The canonical Wnt signaling pathway is involved in a variety of biological processes like cell proliferation, cell polarity, and cell fate determination. This pathway has been extensively investigated as its deregulation is linked to different diseases, including various types of cancer, skeletal defects, birth defect disorders , metabolic diseases, neurodegenerative disorders and several fibrotic diseases like desmoid tumors. In the "on state", beta-catenin, the key effector of Wnt signaling, enters the nucleus where it binds to the members of the TCF-LEF family of transcription factors and exerts its effect on gene transcription. Disease development can be caused by direct or indirect alterations of the Wnt/β-catenin signaling.APC) - and the desmoid-like fibromatosis, a sporadic tumor associated with somatic mutations of the β-catenin gene (CTNNB1).In the first case germline or somatic mutations of the Wnt components are associated to several diseases such as the familial adenomatous polyposis (FAP) - caused by germline mutations of the tumor suppressor adenomatous polyposis coli gene (In the second case, epigenetic modifications and microenvironmental factors have been demonstrated to play a key role in Wnt pathway activation. The natural autocrine Wnt signaling acts through agonists and antagonists competing for the Wnt receptors. Anomalies in this regulation, whichever is their etiology, are an important part in the pathogenesis of Wnt pathway linked diseases. An example is promoter hypermethylation of Wnt antagonists, such as SFRPs, that causes gene silencing preventing their function and consequently leading to the activation of the Wnt pathway. Microenvironmental factors, such as the extracellular matrix, growth factors and inflammatory mediators, represent another type of indirect mechanism that influence Wnt pathway activation. A favorable microenvironment can lead to aberrant fibroblasts activation and accumulation of ECM proteins with subsequent tissue fibrosis that can evolve in fibrotic disease or tumor.Since the development and progression of several diseases is the outcome of the Wnt pathway cross-talk with other signaling pathways and inflammatory factors, it is important to consider not only direct inhibitors of the Wnt signaling pathway but also inhibitors of microenvironmental factors as promising therapeutic approaches for several tumors of fibrotic origin. The Wnt signaling pathway is involved in several essential biological processes in both embryonic development and in adult cell maintenance and regeneration.APC are associated with familial adenomatous polyposis (FAP), and somatic mutations of the β-catenin gene (CTNNB1) are associated with sporadic desmoid tumors. In the first case the disease is caused by a transmissible genetic defect, in the second case the pathology is linked to a somatic mutation that makes β-catenin unable to be completely phosphorylated and degraded.The canonical or Wnt/β-catenin dependent pathway controls key developmental gene expression programs by modulating the amount of β-catenin through regulating its degradation or accumulation and its translocation from the adherens junction and cytoplasm to the nucleus. In the absence of a Wnt signal, the cytoplasmic β-catenin is tightly maintained at a low level by a multiprotein destruction complex consisting of Axin, the adenomatous polyposis coli protein (APC), casein kinase 1α (CK1α), and Glycogen Synthase Kinase 3β (GSK3β). The complex phosphorylates cytoplasmic β-catenin leading to its degradation by the ubiquitin-proteasomal system. The continuous elimination of β-catenin prevents its accumulation in the cytoplasm and the consequent translocation into the nucleus. In the presence of a Wnt signal, the destruction complex is disassembled leading to an increment of β-catenin levels and allowing its translocation into the nucleus where it activates Wnt target gene expression. The aberrant regulation of the Wnt/β-catenin pathway plays a role in the pathogenesis of several diseases including cancer, birth defect disorder, skeletal diseases, and fibrotic diseases. For this reason Wnt/β-catenin signaling is tightly regulated and kept under strict control at different levels of the Wnt cascade. Wnt activation is temporally and spatially tuned by autocrine Wnt signaling that is associated with extracellular Wnt agonists and antagonists. The agonists activate the Wnt cascade while the antagonists inhibit Wnt signaling at the level of ligand/receptor . HoweverWnt/β-catenin signaling can be also indirectly altered by epigenetic modifications that cause silencing of Wnt endogenous brakes, and by the effect of microenvironmental factors, such as the extracellular matrix, hormones and growth factors. Of particular interest is the involvement of inflammatory factors in the modulation of the Wnt/β-catenin pathway and its association with fibrotic disease as well as tumor development.Either direct or indirect Wnt pathway alterations can cause an increase of β-catenin levels and its accumulation into the nucleus, activating the signaling cascade. The cross-talk between these extracellular stimuli and intracellular signals highlights the complex interaction of the numerous factors involved in the development of the Wnt pathway linked pathologies and are well represented in fibrotic disease and in particular in the sporadic desmoid tumors.Many studies describe the use of small synthetic molecules for inhibiting the β-catenin as therapeutic approach. Among these, there are molecules that target the interaction of β-catenin with co-activators disabling the formation of an active transcriptional complex. Recently GSK3β inhibitors have been described as promising drugs for several pathologies such as diabetes, stroke, mood disorders, inflammation, and Alzheimer’s disease. The use of specific inhibitors of the Wnt signaling molecules or/and inhibitors of other signaling pathways associated to β-catenin pathway may help to find the key steps of the different pathologies linked to the Wnt pathway.The Wnt pathway is one of the evolutionarily-conserved cell signaling pathways used both during embryogenesis and in developed organism’s homeostasis to regulate cell proliferation, cell polarity, and cell fate determination -6. The e2+, and Wnt-atypical protein kinase C pathways. These pathways have been reported to contribute to developmental processes such as planar cell polarity (PCP), convergent extension movements during gastrulation, neuronal and epithelial cell migration [The non-canonical Wnt pathways, defined as Wnt- or Frizzled-mediated (Fzd) signaling independent of β-catenin transcriptional activity , are divigration -13.2+ signaling, in particular, activates heterotrimeric G proteins that stimulate phospholipase C (PLC). The signaling activation results in intracellular Ca2+ mobilization with activation of Ca2+-dependent effectors that include protein kinase C (PKC), calcium calmodulin mediated kinase II (CAMKII), and calcineurin [Wnt/Cacineurin .The canonical pathway is the most studied Wnt signaling pathway as it is involved in a variety of biological processes and integrates signals from other cellular pathways. It controls different processes throughout embryonic development, such as stem cell pluripotency, cell proliferation, differentiation, and cell migration. In adult cells, Wnt signaling contributes to maintain somatic stem cells, regulates cell fate decisions and it is involved in tissue regenerative processes following injury .The hallmark of the canonical Wnt pathway is transcriptional activation by β-catenin. The pathway regulates the amount of β-catenin through its degradation or its accumulation and translocation from the adherens junction and cytoplasm into the nucleus. In this way it controls key developmental gene expression programs ,16,17. IThe Wnt/β-catenin pathway is activated when specific extracellular molecules, Wnt ligands, bind to a receptor complex consisting of a seven-pass transmembrane Frizzled (Fzd) receptor and its co-receptor, low-density lipoprotein receptor related protein 6 (LRP6) or its close relative LRP5. The activated receptors recruit the scaffolding protein Dishevelled (Dvl), which leads to LRP5/6 phosphorylation, mediated by either CK1γ or GSK3β. These events trigger the translocation of Axin to the membrane where it binds to a conserved sequence in the cytoplasmic tail of LRP5/6 ,25. ConsThe Wnt/β-catenin pathway’s complexity derives from the high number of ligands and receptors involved in Wnt signal transduction that can elicit a variety of intracellular responses ,29.Wnt ligands comprise a large family of 19 highly conserved cysteine-rich proteins of approximately 350–400 amino acids that contain an N-terminal signal peptide for secretion .Wnt ligands are involved in both the canonical and the non-canonical pathways. Traditionally some ligands  have been classified as canonical ligands and others  as non-canonical ligands but this classification is now viewed as obsolete. Single Wnt ligands can be involved in multiple intracellular cascades and activate both types of pathways with opposing outcomes. The Wnt outcome depends on the receptor status and on the cellular and microenvironmental context ,31.The activation of Wnt/β-catenin signaling requires the cooperation and the aggregation of two types of transmembrane receptors: the Frizzled (Fzd) seven-pass transmembrane G-protein-coupled receptors  and the Dishevelled (Dvl) proteins are multifunctional intracellular proteins involved in both canonical and non-canonical pathways and have numerous putative binding partners . In mammAxin is a scaffold protein that acts as a constitutive negative regulator of Wnt signaling by forming a complex with β-catenin, APC, and GSK3β. In particular, this function is carried out by the Axin C-terminal DIX domain (DAX domain) -49. The APC is a tumor suppressor gene located on the long arm of chromosome 5 (5q21). APC has multiple domains that mediate oligomerization as well as binding to a variety of other proteins [et al. speculated that APC competes with Dvl for association with Axin, displacing it from Axin protein complex. Wnt signaling may overcome the competition between APC and Dvl for their binding to Axin, allowing simultaneous interaction of all three proteins [proteins , which hproteins . APC is proteins . APC mayproteins , or indiproteins . Mendozaproteins ,51,60,61APC can be phosphorylated