As a major alternative therapy in Traditional Chinese Medicine, it has been demonstrated that moxibustion could generate a series of molecular events in blood, spleen, and brain, and so forth. However, what would happen at the moxibustioned site remained unclear. To answer this question, we performed a microarray analysis with skin tissue taken from the moxibustioned site also Zusanli acupoint (ST36) where 15-minute moxibustion stimulation was administrated. The results exhibited 145 upregulated and 72 downregulated genes which responded immediately under physiological conditions, and 255 upregulated and 243 downregulated genes under pathological conditions. Interestingly, most of the pathways and biological processes of the differentially expressed genes (DEGs) under pathological conditions get involved in immunity, while those under physiological conditions are involved in metabolism. In acupuncture research, microarray analysis has been widely employed to uncover gene expression profiles at different tissues or organs –19. Basead libitum and randomly assigned to the various experimental groups . The experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and all experimental protocols were approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine.Adult male Sprague-Dawley rats weighing 200–220 g obtained from Chengdu University of Traditional Chinese Medicine, Experimental Animal Centre, were utilized in this study. Maintained in animal room of automatically controlled day cycles (12 : 12 = light : dark cycle) at 24 ± 2°C, all rats were allowed to freely take food and water In this study, we aimed to explore what would take place at the moxibustioned site in the view of potential molecular target under physiological and pathological conditions. Thus, we designed two different parts of microarray experiment. One is designed for uncovering the gene expression profile at physiological condition in which it consisted of 2 group: healthy control group (C) and healthy control with moxibustion stimulation group (CM). The other one is designed for revealing the gene expression profile at pathological condition in which it composed of model control group (M) and model control with moxibustion stimulation group (MM).Under physiological condition, the rats in CM group received moxibustion at the left acupoint Zusanli (ST36), at the depression below the knee from the anterior crest of the tibia  for 15 mFirstly, the pathological condition was established by injecting subcutaneously with 0.1 mL Freund's Complete Adjuvant  into the plantar surface of the left hind paw of the rat . The CFA2 inhalation. The cutaneous tissue (0.5 cm × 0.5 cm × 0.2 cm) located at moxibustioned site were immediately removed and preserved in RNAlater  to prevent RNA degradation. Total RNA was extracted using TRIzol Reagent  and purified with RNA clean-up Kit  following the instructions of manufacturers, respectively. Total RNA was quantitated by spectrophotometry, and the integrity was assessed by formaldehyde denatured agarose gel electrophoresis. Two hours after one time of moxibustion stimulation was completed, rats was euthanized by COThe microarray analysis service provided by CapitalBio Corporation  was performed as described , 31. BriThe detected signal intensities of all probes on the chip ≥400 were included for comparison analysis. We applied two-class unpaired algorithm in the Significant Analysis of Microarray software  to identify significantly differentially expressed genes between CM and C groups, and MM and M groups. DEGs were determined with the threshold of false discovery rate, FDR ≤5% and fold change ≥2.0 or ≤0.5.http://bioinfo.capitalbio.com/mas3/) which integrated with KEGG and Gene Ontology (GO) database to perform pathway and GO Biological Process term enrichment analysis and calculate the statistical significance as described [P  value <0.001 was considered statistically significant.We employed the online Molecule Annotation System (MAS) established by CapitalBio Corporation  to detect the expression of four DEGs, Hspa1a, Mcpt8, Slpi, and Clqa, which were randomly selected from the 27K Rat Genome Array. http://dx.doi.org/10.1155/2013/890579). While under pathological condition, the results displayed 255 upregulated and 243 downregulated DEGs (Supplementary Table 2).Different numbers of DEGs at moxibustioned site were obtained from different condition. Under physiological condition, we obtained 145 upregulated and 72 downregulated DEGs  at moxibustioned site. Under physiological condition  at moxibustioned site. Under physiological condition  of the involved pathways at moxibustioned site were related to metabolism under physiological condition . HoweverAccording to the biological processes analysis, we also could find out the difference from different state. Without FCA as the pathological stimulation, the biological progresses following moxibustion administration at moxibustioned site were most composed of oxidation reduction, potassium ion transport, and so forth. However, a variety of biological processes related to immunity, such as immune response, antigen processing and presentation, and antigen processing and presentation of peptide antigen via MHC class I, and were exhibited in this study. Taken together, it seemed to be concluded that a series of molecular events would happen at moxibustioned site. Moreover, different pathways and biological progresses at moxibustioned site would be involved in different condition. However, this conclusion should be seriously taken for granted given the following limitations. Firstly, only one stimulation time point was used in this study. How about the time course or different time points, such as 5, 10, 15, 20, and 30 minutes, which were frequently practiced in moxibustion clinic, which needs to be answered in future study? Secondly, the pathological condition was induced by FCA injection in this study. To our knowledge, FCA injection will generate adjuvant arthritis through a series of immune actions , 34. MorAdditionally, in this study the tissue used for RNA extraction and microarray detection was taken from the skin of the moxibustioned site. So far, the skin has been also regarded as an important immune organ , 38 as wThe results suggested that a set of molecular events would have happened at moxibustioned site. Among those molecular events, different genes and different pathways and biological progresses at moxibustioned site would have got involved under different conditions.Supplementary Table 1: The DEGs list under physiological condition (CM versus C)Supplementary Table 2: The DEGs list under pathological condition (MM versus M)Supplementary Table 3: Co-expressed genes list between physiological and pathological condition.Click here for additional data file.


Here we hypothesised the contribution of CX3CR1- CX3CL1 interaction to in vivo neovascularization and studied the functional consequences of genetic and pharmacologic targeting of CX3CR1 in formation, maturation and maintenance of microvascular integrity. Cells functionally deficient in CX3CR1 lacked matrix tunnelling and tubulation capacity in a 3D Matrigel assay. These morphogenic and cytokinetic responses were driven by CX3CL1-CX3CR1 interaction and totally abrogated by a Rho antagonist. To evaluate the role of CX3CR1 system in vivo, Matrigel plugs were implanted in competent CX3CR1+/gfp and functionally deficient CX3CR1gfp/gfp mice. Leaky microvessels (MV) were formed in the Matrigel implanted in CX3CR1gfp/gfp but not in CX3CR1+/gfp mice. In experimental plaque neovascularization immature MV phenotype was observed in CX3CR1gfp/gfp mice, lacking CX3CR1 positive smooth muscle-like cells, extracellular collagen and basement membrane (BM) laminin compared to competent CX3CR1+/gfp mice. This was associated with increased extravasation of platelets into the intima of CX3CR1gfp/gfp but not functionally competent CX3CR1 mice. Pharmacologic targeting using CX3CR1 receptor antagonist in wild type mice resulted in formation of plaque MV with poor BM coverage and a leaky phenotype. Our data indicate a hitherto unrecognised role for functional CX3CR1 in Matrigel and experimental plaque neovascularization in vivo, which may buttress MV collectively in favour of a more stable non-leaky phenotype.Monocyte/Macrophages are implicated in initiation of angiogenesis, tissue/organ perfusion and atherosclerosis biology. We recently showed that chemokine receptor CX The monocyte–macrophage system (MPS) exhibits numerous functions with respect to vascular remodeling which include classical phagocytosis, regulation of antigen presentation and atherosclerotic plaque formation 3CR1 monocyte subpopulation capable of phagocytic functions typical of classical MPS as well as mural vascular smooth muscle cell-like functions that occur subsequent to CX3CL1-CX3CR1 interaction in the injured vessel wall 3CL1-CX3CR1 interaction was also observed to decrease neointima formation and atherosclerosis in murine animal models 3CL1-CX3CR1 axis in atherosclerotic plaque development. Moreover polymorphisms in CX3CR1 receptors have been associated with variability in prevalence of human atherosclerosis and coronary artery disease 3CL1-CX3CR1 signaling may also be implicated in mural cell fate determination during plaque microvessel formation both in terms of cell recruitment to the perivascular space but also in cytoskeletal re-organization of recruited mural cells that integrate into nascent and maturing microvessels. In the current study we selected the chemokine receptor CX3CR1 as a transgenic marker allowing baseline and loss of function status to be examined in mice that had green fluorescence protein knocked in at one or both CX3CR1 alleles 3CR1 function effects on CX3CR1 cell recruitment to the perivascular space, mural cell integration into evolving microvasculature, microvessel maturation and extracellular matrix production and microvessel haemorrhage and permeability. In vitro studies evaluated the signaling pathway downstream of CX3CR1 activation and its effects on cytoskeletal re-organization and solid matrix tunneling of CX3CR1 positive cells.We have previously identified a CX3CL1 and CX3CR1 initiates signaling through a Rho dependent pathway that promotes cell tubulation and tunneling through extracellular matrix. Moreover, functional competence of CX3CR1 to interact with its cognate ligand CX3CL1 is essential for microvessel maturation and expansion, and monocyte/macrophage-derived mural cell integration into evolving vasculature during neovascularization in vivo. Finally, CX3CR1 deficiency or therapeutic targeting with a peptide-antagonist reduces experimental plaque and Matrigel neovascularization but favours a leaky, haemorrhagic microvessel phenotype.We show here for the first time that interaction between CXin vivo procedures were approved by University College Cork Animal Experimentation Ethics Committee. Mice  employed in experiments were 8–12 weeks of age. Initial C57BL/6J breeding colonies were from Charles River UK. Transgenic CX3CR1gfp (C57BL6/J background) mice 3CR1gfp/+) or both (CX3CR1gfp/gfp) copies of the 3CR1CX gene were interrupted by enhanced gene fluorescent protein (eGFP) and were originally from the European Mutant Mice Archive (EMMA).All 3CR1 peptide antagonist (F1) was engineered from phage library of CX3CL1 mutants consisting of modified N terminus (QHHGVT sequence of the native CX3CL1 replaced with ILDNGVS). F1 peptide was purified from E. Coli inclusion bodies using standard procedures and its specificity to CX3CR1 receptor and pharmacodynamics has previously been described nd to 4th week post carotid artery ligation.The CX3CL1  for 1 hr. Since recombinant CX3CL1 is expressed with a C-terminal 6-histidine tag to facilitate purification using nickel agarose, an anti-His tag antibody was coated onto the plate to assist CX3CL1 capture and orientation 3CR1 cells isolated from bone marrow of CX3CR1gfp/+ mice were pre-treated with or without CX3CR1 peptide antagonist  and were allowed to adhere to wells and then washed with PBS (3×). Attached cells were then labelled with Hoechst dye and counted using UV microscopy. The number of cells per field (10× objective) was expressed as mean ± SEM.48-well plates were coated for 1 hour with a 1∶200 dilution of a mouse antipolyHis tag antibody  in bicarbonate buffer, pH 9.6 with 0.1% BSA. Subsequently, selected wells were coated with 100 nM rmCXet alA murine plaque angiogenesis model previously employed by Sasaki Griffonia (Bandeiraea) Simplicifolia Lectin I  was administered to mice by tail vein 24 hrs before culling. The Evans blue content of Matrigel was determined by dissolving the Matrigel in formic acid and then by reading the absorbance at 620 nm. While the heme content was quantified by pyridine hemochrome spectrophotometric heme assay 500 µl of Matrigel  fortified with 1 ng/ml FGF was subcutaneously implanted into the right and left flank region of mice. At 4 weeks post Matrigel implantation the mice were injected i.v. with 100 µl of 0.05% Evans blue or saline via tail vein and the mice were sacrificed 4 hrs post injection by overdose of