complex. Of course, now we are unable to exclude, at least in HCT116, a potential interaction between Rimonabant and ErbB4, but we are testing this hypothesis in in vivo models.ErbB4/Her4  are transcriptional coactivators that influence Wnt/β-Catenin signalling through specific interaction with β-Catenin. Despite their high homology, extensive genome-wide surveys demonstrated that the two coactivators CBP/KAT3A and p300/KAT3B exert different functions in β-Catenin dependent gene regulation58. Moreover, the small molecule windorphen, a selective inhibitor of p300-HAT, showed a robust anti-tumor effect in cancer cells harbouring stable WNT signalling activation59.Moreover, among a large set of tested proteins, Inverse Virtual Screening In our study, although further experiments are needed to clarify the specific mechanisms involved and to dissect selective HAT-inhibiting properties, the observed downregulation of Cyclin-D1, COX-2 and c-Myc seems very promising data.2, involved in inflammation and tumor framework. Transcription of COX-2 gene is controlled by several consensus sequences in the promoter region, and a TCF-binding elements have been identified as functional Wnt/β-Catenin responsive elements within the human COX-2 promoter in both colorectal and gastric cancer cell lines62. On the other hand, COX-2/PGE2 pathway can inactivate the GSK3 β-mediated phosphorilation of β-Catenin and then trigger the activation of Wnt/β-Catenin signalling63. COX-2 is frequently overexpressed in CRCs, controlled by the hypoxia-inducible factor (HIF)-1 binding to an HIF-responsive element on its promoter, and triggers the expression of proangiogenic factors such as vascular endothelial growth factor (VEGF). Moreover, COX-2-selective inhibitors exert significant antitumor effects both in vitro and in vivo60.An impressive amount of works analyzed the role of COX-2 in the onset and progression of CRC /2. Tumor volumes were analyzed using one-way ANOVA. At the end of the study, mice were humanely euthanized and tumors were resected and frozen immediately or fixed in 10% formalin for further analysis.HCT116 cells .Supplementary Information


In this paper, we examined brain activation in subjects during two music listening conditions: listening while simultaneously rating the musical piece being played [Listening and Rating (LR)] and listening to the musical pieces unconstrained [Listening (L)]. Using these two conditions, we tested whether the sequence in which the two conditions were fulfilled influenced the brain activation observable during the L condition (LR → L or L → LR). We recorded high-density EEG during the playing of four well-known positively experienced soundtracks in two subject groups. One group started with the L condition and continued with the LR condition (L → LR); the second group performed this experiment in reversed order (LR → L). We computed from the recorded EEG the power for different frequency bands . Statistical analysis revealed that the power in all examined frequency bands increased during the L condition but only when the subjects had not had previous experience with the LR condition . For the subjects who began with the LR condition, there were no power increases during the L condition. Thus, the previous experience with the LR condition prevented subjects from developing the particular mental state associated with the typical power increase in all frequency bands. The subjects without previous experience of the LR condition listened to the musical pieces in an unconstrained and undisturbed manner and showed a general power increase in all frequency bands. We interpret the fact that unconstrained music listening was associated with increased power in all examined frequency bands as a neural indicator of a mental state that can best be described as a mind-wandering state during which the subjects are “drawn into” the music. The capacity to appreciate music is a universal human phenomenon that helps inspire individual and social life. Neuroscientific studies have shown that music is processed in a cascade of steps that begins with segregation within the auditory stream, followed by the extraction and integration of a variety of acoustic features, and leading to cognitive memory-related processes that induce personal, often emotional, experiences. In the past 25 years, a wealth of brain-imaging studies has explored critical components of music and how they are processed in the underlying neural networks . During the second condition, the subjects were asked to continuously rate the musical pieces using the computer mouse. Thus, they simultaneously performed the rating task while listening to the musical pieces [Listening and Rating (LR)]. These ratings were done on a computer monitor placed in front of the subjects, on which they were shown a horizontal line for valence and a vertical line for arousal. The SAM for valence ranged from a