by CK1/GSK3 increasing its affinity for the same β-catenin domain as Axin, suggesting the role of APC in removing the phosphorylated β-catenin molecules from the complex ,22,62. ACK1 is a monomeric serine/threonine kinase involved in many different cell functions. There are seven members with high homology: α, β, γ1, γ2, γ3, δ, and ε. Each isoform is involved in different steps of Wnt pathway, with different effects. CKIα is the kinase that first phosphorylates β-catenin at S45, preparing the molecule for the following phosphorylations by GSK3β ,63,64. CGSK3β is a serine/threonine kinase that is highly conserved from yeast to mammals. In mammals two distinct genes encode two GSK3 isoforms, α (51 kDa) and β (47 kDa), which share 97% amino acid sequence identity within their catalytic domains. The two GSK3 isoforms are ubiquitously expressed and they are involved in a wide variety of essential biological processes such as tissue patterning, glucose metabolism, apoptosis, stem cell homeostasis, and cell cycle regulation . GSK3 haβ-catenin has a dual role in the cells: (a) it is a structural protein, stabilizing cell-cell adhesions, which are essential for normal cell physiology and tissue architecture ,74; (b) β-catenin protein has three domains: the N-terminal domain, the armadillo domain consisting of 12 armadillo repeats (residues 141–664), and a C-terminal domain. The positively charged armadillo (Arm) repeat is the binding site for most β-catenin binding partners ,77. LocaWnt/β-catenin signaling is endogenously regulated by secreted proteins that antagonize the Wnt ligands and act at the cell surface level in order to inhibit the pathway . Among tDeregulations of the Wnt pathway are linked to several human diseases comprising various types of cancer , skeletal defects, birth defect disorders , fibrotic diseases, metabolic diseases, neurodegenerative disorders and others ,83. SeveAPC gene and somatic CTNNB1 gene mutations [Axin2 has been described in adrenocortical carcinoma [LRP6 are linked to hereditary disorders as osteoporosis, coronary artery disease, and metabolic syndrome [LRP5 and TCF7L2 genes may lead to the development of obesity and mellitus diabetes [The Wnt pathway has been extensively investigated for its involvement in many types of cancer . Severalutations ,72,75. Garcinoma , hepatocarcinoma ,85. Patisyndrome . Mutatiodiabetes ,87.APC gene at this chromosomal site [The association between colon cancer and the aberrant regulation of the Wnt pathway has been known since the identification of alterations of chromosome 5q as an early event in the carcinogenic process for hereditary colon tumors , and the discovery, through different linkage studies, of the mal site ,89.FAP is a colon cancer predisposition syndrome, which is inherited in an autosomal dominant manner. Clinical diagnosis of FAP can be made when more than 100 adenomatous polyps are identified in the colorectum. FAP patients present not only colorectal adenomas but also various extracolonic manifestations, including desmoid tumors, osteomas, dental abnormalities, congenital hypertrophy of the retinal pigment epithelium, lipomas, epidermoid cysts and upper gastrointestinal polyps.APC gene mutations are recognized as the cause of FAP. Most of these mutations  cause a truncated or inactive protein [APC mutations have been subsequently found in ~80% of sporadic colorectal tumors, confirming that APC acts as a central gatekeeper protein in colorectal tumorigenesis [To date, more than 300 different  protein . APC mutigenesis .CTNNB1 and AXIN2, though these are significantly less frequent than mutations in APC [Inherited or somatic mutations that inactivate or destroy APC function prevent effective degradation of β-catenin, promoting the aberrant activation of canonical Wnt signaling. This leads to the development of non-invasive colonic adenomas (polyps) because β-catenin nuclear accumulation causes the overexpression of growth-promoting genes . The sams in APC .In addition to the genetic mutations, epigenetic modifications can contribute to aberrant activation of the canonical Wnt pathway. This can occur at various levels and determines the silencing or promoting of specific genes. In particular, aberrant methylation of CpG islands within gene promoter regions represents one of the most studied mechanisms of gene silencing and it is associated with selective transcriptional inactivation.Many evidences indicate, for example, that almost complete loss of SFRPs at the protein levels are frequently correlated with gene promoter hypermethylation in several pathologies such as colon carcinomas, hepatocarcinomas , prostatA loss of SFRP expression, through epigenetic silencing, contributes to the constitutive activation of autocrine Wnt signaling affecting cell proliferation and potentially enhancing the cell growth and promoting malignant transformation and cancer cell survival ,2,97,98.Regulators of the microenvironment, such as the extracellular matrix, growth factors and inflammatory factors, are associated with the aberrant activation of Wnt pathway and the promotion of several diseases.Inflammation is a critical defense mechanism against various harmful stimuli, although aberrant regulation may lead to diseases. The inflammation process, caused by injury, leads to wound healing, tissue repair and regeneration. Damaged epithelial and endothelial cells release inflammatory factors, growth factors, cytokines, and chemokines, which subsequently initiate an influx of neutrophils and monocytes to the site of the damaged tissue. Macrophages secrete platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF) and transforming growth factor-β (TGF-β). They also activate and convert fibroblasts into myofibroblasts, which are engaged in extracellular matrix (ECM) deposition and scar formation ,100. CytGSK3 has a crucial role in inflammation because it promotes pro-inflammatory cytokine production  and cell migration ,105,106.The development and progression of several fibrotic diseases is the outcome of the Wnt pathway cross-talk with other signaling pathways and pro-inflammatory mediators .In general, aberrant Wnt/β-catenin signaling activation drives fibrogenesis through interaction with profibrotic growth factors, epithelial cell transformation, myofibroblasts activation and proliferation . Mutant The cross-talk between Wnt/β-catenin and TGF-β pathways has been demonstrated in several fibroproliferative disorders such as Dupuytren’s disease and pulmonary fibrosis ,113. TGFDesmoid-type fibromatosis (DFs) can be an example of pathology arising from direct Wnt/β-catenin signaling alteration (Wnt mediator mutations) as well as indirect Wnt deregulation by involvement of the microenvironment. DF is a rare myofibroblastic neoplasm arising from a defect in connective tissue regulation, the neoplasia is unable to metastasize but it shows marked local aggression and a high recurrence rate. Some DFs are consequence of local trauma including pregnancies and surgical treatments ,115; repAPC gene linked FAP but they are generally sporadic tumors. A range from 50% to 87% of sporadic DFs are characterized by mutations in codons 41 and 45 of exon 3  of the gene encoding β-catenin, CTNNB1. These codons are the serine and threonine phosphorylation sites required for β-catenin degradation [CTNNB1 mutations, suggesting that other factors might be involved in the alteration of the Wnt/β-catenin pathway in DFs. Intriguingly, in all DF cells, we have also noticed a very marked increase in nuclear GSK3β (95%) associated to β-catenin, suggesting that other changes involving the multiprotein complex are involved with the disease  have been identified to Furthermore, the level of Axin in the β-catenin destruction complex is controlled by tankyrases, members of the PARP-family of poly-ADP-ribosylation enzymes. Small molecules, inhibiting the tankyrase 1 and tankyrase 2 enzymes, stabilize the level of Axin and promote the phosphorylation-dependent degradation of β-catenin by increasing the activity of the destruction complex . Among tMoreover, as GSK3 is a vital factor in inflammatory processes, inhibitors of GSK3 provide strong anti-inflammatory protection. GSK3 inhibitors were reported to reduce the inflammatory response in induced colitis in rats, as well as in arthritis and peritonitis in mice highlighting the potential therapeutic treatments in pathological conditions associated to inflammation ,136,137.in vitro desmoid cells. The results showed the reduction of receptor number only in desmoid cells, suggesting that Toremifene may reduce TGF-β's affinity for its receptors [DF treatment is complicated by its recurrence, invasiveness, and persistence. Due to the heterogeneity of the desmoid-type fibromatosis and to the unpredictable clinical course, at the moment, the treatment is given on a case-by-case multimodal basis -140. Foreceptors ,152. TorAn experimental animal model demonstrated that Apc(+)/Apc(1638 N) mice treated with Triparanol, an inhibitor of Hedgehog (Hh) signaling, develop few and smaller desmoid tumors compared with the untreated mice . These dThe Wnt/β-catenin pathway is a great example of heavily context dependent cellular pathways with several ligands, receptors, transmitters and modulators. In general the interactions of the Wnt/β-catenin pathway with the other cellular processes clearly state its importance for the cell and the entire organism.The most direct therapeutic approach against the deregulation of the Wnt/β-catenin pathway is to target the components of the pathways themselves or their closest interactors.The dependence of the Wnt/β-catenin pathway on its microenvironment can be exploited as a potential target for therapeutic approaches in particular the host’s response to pathological cells including inflammation and the various growth factors produced in the attempt to “heal” the organism. This approach could also lead to a faster individuation of a valid treatment as several compounds are already commercialized, even if they are developed for completely unrelated diseases.In synthesis, we need to continue studying on two fronts in order to find effective treatments for Wnt/β-catenin pathway related pathologies: the Wnt/β-catenin pathway itself and its role in the network comprising other pathways associated to the microenvironment.An informed written consent was obtained from the persons whose cell culture images were included in this review.