sodium pentobarbital (i.p.). In a separate set of studies 200 µg of Rhodamine 5 µM thick frozen sections from carotid artery were cut and stained with hematoxylin-Z and eosin and Trichrome stains . Tissue images were acquired using Nikon CCD camera attached to a Nikon microscope. Morphometric analysis was performed using NIH ImageJ software for data quantification.3CR1 , F4/80 , Laminin  and/or CD42b . Goat anti-rabbit, anti-mouse or anti rat Alexa Fluor 546 or 488  was used as secondary antibody and DAPI was used for nuclear staining. Rabbit, mouse or rat IgG  were used as isotype controls. No additional labelling was required for GFP. Several thin optical slices (thickness<0.7 µm) of high-resolution sequential confocal scans  of carotid artery sections were acquired to identify single GFP+ cells co-expressing smooth muscle marker and to avoid false positive images due to overlapping cells. The acquired images were coded and analysed by two observers blinded to the codes. The 3D reconstruction and analysis of the images was performed using IMARIS software .For immunofluorescence detection of smooth muscle specific protein and GFP expression carotid artery/Matrigel sections were stained for calponin , CX3CR1 positive cells were FACS sorted from whole bone marrow of CX3CR1gfp/+ and CX3CR1gfp/gfp mice and were sandwiched between DQ-red BSA  fortified Matrigel layers with CX3CL1 gradient in a 15 μ-angiogenesis slide . CX3CL1 gradient was created by serially layering two layers of Matrigel consisting of 50 and 100 nM rmCX3CL1 respectively. The cells were imaged using high-resolution sequential confocal scans  at 3 hrs and every day up to day 7 post seeding. A separate series of experiments were conducted in the absence and presence of Rho inhibitor . The acquired confocal images were 3D reconstructed and analysed using IMARIS software.CX3CR1 positive cells were FACS sorted from whole bone marrow of CX3CR1gfp/+ and CX3CR1gfp/gfp mice and were grown on 6 well plates at a seeding density of 7×105 cells per well for 5 days with or without 100 nM rmCX3CL1 stimulation. In a parallel set of experiments the cell viability was assessed using FACS at day 5 using 7AAD staining. On day 5 the cells were removed by treating with 500 µl of 20 mM Ammonium Hydroxide +0.1% Triton X-100 incubated for 5 mins at 37°C. Following six washes with 1 ml TBS buffer , ECM on the plate was removed in the presence of 1% SDS for Western blot analysis. Equal total protein was loaded for Western blot analysis and β-actin was probed as a loading control. The Laminin bands were quantified by digital densitometry normalised to density of respective β-actin bands.CX3CR1 positive cells FACS sorted from whole bone marrow of CX3CR1gfp/+ mice were plated in 6 well plates at a density of 7×105 cells per well and were stimulated with 100 nM rm CX3CL1 for 30 mins. The cells were then lysed (Mg2+ lysis/wash buffer) and the lysates were incubated with GST-tagged Rhotekin Rho binding domain for affinity precipitation of GTP-Rho. The bound GTP-Rho protein was pulled-down and resolved on 12% SDS-PAGE. The gel was transferred onto nitrocellulose membrane and detected by immunoblotting using 1∶250 anti RhoA primary antibody (clone 55) and the appropriate peroxidase-conjugated secondary antibody and luminol based chemiluminescent HRP substrate . Whole cell lysate samples from all groups were also analysed by SDS-PAGE as total RhoA controls. The active-RhoA bands were quantified by digital densitometry normalised to density of respective total RhoA bands.Active RhoA expression during the in vitro cell tubulation and tunneling process was assessed using a commercial RhoA activation assay kit . Briefly, CXAll data are represented as mean ± SEM and analysed by unpaired Student t-test or analysis of variance (ANOVA) using Graphpad Prism Version 4 . Animal experiments with tissue section analysis were compared based on intra-experiment variation involving 3–4 mice/group that were not littermates.3CR1 expressing cells was established by FACS and tissue immunofluorescence (3CR1 transgenic models selected would be suitable for assessing structure function relationships between monocyte/macrophages and evolving neovascularization networks. We first assessed whether CX3CR1 positive cells contribute to microvasculature in a well-established in vivo model of Matrigel plug neovascularization. Following implantation of Matrigel (500 µl) in functional CX3CR1gfp/+ and functionally incompetent CX3CR1gfp/gfp mice microvascular association (perivascular) and integration(vascular) of CX3CR1 positive cells was assessed by immunofluorescence imaging, high resolution confocal microscopy with Z stacking and volume rendering. Functioning microvessels (MV) were identified by intravital staining of rhodamine conjugated lectin injected via tail vein. CX3CR1 positive cells were noted in the perivascular region of MV and integrated within the walls of MV  compared to Matrigels implanted in CX3CR1gfp/gfp mice (1.33±0.05 cells/Matrigel MV/HPF)  , p<0.05 gfp mice . Further content , p<0.05, content , p<0.05, content , p<0.05 3CR1 cells with angiogenic MV in Matrigel, we investigated whether such an association exists with intimal MV using a recently described carotid artery ligation model of plaque neovascularization in mice 3CR1gfp/+ compared to functionally deficient CX3CR1gfp/gfp mice  compared to CX3CR1gfp/gfp mice . Furthermore 11.1±2.3% MV were positive for CX3CR1 cells co-expressing smooth muscle markers  and this was significantly impaired in the CX3CR1gfp/gfp mice (1.8±1.3% MV)  inhibitor of CX3CR1 receptor. The in vivo dose of F1 peptide was optimised based on the capacity of F1 peptide to inhibit binding of CX3CR1 positive cells to CX3CL1 coated plates in a static adhesion assay  in all groups at day 5 in culture. Moreover CX3CR1 positive cells from CX3CR1gfp/+ mice developed larger tubular structures of greater length and with greater external and internal luminal diameter compared to their counterparts from CX3CR1gfp/gfp mice  formation  cell types. Consistent with our previous evidence for CX3CR1 differentiation into SM-like cells we show that almost all mural CX3CR1 positive cells within MVs co-express smooth muscle markers 3CR1 markedly reduced in vivo mural and perivascular association of CX3CR1 cells within plaque MVs and mural association within Matrigel MV. Functional deficiency of CX3CR1 also reduced MV associated extracellular matrix production with more than 70% reduction in MV-associated laminin and collagen staining in CX3CR1 deficient mice. The functional consequences of aberrant CX3CR1 phenotype included increased microvascular leakage of blood, platelets, and intravascularly administered solutes (Evans Blue dye). It is tempting to speculate whether failure of CX3CR1 deficient cells to tubulate, or initiate differentiation into mural smooth muscle-like cells or impairment of extracellular matrix production may be significant contributors to increased microvessel porosity to blood and solutes in vivo thus providing a mechanism underlying the leaky MV phenotype. It is also conceivable that reduced mural CX3CR1 positive cells may have effects on microvascular endothelial permeability Consistent with these ormation . Monocyt3CR1 positive cells contributing to formation of stable MV within neovascular networks and within experimental plaque. The monocyte/macrophage contribution to stable MV may thus occur via a number of mechanisms including inter alia differentiation of myeloid cells into smooth muscle cells, intra-mural and perivascular cell integration, synthesis of basement membrane, and paracrine secretion of other extracellular matrix support factors by these cells. Additionally CX3CR1 positive cells contribute to formation of stable MV in a plaque-independent setting, as indicated by our in vivo Matrigel induced neovascularization data.Our current findings contribute to emerging literature on the direct involvement of monocyte/macrophages to smooth muscle cell differentiation in vivo neovascularization, controversy remains on the appropriateness of current plaque neovascularization models used in mice. This limitation was recently addressed by Sasaki et al3CR1 studies and provide clear evidence of functional MV formation in this model using high resolution 3D confocal imaging.While the implantation of a subcutaneous Matrigel plug is accepted as an excellent model of 3CR1 would alter MV structure and function. Remarkably, treatment of wild type mice with a CX3CL1 peptide-mimetic (F1) that specifically antagonized CX3CR1 recapitulated the leaky microvessel phenotype seen in CX3CR1 deficient mice. These F1 treated animals also exhibited marked reduction in basement membrane-laminin MV coverage, increased platelet extravasation and excess leakage of intravascularly administered microspheres compared to control treated animals. These data confirm that CX3CR1 deficiency or specific small molecule targeting of CX3CR1 reduces neovascularization  Given that microvessel integrity and function is increasingly investigated as a potential contributor to plaque growth and stability we examined whether therapeutic targeting of CXrization , S8 and Figure S13CR1-GFP cells. A, Flow cytometric analysis of the CD45/CX3CR1-GFP dual positive cell population of mouse bone marrow cells showing ∼85% or greater coexpression of GFP cells with cfms, CD11b or F4/80, with lower levels of coexpression with Gr-1. Data is representative of 3 independently performed experiments, the values inset are mean ± SEM of 3 independently performed experiments. B, CX3CR1-GFP and F4/80 co-staining in plaque (upper panels) and Matrigel angiogenesis (lower panels) models showing majority of CX3CR1-GFP cells positive for myeloid marker (F4/80). Data is expressed as mean ± SEM of 10 carotid artery/Matrigel cross sections/mice (n = 3 independently performed experiments).Myeloid phenotype of CX(TIF)Click here for additional data file.Figure S23CR1+/gfp and CX3CR1gfp/gfp mice. Data is expressed as mean ± SEM of 20 carotid artery cross sections/mice . D, Representative cross section image of carotid artery from CX3CR1+/gfp and CX3CR1gfp/gfp mice were stained with α-smooth muscle actin  and DAPI . CX3CR1 positive cells  integrated into microvascular wall and were also present in perivascular region and co-expressed smooth muscle marker  . E, In the CX3CR1 functionally deficient (CX3CR1gfp/gfp) mice the number microvascular CX3CR1 positive cells co-expressing smooth muscle marker were significantly reduced. Data is expressed as mean ± SEM of 20 carotid artery cross sections/mice (n = 4 independently performed experiments). * denotes p<0.01.Contribution of myeloid cells to formation of microvessels in neointima. A, Representative cross section image of carotid artery from wild type mice stained with myeloid (F4/80) and smooth muscle  marker. Nuclei are stained with DAPI (Blue). B, ∼30% of microvessels in the neointima were positive for myeloid marker and ∼15% of microvessels co-expressed myeloid and smooth muscle marker. C, The total number of GFP positive cells in the neointima was similar in CX(TIF)Click here for additional data file.Figure S33CR1gfp/gfp mice show increased intra plaque haemorrhage. A, Representative cross section image of carotid artery from CX3CR1+/gfp and CX3CR1gfp/gfp mice stained with Hematoxylin and Eosin, showing increased intraplaque haemorrhage (Dark red staining). B, Data is expressed as mean ± SEM of 20 carotid artery cross sections/mice (n = 4 independently performed experiments); * denotes p<0.01.Neointimal lesions from CX(TIF)Click here for additional data file.Figure S43CR1 cells to CX3CL1 coated plates by selective CX3CR1 inhibitor (F1) in a static cell adhesion assay. Data is represented as mean ± SEM of 4 independently performed experiments; * denotes p<0.01.Dose dependent inhibition of CX(TIF)Click here for additional data file.Figure S53CR1 receptors using CX3CR1 inhibitor (F1) reduces collagen staining (light blue on trichrome) in the neointima. B, Data is expressed as mean ± SEM of 20 carotid artery cross sections/mice (n = 4 independently performed experiments); * denotes p<0.01.A, Pharmacological inhibition of CX(TIF)Click here for additional data file.Figure S63CR1+/gfp or CX3CR1gfp/gfp mice cultured for 5 days in Matrigel sandwich with 10 ng/ml CX3CL1 gradient. Data is represented as mean ± SEM of 4 independently performed experiments;* denotes p<0.01.Cell length (A), cell diameter (B) and lumen diameter (C) of CX3CR1 cells isolated from CX(TIF)Click here for additional data file.Figure S73CR1gfp/gfp mice are significantly reduced compared to CX3CR1+/gfp mice. Data is expressed as mean ± SEM of 20 carotid artery cross sections/mice (n = 8 independently performed experiments); * denotes p<0.01.Neointimal lesions from CX(TIF)Click here for additional data file.Figure S83CR1 receptors using CX3CR1 inhibitor (F1) significantly reduces the neointimal area. Data is expressed as mean ± SEM of 15 carotid artery cross sections/mice (n = 4 independently performed experiments); * denotes p<0.01.Pharmacological inhibition of CX(TIF)Click here for additional data file.