frowning, unhappy figure (left) to a smiling, happy figure (right). The subjects were instructed to place/move the mouse cursor to the position of this horizontal valence line that best represents their actual valence rating. The values for each position on the valence line ranged from -1000 (left = unhappy) to +1000 (right = happy). Thus, values > 0 point to positive valence while values < 0 point to negative valence. For arousal rating we used a vertically arranged analog scale ranging from 0 (bottom = relaxed) to 1000 (top = excited). These rating data were collected with a sampling rate of 500 Hz. Twenty-six subjects started with the passive listening condition (G1) while the second group (G2) started with the LR condition. During the music presentation, we also collected EDA and HR. EDA and HR were recorded with a Biopack MP100 amplifier. The sampling rate for the vegetative data was 200 Hz. The subjects were asked not to clench their teeth and to avoid any kinds of movements other than mouse movement during the entire recording time. The entire experiment lasted about 1.5 h including fixing of electrodes, instruction, conducting the EEG measurements during the two listening conditions, debriefing and removing of electrodes.We would like to explicitly mention that we refrained from employing further experimental conditions for this study. For example, it would have been possible to use a L-L and a LR-LR condition. However, we refrained from using them because in our previous study  we showeElectroencephalograms were recorded using a high-density Geodesics EEG system  with a 128-Channel HydroCel Geodesic Sensor Nets@ (HCGSN120). Data was sampled at 500 Hz and bandpass filtered at 0.1–30 Hz. The vertex electrode (Cz) served as an on-line reference. Impedance was maintained below 50 kOhm. For the exact positioning of the onset of music in the EEG, a marker channel was used to indicate the start and end of the musical piece. EEG analysis was conducted to identify the spectral correlates of music-induced fluctuations in cortical activations.Brain Vision Analyzer version 2.0.1 . In a first step, raw EEG data were bandpass filtered (1–30 Hz) including a notch-filter of 50 Hz to eliminate even very small oscillations leaking above 30 Hz. Eye movements and muscle artifacts were corrected by applying independent component analysis  for the entire EEG recorded during each musical piece, and the EO and EC condition for the theta band (4–8 Hz), the lower alpha band , the upper alpha band , a lower beta band (beta-1: 13–20 Hz), and an upper beta band (beta-2: 20–30 Hz). In this paper we focus on the average EEG response during the entire music presentation in order to uncover general and tonic brain activations induced by the musical pieces in the different conditions. In order to gain statistical power, we defined nine electrode clusters of interest (EOI) for which the mean power of the frequency bands was calculated: three frontal, three central, and three parietal . The EOIs comprised the following sensors in the Geodesics space :For the artifact-free EEG data we computed spectral amplitudes (μV(1)left frontal (LF) = 23 (F3), 24, 26, 27, and 33 (F7);(2)midline frontal (MF) = 4, 5, 10, 11 (Fz), 12, 16, 18, and 19;(3)right frontal (RF) = 2, 3, 122 (F8), 123, and 124 (F4);(4)left central (LC) = 36 (C3) and 41;(5)midline central (MC) = 31, Cz, and 80;(6)right central (RC) = 103 and 104 (C4);(7)left parietal (LP) = 47, 51, 52 (P3), 58, and 59;(8)midline parietal (MP) = 61, 62 (Pz), and 78;(9)right parietal (RP) = 91, 92 (P4), 96, 97, and 98.Region: Frontal, Central, Parietal; Hemisphere: Left, Central, Right; Condition: Listening (L), Listening and Rating (LR)] and one grouping factor . Arousal and valence ratings were subjected to t-tests comparing both groups . The HR and EDA measures were subjected to three-way ANOVAs with two repeated measurements factor  and one grouping factor . For calculating the ANOVAs we used the afex R package for mixed and repeated measurements designs . The three-way ANOVA for HR revealed a significant main effect for Condition , which is qualified by HR decreases during the LR condition, while there was a slight HR increase during the L condition. There was no further significant main effect nor interaction with respect to the HR changes. The ANOVA for the EDA measures only revealed a trend toward a significant interaction between Group and Condition . This interaction is qualified by differently changing EDA measures for both groups in the LR and L conditions. Those subjects starting with the L condition demonstrated reduced EDA values during the LR compared to the L condition. Subjects starting with the LR condition demonstrated slightly increased EDA measures during the LR condition.The Figure 1 EEG topoplots are shown for each frequency band broken down for the major conditions (L and LR) and for the two groups with the different