MMP-7 and c-Myc. Fibulin-5 impedes Wnt/β-catenin signaling by inhibiting extracellular signal-regulated kinase (ERK) to activate glycogen synthase kinase-3 β (GSK3β), which downregulates β-catenin and prevents its nuclear accumulation, leading to suppression of MMP-7 and c-Myc expression. These effects of fibulin-5 are mediated by its amino-terminal integrin-binding RGD motif. Fibulin-5 also blocks Wnt/β-catenin signaling in vivo in H460 metastasis and H1299 tumor models. Furthermore, knockdown of β-catenin suppresses metastasis of H460 tumors, while knockdown of GSK3β promotes metastasis of fibulin-5-expressing H1752 tumors. Together, our results suggest that fibulin-5 functions as a metastasis suppressor in lung cancer by modulating tumor microenvironment to suppress Wnt/β-catenin signaling.Metastatic lung cancer is incurable and a leading cause of cancer death in the United States. However, the molecular mechanism by which lung cancer cells invade other tissues has remained unclear. We previously identified fibulin-5, an extracellular matrix protein, as a frequently silenced gene in lung cancer and a suppressor of cell invasion. In this study, we found fibulin-5 functions by inhibiting the Wnt/β-catenin pathway. The Cancer Genome Atlas (TCGA) datasets show a strong association between loss of fibulin-5 expression and poor outcomes of lung cancer patients, and also activation of the Wnt target genes  Lung cancer, accounting for nearly 27% of cancer deaths, is the leading cause of cancer death in the United States . The higfibulin-5 and fibulin-3 as two genes that are most frequently silenced by promoter hypermethylation in non-small cell lung cancer (NSCLC) [The fibulin family, including fibulin-1–7, is a group of widely expressed extracellular matrix (ECM) proteins involved in cell motility, cell adhesion, elastogenesis, as well as cell-to-cell and cell-to-matrix communication . Aberran (NSCLC) , 11. Fib (NSCLC) , which p (NSCLC) . HoweverAPC mutations in colon cancer, result in the stabilization and translocation of β-catenin to the nucleus, where it binds to the T-cell factor (TCF) family of transcription factors to induce the expression of downstream target genes, such as c-Myc, CCND1, and MMP-7. The Wnt/β-catenin pathway is frequently activated in lung cancer [The Wnt/β-catenin pathway plays a critical role in tumor initiation, progression and metastasis . The ceng cancer , 17, andg cancer . HoweverMMP-7 and c-Myc. Fibulin-5, via its integrin-binding RGD motif, inhibits ERK to activate GSK3β and suppress β-catenin expression. Our results indicate that fibulin-5 functions as a metastasis suppressor in lung cancer by inhibiting Wnt/β-catenin signaling, and provide new insights on lung cancer invasion and metastasis.In this study, we found that loss of fibulin-5 expression in lung cancer is strongly associated with accumulation of β-catenin and activation of Fibulin-5 is known to be epigenetically silenced in lung cancer by promoter hypermethylation [fibulin-5 in large datasets from The Cancer Genome Atlas (TCGA) databases. TCGA RNA Seq data showed marked downregulation of fibulin-5 in over 50% of 1081 lung tumors analyzed, compared to normal control tissues, which all express high levels of fibulin-5     Fig. , earlier−4) Fig. , and low= 0.042) . These rfibulin-5 with other genes implicated in lung cancer progression. TCGA data showed an obvious inverse correlation between fibulin-5 and MMP-7 expression  association between loss of fibulin-5 expression and accumulation of nuclear β-catenin. While 64.6% (64/99) of tumors expressed either fibulin-5 or nuclear β-catenin, only 14.1% (14/99) were positive for both  association of fibulin-5 promoter methylation and β-catenin expression, with 76.9% (10/13) of tumors having fibulin-5 promoter methylation and nuclear β-catenin staining, compared to 35.3% (6/17) lacking fibulin-5 promoter methylation but expressing nuclear β-catenin  TCF-4 reporter, without affecting the control FOPFlash (OF) reporter  in the promoter region of MMP-7 . FibulinBEs Fig. , suggestMMP-7 reporter  was unable to suppress the ter Fig. , GSK3β Ster Fig. . We thenter Fig. , ΔRGD coter Fig. . Therefoin vivo. Fibulin-5 expression significantly (P = 0.0007) suppressed the growth of H1299 xenograft tumors in nude mice  databases were analyzed by using The UCSC Cancer Genomics Browser (https://genome-cancer.soe.ucsc.edu/proj/site/hgHeatmap/) [N = 1081) and DNA methylation  datasets were used to compare fibulin-5, MMP-7 and c-Myc expression and fibulin-5 DNA methylation, because these datasets include results from control normal tissues. Heatmap mode was used to display the results. Kaplan-Meier curves were generated using the Kaplan-Meier plotter program  as described [eatmap/) . The TCGescribed .fibulin-5 promoter methylation status, including 30 randomly selected lung tumors and the matched histologically normal lung parenchyma samples, were previously described [Tissue microarray slides containing 99 NSCLC samples were purchased from US Biomax . The information of these samples was summarized in escribed . Fibulinescribed . The sta2, and cultured in RPMI 1640 medium  supplemented with 10% defined fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin . Transfection of plasmids and small interfering RNA (siRNA) was performed using Lipofectamine™ 2000 (Invitrogen) as described [β-catenin and GSK3β in A549 and H1299 cells were knocked down using pre-made siRNA . β-catenin and GSK3β in H460 and H1752 cells were stably knocked down by transduction with pre-made lenti-viral short hairpin RNA (shRNA) vectors  as previously described [β-catenin (target sequence: 5′-TTGGAATGAGACTGCTGAT-3′) and shRNA #9374 for GSK3β (target sequence: 5′-ACTGATTATACCTCTAGTA-3′). The infected cells were selected by puromycin at 5 μg/mL for H460 cells, and 3 μg/mL for H1752 cells. Cell viability was analyzed for cells seeded at 2 × 103/well in 96-well plates using MTT assay kit  according to the manufacturer's instructions.Lung cancer cell lines were obtained from American Type Culture Collection . Cells were maintained at 37°C and 5% COescribed . Expressescribed . The shRCollection of cell lysates and western blotting were done as previously described (24). The antibodies used for western blotting included those for p-GSK3β (Ser9), ERK, p-ERK (Thr202/Tyr204) , cyclin D, c-Myc (9E10) (Santa Cruz Biotechnology), MMP7 , V5 (Invitrogen), β-actin , and β-catenin .For immunoprecipitation (IP), cells were harvested by centrifugation and washed once with PBS on ice. Cells were lysed for 15 min with RIPA buffer  containing 1:100 protease/phosphatase inhibitors (Sigma). After vortexing, the lysates were cleared by centrifugation, and then frozen at −80°C or used immediately for IP. For each IP reaction, lysate containing 500–600 mg proteins was used. Briefly, cell lysates were incubated with 2 mg of anti-FLAG (Sigma) or anti-Myc  at 4°C overnight, and then incubated with protein-G sepharose  at room temperature for 2 hr with rocking. After centrifugation, the beads were washed once with 500 μL RIPA buffer containing protease/phosphatase inhibitors, 3 times with 1 mL of RIPA buffer, and then boiled in 2× Laemmli sample buffer. Whole cell lysates prepared before IP were used as a control for input. Samples were run on an SDS-PAGE pre-cast gels at 100 V for 1 hr , blotted onto Immobilon-P PVDF membranes (EMD Millipore), and then blocked with either 5% non-fat dry milk or 5% BSA diluted in wash buffer (0.1% tween-20 in TBS). After incubation with primary and secondary antibodies at 4°C, blots were developed using the ECL chemiluminescent detection kit .CCND1, 5′-CCCTCGGTGTCCTACTTCAAA-3′/5′ CCAGGTTCCACTTGAGCTTGT-3′ and c-Myc, 5′-CCTCAACGTTAGCTTCACCAA-3′/5′-TTTGATGAA GGTCTCGTCGTC-3′. PCR reactions were performed on an MJ Mini Personal thermo cycler (Bio-Rad) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. The cycle conditions are available upon request.Total RNA was isolated from lung cancer cells using the RNAgents Total RNA Isolation System (Promega). First-strand cDNA was synthesized from 10 μg of total RNA using Superscript II reverse transcriptase (Invitrogen). The PCR primers include those for 6 cells. After incubation for 36 hr, cells passing through the filters into bottom wells were fixed with formalin and stained with crystal violet (Sigma). Cell numbers in 10 randomly selected fields (×200) from each well were counted.Invasion assays were performed in triplicate in six-well trans-well units with 8-μm filters coated with Matrigel (BD Biosciences) at 1:6 dilution. Each well was loaded with ~2 × 10MMP-7 luciferase reporters constructed using the pBV-Luc vector were previously described [MMP-7 reporter. In some experiments, cells were transfected with mutant fibulin-5 with deletion of its RGD motif (ΔRGD). After transfection, collection of cell lysates and measurement of luciferase activities were done as described [WT and mutant escribed . For repescribed . All repNuclear β-catenin in H1299 and A549 cells was analyzed by western blotting of β-catenin in nuclear fractions isolated from transfected cells using the NE-PER nuclear/cytoplasmic extraction kit (ThermoFisher) according to the manufacturer's instructions. Nuclear β-catenin in SW480 colorectal cancer cells and H1752 lung cancer cells were analyzed using immunofluorescence as previously described . Mountedad libitum. To analyze tumor growth, mice were injected subcutaneously in both flanks with parental and fibulin-5-expressing H1299 cells (5 × 106 cells/injection). Tumor volumes were calculated according to the formula 0.5 × length × width2. To analyze tumor metastasis, stable H460 and H1752 cells expressing lenti-viral β-catenin, GSK3β, or control scrambled shRNA were injected intravenously (i.v.) by tail vein into BALB/c nude mice. For each injection, 1 × 106 cells suspended in 200 μl PBS were used. Following sacrifice of mice at 6 weeks after injection, lung metastasis nodules were analyzed as previously described [All animal experiments were approved by the Institutional Animal Care and Use Committees at the University of Pittsburgh and Tianjin Medical University. Female 5- to 6-week-old BALB/c nude mice  were housed in a sterile environment with microisolator cages and allowed access to water and chow escribed . Tumors escribed . Tissuesescribed .P values < 0.05 were considered to be statistically significant. Means + one standard deviation were displayed in the figures.Statistical analyses were performed using GraphPad Prism V software.