In the liver, it was unravelled as both an oncogenic pathway involved in a subset of liver cancers and a physiological signalling identified as the “zonation-keeper” of the quiescent liver lobule. This duality has encouraged to explore the role of canonical Wnt in liver regeneration and liver-cell proliferation mainly using murine genetic models of β-catenin overactivation or inactivation. These studies definitely integrate Wnt signalling within the hepatic network driving regeneration and proliferation. We will review here the current knowledge concerning the mitogenic effect of Wnt, to switch on its specific role in the liver, which is quiescent but with a great capacity to regenerate. The duality of β-catenin signalling, associated both with liver quiescence and liver-cell proliferation, will be brought forward.Canonical Wnt signalling, governed by its effector  Since 1982 and the initial discovery of Int1 (Wnt1a) being an oncogene in murine breast cancers, Wnt signalling has been strongly associated with cancer and therefore with cell proliferation , it now β-catenin is the main effector of the canonical signalling  and the glycogen synthase kinase 3 (GSK3). This occurs within a so-called “destruction complex” scaffolded by the tumor suppressors AXINS. The tumor suppressor APC (Adenomatous polyposis coli) is required within this destruction complex for an efficient degradation of β-catenin. Upon Wnt ligand binding to its frizzled receptor and LRP5/6 coreceptor at the membrane, a cascade of events impedes GSK3 kinase activity, through LRP5/6-dependent sequestration of GSK3 within endosomal vesicles [β-catenin triggers its nuclear translocation and its association with a Lef/Tcf DNA-binding partner. This leads to the transcription of a genetic program specific for the temporal, spatial, and tissue contexts ([β-catenin gene- (CTNNB1-) activating mutations, loss-of-function mutations in APC, AXIN1, or AXIN2 genes, induce a constitutive activation of β-catenin signalling and are found in a large number of human cancers .β-catenin, and GSK3 accumulate at the centrosomes, which align the mitotic spindle. Therein, they regulate MT growth  peaks at G2/M [β-catenin occurs at G2/M [Wnt signalling influences the cell cycle but conversely the cell cycle has an impact on Wnt signalling. It had been observed that  mitosis . Similar at G2/M . This ph at G2/M .β-catenin activating mutations occur in 20 to 40% of hepatocellular carcinoma (HCC) [β-catenin partners predominate, 32.8% of HCCs being mutated in CTNNB1, 15.2% in AXIN1, and 1.6% in APC genes [β-catenin pathway, suggesting that its tumor suppressor function is mediated through other partnerships [β-catenin signalling triggers hepatocarcinogenesis is somehow different from that which is implemented following P53 mutations [β-catenin-activated liver tumors, several Wnt target genes have been described with a potential role in hepatocyte proliferation in a cancerous context: these are Cyclin D1, but not c-myc, the regenerating islet-derived 3- and 1-α genes (REG1A and REG3A), Tgf-α [β-catenin induces proliferation in liver cancers remain elusive.In 1998, the link between Wnt signalling and the liver was initially established, through the demonstration that ma (HCC) , 31. HCCPC genes . This prPC genes , 35. Unenerships . Interesutations . From st), Tgf-α , 38, 39.β-catenin, an unexpected Wnt signalling was detected in 2006 in a subset of quiescent hepatocytes located within the pericentral area [After the initial discovery of the oncogenic role for liver ral area . It shoural area . β-catenin is physiologically activated in pericentral hepatocytes [β-catenin signalling is also due to the low amount in this area of APC, further defined as the “zonation-keeper” of the liver: as a consequence, its liver-specific loss enables a loss of zonation together with hepatocyte hyperproliferation, with a dramatic metabolic phenotype leading to the death of the mice [β-catenin inducing the transcription of genes encoding metabolic enzymes in the pericentral area, whereas in the same zone it directly represses the transcription of genes encoding periportal enzymes or transporters  or close to the central vein . This is the concept of metabolic zonation –43. We fatocytes . This pratocytes –46. Thisthe mice . This zoporters  and somatic stem cells (reviewed in ). It wasIt must be understood that liver homeostasis is not dictated by its self-renewal, due to the quiescence of the hepatocytes with a lifespan of 300–400 days . MoreoveThe role of Wnt in the adult liver is paradoxical, due to its dual role as patterning its metabolic zonation, while being engaged in liver-cell proliferation both physiologically in regeneration processes, and pathologically during oncogenesis. In fact, the balance between quiescence and proliferation has to be fine-tuned in order to avoid either a fatal loss or tissue regeneration or neoplasia , 62. So


Several novel findings regarding the role of ROS in Notch and Wnt/β-catenin modulation prompted us to review their emerging function in the cardiovascular system during embryogenesis and postnatally.Reactive oxygen species (ROS), traditionally viewed as toxic by-products that cause damage to biomolecules, now are clearly recognized as key modulators in a variety of biological processes and pathological states. The development and regulation of the cardiovascular system require orchestrated activities; Notch and Wnt/ It has In the last decade the scientific community clarified the importance of low levels of ROS as key signaling molecules in physiological functions such as the regulation of cell signaling, proliferation, and differentiation . DiffereThe purpose of this review is to describe how ROS regulate Notch and Wnt pathways. Understanding the molecular mechanism regulated by ROS could lead to the development of new therapeutic approaches for cardiovascular diseases.Vascular ROS formation can be stimulated by mechanical stress, environmental factors, platelet-derived growth factor (PDGF), angiotensin II (AngII), and low-density lipoproteins –7. BecauA particularly important mechanism for ROS-mediated cardiovascular disease appears to be via stimulation of pro-inflammatory events . These, It has been previously reported that, in response to ROS, VSMC may secrete proteins that participate in autocrine/paracrine growth .ROS are produced at low levels prevalently as byproducts of the mitochondrial electron transport chain and through NAD(P)H oxidase family. They play a physiologically important role in the regulation of various biological responses such as cell migration, proliferation, gene expression, and angiogenesis , 18.2−∙ that can further disproportionate forming hydrogen peroxide (H2O2) or, in presence of nitric oxide (∙NO), creating peroxynitrite (ONOO–).NAD(P)H oxidase (Nox) enzymes are membrane-associated enzymatic complexes and structural homologues of phagocytic Nox (gp91phox/Nox2) and consist of both single (Nox1–Nox5) and dual (Duox1 and Duox2) oxidases . ROS areNox2 and Nox4 are critical ROS-generating complexes in ECs; they are activated by various stimulants and agonists, for instance, VEGF. Nox1 isoform is more abundant in epithelium, even if it was detected also in ECs and VSMCs .VEGF stimulates ROS production from Nox2 that in turn promotes VEGFR2 autophosphorylation through oxidation/inhibition of phosphatase enzymes. The result is an enhancement in cell proliferation, migration, and angiogenesis , 22.Khatri et al.  have shoUrao and Ushio-Fukai  demonstrFurthermore, Nox1-dependent redox signaling pathway modulates the phosphatase PTP-PEST/PTPN12, an important regulator of endothelial cell migration and adhesion . In partγ-secretase, a cleaving protease complex. γ-secretase in turn generates the active form of Notch  which translocates to the nucleus where it binds the transcriptional factor CSL  also known as recombinant signal binding protein 1 for Jκ (RBP-Jκ). Such NIC binding displaces repressor molecules and promotes the recruitment of coactivator molecules. This in turn activates the transcription of specific Notch target genes such as Hes (hairy/enhancer of split), Hey (Hes-related proteins), Nrarp (Notch-regulated andrin repeat protein), cMyc, cyclin D1, and many other genes that control ECs proliferation, differentiation, and apoptosis as well as stem cells maintenance and angiogenesis [Notch pathway is a highly conserved signaling system that controls cell fate decisions . It is aogenesis .Notch receptors 1, 2 as well as Jagged1, Delta-like ligand 1 and 4 (Dll1 and Dll4), are preferentially expressed in ECs and have a key role in developmental and postnatal angiogenesis. During early embryogenesis, Notch induces differentiation of angioblasts to ECs, whereas at later stages it controls specification of ECs into arterial and venous identity . Mice emIn the context of sprouting angiogenesis, Notch signaling regulates not only the frequency of sprouts but also their ability to anastomose. Retinas of mice heterozygous for a myeloid-specific Notch1 mutation were characterized by long sprouts unable to anastomose and by lack of macrophages at the edge of vascular branch points .Several studies have recognized the central role of Notch-mediated signaling also in cardiac development . In cardβ-catenin signaling, plays a key role in vascular biology [β-catenin gene in ECs caused aberrant vascular patterning and increased vascular fragility [Notch signaling modulates endothelial homeostasis by crosstalking with other signaling pathways such as receptor tyrosine kinases  –43 and e biology , 48. Mic biology . Knock-o biology . Defectsragility .β-catenin, a scaffold protein, linking the cytoplasmic tail of classical cadherins in the endothelium  cadherin and N-cadherin) via α-catenin to the actin cytoskeleton. Without Wnt stimulation, cytoplasmic β-catenin levels are kept low by a degradation complex, consisting of Axin, Adenomatous polyposis coli (APC), Casein kinase1a (CK1a), and Glycogen synthase kinase 3-β (GSK3-β). Binding of Wnt to its receptors Frizzled and lipoprotein receptor-related protein (LRP) leads to inhibition of the degradation complex function, enabling β-catenin signaling. Wnt allows β-catenin to accumulate and translocate to the nucleus where it binds to several transcription factors, for example, T-cell factor (TCF) and LEF-1 [The canonical Wnt signaling pathway is driven by nd LEF-1 , 53.β-catenin degradation complex [Dishevelled (Dvl) is an essential adaptor protein for Wnt signaling that interacts with several molecules, including Axin, inactivating the  complex –52. Dvl  complex .2+ and Wnt/planar cell polarity (PCP) pathways. The Wnt/Ca2+ pathway, mediated by G-protein signaling, stimulates the release of intracellular Ca2+ and activation of Ca2+-sensitive kinases, such as the protein kinase C and Ca2+-calmodulin kinase II. PKC is a family of Ca2+-dependent and Ca2+-independent isoforms that have different distributions in various blood vessels, and individual members can have different roles in a plethora of biological and pathological events [The noncanonical Wnt pathway can be categorized largely into two classes, the Wnt/Cal events –56. The l events . This paNotch and Wnt signaling pathways can have opposing effects on cell-fate decisions with each pathway promoting an alternate outcome. For example, in both the skin and mammary gland, Wnt signaling promotes the maintenance of the stem cell fate whereas Notch signaling promotes lineage commitment and differentiation , 58–60. β-catenin induces activation of the Notch pathway by increasing the transcription of Dll4. This, in turn, prevents a correct endothelial cell differentiation, altering vascular remodeling and arteriovenous specification. Sustained Wnt/β-catenin signaling increases Dll4 in tumor vessels and induces Notch signaling that mediates a reduced angiogenic response and normalizes tumor vasculature [β-catenin signaling leads to alterations of vascular morphology by inhibiting angiogenesis, through stimulation of Dll4/Notch signaling. These effects of β-catenin were detectable only during embryonic vasculogenesis and angiogenesis but were lost at late stage of development or postnatally [Disregulation of Wnt-Notch signaling crosstalk alters early vascular development. Corada et al.  found thculature . Overexptnatally .