sequences of L and R (L → LR and LR → L). Table 1 lists all significant effects obtained from the mixed ANOVAs for the five frequency bands. The detailed tabulation of these results is given in the Appendix at the end of this manuscript including the degrees of freedom and the general effect size measures. Here, we will only discuss the findings based on the reported p-values.In Group × Condition interactions are most important for our study we will present them first. These interactions are graphically presented in Figure 2. Post hoc tests revealed stronger power values during the L condition than during the LR condition but only for those subjects who started with the L condition (Group L → LR). The other group who started with the LR condition showed basically similar power values during both conditions. There are also two partly significant three-way Group × Condition × Region interactions, which are graphically shown in Figure 3. These interactions are qualified by slightly stronger differences between L and LR for the central and parietal EOIs than for the frontal EOIs. There is also a three-way Group × Condition × Hemisphere interaction which became significant for the theta band power only Figure 4. This interaction is qualified by the stronger theta power values during the L than during the LR condition only for the subjects from the L → LR group. These L vs. LR differences are particularly strong for the central and parietal EOIs.Since, the Figure 5 the significant three-way Condition × Hemisphere × Region interaction plots are shown. As one can see from these plots the frontal to parietal profiles of the power values are different across the hemispheres and conditions. For lower and upper alpha, there are preponderances for the parietal EOI. For theta, the strongest power values are found for frontal midline EOIs. Beta1 power is mostly stronger frontally. In addition, the power values for the L condition are significantly stronger except for beta1. The two-way Condition × Region interactions revealed significant differences between the power values obtained during the L compared to the LR conditions for all regions and the four frequency bands except for the theta band at frontal EOIs.In Figure 6 the intercorrelations between the power values of the different frequency bands are shown. As one can see from this Figure the correlations are generally strong with the strongest correlation between adjacently located frequency bands. This speaks for a general mechanism driving and/or modulating the power in the different frequency bands.In In this study, we show that passive unconstrained music listening (L condition) is associated with increased power values in all examined frequency bands . However, this power increase was only apparent when the subjects followed the L condition first. Thus, after following the LR condition first and the L condition second, the subjects obviously did not listen to the music during the L condition in an unconstrained manner. They most likely applied the same (or similar) mental state as used during the LR condition when listening to the music in the L condition. Possibly, they implicitly rated the musical pieces although they are not required to do so.We also observed HR increases during the L condition but HR decreases during the LR condition. HR decreases are often reported during the performance of long-lasting and partly boring tasks, while HR increases are generally observed during emotionally arousing situations  . We inteNevertheless, what is the functional meaning of our findings? An initial approach to interpreting our findings is interpreting the power in the EEG frequency bands as indicators of the activity of different cortical and subcortical networks. The best-elaborated frequency band in this context is the alpha band. Meanwhile, several studies support the notion that alpha band oscillations are strongly related to cortical inhibition, especially of task-irrelevant brain areas . It has Explaining the functional meaning of the other frequency bands during music listening is less easy and straightforward. In our study, we also identified an increase in theta band power. Increased theta band power has been associated with three psychological functions: (a) the so-called frontal midline theta (Fm theta), which is generally related to cognitive effort, working memory, and emotion processing ; (b) theIncreased beta power during unconstrained music listening is, on one hand, difficult to explain, as tonic beta band power increases are generally reported during conditions of increased tonic alertness. During the resting state, upper alpha and beta band power both positively correlate with tonic alertness network activity . Thus, iOne striking finding is that the power values in all examined frequency bands in our study were highly intercorrelated, suggesting strong functional interplay between the frequency