PLOS Computational Biology, the premier journal in computational biology, is approaching its tenth anniversary. The task is daunting; not only has the field come a long way in ten years but it is broad with many advances to consider. In addition, since computational biology has become closely tied to experimental research, progress is not purely computational; it is tied to experiment. And that's as it should be. Ten years ago, computational biology was not entirely trusted by experimental biologists. By contrast, today computational biology is integrated in the community. It's easier for computational biologists to collaborate across disciplines. Laboratory scientists have a better understanding of the merit of computational models for hypothesis generation as well as the need to iterate between modeling and laboratory testing Computational biology has soared from being an auxiliary discipline to being a crucial element for progress in practically all aspects of the biological sciences. In this annual Editorial, I would like to step back, consider significant computational biology advances of the last decade, and reflect on some key challenges ahead. The timing is particularly appropriate. We have witnessed huge leaps in biological computing Formidable challenges include: the establishment of computer networks for surveillance of disease; mapping the pathways and biological networks associated with the initiation, growth and spread of cancer; predicting function and mutational dysfunction in disease from the structure of complex molecules; resolving the mechanisms of oncogenic mutations and the cellular network which is rewired in cancer; achieving accurate, efficient, and comprehensive dynamic models; and moving from artificial intelligence to the “connectome”—the connections among all of the neurons of the brain. Multiscale biological modeling—an area where vast progress has been made during the last decade—still faces major challenges. To tackle this aim, hybrid methods across disciplines, scales, and sources are essential. Hybrid methods integrate data from, for example, serial crystallography and time-resolved wide-angle X-ray scattering, micro- and nano-crystals for (future) free-electron lasers, electron microscopy, fluorescence resonance energy transfer (FRET), cross-linking data, small-angle X-ray scattering, crystallography, nuclear magnetic resonance (NMR), and more. Equally important is the development of protocols for model validation. We may expect an influx of models based on experimental data integration. If these are to be deposited in a public archival system, which is now a community aim, such clear protocols are essential for maintaining quality control. Finally, studying the dynamics of large integrated models is increasingly used to improve our understanding of how large complexes function in the cell and how they are regulated. The dynamics of such large associations provides an additional hugely complex layer; to date, we are still struggling to comprehend the dynamics of single molecules and their associations. This is compounded by the fact that large regions of the molecules can be disordered, and multiple temporal post-translational modifications take place, with different combinations spelling distinct functions. On a different level, improved tumor mutational analysis platforms and knowledge of the redundant pathways, which can take over in cancer, may not only supplement known actionable findings but forecast possible cancer progression and resistance. Such forward-looking can be powerful, endowing the oncologist with mechanistic insight and cancer prognosis, and consequently more informed treatment options.Lastly, the community faces the global challenge of linking genetics to phenotype, including the genetics of cancer. Genetics is mediated by dynamic conformational ensembles. Powerful ideas such as that of the free energy landscape PLOS Computational Biology has contributed to each advance and challenge. Manuscripts can be declined, for example, because of the absence of substantiating experimental data at the time, lack of sufficient rigor, or if the manuscripts included new experimental data, the authors may have opted for alternative journals. At the same time, it may also suggest that PLOS Computational Biology needs to be more open and receptive to new concepts. Differentiating between novel ideas that may lead to key advances and speculative propositions can, however, be challenging.The significance of any research advance and challenge—achieved or aspired to—is a matter of opinion. The list above is partial, incomplete, and possibly biased toward structural biology and cancer. Nonetheless, this list does indicate the magnitude of the tasks confronting computational biology as a discipline. In the absence of a meaningful way to quantify a journal's contribution to a field, it is unclear whether, and to what extent, PLOS Computational Biology aims to serve the biological community and welcomes manuscripts addressing all areas of computational biology. We encourage submission of research papers describing novel results that provide significant new insights into biological processes and of methods papers presenting new protocols for tackling key problems that have been shown, or have the promise to provide, new biological insights. We aspire to be the journal that will publish key computational advances in the next decade with the rigor that PLOS Computational Biology is known for. The PLOS Computational Biology editorial team seeks to identify and publish only the most outstanding papers, aiming to consider only those that are of exceptional quality. Our goal of furthering our understanding of living systems through the application of computational methods is shared with the International Society for Computational Biology (ISCB); together, we hope to meet the challenge.PLOS Computational Biology plans to publish a series of “Focus Features” addressing key areas of computational biology. We welcome suggestions from our community.Finally, for 2015, our tenth anniversary year,


Rac1 GTPase is known to stimulate β-catenin-dependent transcription of Wnt target genes and we confirmed this activity. Here we tested the recent hypothesis that Rac1 augments Wnt signaling by enhancing β-catenin nuclear import; however, we found that silencing/inhibition or up-regulation of Rac1 had no influence on nuclear accumulation of β-catenin. To better define the role of Rac1, we employed proximity ligation assays (PLA) and discovered that a significant pool of Rac1–β-catenin protein complexes redistribute from the plasma membrane to the nucleus upon Wnt or Rac1 activation. More importantly, active Rac1 was shown to stimulate the formation of nuclear β-catenin–lymphoid enhancer factor 1 (LEF-1) complexes. This regulation required Rac1-dependent phosphorylation of β-catenin at specific serines, which when mutated (S191A and S605A) reduced β-catenin binding to LEF-1 by up to 50%, as revealed by PLA and immunoprecipitation experiments. We propose that Rac1-mediated phosphorylation of β-catenin stimulates Wnt-dependent gene transactivation by enhancing β-catenin–LEF-1 complex assembly, providing new insight into the mechanism of cross-talk between Rac1 and canonical Wnt/β-catenin signaling. Summary: Evidence is presented for a novel role of Rac1 in stimulating β-catenin–LEF-1 complex formation. The canonical Wnt/β-catenin pathway plays a central role in embryonic development and adult tissue homeostasis in intestinal stem cell crypts, where it regulates the equilibrium between cell differentiation and proliferation. Wnt glycoprotein ligands attach to Frizzled transmembrane receptors and this culminates in the stabilization and nuclear translocation of β-catenin, leading to transactivation of Wnt target genes . In the Interestingly, cross-talk between Rac1, a small GTPase and critical regulator of actin polymerization and cell migration, and dysregulated Wnt signaling in colon adenocarcinoma cells has been reported . Rac1 isThe evidence for a role of Rac1 in controlling the nuclear localization of β-catenin is intriguing but controversial. A few studies reported a Rac1-dependent shift in β-catenin nuclear localization , while oTo evaluate the sensitivity of canonical Wnt pathway induction by Rac1, we used a LEF-1/TCF reporter assay called pTOPflash. HEK293T cells were transfected with plasmids encoding GFP, Rac1-WT-GFP (wild-type) or Rac1-Q61L-GFP (constitutively active) and assayed for their responses to LiCl, a Wnt agonist, in up-regulating expression of the luciferase reporter A. In theThere are two likely pathways (but not necessarily the only ones) by which Rac1 might increase Wnt/β-catenin-dependent gene activation as shown above. The favored mechanism currently proposed in the literature is that Rac1-mediated phosphorylation of β-catenin drives the nuclear translocation and accumulation of β-catenin . HoweverFig. S1A). Similar results were observed using a different Rac1 inhibitor, EHT 1864 . Furthermore, even when Rac1 expression was silenced by siRNA treatment we observed no change in β-catenin nuclear/cytoplasmic distribution or protein levels, as revealed by cell fractionation and western blot analysis  and fibroblastic (NIH 3T3) cell lines using an Olympus FV1000 confocal microscope. SW480, NIH 3T3 and HCT116 cells were treated for 6 h with 50 µM of the Rac1 inhibitor, NSC23766, which inhibits Rac1 by trapping it in a predominantly GDP (inactive) form, and then cells were fixed and immunostained for microscopy B. The Raanalysis D. TogethFig. S1C). Fluorescence confocal microscopy and visual scoring of the immunostained cells revealed that all mutants showed predominantly a nucleo-cytoplasmic distribution similar to that observed for WT-β-catenin . This result was striking given that SW480 cells express a mutant form of APC (amino acids 1–1337), which has been implicated as a prerequisite for Rac1-dependent nuclear targeting of β-catenin  and T17N mutants were transiently expressed in HCT116 cells in the absence