β-catenin signaling can constructively reproduce the induction processes of arterial ECs from vascular progenitors expressing VEGFR2 through the formation of an arterial-specific protein complex. In particular these authors reported that RBP-Jk, NICD, and β-catenin formed a protein complex that promoted the transcription of genes specifically expressed in arterial but not venous districts in ESCs both in vitro and in vivo. Moreover, dual induction of NICD and β-catenin enhanced promoter activity of Notch target genes in vitro  and arterial gene expression during in vivo angiogenesis in adult mice.Yamamizu et al.  investigThe finding that a baseline level of Wnt and Notch activity can be detected during vascular development –74 and aβ-catenin signaling by reducing levels of active β-catenin in CPCs.Wnt signaling has been identified as a downstream target of Notch1 that regulates expression of cardiac transcription factors during mouse cardiogenesis and is essential for cardiac development facilitating transcription of target genes involved in cell fate regulation , 76. Kwoβ, a multifunctional kinase that regulates many cellular processes including proliferation, differentiation, and apoptosis [β is constitutively active in resting cells and is negatively regulated in response to external stimuli by phosphorylation on serine via activation of several kinases, including Akt and protein kinase C (PKC) [β is involved in a mechanism of accelerated atherosclerosis in hyperglycemic, hyperhomocysteinemic, and high-fat-fed apolipoprotein E-deficient (apoE(−/−)) mouse models; McAlpine et al. showed that atherosclerosis can be attenuated by promoting GSK3β phosphorylation [β activity contributes to accelerated EPC cellular senescence, effect reversed by small molecule antagonism of GSK3β which enhances cell-based therapy following vascular injury [Crosstalk between Notch and Wnt pathways may be partially mediated by specific regulation of GSK3-poptosis . GSK3-β  C (PKC) . Endoplarylation . In diabr injury .β directly binds to Notch and inhibits transcriptional activation of different Notch target genes [β reduced NICD degradation by the proteasome, while low GSK3-β activity and high Notch signaling correlate with the highly proliferative, undifferentiated nature of EPCs [β is reversed in the presence of Wnt1, resulting in the upregulation of Hes1 [GSK3-et genes –83. Acti of EPCs . Phospho of Hes1 .in vitro, Integrin-Linked Kinase (ILK) could phosphorylate GSK3-β and, in turn, activate Wnt signaling. ILK can also activate Notch signaling. Therefore, the phosphorylation of GSK3-βvia ILK directs Wnt and, thereby, Notch signaling activation [Integrin signaling is linked to Wnt signaling. Rallis et al. showed that, tivation .β positively regulates the activity of Notch1 and 3 in VSMCs. Ectopic expression of GSK3-β in VSMCs increased NICD levels, promoted CBF-1/RBP-Jκ transactivation, and enhanced Notch target genes expression. Coincidentally, inhibition of GSK3-β activity using a pharmacological inhibitor or reduction in GSK3-β levels by siRNA knockdown resulted in attenuation of Notch activity.On the other hand a recent report  showed tβ-catenin pathways are regulated at least in part by Nox-derived ROS [Studies conducted in the last several years have provided clear evidences that both Notch and Wnt/ived ROS , 87.2O2, contribute to the activation of arteriolar myogenic response, contraction, and reorientation, through p38 MAPK signaling activation in VSMCs [2O2, prevented the stretch-induced translocation of Notch3 to the nucleus, increased Hes1 expression, and decreased Notch3 extracellular domain.Stretch-induced mechanotransduction in VSMCs is known to be regulated by redox signaling initiated by stretch-induced activation of Nox and consequently increased ROS level , 89. In in VSMCs –92. Zhu in VSMCs  showed t2O2. These results are in line with other authors: an increase in Wnt11 expression after oxidative stress injury induced cardiomyogenic differentiation of ESCs and mouse bone marrow mononuclear cells [In bone marrow-derived mesenchymal stem cells Boopathy et al.  observedar cells .2O2 decreases the amount of nuclear β-catenin and TCF/LEF-dependent transcription in human embryonic kidney cells. Overexpression of Dvl1 abrogated H2O2-induced downregulation of β-catenin suggesting that H2O2 negatively modulates Wnt signaling pathway through downregulation of β-catenin [β-catenin signaling through dissociation of NRX from Dvl. Nox1 siRNA inhibits cell response to Wnt, including stabilization of β-catenin, expression of cyclin D1, and c-Myc via the TCF transcription factor.H-catenin . A recen-catenin , 97 iden-catenin  demonstrα (TNFα) in arterial myofibroblast via tumor necrosis factor receptor 1 (TNFR1). In vitro and in vivo gene expression studies established that Nox/mitochondria-derived ROS inhibition reduces Msx2 induction by TNFα. Wnt7b as well as β-catenin levels were also reduced, suggesting that ROS metabolism contributes to TNFα induction of Msx2 and Wnt signaling in myofibroblasts via TNFR1 [Msx2, a profibrotic, proosteogenic transcription factor upregulates the expression of multiple Wnt ligands during angiogenesis  and enhaia TNFR1 .κB activation. Accordingly, increased ROS might contribute to tumorigenesis by activating specific signaling pathways in different cell types [APC mutation causes Wnt signaling activation and is commonly found in colorectal cancer . Myant ell types , one of β [via ROS that promote activation of Akt and inhibition of GSK3-β. The disruption of the Wnt pathway modulates this loop and induces GSK3-β activation [The cardioprotective effect of ischemic preconditioning (IPC) is abolished by overexpression of secreted frizzled protein 1 (sFRP1), an antagonist of the Wnt/Frizzled pathway, and this effect is related to the ability of sFRP1 to decrease the phosphorylation/inhibition of GSK3-β . Cardioptivation .β and β-catenin translocation into the nucleus as well as Notch1 activation. They show that loss of Nox1 results in increased PTEN activity that in turn inhibits Akt signaling pathway, as well as Wnt/β-catenin and Notch1 signaling. As previously discussed, GSK3-β is an important mediator of Notch-Wnt crosstalk; therefore, it could be a key player by which ROS regulate both pathways.Coant et al.  reportedβ-catenin signaling could provide further insight into their involvement in vascular biology.Taken together these results suggest that redox-dependent regulation of Notch and Wnt/β-catenin signaling modulation. A clear distinction between Notch and Wnt responses is vital for appropriate and robust cell-fate decisions, and ROS modulation of these signaling pathways would provide clues for clinical strategies and drug discovery targeting cardiovascular diseases and cancer.ROS have long been deemed as noxious molecules in cardiovascular diseases, including systemic and pulmonary hypertension, atherosclerosis, cardiac hypertrophy, and heart failure. With years of efforts, ROS are becoming increasingly recognized as important modulator for a variety of biological functions and pathophysiological states. Recent evidences suggest an even more significant role of ROS: Notch and Wnt/β-catenin signaling pathways could have therapeutic effects repressing angiogenesis in tumours or favouring it in ischemic tissues.Above we have discussed how ROS modulation of Notch and Wnt signaling regulates vascular development in different aspects, including stem cells differentiation, angiogenesis, VEGF signaling, endothelial as well as cardiac progenitor cells recruitment, and vascular cell migration. Nonetheless, more details regarding the ROS signaling and pathophysiological functions remain to be elucidated. A deeper insight into the mechanism of how ROS affect normal vascular development, especially CPCs, VSMCs, and ECs differentiation from stem cells, could contribute to a brighter future for regenerative medicine in cardiovascular therapies. Factors that selectively control ROS modulation of Notch and Wnt/


Recent studies have shown that tumor immune evasion mechanisms significantly contribute to immunotherapy failure. Emerging data is indicating that tumor-associated dendritic cells (DCs) undergo phenotypic tolerization and promote the differentiation and activation of regulatory T cell (Treg) populations to generate local immune subversion. The critical role of DCs in orchestrating anti-tumor immunity suggests that the signaling pathways that regulate DC tolerogenesis may be promising targets for pharmacologically augmenting immunotherapy efficacy. Previous work has shown that the β-catenin signaling pathway plays a potential role in the DC tolerization process. Others have demonstrated that the DC-expressed enzyme, indoleamine 2,3-dioxygenase (IDO), promotes tumor-induced immune tolerance by catalyzing the conversion of the amino acid tryptophan into kynurenine. Other investigators have demonstrated melanomas to upregulate the expression of several Wnt ligands during their malignant transformation. Using a transgenic murine model of melanoma, we have determined that melanoma-associated DCs exhibit elevated levels of expression of IDO and other β-catenin target genes and demonstrate diminished T cell stimulatory capacity in vitro. Further studies have shown that Wnt3a and Wnt5a induce the expression and enzymatic activity of IDO in myeloid DCs (mDCs) in a β-catenin-dependent fashion while IDO reporter assays suggest this process to be mediated by direct promoter activation. This data suggest the Wnt-β-catenin signaling pathway to be a potential target for suppressing melanoma-induced DC tolerization and enhancing anti-tumor immunity. Indeed, both small molecule inhibition and genetic silencing of the PORCN acyl transferase enzyme which is necessary for Wnt secretion, abrogated B16 melanoma paracrine induction of β-catenin signaling and IDO expression by local mDCs. Further studies have shown PORCN-silenced B16 melanomas to exhibit reduced tumorigenecity in vivo and that this effect correlated with decreased mDC IDO expression and reduced levels of local Tregs. We therefore hypothesize that melanoma-expressed Wnt ligands induce DC tolerogenesis and stimulate β-catenin-dependent IDO expression and subsequent Treg generation in the tumor microenvironment, resulting in the development of an immunotolerant state and ultimately allowing for disease progression. Further, we propose that the pharmacological inhibition of this pathway can reverse this mechanism of immune evasion and enhance other immunotherapeutic approaches. Future work will include testing the ability of the small molecule PORCN inhibitor to stimulate the generation of tumor antigen-specific T cell responses in an autochthonous transgenic melanoma model.