bands  coherences during unconstrained music listening e.g., .Our most important finding, however, is that the pattern of brain activation during unconstrained and undisturbed music listening depends on whether the subjects had followed the L condition before or after the LR condition. The LR condition is a multitasking condition wherein subjects were required to listen to the music, evaluate their emotional feelings, transfer these feelings to reportable  categories, and to indicate these feelings with the appropriate motor output. These psychological functions obviously disrupt unconstrained music listening and the associated brain activations. As mentioned above, we speculate that during unconstrained music listening, the subjects employed increased internal attention, accompanied by reduced external attention, increased inhibition of brain networks uninvolved in generating this internal state, and a mind-wandering state. Thus, subjects engaged in the LR condition could not let their mind wander; rather, they had to focus their attention on the manipulandum with which they indicated their internal state. They also focused their attention on their feelings and on the processes necessary for transferring the feelings to reportable categories. These different processes would prevent the development of mind-wandering as in unconstrained music listening. Interestingly, when the subjects were confronted with this particular state, it was obviously not that easy to switch to the unconstrained listening style.A possible limitation of our study is that we did not explicitly examine whether repeated exposure to the L and LR conditions  might have revealed similar or different results to the L → LR and/or LR → L conditions. However, we refrained from using these conditions in our experiment because: (1) Previously , we showTaken together, we show that the power in all examined frequency bands increases during the L condition, but only when subjects have not had previous experience with the LR condition. During the LR condition, the power values are all substantially lower. We interpret the association between unconstrained music listening and increased power in all examined frequency bands as a neural indicator of a mental state, which can best be described as a mind-wandering state, during which subjects are “drawn into the music.” Further studies are needed to delineate possible characteristic differences (if they exist) between what has typically been described as mind-wandering or meditation.LJ designed the experimental paradigm, performed the statistical analysis and drafted the manuscript. AM conducted the experiments, did the analysis of the EEG, vegetative, and behavioral data, and drafted the manuscript. JK designed the experimental paradigm, performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.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.


The recent shift of computational biologists from bioinformatics service providers to leaders of cutting-edge programs highlights the accompanying cultural and conceptual changes that should be implemented by funding bodies and academic institutions. Computational approaches began to revolutionize the life sciences a generation ago, when DNA sequences became more widely available . SequencAs a consequence of these changes, computational biologists must invent analytical algorithms and deploy them to make scientific breakthroughs. Here, we describe five specific areas in which the role of computational biologists has shifted over the past 20 years Table . Within Computational biology originated as a tool rather than as its own discipline as it did not embody a set of core questions. Thus, bioinformaticians have historically had a supportive role in research programs led by other scientists who decided on the tractability and value of the scientific questions to be pursued. Bioinformatics, therefore, belonged more as a subheading in the Methods section than as a title of a manuscript. Yet those who are able to make sense of the richness of data in the modern life sciences have now been put in the driver’s seat. As a result, computational biologists are now often principal investigators on grants, rather than co-investigators, and they are also last authors on groundbreaking publications.This change occurred because many fields now present opportunities for computational explorations of patterns and integration across diverse and rich data sets. An example is the development of RNA-sequencing (RNA-seq). Bioinformaticians were involved in setting up the statistical framework for analyzing the data and producing a gene expression matrix . HoweverMuch of the power of computational biology follows from its use in collaboration with experts in other fields. How then are computational biologists to collaborate as equals with more experimental biologists? We have found that a key aspect in starting a