and presence of LiCl or Wnt3a conditioned medium B. AccordIf Rac1 does not stimulate nuclear import of β-catenin, then how does it augment the Wnt pathway? We speculated that the site of Rac1–β-catenin complex assembly might provide some insights. To address this we investigated the subcellular locations at which Rac1–β-catenin complexes form, and asked whether this might change in response to Rac1 or Wnt activation. These issues have not previously been addressed.Fig. S2A); however, a more sensitive and consistent approach was required. To more effectively investigate Rac1–β-catenin complex localization we used an in situ microscopy approach using a proximity ligation assay (PLA). PLA is an antibody-based method in which two proteins are immunolabeled: first with primary antibodies and then with secondary antibodies conjugated to complementary oligonucleotides . Positive interactions were observed for both types of complex but their distribution patterns were significantly different  . This increase in nuclear complexes did not result from a direct effect of Wnt on Rac1 expression or distribution  but appeared to result indirectly from stabilization and increased levels of β-catenin.Next, to determine if Wnt stimulation affected the distribution of Rac1–β-catenin complexes, the PLA was repeated in the presence and absence of the Wnt agonist LiCl in HEK293T and NIH 3T3 cells. Cells were fixed and subjected to the Duolink PLA method using anti-β-catenin and anti-active Rac1 antibodies. In the untreated sample we observed a nucleo-cytoplasmic distribution of active Rac1–β-catenin complexes ; howeverFig. S3D–F). The reduction observed in LEF-1–β-catenin complex formation was not due to altered LEF-1 levels . Note that we were unable to detect an interaction between LEF-1 and Rac1 , so it is unlikely that all three proteins are in complex at chromatin. These results, when considered together, suggest that active Rac1 can stimulate complex formation between β-catenin and LEF-1 and contributes to the positive effect of Wnt on the assembly of β-catenin–LEF-1 complexes in cells  and treated for 6 h with: (i) a Wnt stimulus (Wnt3a conditioned media or 40 mM LiCl), (ii) a Rac1 inhibitor (50 µM NSC23766), or (iii) combination of both 50 µM NSC23766+Wnt3a. Cells were then fixed and subjected to Duolink PLA using rabbit anti-β-catenin and mouse anti-LEF-1 antibodies, and endogenous complexes between β-catenin and LEF-1 were then detected as red dots by fluorescent microscopy . This suggested that S191 and S605 may contribute to binding of LEF-1. To compare the interaction between these Rac1-specific mutants of β-catenin and LEF-1, a PLA was used. NIH 3T3 cells were co-transfected with myc-tagged β-catenin constructs  and HA-LEF-1, and then anti-myc and anti-HA antibodies were used for the assay. To confirm specificity of the in situ assay each antibody was used in isolation to ensure that no cross-reactivity was present (P<0.0001) and even more substantially disrupted by the S605A mutation (P<0.0001)  and pHA-LEF-1. When analyzed by fluorescence microscopy, we observed that ectopically expressed LEF-1 (stained red) caused a stable retention of WT-β-catenin (stained green) in the nuclei of >90% of transfected cells (see images in <0.0001) C,D. It iWe next used immunoprecipitation assays to measure binding efficacy between myc-tagged forms of β-catenin and LEF-1, using an anti-HA antibody to pull down ectopic HA-LEF-1. Remarkably, the trends observed in the immunoprecipitation experiments E were alIn this study we demonstrate that Rac1 does not affect the nuclear-cytoplasmic distribution of β-catenin in the absence or presence of Wnt, contrary to current theories . Our finFig. S1C) or nuclear import rate  coupled with confocal microscopy to image the localization patterns of hundreds of endogenous Rac1–β-catenin complexes throughout the cell. In untreated cells, Rac1–β-catenin complexes were mainly located at the plasma membrane, which was unsurprising given their roles in cell adhesion and migration . Wnt stiInteractions between active Rac1 and β-catenin have been previously reported in the nuclei of cells ; howeverThe overexpression of LEF-1 drives nuclear accumulation of wild-type β-catenin . NotablyDue to the tumor-promoting and cell migration-inducing activities of Rac1 and accumulated β-catenin in colon cancer   and the 2-humidified incubator. LiCl  was dissolved in water and made fresh at each use. NSC23766  or EHT1864  were dissolved in water and stored at −20°C in aliquots to avoid repeated freeze–thaw cycles. Drugs were diluted to the required concentration in media immediately prior to administration. The optimal non-toxic and effective dose was determined for each drug by titration, starting with functional concentrations reported in the literature. For transfection, cells were seeded onto coverslips or in 8-well chamber slides 24 h prior to transfection. Transfections were carried out using Lipofectamine 2000  according to manufacturer's instructions and media were replaced 6 h post-transfection and incubated for a total of 48 h.SW480 and HCT116 human colon cancer epithelial cells, NIH 3T3 mouse fibroblasts and HEK 293 embryonic kidney cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin) at 37°C in a 5% COFor the preparation of L and Wnt3a media, cells were seeded into T150 flasks (1:10 dilution) with complete DMEM. Cells were incubated at 37°C and the first batch of media was harvested after 4 days. Fresh DMEM was added to the cells and the second batch of media was harvested after 3 days. The first and second batches of harvested media was combined, filtered and stored at −20°C.HEK293T cells were transiently co-transfected with GFP-tagged Rac1 constructs (WT or Q61L) and β-catenin–TCF luciferase reporter constructs, TOPflash, which contains multiple optimal TCF/LEF-1 binding sites that induce transcription of a luciferase reporter gene when activated by β-catenin, or a negative control FOPflash, which contains mutant and inactivated TCF/LEF-1 binding sites. Forty-eight hours after transfection, cells were treated with 40 mM LiCl for 6 h prior to harvesting. Cells were then washed with 1× PBS and lysed with 50 µl luciferase buffer . Lysates were collected after centrifugation  and assayed for luciferase activity according to manufacturer's instructions . The luciferase activities were normalized against the levels of GFP expression and presented as a TOPflash/FOPflash ratio. Each experiment was performed in duplicate and repeated three times.2 First Strand Kit  according to manufacturer's instructions. Gene transcripts were quantified by real-time (RT) PCR using RT2 SYBR Green  and GAPDH was used as a control. The following primer sets were used: human β-catenin forward primer 5′-CAGAAGCTATTGAAGCTGAGG-3′ and the reverse primer 5′-TTCCATCATGGGGTCCATAC-3′; human c-myc forward primer 5′-CTTCTCTCCGTCCTCGGAT- TCT-3′ and the reverse primer 5′-GAAGGTGATCCAGACTCTGACCTT-3′; human Axin2 forward primer 5′-ACTGCCCACACGATAAGGAG-3′ and the reverse primer 5′-CTGGCTATGTCTTTGGACCA-3′; human Cyclin D1 forward primer 5′-AAGTGCGAGGAGGAGGTCTT-3′ and the reverse primer 5′-GGATGGAGTTGTCGGTGTAGA-3′; human LEF-1 forward primer 5′-CTTTATCCAGGCTGGTCTGC-3′ and the reverse primer 5′-TCGTTTTCCACCATGTTTCA-3′; human GAPDH forward primer 5′-GAGTCAACGGATTTGGTCGT-3′ and the reverse primer 5′-GACAAGCTTCCCGTTCTCAG-3′.HEK293T cells were transfected with GFP alone or Rac1-WT-GFP and treated with Wnt3a conditioned media (6 h) before being sorted into a GFP-rich pool using fluorescence-activated cell sorting (FACS). Total RNA was extracted using the RNAeasy mini kit , quantified by UV spectroscopy and the integrity was confirmed by gel electrophoresis. One microgram of RNA was reverse transcribed using the RTAll RT-PCR experiments were performed in triplicate and repeated twice on a Stratagene Mx3000 qPCR system .SW480 cells treated with 50 µM NSC23766 for 6 or 24 h or SW480 cells 72 h after siCONTROL or siRAC1 transfection were harvested and washed with PBS. The nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents  according to the manufacturer's manual. Total lysates were prepared using Pierce RIPA buffer  according to manufacturer's protocol. Samples were quantified by Bradford analysis and measured at 595 nm with a microplate reader. Normalized amounts of the samples were denatured in SDS sample buffer for 10 min at 95°C before being separated by SDS-PAGE and blotted onto polyvinylidene (PVDF) membranes (Millipore). Membranes were blocked with 5% non-fat dry milk-TBS before incubating with primary antibodies at room temperature for 2 h. Topoisomerase II and β-tubulin antibodies were used as nuclear-cytoplasmic loading controls, respectively. After being washed with 0.1% Tris-buffered saline and Tween 20 (TBST), membranes were incubated with anti-mouse/rabbit horseradish peroxidase conjugated (HRP) secondary antibodies (Sigma-Aldrich) for 1 h at room temperature. Western Bright ECL Western Blotting Kit  was used to detect chemiluminescence, and blots were imaged using the ChemiDoc MP Imaging system .At 48 h post-transfection, NIH 3T3 cells or HEK293 cells were harvested and lysed in Pierce IP lysis buffer . The lysates were centrifuged for 10 min at 13,000 r.p.m. at 4°C. One milligram of lysate was incubated with specific antibodies [e.g. anti-HA monoclonal antibody  or mouse IgG ] overnight at 4°C with continuous end-over-end mixing. Thirty microliters of protein-G–agarose  beads were added to the samples and incubated for a further 2 h at 4°C with continuous end-over-end mixing. Samples were then washed once with Pierce IP buffer and then with Tris-buffered saline (TBS) before being denatured in SDS sample buffer for 7 min at 95°C. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes . Membranes were blocked with 5% non-fat dry milk for 1 h and then incubated with anti-myc , anti-HA  or anti-GFP (Invitrogen) polyclonal antibodies for 2 h at room temperature. After being washed three times in 0.1% TBST, membranes were incubated for 1 h at room temperature with anti-rabbit HRP secondary antibody . Results were visualized by the Chemidoc MP imaging system (Bio-Rad).Cells were washed three times with phosphate-buffered saline (PBS), fixed with 3.7% Formalin-PBS  then permeabilized with 0.2% Triton-X-100-PBS  for 10 min, blocked with 3% BSA-PBS  and incubated with primary antibodies for 1 h at room temperature. Cells were washed twice with PBS before secondary antibodies were added for 40 min. Cells were subsequently mounted with Vectashield  and visualized by fluorescence microscopy and imaged and scored using an Olympus FV1000 confocal laser microscope. Fluorescence recovery after photobleaching (FRAP) assay to measure nuclear import rates of β-catenin-GFP in live NIH3T3 cells was as described previously , using aBackground values were subtracted from cytoplasmic and nuclear fluorescence intensities, which were then expressed as a nuclear/cytoplasmic ratio. For each cell data set, the pre-bleach ratio was set to 100%, the time for the first post-bleach image was set to 0 s, and the recovery curve was adjusted to start at 25% recovery at time 0 . The average of the data from two experiments was plotted. Initial nuclear import rates for the first 30 s were analyzed with GraphPad Prism 5.0 software using linear regression analysis.z-stack images were taken using an Olympus FV1000 confocal laser scanning microscope with 60× water objective at 0.2 µm. Over 100 cells were imaged for analysis and each red puncta within the nuclei of cells was scored as a single interaction site.Cells cultivated for 48 h in 8-well chamber slides  were fixed with 3.7% formalin in PBS for 30 min before being permeabilized with 0.2% Triton-X-100 in PBS for 10 min. Cells were then subjected to the PLA assay using the Duolink red kit  in a humidity chamber according to the manufacturer's instructions. Briefly, plastic wells were removed from the chamber slides prior to blocking and incubation with primary antibodies and PLA probes (containing oligonucleotides) for 1 h. Ligase was then added to hybridize PLA probes and generate a closed circle before DNA amplification and conjugation to Alexa Fluor 594 for 100 min. After the PLA protocol, cells were then counterstained with anti-β-catenin mAb, myc pAb or HA mAb and Alexa Fluor 488 or 405 to visualize β-catenin and HA staining. Cells were then mounted onto coverslips with Duolink mounting media with 4′,6-diamidino-2-phenylindole  or Vectashield . t-test. Differences were considered to be statistically significant when P<0.05.Data analysis was performed using Statview  or GraphPad Prism 5 software by one-way ANOVA or Student's


Here, we report the biphasic change of renal β-catenin expression in mice with overload proteinuria in which β-catenin was upregulated at the early stage (4 weeks after disease induction) but abrogated at the late phase (8 weeks). Acute albuminuria was observed at 1 week after bovine serum albumin injection, followed by partial remission at 4 weeks that coincided with overexpression of renal tubular β-catenin. Interestingly, a rebound in albuminuria at 8 weeks was accompanied by downregulated tubular β-catenin expression and heightened tubular apoptosis. In addition, there was an inverse relationship between Dickkopf-3 (Dkk-3) and renal tubular β-catenin expression at these time points. In vitro, a similar trend in β-catenin expression was observed in human kidney-2 (HK-2) cells with acute (upregulation) and prolonged (downregulation) exposure to albumin. Induction of a proapoptotic phenotype by albumin was significantly enhanced by silencing β-catenin in HK-2 cells. Finally, Dkk-3 expression and secretion was increased after prolonged exposure to albumin, leading to the suppression of intracellular β-catenin signaling pathway. The effect of Dkk-3 on β-catenin signaling was confirmed by incubation with exogenous Dkk-3 in HK-2 cells. Taken together, these data suggest that downregulation of tubular β-catenin signaling induced by Dkk-3 has a detrimental role in chronic proteinuria, partially through the increase in apoptosis.Studies on the role of Wnt/ The stabilized non-phosphorylated β-catenin translocates into the cell nucleus and works together with T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) transcription factors to trigger the transcription of Wnt target genes. Hence, the persistence of intracellular β-catenin accumulation increases the activity of Wnt/β-catenin signaling pathway. Recently, the Dickkopf family has been recognized as an important regulator of this signaling pathway, but little is known for its involvement in proteinuric nephropathy.23The canonical Wnt/β-catenin signaling pathway has been implicated in cystic kidney disease,25 acute kidney injury (AKI) and diabetic nephropathy (DN).26 Recent evidence have indicated that aberrant Wnt/β-catenin activities in renal cells contributed to epithelial–mesenchymal transition (EMT),27 fibrosis29 and apoptosis during nephropathy.30 However, data on the role of Wnt/β-catenin signaling in proteinuric nephropathy remains scarce. In this study, we presented in vitro and in vivo evidence that protein overload induced apoptosis in proximal tubular epithelial cells (PTECs) via downregulation of Wnt/β-catenin signaling pathway. In addition, the upregulation of Dickkopf-3 (Dkk-3) may act as a novel autocrine mechanism that suppresses Wnt/β-catenin signaling upon protein overload in PTECs.In the kidney, canonical Wnt/β-catenin signaling was evident by the translocation of stabilized β-catenin into cell nuclei. After 2 days of treatment, HSA-treated cells showed significant downregulation of active β-catenin expression in the cell nucleus. By day 4, the reduction of both nuclear and cytosolic β-catenin expression was further decreased to approximately 50% compared with untreated HK-2 cells  cells after 60-min incubation with fluorescein (FITC)-labeled human serum albumin HSA; . In HSA--2 cells . Immunofed cells . Lucifered cells . These dβ-catenin signaling. We postulated that these molecules would be upregulated by protein overload in HK-2 cells. As shown in β-catenin signaling.31 We also showed a twofold increase in secreted Dkk-3 protein levels in culture medium by ELISA assay -mediated gene silencing of β-catenin. The expression of β-catenin was decreased gradually after 2 days in HSA-treated HK-2 cells, whereas the suppression of β-catenin was markedly enhanced and sustained in cells transfected with siRNA  or saline injection for 8 weeks . UninephRenal cortical expression of AKI markers  were upregulated after BSA injection for 1 week. Notably, the expression of KIM-1 and NGAL was further upregulated after 8 weeks of BSA injection .β-catenin signaling pathway was investigated in the kidney of mice after 4 and 8 weeks of BSA injection. Nuclear β-catenin expression in cortical lysates was increased significantly at 4 weeks but decreased at 8 weeks as shown by western blot  or repair  – has been a subject of intense debate.32 Here, we reported for the first time the dynamic correlation between protein overload and Wnt/β-catenin signaling pathway and suggested that the regulatory protein Dkk-3 may mediate renal tubular apoptosis and the ensuing renal injury.Progressive CKD is effected through a complex multiplicity of tubular cell signalings,β-catenin is one of the crucial scaffold proteins that forms a complex with E-cadherin to support the tubular epithelial architecture. It is postulated that β-catenin could dissociate from the cell membrane and contribute to the cytosolic-free β-catenin equilibrium pool.33 Once the cytosolic β-catenin is stabilized, it translocates to the cell nucleus and triggers target gene transcription. To mimic the persistent proteinuric state in renal tubules of CKD patients, we used an in vitro HK-2 cell culture system with prolonged exposure to HSA treatment (4 days). We first demonstrated that the membrane-bound β-catenin expression was reduced in protein overloaded HK-2 cells. The expression of active nuclear β-catenin, which drives TCF/LEF promoter activity, was also diminished in PTECs upon the protein stress. These data confirmed that stress from protein overload downregulated Wnt/β-catenin signaling pathway in renal tubular cells. In agreement with this, stress from exposure to cyclosporine A or high glucose has also been associated with the suppression of Wnt/β-catenin signaling pathway.35Besides acting as a transcription factor, β-catenin signaling pathway in different disease models,37 however, the role of Dkk-3 is largely unknown. Here, we found that protein overload upregulated Dkk-3 synthesis and secretion in HK-2 cells. Exogenous Dkk-3 protein also inhibited intracellular β-catenin expression in an autocrine manner. Our finding that Dkk-3 suppressed tubular Wnt/β-catenin signaling in proteinuric nephropathy was in line with other experiment, which showed that Dkk-3 is an inducer of apoptosis38 and inhibition of this protein leads to the activation of Wnt/β-catenin signaling in lung cancer.39 Our findings should ideally be confirmed using cell-specific Dkk-3 knockout animal models, or using a Dkk-3 neutralizing antibody. However, both are currently not readily available.Dkk-1 is the most well-known endogenous inhibitor of Wnt/β-catenin signaling pathway is known to regulate cell fate, and it is not surprising that its activation allows continuous cell growth as observed in carcinogenesis.40 In chronic kidney injury, protein overload causes tubular injury via induction of cell death. Hence, we postulated that downregulation of tubular Wnt/β-catenin expression by protein overload promoted tubular apoptosis. Indeed, we found that gene silencing of β-catenin in HK-2 cells enhanced HSA-induced apoptosis as demonstrated by the upregulation of apoptotic markers, including the Bax/Bcl-2 gene expression ratio, caspase-3 activity and TUNEL-positive cells.Wnt/β-catenin signaling has been implicated in other disease models that are associated with interstitial inflammation such as diabetes35 and the murine model of unilateral ureter obstruction.37 In AKI, ablation of β-catenin in tubular cells aggravated kidney damage with increasing cell death. Conversely, tubular cells were protected from apoptosis by Wnt1 or stabilization of β-catenin30 demonstrating the differential roles of β-catenin in kidney injury and repair. Here, we showed evidence for the first time that β-catenin may have dual roles during longitudinal disease progression in the protein overload model.Wnt/In addition to apoptosis, expression levels of AKI markers  were also upregulated in the renal cortex of mice with the highest proteinuria at 1 week after protein overload. Interestingly, intrarenal KIM-1 and NGAL expression levels were sustained up to 8 weeks after BSA injection, indicating that the persistent acute injury response was extended to the chronic phase.