IFNs are a group of cytokines that possess potent antiviral and antitumor activities, while β-catenin pathway is a proliferative pathway involved in carcinogenesis. Interaction between these two pathways has not been well elaborated in hepatocellular carcinoma (HCC).HCC cell lines, HepG2 and Huh7, were used in this study. β-catenin protein levels and corresponding signaling activities were observed by flow cytometry and luciferase assay, respectively. Cell proliferation was quantified by counting viable cells under microscope, and apoptosis by TUNEL assay. DKK1 and GSK3β levels were determined by flow cytometry. Secreted DKK1 was tested by ELISA. FLUD, S3I and aDKK1 were used to inhibit STAT1, STAT3 and DKK1 activities, respectively.Our findings show that all three types of IFNs, IFNα, IFNγ and IFNλ, are capable of inhibiting β-catenin signaling activity in HepG2 and Huh7 cells, where IFNγ was the strongest (p<0.05). They expressed suppression of cellular proliferation and induced apoptosis. IFNγ expressed greater induction ability when compared to IFNα and IFNλ (p<0.05). All tested IFNs could induce DKK1 activation but not GSK3β in HepG2 and Huh7 cells. IFNs induced STAT1 and STAT3 activation but by using specific inhibitors, we found that only STAT3 is vital for IFN-induced DKK1 activation and apoptosis. In addition, DKK1 inhibitor blocked IFN-induced apoptosis. The pattern of STAT3 activation by different IFNs is found consistent with the levels of apoptosis with the corresponding IFNs (p<0.05).In hepatocellular carcinoma, all three types of IFNs are found to induce apoptosis by inhibiting β-catenin signaling pathway via a STAT3- and DKK1-dependent pathway. This finding points to a cross-talk between different IFN types and β-catenin signaling pathways which might be carrying a biological effect not only on HCC, but also on processes where the two pathways bridge. Hepatocellular carcinoma (HCC) is known to be the third most common cause of cancer-related death in the world. Its prevalence is peaking rapidly compared to other cancers possibly due to a high prevalence of hepatitis B and C viral infections Interferons (IFNs) are a group of pleiotropic cytokines involved in anti-microbial and anti-tumor immunity by enhancing antigen presentation through major histocompatibility complex (MHC) class I and class II interactions, regulating a variety of genes, and facilitating pre-apoptotic responses of infected cells The significant effects of most IFNs are mediated by the JAK-STAT  signaling pathway The β-catenin is a key component of the canonical pathway of β-catenin (Wnt) signaling. It is an essential regulator of proliferation, differentiation, and carcinogenesis Interruption of apoptosis is a crucial step in the development of cancer In this study we aimed to find out and compare the effects and mechanisms of IFNα, IFNγ and IFNλ in suppressing β-catenin signaling and promoting apoptosis induction in HCC cells. Our study demonstrates different characteristic potentials of various types of IFNs, and unfolds a link between IFNs and β-catenin signaling pathways that impact apoptosis induction in HCC cells and may have a larger biological impact on mechanisms of carcinogenesis in liver, and benefit liver cancer therapy.The hepatocellular carcinoma cell lines HepG2 and Huh7 were obtained from PriCell Research Institute. They were propagated in DMEM  supplemented with 10% heat-inactivated FBS  and 1% penicillin-streptomycin (Gibco Invitrogen). The cells were used at approximately 80% confluence. Human recombinant IFNγ, GSK3β antibody (pY216), pSTAT1 (S727)-AF647 mAb, pSTAT3 (pY705)-AF488, Caspase3-AF488 were purchased from BD Pharmingen . APC and FITC-conjugated goat anti-mouse antibodies and FITC-bovine anti-goat IgG (H+L) antibody were purchased from Jackson ImmunoResearch Lab Inc . Human recombinant IFNα, IFNλ  and Fludarabine (FLUD) were purchased from Sigma-Aldrich . STAT3 inhibitor V, Stattic, STAT5 inhibitor, and GSK3β inhibitor IX were purchased from CalBiochem/EMD biosciences . hDKK1 neutralizing antibody was purchased from R&D Systems  while DKK1 detection antibody was purchased from Abcam . DKK1 ELISA was purchased from RayBiotech  and used as recommended.6 cells were transfected with 10 µg TOPflash reporter construct . TOPflash construct consists of two sets of three TCF/LEF binding sites linked to a luciferase reporter. The cells were also co-transfected with 1 ng Renilla construct  to normalize for transfection efficiency and green fluorescent protein (GFP)  to equalize the amount of total DNA used per transfection condition. Firefly and Renilla luciferase activity were measured using dual luciferase assay reporter system .HepG2 and Huh7 hepatocellular carcinoma cell lines were transiently transfected using the LT-1 transfection reagent  as recommended by the manufacturer. To measure β-catenin-dependent signaling activity, 5×10To detach cells without cleaving surface proteins, cells were incubated with 1 mM EDTA for 5 minutes then washed and suspended in 1×PBS. Cells were stained with appropriate target antibodies and isotype antibodies using conventional surface and/or intracellular staining methods. When both surface and intracellular staining were prepared, cells were first fixed and made permeable using BD Cytofix/Cytoperm,Fixation and Permeability Solution , followed by staining for intracellular proteins. Cells were then washed extensively with 1×PBS in order to remove excess antibodies, stained for extracellular targets, and fixed with 2% formaldehyde. Fluorescence was evaluated with a FACSCalibur flow cytometry and data analyzed using FlowJo software .5/well) were plated in wells of 6-well plates and transfected and/or treated, as indicated in the text. Dead cells lost their attachment and were washed away by 1×PBS. Viable (adherent) cells were released from the wells by trypsinization before cell counting.Cell viability assays were performed as described previously 6 cells were briefly trypsinized, washed twice with cold PBS, fixed in 4% paraformaldehyde at 4°C for 20 minutes, and then washed again with PBS and made permeable with 0.5 ml 0.5% saponin at 22°C for 5 minutes. The cells were washed with PBS, incubated with 80 µl equilibration buffer at 22°C for 5 minutes, washed with PBS, re-suspended in 50 µl Nucleotide Mix and incubated in the dark at 37°C for 1 hr. Cells were washed again with PBS and analyzed by fluorescence microscopy.TUNEL assay to determine DNA fragmentation in apoptotic cells was performed according to the manufacturer’s suggested protocols (Promega). 3–5×10t-Test with the data normally distributed. Abnormally distributed data had the two groups compared using the non-parametric Mann-Whitney test. All tests were two-tailed and a p-value <0.05 was considered significant.Statistical analyses were performed using Prism software . Untreated and treated groups were compared using the Student's We evaluated whether IFNα, IFNγ and IFNλ down-regulate β-catenin in hepatocellular carcinoma (HCC) cell lines, HepG2 and Huh7. HepG2 and Huh7 cells were co-transfected with a TCF/LEF firefly luciferase construct (TOPflash) and a control reporter (Renilla luciferase), and then left untreated or treated with IFNα (100 ng/ml), IFNγ (100 ng/ml) and IFNλ (100 ng/ml), respectively. The TOPflash reporter is an indicator of basal and inducible levels of β-catenin-dependent signaling. At 24 hrs post IFN treatment, IFNα, IFNγ and IFNλ markedly reduced β-catenin signaling by approximately 36%, 55% and 50%, respectively. And the reduction in Huh7 cells is comparable to that observed in HepG2 , IFNγ (100 ng/ml) and IFNλ (100 ng/ml) for 72 hrs, and viable cells were counted under microscope. The results demonstrated that, if compared with untreated controls, IFNα, IFNγ and IFNλ reduced viable cell counts in HepG2 cells by approximately 20%, 40%, and 20%, respectively, where IFNγ was the strongest (p<0.05) . Similar. Apoptosis was confirmed via detecting activation of caspase 3 by flow cytometry using specific antibody against activated caspae 3 . Anti-proliferative effects and apoptosis induction by different IFNs in HepG2 cells were similar to those in Huh7 cells, except that the apoptosis induced by IFNα was stronger than that of IFNλ in HepG2 cells, while it expressed contrary to that found in Huh7 cells (p<0.05) . IFNγ was always the most potent one when compared with IFNα and IFNλ in both cell lines (p<0.05). Apparently, IFNs suppressed cell proliferation and induced apoptosis in HCC cell lines.To determine whether apoptosis was induced by IFNs in HepG2 cells, TUNEL assay was used to detect DNA fragmentation in apoptosis, and was performed on HepG2 cells, treated with or without different IFNs for 72 hrs, respectively. The fluorescence was increased by 1.8-, 2.5-, and 1.5-folds when treated with IFNα, IFNγ and IFNλ, respectively. IFNγ was still the most potent among the three IFNs (p<0.05) . Apoptos. ApoptosTo determine how IFNs down-regulate β-catenin signaling activity, we evaluated the impact of different IFNs on two potent endogenous antagonists of the β-catenin pathway, DKK1 and GSK3β. DKK1 antagonized β-catenin signaling by depleting frizzled co-receptors (LRP) and thus, inhibiting frizzled activation through Wnt ligands. Following CK1-mediated phosphorylation of β-catenin at serine 45, GSK3β phosphorylates β-catenin at Thr 41, Ser 33, and Ser 37, which tags β-catenin for ubiquitination by βTrCP and proteomic degradation , black bars). Similar results were observed in Huh7 cells (data not shown).JAK-STAT pathway is used by all IFNs to conduct downstream signaling, where STAT family proteins are induced selectively by different IFNs. Among proteins in the STAT family, STAT1 and STAT3 can be activated by all types of IFNs , grey and open bars) and Huh7 cells (data not shown). IFN-induced DKK1 in HepG2 and Huh7 cells were abrogated by S3I, but not FLUD, indicating that STAT3 but not STAT1, is required for IFN-induced DKK1 up-regulation , STAT1 inhibitor (FLUD), and STAT3 inhibitor (S3I). Addition of neutralizing antibody against DKK1 abrogated the ability of IFNs to induce DKK1 and reduced the level of DKK1 from untreated cultures in both HepG2 and Huh7 cells Fig. 5. Since DKK1, STAT1 and STAT3 could be induced by all three types of IFNs, we next tested the contribution of DKK1, STAT1 and STAT3 to IFN-induced apoptosis in HCC cell lines. Addition of αDKK1 or S3I abrogated the capability of IFNα, IFNγ and IFNλ to induce apoptosis in HepG2 and Huh7 cells, determined by TUNEL assay. However, FLUD showed no effects on blocking IFN-induced apoptosis, indicating that STAT1, although activated by IFNs, had no role in this regulation Fig. 6. in vitro and in vivo pegylated-IFNα treatment leads to increased export of nuclear β-catenin in multiple HCC cell lines in vivo IFNα2b treatment inhibits Wnt/β-catenin/TCF pathway and promotes programmed cell death possibly providing a link with FOXO (forkhead box containing protein class O) pathway IFN signaling, as a critical component of the innate immunity, has a broad effect on the immune system. Canonical β-catenin signaling is involved in cell proliferation and differentiation. These two pathways have been reported to have a cross-regulation at certain levels. Nava et al discovered that IFNγ exerted a biphasic effect on intestinal epithelial cell proliferation and apoptosis by regulating DKK1, and in turn β-catenin signaling Our results demonstrated that, in different HCC cell lines, all three types of IFNs suppress the level of activated β-catenin and β-catenin signaling activity. The result of IFNγ is consistent with our findings in astrocytes β-catenin pathway is tightly regulated to avoid aberrant activation. A number of proteins in this signaling are regulated at gene level by β-catenin/TCF transcriptional regulation, including DKK1, an important secreted endogenous inhibitor against β-catenin pathway The two cell lines used in this study, HepG2 and Huh7, are not identical in terms of β-catenin signaling. There is a truncated form of β-catenin, which is constitutively active, in HepG2 cells in addition to the wild-type HCC is a devastating disease and among the most fatal cancers in the world. Treatment options are limited mainly due to inefficiency of existing anticancer chemotherapeutic drugs against HCC. IFNα has been used to treat HBV and HCV infections, and also indicated for HCC. In addition, there are many completed and ongoing clinical trials on IFNα treating HCC Given the link we demonstrate here between IFNs and β-catenin signaling pathway, IFNs may have broader roles than that was previously appreciated. Understanding the interactions between IFNs and β-catenin signaling will have a broader impact not only on carcinogenesis but also on understanding the normal biology of liver cells at the interface of IFNs and β-catenin-dependent pathways.