collaboration is finding a “win-win” situation to working together. Often this comes from establishing not one but two parallel projects; with each lab leading one project. Thus, neither lab is second to the other in the collaboration and both projects gain from the interaction.PhD training programs now enable the development of leading scientists who are not only interested in algorithm creation and data analysis but who are also keenly aware of the most pressing questions in biomedical research. With the “feeling for the biology” gained by such training comes a drive to address the central problems of the field in question. For example, to have a feeling for cancer one must understand that cancer has a somatic evolution, and to have a feeling for a host–pathogen interaction one must understand that the interaction unfolds over time. Without these insights, an analysis of the data cannot be connected to the biology in a way that elicits discoveries. Similar to the experimental biologist, in order for the computational biologist to make the most groundbreaking discoveries in a particular field, they must be tormented by the most pressing biological problems in that field of study.In order for computational biologists who are currently in training to obtain such a feel for the biology, they must have ample opportunities to discuss key biological concepts with life scientists who focus on the same field, such as cancer or immunology. Moreover, future training programs will need to teach computational biology approaches to all students, not only those with a more computational biology bent, such that computational approaches increasingly become part of the general language of science.By virtue of the classical university departmental structure, when computational researchers began launching independent labs they would often be surrounded by traditional life science researchers who could not speak the language of quantitative biology. This isolated structure did not allow for computational biologists to learn from each other or provide a critical mass for computational thinking. Examples of an analogous shift can be seen when looking at stem cell researchers, who were previously isolated within separate departments: a skin stem cell researcher would have been in a dermatology department, and a neural stem cell researcher would have been in a neuroscience department. But with the launch of many stem cell institutes in the 1990s, these investigators had more opportunities to stimulate and inspire each other.The specific topics of a microbiome computational biologist and a cancer computational biologist, for example, will vary, but by increasing their interactions they will inspire and cross-fertilize each other’s fields. This is because of a common set of tools from which all computational biologists draw, such as modeling methods for systems-level analyses, statistical methods for assaying significance of specific hypotheses, and visualization for reducing the high-dimensionality of data sets.In the past, by virtue of their focus on computational approaches and lack of expertise at the bench, computational biologists would focus on data sets that were available in the public domain or from collaborators. This constrained computational researchers from creating their own data sets, and from being able to validate any hypotheses arising from their computational approaches.However, computational biology is now more integrative both in how it generates and in how it interprets data. From this perspective, the typical computational biology lab may look very similar to a molecular biology lab in that its members have both bench space and private computer workspace. Moreover, novel data sets generated in the lab are often best studied with respect to other data sets. This requires the computational biologist to be a “Renaissance scientist”, with multiple experimental and computational tools, as well as to have the ability to take advantage of theoretical models and data described in the literature.The quality of the findings derived from a complex data set likely requires both a deep understanding of biological questions and algorithm innovation. Therefore, we may be witnessing a change in what “ownership” means for data. A de facto rule in the culture of life sciences has been that data is “owned” by those who generated it. Thus, an RNA-seq data set is owned by the person who generated it using commonly available methods and only requiring a short period of work. The data set may have been subsequently intensely analyzed for a considerably longer period by another person, who used specialized computational tools and biological insight to make a discovery. However, as we have witnessed many times, the person who generated the data has an inherent advantage in becoming the first author on the subsequent paper. This often unfair assignment of credit is now changing, however, as