β-catenin expression. Notably, amelioration of UACR level coincided with the overexpression of tubular β-catenin at the early phase (4 weeks). Conversely, elevation of UACR level correlated with the decline in tubular β-catenin expression at the late phase (8 weeks). It is noteworthy that the trend of tubular β-catenin expression under protein overload was also in line with our in vitro results. Together with the observation of increased tubular apoptosis toward the late phase of murine protein overload model, we speculate that downregulation of Wnt/β-catenin signaling is a driving force for both proteinuria and tubular cell death. This phenomenon suggests that heightened tubular cell death casts a negative impact on the degree of proteinuria. One possible underlying mechanism of suppressed Wnt/β-catenin signaling is the induction of Dkk-3. The expression of Dkk-3 was downregulated at the early stage of the protein overload and upregulated thereafter. In agreement with our in vitro data, renal Dkk-3 expression was negatively correlated with tubular β-catenin expression at the respective time point, suggesting that prolonged protein overload induced tubular Dkk-3 expression and suppressed tubular β-catenin expression.Fluctuations in UACR appeared to be associated with the level of β-catenin signaling pathway that unleashes the downstream detrimental events. Maintenance of tubular β-catenin signaling could be renoprotective as it has anti-apoptotic property and partially ameliorates proteinuria. Further investigation is warranted to exploit this pathway in order to unveil new therapeutic targets for proteinuric CKD.Taken together, our data support the notion that upregulation of renal Dkk-3 expression in chronic proteinuric nephropathy inhibits tubular Wnt/μg/ml) and 10% fetal bovine serum , at 37 °C and 5% CO2. HK-2 cells were grown to subconfluence and rendered in medium without FBS for 24 h prior further treatments. HSA (5 mg/ml) was obtained from CSL laboratory  and applied to HK-2 cells for up to 4 days, with culture media changed every 2 days. HK-2 cells were also treated with 0.1 μg/ml human recombinant Dkk-3 .An immortalized human PTEC cell line derived from normal kidney named HK-2 cells was purchased . HK-2 cells were grown in Dulbecco's modified Eagle's medium , supplemented with penicillin (100 IU/ml) and streptomycin  at the dose of 10 mg/g body weight for 5 consecutive days weekly, up to 4 and 8 weeks. Control mice were injected with equal volume of saline. Twenty-four-hour urine and blood serum were collected at 1, 4 and 8 weeks after BSA injection. Mice were killed at 4 and 8 weeks after BSA injection and kidney tissues were collected. Each of the kidney tissue was dissected into half; one half was snap-frozen in liquid nitrogen and stored at −70 °C, the other half was fixed with 10% formalin and embedded in paraffin for immunohistochemistry examinations. RNA and protein were extracted from frozen kidney cortices.This study was approved by the Committee on the Use of Live Animals in Teaching and Research  and was performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The protocol of protein overload model was carried out according to previous study.HK-2 cells were transfected with siRNA targeting CTNNB1  by Lipofectamine 2000 (Invitrogen) in Opti-MEM I Reduced Serum medium (Applied Biosystems) for 6 h, followed by incubation with fresh DMEM/F-12, GlutaMAX medium for another 18  h before they were subjected to HSA treatment. The knockdown efficiency was verified by immunoblotting assay.β-actin expression. All experimental groups were compared with their respective control group using SDS software (Applied Biosystems).Total RNA was isolated from HK-2 cells and mouse kidney cortical tissues using Trizol (Invitrogen) and NucleoSpin Triprep , respectively. RNAs was reversely transcribed according to the manufacturer's instructions (Applied Biosystems). Quantitative real-time PCR was performed using ABI 7500 Real-Time PCR System (Applied Biosystems) with SYBR Green reagent (Applied Biosystems) and specific primers . Relativβ-catenin, total caspase-3, total caspase-8 , Dicckopf-3, TATA-binding protein  and β-actin (Thermo Scientific). The membranes were then incubated with peroxidase conjugated secondary antibodies  accordingly and visualized with ECL prime chemiluminescnece  by using ChemiDoc XRS+ system . Protein expression levels were quantified and normalized to β-actin/TBP by Image Lab software (Bio-Rad).Total protein, cytosolic and nuclear protein were extracted from HK-2 cells using lysis buffer and NE-PER extraction kit  respectively, containing protease inhibitor cocktail (Sigma-Aldrich). For mouse kidney tissue, total protein were extracted by NucleoSpin Triprep or fractionized by the NE-PER extraction kit. Protein concentration was quantified by BCA Protein Assay Kit (Thermo Scientific). Equal amount of protein was resolved by 12% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% non-fat milk, the membranes were subjected to overnight primary antibody incubation using antibody against active The level of Dkk-3 from HK-2 culture medium was measured by a commercial ELISA kit (Abcam). Mouse urinary albumin was measured by mouse albumin ELISA quantitation kit . Urine creatinine and serum BUN were quantified by enzymatic assays .HK-2 cells were subjected to the albumin or siRNA treatment as described and lysed for caspase-3 activity assay, according to the manufacturer's protocol of ApoAlert Caspase Colorimetric Assay Kit . Briefly, the cell lysates were incubated with the chromophore p-nitroaniline (pNA) linked with the specific caspase-3 substrate. Caspase-3 activity was measured by spectrophotometric detection of released pNA after the cleavage by active caspase-3.μm) were de-paraffinized, rehydrated with concentration gradient of ethanol. The sections were subjected to antigen retrieval step, by microwave-based method in 10 mM citrate buffer, or incubation with 20 ug/ml proteinase k  for 10 min Then, the sections were quenched in 3% hydrogen peroxide for 10 min and incubated with 2% BSA to block nonspecific binding. Primary antibodies against β-catenin , aquaporin-1 and active caspase-3 (Abcam) were added to the sections overnight and subsequently with secondary peroxidase conjugated antibodies (Dako) or alkaline phosphatase conjugated antibodies  for 1 h. The sections were developed by using DAB substrate from the Envision Plus system (Dako) or HIGHDEF red chromogen (Enzo Life Sciences), followed by counterstain with CAT hematoxylin . The slides were then dehydrated with ethanol and xylene before mounting.Paraffin-embedded kidney tissue sections (4 β-catenin (BD Bioscience) overnight thereafter incubated with the corresponding FITC-conjugated secondary antibody (Dako). The chamber slides were mounted with ProLong antifade mounting medium (Invitrogen) and examined under fluorescence microscope .HK-2 cells were seeded on chamber slides (Thermo Scientific) and incubated with HSA or Dkk-3 treatments. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich), followed by permeabilization in 0.25% Triton X 100 and 2% BSA (Sigma-Aldrich) incubation. The slides were then incubated in primary antibody against Nano-Glo Dual-Luciferase Reporter Assay System  was used to measure the TCF/LEF promoter activities. HK-2 cells were seeded in 96-well plate and co-transfected with pGL4.53 firefly luciferase (Fluc) and pGL4 (luc2CP/TCF-LEF RE/Hygro) NanoLuc luciferase (Nluc) plasmid using Lipofectamine 2000 (Invitrogen). Transfected cells were treated HSA and Dkk-3 for the respective duration lengths, then lysed with lysis buffer with Fluc substrate and incubated on an orbital shaker for 10 min. Fluc signals were then quenched and following by reaction with Nluc substrate. The signals in arbitrary unit (AU) from both Nluc and Fluc were measured by a luminometer . The promoter activities were calculated by the ratio of respective AU values of Nluc/Fluc.FITC-conjugated HSA, 1 mg/ml  was treated to HK-2 cells for albumin uptake measurement. HK-2 cells were seeded in 96-well black plate with clear bottom and treated with the labeled albumin for the respective time points. Cells were lysed with 0.1% SDS and the fluorescence intensities were measured by a FLUOstar Omega Microplate Reader (BMG Labtech).In Situ Apoptosis Detection Kit . The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with ethanol:acetic acid, 2 : 1 (v : v) and followed by protocol according to the manufacturer's instructions. Cells were counterstained with Methyl Green (Dako) and were examined under light microscopy (200X). Images were taken on non-overlap areas, TUNEL-labeled cells/total cell number ratios were counted for individual image and averaged for each experimental sample.HK-2 cells were seeded in chamber slides and treated with HSA or siRNA transfection. Apoptotic cells were detected by ApopTag Peroxidase In Situ Cell Death Detection kit, POD . For mouse tissue sections, approximately 10 images of cortical area with at least one glomerulus (400X) were taken. TUNEL-labeled cells/total cell number ratios were counted for individual image and averaged for each experimental sample.Deparaffinized, rehydrated and antigen retrieved sections of mouse tissue were subjected to apoptotic analysis by t-test were used to calculate the difference between experimental groups. GraphPad Prism v.4  was used to evaluate the data. P<0.05 was considered statistically significant.All data were expressed as mean± S.D. from three independent experiments. Two/one-way ANOVA with Tukey's multiple comparison test or