Ginkgo possesses anti-inflammatory, blood flow-enhancing, and free radical scavenging effects. Considering strenuous exercise can reduce BPH risks, we hypothesize Ginkgo + exercise (Ginkgo + Ex) could be beneficial to BPH. To verify this, rat BPH model was induced by s.c. 3.5 mg testosterone (T) and 0.1 mg estradiol (E2) per head per day successively for 8 weeks, using mineral oil as placebo. Cerenin® 8.33 μL/100 g was applied s.c. from the 10th to the 13th week, and simultaneously, Ex was applied . In BPH, Ginkgo alone had no effect on T, 5α-reductase, and dihydrotestosterone (DHT), but suppressed androgen receptor (AR), aromatase, E2 and estrogen receptor (ER), and the proliferating cell nuclear antigen (PCNA); Ex alone significantly reduced T, aromatase, E2, ER, AR, and PCNA, but highly raised DHT. While Ginkgo + Ex androgenically downregulated T, aromatase, E2, and ER, but upregulated DHT, AR, and PCNA, implying Ginkgo + Ex tended to worsen BPH. Conclusively, Ginkgo or Ex alone may be more beneficial than Ginkgo + Ex for treatment of BPH.Benign prostatic hyperplasia (BPH), an imbalance between androgen/estrogen,overexpression of stromal, and epithelial growth factors associated with chronic inflammation, has become an atypical direct cause of mortality of aged male diseases.  Benign prostatic hyperplasia (BPH) and low urinary tract symptoms (LUTS) are quite common male diseases. The prevalence of BPH increases with aging  and has α-blockers, 5α-reductase inhibitors , and surgery therapy, or the combined treatment [Ginkgo biloba leaf extract (for simplicity named “Ginkgo” herein) comprise Ginkgo flavone glycosides  and terpene lactone ginkgolides  [Ginkgo possesses antioxidant and anti-inflammatory activity [Ginkgo biloba leaf extract (Ginkgo) has been used for centuries in China for treating asthma and bronchitis. Currently, Ginkgo has been widely used to treat the cerebrovascular and the peripheral vascular insufficiency, neurosensory problems, and disturbances in vigilance, short-term memory, and other cognitive functions that are associated with dementias, ageing, and senility [Gingko causes two main actions: increase of blood flow in central and peripheral vessels and inhibition of platelet aggregation and free radical scavenging. Thus Gingko may be effective in cases of erectile dysfunction due to a decreased blood flow [The clinical care for BPH usually involves reatment . Currentreatment . Other nreatment . The maiobalide) . Ginkgo activity –24. Ginksenility . Many phood flow .A review of Sea et al. (2009) highly supports a clinically significant, independent, and strong inverse relationship between exercise (Ex) and the development of BPH /LUTS . GreaterGinkgo in treating BPH is still lacking. In view of the above mentioned beneficial biological activities of Gingko and Ex, we hypothesize that Ginkgo + Ex therapy as well could be beneficial to BPH. To verify this, we conducted this experiment and the relevant biochemical, immunological, and pathological parameters were examined and compared.The application of ®, is an inject testosterone medication provided by Schering-Plough Company , which in reality contains four testosterone esters. Each ampoule (1 mL) contains testosterone propionate 30 mg, testosterone phenylpropionate 60 mg, testosterone isocaproate 60 mg, and testosterone decanoate 100 mg. The overall androgenic potency in per mL of Sustanon® is equivalent to 176 mg testosterone. The Ginkgo biloba leaf extract, Cerenin®, was purchased from Dr. Willmar Schwabe Arzneimittel GmbH & Co. . Ginkgo consisted of 24% of Ginkgo flavone glycosides, mainly kaempferol and quercetin glucorhamnoside esters, and 6% of the characteristic terpene lactones, the bilobalide, and ginkgolides, namely, A, B, C, and very small quantities of ginkgolide. The inject solution contained in per mL 3.5 mg Ginkgo (240 mg/g flavonoids and 60 mg/g terpenoids), 30 mg of 960 mL/L ethanol, 40 mg sorbitol, and 0.1 mol/L NaOH. All other chemicals, not cited but used in this experiment, were of reagent grade provided by Wako Pure Chemical Co. . The sources of the antibodies used in this experiment were 5α-reductase and androgen receptor , estrogen receptor α (ERα) , aromatase and proliferating cell nuclear antigen (PCNA) , and β-actin from Novus Biologicals .Testosterone, dihydrotestosterone (DHT), estradiol ELISA kits were provided by Cayman Chemical Co. . Free PSA and Total PSA assay kits were provided by Cusabio Biotech . Rat IL-1 ELISA development kit is product of PeproTech Co. . Enhanced Chemiluminescence (ECL) system was provided by Merk Millipore Co. . TEMED is a product of Bio-Rad Co. . Protein Extraction Solution was provided by iNtRON Biotech. Co. . The pharmaceutical preparation of androgen, SustanonGinkgo-only treated control; Group 5, the Ex-only control; and Group 7, the Ginkgo + Ex. The medication treated groups were Group 4, BPH treated with Ginkgo; Group 6, BPH treated with Ex; and Group 8, BPH treated with Ginkgo + Ex, each having 12 rats. The exercise groups were subjected to treadmill exercise protocol. The treatment started from week 10 to week 13.This experiment was proved by the Institutional Animal Care and Ethic Committee of China Medical University . Ninety-six Sprague-Dawley (SD) rats weighing approximately 280 g were purchased from BioLasco Animal Centre, Taiwan. The rats were housed in a controlled environment, 3 in each cage, with 12 h/12 h light/dark cycle, at 28 ± 1°C and under relative humidity 65–75%. These rats were acclimated in such an environment for the first week. Then rats were divided into eight groups. The five controls involved Group 1, the normal control; Group 2, the BPH control; Group 3, the μL mineral oil/head/day as placebo. The BPH groups  were s.c. administered a combined testosterone (Sustanon®) 3.5 mg with estradiol 0.1 mg per head per day successively from week 2 to week 9, that is, a total induction period of 8 weeks.The protocol to induce BPH was conducted according to Suzuki et al. (1994) with slight modification . Briefly® was calculated from the recommended dose for clinical human use, that is, 35 mg (5 mL injection solution) i.v. for 60 kg adults. Thus in the beginning of week 10 immediately prior to the treatment experiment, a dose of Cerenin® 8.33 μL/100 g was recommended daily for each Ginkgo medicated group. The whole treatment course sustained for 4 weeks .The dose of CereninFor treadmill exercise training, the groups 5, 6, 7, and 8 were first acclimated from week 8 to 9, starting with 5 min, 10 min, 15 min, and then 20 min per time per day. The formal exercise therapy was then started from week 10 until week 13, three times per week, 30 min per time at a speed of 30 m/min on a motorized rodent treadmill .At the end of week 13, blood samples were withdrawn from the abdominal aorta under ether anesthesia. The blood was collected and centrifuged at 3000 ×g for 15 minutes to separate the serum. The prostates were excised, immediately frozen with liquid nitrogen and stored in −80°C or fixed by immersion with 10% formalin in PBS (pH 7.4).The prostates were fixed by immersion in 10% formalin-PBS (pH 7.4) at 4°C for 24 h and processed for paraffin embedding. Paraffin sections were dewaxed in xylene and rehydrated in a series of ethanol washes. The nuclei of these specimens were subjected to hematoxylin-eosin stain. Otherwise, the collagen content was stained with Sirius Red  .Serum levels of testosterone, estradiol, dihydrotestosterone (DHT), prostate-specific antigen (PSA), and IL-1 were measured by the ELISA kits. All protocols were performed by following the manufacturer's instruction. The EZ Read 400 Microplate Reader used was a product of Biochrom Co.  .The protein expression of androgen receptor and proliferating cell nuclear antigen were analyzed by IHC according to previous protocol cited . Quantitα-reductase, estrogen receptor α, and aromatase were analyzed. Briefly, frozen prostate tissue samples (approximately 100 mg) were homogenized with the homogenizer  in 1 mL of Pro-PREP lysis buffer (pH 7.2). The homogenate was centrifuged at 12000 ×g for 20 min at 4°C, and the supernatant was collected as tissue sample lysate. The sample lysates were heated at 100°C for 10 min before loading and separated on precasted 7.5% SDS-PAGE. The proteins were electrotransferred onto the PVDF membrane in transfer buffer for 1 h.Levels of 5β-actin was used as the reference protein [The nonspecific binding to the membrane was blocked for 1 h at room temperature with 5% nonfat milk in TBS buffer. The membranes were then incubated for 16 h at 4°C with various primary antibodies. After extensive washing in TBS buffer, the membranes were then incubated with secondary antibody in blocking buffer containing 5% nonfat milk for 1 h at room temperature. Membranes were then washed with TBS buffer and the signals were quantified using the Luminescent Image Analyzer LAS-4000 .  protein .P < 0.05.Data obtained in the same group were treated with ANOVA and Duncan's multiple range tests with computer statistical software SAS 9.0 . Different letters indicate significant differences at Starting from the original body weight (275 to 300 g), the body weight of all groups was seen increasing steadily from week 0 to week 2 (week 1 for acclimation) . When inGinkgo control, and BPH + Ginkgo, respectively. When treated with exercise, the incidence rates were apparently shifted to 0.00, 0.00, 0.00, and 0.18, respectively, for Ex control, BPH + Ex, Ginkgo + Ex, and BPH + Ginkgo + Ex. Usually BPH is accompanied with inflammation. The incidence rate of inflammation was 0.00, 0.64, 0.08, and 0.18, for the normal control, BPH control, Ginkgo control, and BPH + Ginkgo, respectively. After exercise training, the incidence rate of inflammation was improved to give 0.00, 0.46, 0.25, and 0.55, respectively, for Ex control, BPH + Ex, Ginkgo + Ex, and BPH + Ginkgo + Ex , Taipei, Taiwan), implying the fact that Ginkgo was beneficial to curing BPH, and also effective to suppress the inflammation status. The Sirius Red stain revealed huge amount of collagen deposition occurring in BPH group  in normal controls ranged within 1.03 ± 0.12%, and that of BPH within 0.86 ± 0.10%. Ginkgo, exercise, and Ginkgo + Ex all significantly reduced the levels to ranges within 0.48 ~ 0.68 pg/mL  compared with normal control (530.8 ±68.3 pg/mL) (ectively . The DHT3 pg/mL) .α-reductase to be totally unaffected by any of these specific therapies . However strangely, Ginkgo + Ex further upregulated the level of AR in BPH group (IOD 3.97 ± 0.48%)  Figures . Ginkgo  Figures .Ginkgo, Ex, and Ginkgo + Ex  with significantly higher collagen content (P = 0.0038) and lower granularity than that from men without lower urinary tract symptoms  [The effect of  effects –24, 31.  Figures . Tissue or less) . Thus, fGinkgo, exercise, and Ginkgo + Ex  and the ratio of free-to-total PSA are widely used as the tumor markers, but the effect of exercise on these parameters is unclear. Ratio of free-to-total PSA was equally improved by kgo + Ex . LiteratGinkgo + Ex were seen significantly lower than those in the BPH control. Speculatively, the highly upregulated DHT in these two groups implied BPH possibly incurable by these treatments. AR was seen more highly expressed in BPH + Ginkgo + Ex group , an anti-AR, an aromatase inhibitor, and a potent anti-ER and a strong antiestrogenic (regarding the estradiol level). Exercise acts as an androgenic (regarding the DHT level), an anti-AR, a moderate anti-ER, a moderate aromatase inhibitor, and an estrogenic (regarding the estradiol level). The combined therapy behaves as an androgenic (regarding the DHT level), an AR-upregulator, a moderate anti-ER, a moderate aromatase inhibitor, and an antiestrogenic (regarding the estradiol level). Thus, the therapeutic outcomes could be very complicated and different from each other. Overall, pathologically Ginkgo alone and exercise alone may be more beneficial than the combined therapy.In BPH,