the role of computational biologists is gaining recognition. Funding bodies and institutional tenure committees should also create new credit structures to ensure that those who make important discoveries are promoted and funded regardless of whether they generated the data themselves.The ability to generate and test a hypothesis has always been the cornerstone of science. Bioinformaticians have classically used complex statistical methods to contribute to the experimental design of a study and to data analysis for hypothesis testing. However, hypotheses were often generated by the biological experts. Now, with the emergence of large data sets across biomedicine, computational biologists can contribute not only to testing hypotheses, but also to exploring the data in such a way as to generate novel, unexpected, groundbreaking hypotheses that can subsequently be tested and validated in independent data sets, as well as at the bench. Indeed, the recognition that crucial hypotheses arise from data-driven “fishing” expeditions must be communicated to funding bodies and other scientific institutions.A current stumbling block with computational biology, which we hope to see overcome soon, is the validation of hypotheses inferred from complex data sets. Computational biologists tend to lack extensive validations in their papers, which may indicate a more theoretical bent or a lack of expertise in the required experimental methods. Efforts by computational biologists, however, to improve this aspect of their science are likely to improve their grant applications and to increase their publications in top journals.In spite of the transformation that we describe here in how computational biologists now contribute to the scientific enterprise, one aspect that has not changed is the need for computational biologists to collaborate closely with experimentalists and clinicians across the biomedical spectrum. The testing of a hypothesis generated by the analysis of a data set needs to be validated in complex experimental systems, such as mice and organoids, or by using interventional in vivo approaches that require a lifetime of expertise in a particular method of drug administration. In addition, the initial hypothesis may need to be validated by collecting new data from an independent human population. This necessitates close interaction with patients, to whom the computational biologist may not have ready access.The importance of collaborative relationships between wet and dry researchers blurs the lines that define research, and is also reflected in recent changes in how research is funded and organized at the institutional and governmental levels. For example, large collaborative federal grant funding requires clinicians, experimentalists, and computational biologists to come together to interact with data and ideas in ways that are not possible in grants that are awarded to a single principal investigator. Many institutions around the world have launched computational centers and departments, particularly in the field of cancer, and have designed cutting-edge lab spaces where computational and experimental researchers work closely together in collaborative groupings, with many opportunities for frequent daily interactions.World-class computational biologists must innovate in both the computational and the biological realms so that the secrets hidden in biomedical data can be unlocked. Some cultural changes by funding bodies and academic institutions may be necessary, but when these parameters are met, computational biologists will be able to make the most groundbreaking discoveries.


KAT8 mRNA is maternally derived and it is required for successful development of early embryos. An abundance of KAT8 transcripts are expressed in oocytes and its abundance continuously decreases throughout meiotic maturation and preimplantation development. In addition, KAT8 expression is insensitive to RNA polymerase II inhibitor after embryonic genome activation, suggesting its maternal origin. The levels of KAT8 mRNA and H4K16 acetylation were effectively knocked down by siRNA microinjection. Knockdown of KAT8 significantly reduced the blastocyst formation rate and total cell number per blastocyst. Analysis of trophectoderm lineage and marker of DNA double-strand breaks revealed that the impaired developmental competence and quality of embryos might be attributed to defects in both the first two lineages development and genome integrity. Taken together, these results demonstrate that maternal KAT8 is indispensible for porcine early embryo development potentially through maintaining the proliferation of the first two lineages and genome integrity.K (lysine) acetyltransferase 8 (KAT8), an acetyltransferase that specifically catalyzes histone H4 lysine 16 acetylation, is critical for key biological processes including cell proliferation and maintenance of genome stability. However, the role of KAT8 during preimplantation development in pigs remains unclear. Results herein showed that  In. InKAT8 s P < 0.0  to discriminate between maternal and zygotic KAT8 transcripts. qPCR results revealed that the levels of KAT8 mRNA in control 4-cell embryos were significantly lower than that in control 8-cell embryos (P < 0.05). Importantly, the relative abundance of KAT8 mRNA did not change in 8-cell embryos comparing control to α-amanitin treated group , but α-amanitin treatment did not further decrease the expression levels of KAT8 at the same time points ; non-injected oocytes were used as an additional control. The oocytes from three groups were then parthenogenetically activated (PA) and cultured for 2 to 7 days. qPCR results revealed that microinjection of KAT8 siRNA resulted in a more than 90% reduction of KAT8 mRNA levels at 2-cell and blastocyst stages, respectively . Altogether, these results suggest that KAT8 siRNA can carry out the robust reduction of levels of KAT8 and its catalyzed product H4K16ac in early embryos.To elucidate the functional role of y Figure  and 4B.  oocytes . As an astocysts . Immunofstocysts . The quas Figure  . To further ascertain the quality of embryos that developed to blastocyst stage, we determined the number of trophectoderm (TE) lineage-specific and total cells in the resulting blastocysts from each group by immunostaining against CDX2 as marker of TE cells and propidium iodide to counterstain DNA in all cells , and these blastocysts also had fewer ICM . On the other hand, the ratio of ICM: TE cells was similar among three groups  and DNA double-strand breaks (DSBs) repair , we thuss Figure . The quas Figure , suggestKAT8 by microinjecting siRNA to MII oocytes resulted in the lower developmental competence of oocytes developed into blastocysts. The reduced rate of blastocyst formation may be due to defects in the first two lineages (ICM and TE) proliferation and genomic integrity. Therefore, the results here indicate that maternal KAT8 functions during porcine early embryo development.In the present study our results reveal an important role of maternally provided histone acetyltransferase KAT8 in porcine preimplantation embryos. Knockdown of KAT8 have been not cloned before now, in this study we successfully cloned the full-length sequences of porcine KAT8 cDNA. Cloning and acid amino sequence alignment of porcine KAT8 indicates that it shares three similar domain structures and is also highly conserved compared to other species, which imply that KAT8 may have a similar function between different species. The relative expression of KAT8 in porcine different tissues revealed that KAT8 is highly expressed in MII oocytes compared to sperm and other tissues, suggesting that the expression of KAT8 is oocyte-specific. To our knowledge, the outcome is the first report of histone acetytranseferase KAT8 gene present restrictedly in porcine oocytes. The pattern of KAT8 expression during porcine preimplantation embryo development is consistent with that of a maternal effect gene, with high levels in GV and MII oocytes that gradually decrease from pronuclear to blastocyst stage. Microarray analysis indicated that the expression profile of KAT8 in mouse oocytes and preimplantation embryos is similar to that in pigs [KAT8 mRNA observed at 4-cell and 8-cell stage were not significantly diminished by transcriptional inhibition, demonstrating the maternal origin of KAT8 transcripts in porcine early embryos. In contrast, α-amanitin treatment resulted in slightly increased KAT8 transcript abundance, suggesting that degradation or recruitment of KAT8 mRNA in porcine early embryos may be transcription-dependent.KAT8 is a histone acetytransferase that specifically acetylates histone H4 lysine 16 (H4K16) in mammalian cells and plays a role in several cellular biological processes including DNA replication, DNA repair and cell cycle . However in pigs . FurtherDJ-1, VIMENTIN, SEBOX and WDR5, are essential for successful preimplantation development of porcine cloned and in vitro fertilized embryos [KAT8 as a maternal gene in porcine early embryos, we knocked down KAT8 via microinjection of siRNA into MII oocytes and then evaluated developmental efficiency of embryos. Results showed that KAT8 knockdown did not decrease the cleavage rate, but significantly reduced the blastocyst rate. It should be noted that a part of KAT8-siRNA oocytes developed into blastocysts could be due to siRNA degradation or insufficient injection amount of KAT8 siRNA.Previous studies indicated that maternal factors including mRNA, non-coding RNA and proteins play an important role in preimplantation embryo development . Indeed, embryos , 29, 30.KAT8-siRNA injected oocytes revealed that total cell number per blastocyst and cell number of the first two lineages including ICM and TE were apparently decreased, but the ratio of ICM: TE did not