Despite the numerous studies proposing early human population expansions from Africa into Arabia during the Late Pleistocene, no archaeological sites have yet been discovered in Arabia that resemble a specific African industry, which would indicate demographic exchange across the Red Sea. Here we report the discovery of a buried site and more than 100 new surface scatters in the Dhofar region of Oman belonging to a regionally-specific African lithic industry - the late Nubian Complex - known previously only from the northeast and Horn of Africa during Marine Isotope Stage 5, ∼128,000 to 74,000 years ago. Two optically stimulated luminescence age estimates from the open-air site of Aybut Al Auwal in Oman place the Arabian Nubian Complex at ∼106,000 years ago, providing archaeological evidence for the presence of a distinct northeast African Middle Stone Age technocomplex in southern Arabia sometime in the first half of Marine Isotope Stage 5. The Nubian Complex is a regionally distinct Middle Stone Age (MSA) technocomplex first reported from the northern Sudan in the late 1960 s Nubian Complex industries are distinguished by a characteristic and highly standardized method of preferential Levallois reduction, “mass-produced from an elaborate archetype” The early Nubian Complex is distinguished by a higher frequency of Nubian Type 2 cores in conjunction with bifacial foliates and handaxes Taking into account its distinct, regionally-specific characteristics, Marks There are claims for the presence of Nubian technology in eastern Arabia. One “possible” Nubian Type 1 core was reported at Jebel Barakah, UAE, illustrated in Wahida et al. Despite published reports of Nubian At present, the northernmost extent of Nubian Type 1 cores is demarcated by assemblages found in the vicinity of Jebel Urayf and Naquah, in central-east Sinai In light of these tantalizing, yet inconclusive findings, the Dhofar Archaeological Project (DAP) was initiated in 2010 to explore the Late Pleistocene archaeological record of the Dhofar region in southwestern Oman. During the 2010 fieldwork campaign, a surface scatter with Nubian Type 1 and Type 2 Levallois cores was discovered in Wadi Aybut, central Dhofar. Subsequent research by DAP has focused on geoarchaeological investigation of the Aybut drainage system and surrounding landscapes, optically stimulated luminescence (OSL) dating of cemented fluvial sediments at Aybut Al Auwal that contained a handful of diagnostic Nubian Levallois artifacts, systematic survey to articulate the distribution of Nubian Levallois core technology throughout Dhofar, and techno-typological analysis of Nubian Levallois reduction strategies in Dhofar to assess the relationship of these assemblages with the African Nubian Complex.2. The landscape of this region encompasses a variety of geomorphic settings, partitioned into four ecological zones  SalaThe coastal plain, stretching some 90 km in length, is a crescent-shaped landmass up to 15 km wide, bounded by the Arabian Sea and the Jebel Qara escarpment. The plain is composed of alluvial fans up to 100 m thick, which are cut by short wadis draining southward from the escarpment toward the sea. Several springs emerge along the foot of the escarpment, around which grow date and coconut palms, bananas and other tropical fruits, and grasses North of the plain, the Jebel Qara-Jebel Samhan mountain chain rises abruptly to a maximum height of 850 m above sea level. The high escarpment forms an orographic barrier that traps moisture from the Indian Ocean Monsoon (IOM), generating up to 200 mm of annual rainfall across the mountains. Due to moisture brought by the IOM, the Dhofar highlands are covered in a mantle of dark brown clay soil that supports a subtropical cloud forest belonging to the Somalia-Masai center of endemism Northwards, past the current watershed divide, the escarpment flattens out onto a deeply incised limestone plateau called the Nejd, which extends approximately 250 km east-west and 150 km north-south. Around its southern extent, the Nejd is a barren scabland marked by an intricate series of minor wadis dissecting the plateau. These smaller drainage systems converge into larger and more deeply incised canyons that extend up to 100 km northward across the plateau, running parallel to one another. As they reach the northern extent of the Nejd, the wadis empty onto a gently undulating gravel plain flanking the Rub' Al Khali desert.Mudayy member, which is the highest quality on the plateau, outcropping in the southern and central regions. Chert-bearing units within the overlying Rus formation are concentrated in the southern Nejd, including the lower chalky Aybut member and upper marly-carbonate Gahit member. The quality and size of this raw material is quite variable; due to post-depositional displacement, most of the Aybut member chert is highly fractured. Thin, high-quality grey chert plaquettes are found within the Gahit member Three high-quality Eocene chert beds outcrop widely throughout the Nejd plateau, making the landscape appealing for prehistoric toolmakers. Fine-grained, large, banded chert slabs are found within the The drainage channels incising the Nejd plateau formed during wet climatic regimes throughout the Quaternary These data indicate that the monsoon increased in intensity during three intervals within MIS 5. Among these humid episodes, the last interglacial  appears to represent the most significant wet phase within the entire Late Pleistocene, with rainfall surpassing all subsequent pluvials Climate records indicate that MIS 4 (74–60 ka) was a period of rapid global cooling, at which time much of Arabia was beset by prolonged aridity caused by the southward displacement of the IOM. Records from the Arabian Sea attest to a period characterized by cooler sea surface temperatures, low productivity, and increased terrigenous (aeolian) input Ziziphus ziziphus (Jujube), Adansonia digitata (Baobob), Ficus sp. , Calotropis procera (Sodom's Apple), and Adenium obesum (Desert Rose) a fortiori, that the archaeological record will demonstrate cultural affinities at such times.Activation of Arabian fluvial systems during humid phases eg., DAP fieldwork was conducted over the course of two seasons in the winter of 2010 and 2011; required permits to carry out survey and excavation were granted by the Ministry of Heritage and Culture in Oman. To date, DAP has mapped 110 occurrences with Nubian Levallois technology across the Nejd plateau, ranging from occasional isolated cores to high-density scatters . Lithic Surveys were conducted along 40 transects throughout the Nejd plateau, Jebel Qara escarpment, and Salalah coastal plain . TransecFrom the distribution of findspots in Dhofar exhibiting Nubian Levallois technology, it appears that occurrences are confined exclusively to the Nejd plateau, where they are most often found near stream channels and raw material outcrops. Survey transects did not produce evidence for any kind of MSA/MP occupation along the coastal plain or the fringes of the Jiddat Al Harassis gravel plain bordering the eastern Nejd. The westernmost occurrence (TH.102) was an isolated Nubian Type 1 core in Wadi Tanfarut along the Yemeni border, while the easternmost site (SJ.56) was a low density Nubian Levallois scatter in Wadi Qaharir, 250 km to the east. In the north, a small number of Nubian Type 1 and Type 2 cores were discovered around Shisur Farms (TH.38), on an ancient fluvial terrace 7 km east of Wadi Ghadun. Given the logistical difficulties of survey within the Rub' al Khali desert, we were not able to investigate this zone and cannot yet address the northern distribution of such sites in Dhofar. The southernmost occurrence (TH.78) was an isolated Nubian Type 1 core on a low terrace above Wadi Nirin, less than 2 km from the northern slopes of Jebel Qara. In every assemblage encountered, Nubian Type 1 cores were by far the most prevalent, and Nubian Levallois technology was never found in conjunction with a bifacial component.Mudayy member chert beds as the channels developed. The Aybut-Banut-Amut-Ghadun drainage system is unlikely, however, to be the only center of MSA occupation on the Nejd. We have systematically surveyed less than 1% of the 33,000 km2 plateau, so it is likely that there are other catchments with similarly high concentrations of MSA artifacts.Of the MSA sites with Nubian Levallois technology mapped by DAP, 39 findspots (<1 artifact per sq m), 55 low density scatters (1–10 artifacts per sq m), and 16 high density scatters (>10 artifacts per sq m) were recorded. While isolated findspots and low density scatters are found across the entire plateau, evidence for intensive/recurrent settlement is concentrated in the west-central Nejd, around a large catchment system made up of Wadis Aybut, Banut, Amut, and Ghadun. This may be linked to the presence of ancient and modern groundwater-fed springs that emerge around the village of Mudayy, at the confluence of Aybut and Banut. Not only would this zone have provided a considerable amount of water in both its rivers and springs, but also fluvial downcutting would have continually excavated fresh Mudayy chert and chipping debris, and is incised by a series of small stream channels  is an open-air site that contains artifacts on the surface and buried within fluvial sediments in Wadi Aybut, west-central Nejd. The site was found on the second terrace, ∼20 m above a relict tributary channel feeding the main wadi system. The terrace is blanketed in a pavement of naturally occurring channels . Lithic channels , while tchannels . AlthougMudayy member) and Tertiary limestone (Umm Ar Radhuma formation) The Aybut Al Auwal terrace is formed by unconformities within horizontal strata of the underlying bedded chert  limestone and chert clasts within their course.One such lateral channel-fill deposit was excavated to a depth of 92 cm and is comprised of four distinct stratigraphic units, which overlie the limestone channel bed . The uppThe depositional age of the artifact-bearing sediments in Unit 3 was estimated by OSL dating of buried quartz grains 2. An additional 10 pieces were excavated from ∼1 m3 of highly-cemented sediment comprising stratigraphic Unit 3, and 11 desilicified artifacts were collected nearby eroding from the side of the channel  is a Nubian Complex surface scatter situated on a gravel plain some 5 km northeast of Aybut Al Auwal. The site is positioned at the headwaters of two large tributary systems, with prominent views of wadi channels to the east and west . Althoug2. A 10×10 m area was systematically collected in 1 m2 units, and all cores, tools, and a 25% sample of debitage were analyzed. Cores and larger pieces of debitage are only moderately weathered, however, the smaller material is in exceptionally poor condition, due to a combination of taphonomic processes including deflation, winnowing, surface runoff, chemical alteration, and thermal fracturing. While striking platforms and scar patterns are clear and permit technological analysis, the resulting edge damage caused by these destructive processes has obscured possible retouch, hindering typological identification. Given this problem, the Aybut Ath Thani tool type list should be approached with caution.The small lithic scatter observed at Aybut Ath Thani is constrained to no more than 400 mOf the 1,734 artifacts comprising the Aybut Ath Thani assemblage, 157 (9%) are cores . Nubian Although the site is positioned slightly away from a source of raw material, there is a relatively low ratio of non-cortical pieces to cortical pieces . There aSome blanks were identified with sufficiently consistent retouch to be classified as tools, despite the heavy edge damage on many of the pieces in this assemblage. These types, presented in Mudayy member chert beds, as the soft limestone hills were broken down by wind and surface runoff.Mudayy As Sodh (“Mudayy's Rooftop”) is located on a high plateau above the village of Mudayy. The site consists of multiple surface scatters just over 1 km east of Aybut Al Auwal, around a series of shallow basins that debouch into the main Aybut tributary. Small gullies (<50 cm deep) incise the silicate gravel covering the plateau, where a variety of assemblages were observed in discrete patches across the landscape. Nubian Complex scatters were identified closer to the edge of the plateau overlooking the drainage systems below, while less weathered Nejd Leptolithic 2 was systematically collected from one Nubian concentration at Mudayy As Sodh, chosen for its high density of cores and debitage. 965 artifacts were recovered in total, including 92 cores, 69 tools, and 804 pieces of debitage  is situated 6 km southeast of Aybut Al Auwal. The site consists of several concentrated lithic scatters on the first erosional terrace (about 5–15 m wide), perched ∼10 m above a steeply-incised wadi channel . Excepti2. As there are no obvious differences between the two areas, we have combined them into a single assemblage for the purposes of this analysis. The composite assemblage is comprised of 449 artifacts; Nubian core conjoins indicate there has been minimal post-depositional disturbance  and/or steep bilateral (Type 2) removals. Specimens from Dhofar exhibiting this characteristic distal median ridge are shown in cross section in Nubian Complex assemblages in northeast Africa exhibit multiple core types, including Nubian Type 1, Nubian Type 2, preferential centripetal Levallois, bidirectional, and single platform . In DhofThe most common tool types found within Nubian Complex assemblages in Dhofar are Levallois points, flakes, and blades, which show a propensity toward elongation and converging lateral edges. The relatively few retouched tools include sidescrapers, endscrapers, denticulates, notches, and miscellaneous retouched pieces, with a trace number of burins and perforators . This saGiven these technological and typological similarities, we classify the Dhofar assemblages as late Nubian Complex. It is more likely that the high degree of overlap observed in southern Arabian and northeast African Nubian Complex assemblage – a continuous phytogeographic zone divided only by the Red Sea – is the result of cultural exchange, rather than the synchronistic result of concurrent technological evolution. For the time being, the apparent distribution of Nubian Levallois technology in Arabia is limited to the Nejd plateau and, perhaps, Hadramaut valley . ArchaeoWhile any explanation must be speculative, we suggest that the significantly higher frequency of Nubian cores in Dhofar, as opposed to the Nile Valley, may be the result of variations in hunting behavior across the two landscapes. These differences, in turn, are the function of hydrology and the area of exploitable land in Dhofar versus the Nile Valley. In southern Arabia during MIS 5c, there were extensive grasslands cut by drainages, but none so big as to limit faunal distributions or impede hunter-gatherer mobility. In the Nile Valley, on the other hand, exploitable land was limited to the valley itself and to a narrow strip of land along its sides. Both to the east and west of the Nile Valley, the flat gravel plains would not have been appropriate hunting terrain, as confirmed by the lack of sites even a few kilometers from the valley Homo sapiens found in the Omo river valley, Ethiopia  from Africa is the subject of considerable debate If MSA inhabitants of northeast Africa were AMHs, then the presence of a regionally-specific African MSA industry in Dhofar is relevant to the question of modern human expansion. The route and timing of Movement through the northern dispersal route is based on AMH remains discovered at Skhul and Qafzeh in Israel dating to early MIS 5 Genetic studies of human mtDNA favor the southern dispersal route as the primary conduit for early modern human expansion(s) out of Africa To some degree, the discovery of late Nubian Complex assemblages in Dhofar upholds this model. The distribution of this technocomplex in the middle and lower Nile Valley, the Horn of Africa, Yemen, and now Dhofar provides a trail of diagnostic artifacts - stone breadcrumbs - spread across the southern dispersal route out of Africa. The close similarity between African and Arabian late Nubian Complex assemblages suggests that these sites are more or less contemporaneous; they were separated for an insufficient amount of time for independently derived technological traits to develop between regions. As the late Nubian Complex at Aybut Al Auwal is dated to MIS 5c, slightly earlier than the late Nubian Complex in Africa Coalescence ages for non-African mtDNA lineages range from 70 to 45 ka, depending on the use of different mutation rates, calibration methods, and statistical models A second possibility is that the mtDNA coalescence age of L3 would appear younger than the time of initial expansion if pioneering groups moving into Arabia had been sex-biased toward a low number of females The archaeological evidence does not yet permit us to evaluate what became of the late Nubian Complex in Arabia. Our study only documents the presence of this industry in Dhofar during MIS 5c; we do not yet know when Nubian Complex toolmakers arrived on the subcontinent or what became of them over the course of the Late Pleistocene. The eastern distribution of the Nubian Complex appears to terminate at the edge of Nejd plateau. Surveys throughout the rest of Oman and eastern Arabia have not produced any evidence of Nubian Complex technology. Assemblage C from the last interglacial site of Jebel Faya is classified as a generalized East African MSA technological complex  and is ascribed to AMH toolmakers. Its small assemblage size and limited workshop characteristics, however, preclude attribution to any specific, contemporaneous East African industry The presence of seemingly Nubian-derived assemblages around the Wadi Aybut-Banut-Amut-Ghadun drainage systems, discovered during the DAP 2011 fieldwork campaign, hints at the survival of some aspects of the Nubian Complex technological tradition within Dhofar. These ‘Developed Nubian’ assemblages exhibit a suite of core reduction strategies including Nubian Levallois, ‘microlithic’ Nubian, and flat cores with bidirectional blades struck from faceted platforms. Such assemblages, however, must still be adequately defined and placed within a chronological framework.Although southern Arabia experienced successive periods of extreme aridity after MIS 5, terrestrial archives document another increase in precipitation across the interior of Arabia during early MIS 3 Figure S1Example OSL decay and dose-response curves from AYB1-OSL1. Decay curve (a) and dose-response curve (b) for a single aliquot of quartz (∼50 grains). The De of ∼70 Gy is obtained by interpolation of the sensitivity-corrected natural OSL signal, shown in red on the y-axis of the inset plot. The data in (a) and (b) were collected after preheating the natural and regenerative doses at 260°C for 10 s. Panel (c) shows the De values obtained from aliquots preheated at a range of temperatures , along with the extent of recuperation ; these data indicate that the measured De value is not sensitive to the chosen preheat temperature. The De values obtained from 42 separate aliquots of AYB1-OSL1 are displayed in (d); each aliquot was preheated at 260°C for 10 s. The filled circles and open triangles denote the values obtained using the ‘late light’ and ‘early background’ subtraction approaches, respectively, and the shaded band is centred on the weighted mean De value (∼58 Gy) used to calculate the OSL age of this sample. Plot (e) shows the De values obtained from 22 single aliquots of AYB1-OSL2: the symbols are the same as in (d) and the shaded band is centred on the weighted mean De value (∼61 Gy) used to estimate the sample age.(TIF)Click here for additional data file.Table S1e) values, environmental dose rates, and OSL ages of the sediment samples from Aybut Al Auwal.Equivalent dose  uncertainty, calculated as the quadratic sum of the random and systematic uncertainties. The De uncertainty includes a relative error of 2% to allow for possible bias in the calibration of the laboratory beta source.(DOC)Click here for additional data file.Appendix S1Optically stimulated luminescence (OSL) dating.(DOC)Click here for additional data file.