change. It has been reported that the acetylation of histone H4 at K16 by KAT8 is an essential epigenetic signature of cellular proliferation in mouse embryogenesis [KAT8 knockdown-induced reduction of H4K16ac levels may cause cell cycle arrest that could likely account for the lower number of total cells and ICM and TE lineage cells in these impaired blastocysts. On the other hand, knockout of KAT8 led to differentiation and pluripotent state loss of mouse embryonic stem cells [KAT8 depletion also resulted in loss of epithelial cell features [A closer analysis of the quality of the resulting blastocysts produced from ogenesis . Moreoveogenesis . Thus, Kem cells  and KAT8features , which mKAT8 depleted embryos; results revealed that H4K16ac levels were significantly reduced in KAT8 knockdown embryos. Given reduction of H4K16ac levels is correlated with DDR and DSBs repair [KAT8 depleted embryos might accumulate many DSBs. Histone H2AX is phosphorylated at the site of DNA damage and then phosphorylated form of H2AX  is usually used as a marker of DNA double-strand breaks [KAT8 knockdown blastocysts, suggesting that KAT8 depletion indeed resulted in unrepaired DNA damage in porcine blastocysts. Consistent with our work, KAT8 knockout oocytes and embryos also exhibited a large number of γH2AX in mice [KAT8 knockdown porcine embryos observed in the present study.Two previous studies showed that KAT8 in mouse oocytes and blastocysts specifically acetylates H4K16, but not other lysine residues of histone H3 and H4 , 25. Bass repair , we thusd breaks . As expe in mice , 25. Imp in mice , which mIn conclusion, maternally inherited KAT8 is indispensible for porcine preimplantation embryo development probably through maintaining the first two lineages proliferation and genome integrity.All chemicals in this study were purchased from Sigma  unless otherwise stated. All experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines under current approved protocols at Anhui Agricultural University. This study had been fully reviewed and approved by IACUC.KAT8) gene in the National Center for Biotechnology Information Database (NCBI). Primer information is shown in KAT8 was amplified from porcine reference cDNA synthesized from blastocysts. The amplified PCR products were cloned using a TOPO-TA cloning kit (Invitrogen) and completely sequenced.PCR primers were designed based on the predicted cDNA sequence for the pig KAT8  were selected for subsequent experiments.The experiment was conducted as described previously . Briefly2, 0.1 mM MgCl2 and 0.01% polyvinyl alcohol) and then treated with two pulses of direct current (1.56 kV/cm for 80 μs) by using cell fusion instrument . Subsequently, embryos were washed in porcine zygote medium (PZM-3) [2 and 95% air with saturated humidity.Oocytes with the first polar body were washed with activation medium (280 mM mannitol supplemented with 0.1 mM CaCl (PZM-3)  three tiKAT8 mRNA, small interfering RNA (siRNA) of KAT8 was microinjected into the cytoplasm of porcine MII stage oocytes. Two KAT8 siRNAs were purchased to target different coding regions of porcine KAT8 and a non-specific siRNA was used as a negative control . All siRNA sequence information used in this study is shown in in vitro. Two control groups  were designed to test potential effects of the microinjection technique and siRNA toxicity on embryonic development.To deplete endogenous The experiment was performed as described previously . BrieflyThe signal intensity for H4K16ac in embryos was analyzed as described previously . BrieflyFor each biological replicate, total RNA was isolated from the pooled oocytes, embryos (n=10) or other tissues using RNeasy Micro Kit . The extracted RNA was quantified with spectrophotometry at 260/280 nm with a NanoDrop 2000 instrument . Reverse transcription was immediately performed using QuantiTect Reverse Transcription Kit . The cDNA was aliquoted and stored at -80°C until ready for use. The samples were collected three times and three biological replicates were conducted.EF1α1 was used as an endogenous reference. The following amplification conditions were used: pre-denaturation at 95°C for 10 min, followed by 45 amplification cycles of 95°C denaturation for 15 seconds, 60°C annealing for 10 seconds, and 72°C extension for 20 seconds. Three technical replicates were conducted in each PCR reaction.The primers used in the present study are shown in P<0.05 was considered to be statistically significant.All experiments were repeated at least three times. Prior to statistical analysis, the percentage data were subject to arc-sine transformation. SPSS (version 17.0) software was used to do one-way ANOVA of the cleavage rate, blastocyst rate, total cell number per blastocyst, gene expression, fluorescence intensity of H4K16ac and ratio of γH2AX positive nuclei. All data were presented as mean ± standard error of mean (mean ± S.E.M) and