AbstractThis paper is an addendum for the availability of the names of 11 new species proposed in  PageBreakEupelmus, but only the acronyms of the depositories where the holotypes are deposited were given in the publication. The full names and location of the depositories were provided, but only in a Word document (Appendix S2) as part of the Supplementary Information (SI) available on the website of Systematic Entomology (http://onlinelibrary.wiley.com/doi/10.1111/syen.12089/suppinfo). Article 16.4 of the International Code of Zoological Nomenclature states that “Every new specific and subspecific name published after 1999,... must be accompanied in the original publication... where the holotype or syntypes are extant specimens, by a statement of intent that they will be (or are) deposited in a collection and a statement indicating the name and location of that collection” confususHolotype ♀. FRANCE: Var, Fayence, 43.61774°N, 06.69774°E, 17.iii.2012, emerged 25.iii.2012, ex PageBreakPageBreakDiplolepisrosae on Rosacanina (N. Ris) (1 ♀) [FAL1195/10206] (in MNHG). Paratypes. CYPRUS: Lemesos, 6 km N of Lemesos, 24–25.V.2009, N34.73189°, E33.05175°, pods of carob tree with Apomyeloisceratoniae & Asphondyliagennadii (Fusu L. & Popovici O.) (5 ♀ 6 ♂ not sequenced) (in AICF) (1 ♀) [LF.ma.CY 01/10427] (in AICF); 6 km N of Lemesos, 25. V. 2009, sweep net, N34.727028°, E33.052278° (Fusu L. & Popovici O.) (1 ♀ 10 ♂ not sequenced)(in AICF). FRANCE: Alpes-Maritimes, Biot, N43.63455°, E7.08249°, 11.iii.2012, emerged 27.iii.2012, ex Andricuskollari on Quercuspubescens (N. Ris) (2 ♀)  (in FALPC); Alpes-Maritimes, Opio, N43.64479°, E6.99957°, 04.x.2012, (F. Al khatib & P. Gory) (1 ♀) [FAL1485/10313] (in FALPC); Alpes-Maritimes, Pégomas, N43.58844°, E6.93612°, 08.vi.2012, emerged 11.vi.2012, ex Myopitesstylata on Dittrichiaviscosa (F. Al khatib & P. Gory) (1 ♀) [FAL1429/10433] (in GDPC); Alpes-Maritimes, Sophia-Antipolis, N43.62443°, E7.03667°, 21.ii.2012, emerged 27.ii.2012, ex Myopitesstylata on Dittrichiaviscosa (F. Al khatib) (1 ♀ 1 ♂)  (in MNHG); Alpes-Maritimes, Sophia-Antipolis, N43.61669°, E7.03722°, 07.vi.2012, emerged 16.vi.2012, ex Biorhizapallida on Quercuspubescens (F. Al khatib & P. Gory) (1 ♀) [FAL1338/10227] (in MNHN); Alpes-Maritimes, Villars-sur-Var, N43.93730°, E7.08068°, 14.iii.2012, emerged 18.iii.2012, ex Diplolepisrosae on Rosacanina (F. Al khatib & N. Ris) (2 ♀) ; Ardèche, Saint-Georges-les-Bains, N44.85028°, E4.82433°, 13.vi.2012, emerged 14.vi.2012, ex Biorhizapallida on Quercuspubescens (F. Al khatib & M. Thaon) (2 ♀)  (in CBGP); Ardèche, Saint-Georges-les-Bains, N44.85028°, E4.82433°, 13.vi.2012, emerged 11.vii.2012, ex Dryocosmuskuriphilus on Castaneasativa (M. Thaon) (1 ♀ 1 ♂)  (in GDPC); Ardèche, Saint-Georges-Montpellier, N43.6104°, E3.77227°, ix.2011, emerged ix.2011, ex Bactroceraoleae on Oleaeuropaea (L. Brancaccio & M. Thaon) (1 ♀ 1 ♂)  (in MNHN); Aude, Durban-Corbières, N42.99825°, E2.80690°, 27.iii.2012, emerged 31.iii.2012, ex Myopitesstylata on Dittrichiaviscosa (F. Al khatib & N. Ris) (1 ♀) [FAL1122/10175] (GDPC); Bouches-du-Rhône, La Ciotat, garden, 09.I.2011 emerged 13-30.IV.2011, Lasiopteracarophila on Foeniculumvulgare (H. Dumas)  (1♀) [LF.ma.FR 01/10422] (in AICF); Gard, Garons, N43.76371°, E4.42588°, 11.i.2012, emerged 27.ii.2012, ex Myopitesstylata on Dittrichiaviscosa (N. Ris) (1 ♀) [FAL1092/10162] (in MNHG); Gard, Roquemaure, N44.03148°, E4.72747°, x.2011, emerged x.2011, ex Bactroceraoleae on Oleaeuropaea (N. Borowiec) (1 ♀) [FAL1274/10447] (in CBGP); Haute-Corse, Aléria, N42.12861°, E9.46555°, 22.ix.2011, ex seeds of Asphodelusramosus infested by Bruchophagus sp.  (2 ♀ 1 ♂)  (in MNHG); Haute-Corse, Lumio, N42.55879°, E8.81299°, 23.ix.2012, emerged 28.ix.2012, ex Bactroceraoleae on Oleaeuropaea  (2 ♀)  (MNHN); Haute-Corse, Piedicorte di Gaggio, N42.22166°, E9.26527°, 22. ix.2011, ex seeds of Asphodelusramosus infested by Bruchophagus sp.  (2 ♀ 3 ♂)  (in MNHN); Hérault, Causses-et-Veyran, N43.47131°, E3.08508°, x.2011, emerged x.2011, ex Bactroceraoleae on Oleaeuropaea (A. Auguste-Maros) (1 ♀) [FAL1254/10453] (in FALPC); Hérault, Frontignan, N43.43926°, E3.74145°, 17.vi.2012, emerged 19.vi.2012, ex Myopitesstylata on Dittrichiaviscosa (F. Al khatib & N. Ris) (1 ♀) [FAL1446/10309] (FALPC); Hérault, Laroque, 250-400 m, N45.91722°, E3.74361°, 05.vii.2013, sweeping on Quercuspubescens (G. Delvare), (1 ♀) [4173/10596] (in GDPC); Hérault, Mèze, N43.41670°, E3.6000°, x.2011, emerged x.2011, ex Bactroceraoleae on Oleaeuropaea (N. Ris) (2 ♀) ; Monaco, Monaco, N43.73263°, E7.41369°, x.2010, ex Bactroceraoleae on Oleaeuropaea  (1 ♀) [FAL1247/10436] (in CNC); Pyrénées-Orientales, Argelès-sur-Mer, N42.581000°, E3.010910°, x.2011, emerged x.2011, ex Bactroceraoleae on Oleaeuropaea (N. Ris) (4 ♀ 1 ♂) ; Pyrénées-Orientales, Banyuls-sur-Mer, 04.ii.2012, emerged 20.ii.2012, ex Myopitesstylata on Dittrichiaviscosa (J. Lecomte) (1 ♀) [FAL1100/10164] (in CBGP); Pyrénées-Orientales, Banyuls-sur-Mer, N42.47194°, E3.14333°, 250 m, 21.iii.2010 ex galls of Timaspisphoenixopodos on Lactucaviminea (G. Delvare & J. Lecomte) (2 ♀)  (in GDPC); Pyrénées-Orientales, Banyuls-sur-Mer, N42.46972°, E3.12388°, 10 m, 21.iii.2010 ex galls of Timaspisphoenixopodos on Lactucaviminea (G. Delvare & J. Lecomte) (1 ♀) [GDEL4003/3302] (in GDPC); Pyrénées-Orientales, Calce, N42.7348°, E2.75471°, x.2011, emerged x.2011, ex Bactroceraoleae on Oleaeuropaea (N. Borowiec & L. Brancaccio ) (2 ♂) ; Pyrénées-Orientales, Perpignan, N42.67720°, E2.86912°, 18.vi.2012, emerged 19.vi.2012, ex Myopitesstylata on Dittrichiaviscosa (F. Al khatib & N. Ris) (1 ♀) [FAL1455/10312] (in FALPC); Var, Rians, N43.57352°, E5.77148°, 31.ii.2012, emerged 09.iii.2012, ex Diplolepisrosae on Rosacanina, (N. Ris) (2 ♀ 1 ♂) ; (1 ♀) [FAL1195/10207] (in MNHN), same data as holotype. GREECE: Seres, Kerkini Lake Nat.Park, Kerkini Mts near Vironeia, 300 m, N41.27833°, E23.21955°, sweep net 22.VI.2008  (1 ♀) (in AICF) (1 ♀) [LF.ma.GR 01/10425] (in AICF); Seres, Kerkini lake, Krousia Mts. Site, N41.20180°, E23.07747°, Malaise trap, 12-18.IX.2007 (G. Ramel) (1 ♀) [LF.ma.GR 02/10426] (in AICF); Seres, Kerkini Mts., Plateaux Beech, N41.28580°, E23.03368°, Malaise trap, 08.VIII to 13.VIII.2007, (G. Ramel) ; Seres, Kerkini Lake N. Park, nr Kerkini, Pumping St. Site, N41.19760°, E23.08883°, 13.VI to 19.VI.2007, Malaise trap (G. Ramel) ; Seres, Kerkini Lake N. Park, Kerkini, Krousia Mts site, 190 m, N41.20180°, E23.07747°, 06.VI-12.VI.2007, Malaise tr. (G. Ramel) ; same data but 13.VI-19.VI.2007 ; same data but 20.VI-26.VI.2007 . IRAN: Kerman Prov., Bidkhan, 2897 m, N29°34.956’ E 56°30.612’, 11.v.2012, ex galls on Salixalba (M. Mahdavi) (1 ♀) [LF.ma.IR 05/10424] (in AICF). ITALY: Liguria, Bussana-Vecchia, N43.84026°, E7.82905°, 02.i.2012, emerged 20.ii.2012, ex Myopitesstylata on Dittrichiaviscosa (E. Spagnol) (4 ♀)  and (3 ♂ not sequenced)  (in MNHN); (3 ♀ not sequenced)  (in FALPC) and (2 ♂ not sequenced)  (in CNC) same data but 06.vi.2012, emerged 07.vi.2012 (N. Ris). SPAIN: Logroño, La Rioja, 15.iii.2012, emerged 16.iii.2012, ex Myopitesstylata on Dittrichiaviscosa (R. Cantera Rioja) (2 ♀) . SWEDEN: Skåne, Sk, Höganäs kommun, Kullabergs naturreservat, between Hjortstugan and Ransvik, Oak forest in southern slope, N56.29421°, E12.48399°, 27.vi to 30.vii.2005, Trap ID 1004, Coll. event 1797 (SMTP) [LF.u.SW.03/10660] (in NHRS).Taxon classificationAnimaliaHymenopteraEupelmidaeAl khatibhttp://zoobank.org/4BCB5C66-7AC7-431B-8F51-C76CF7D56AD4Eupelmus(Eupelmus)gemellusHolotype ♀. FRANCE: Haute-Corse, Calenzana, 11.ix.2012, emerged 18.ix.2012, ex PageBreakBactroceraoleae on Oleaeuropaea  (1 ♀) [FAL1515/10408] (in MNHG). Paratypes. FRANCE: Alpes-Maritimes, Biot, N43.63455°, E7.082490°, 29.x.2012, emerged 01.iii.2013, ex Megastigmuspistaciae on Pistacialentiscus (F. Al khatib & N. Ris) (2 ♀ 1 ♂)  (in MNHN); Alpes-Maritimes, Biot, N43.63455°, E7.082490°, 11.iii.2012, emerged 27.iii.2012, ex Mesophlepsoxycedrella on Juniperusoxycedrus (N. Ris), (2 ♀) ; Alpes-Maritimes, Mont-Chauve, 476 m N 43.76578°, E7.27024°, 01.xi.2012, emerged 18.xii.2012, ex Bactroceraoleae on Oleaeuropaea (M. Thaon) (1 ♀) [NB29/10413] (in FALPC); Alpes-Maritimes, Sophia-Antipolis, N43.624423°, E07.03667°, 21.ii.2012, emerged 27.ii.2012, ex Myopitesstylata on Dittrichiaviscosa (F. Al khatib) (1 ♀) [FAL1089/10143] (in FALPC); Ardèche, Saint-Georges-Montpellier, N43.6104°, E3.77227°, x.2011, ex Bactroceraolea on Oleaeuropaea (L. Brancaccio & M. Thaon) (1 ♀) [FAL1279/10444] (in MNHG); Aude, Bize-Minervois, N43.32692°, E2.870750°, 27.iii.2012, emerged 11.iv.2012, ex Mesophlepsoxycedrella on Juniperusoxycedrus (F. Al khatib & N. Ris), (1 ♀) [FAL1360/10233] (in MNHN); Aude, Durban-Corbières, N42.99825°, E2.80690°, 27.iii.2012, emerged 06.iv.2012, ex Mesophlepsoxycedrella on Juniperusoxycedrus (F. Al khatib & N. Ris), (1 ♀) [FAL1362/10234] (in FALPC); Aude, Gruissan, N43.12105°, E3.09539°, x.2011, ex Bactroceraoleae on Oleaeuropaea (N. Ris) (1 ♀) [FAL1266/10446] (in CBGP); Bouches-du-Rhône, Lançon-de-Provence, 33 m, N43.54818°, E5.16727°, x.2011, ex Bactroceraoleae on Oleaeuropaea (A. Auguste-Maros) (1 ♂) [FAL1269/10439] (in MNHN); Bouches-du-Rhône, La Ciotat, 53 m, N43.19011°, E5.65905°, x.2010, ex Bactroceraoleae on Oleaeuropaea (A. Auguste-Maros) (1 ♂) [FAL1243/10437] (in MNHG); Haute-Corse, Bisinchi, 593 m, N42.48983°, E9.32797°, 18.vi.2012, emerged 03.vii.2012, ex Dryocosmuskuriphilus on Castaneasativa (N. Borowiec & M. Thaon) (4 ♀) ; Haute-Corse, Lumio, N42.55879°, E8.81299°, 13.i.2012, emerged 27.ii.2012, ex Myopitesstylata on Dittrichiaviscosa  (1 ♀) [FAL1013/10137] (in AICF); Haute-Corse, Muratu, 750 m, N42.5559°, E9.29929°, emerged 23.i.2013, ex Dryocosmuskuriphilus on Castaneasativa (N. Borowiec & M. Thaon) (1 ♀) [2013CYN355/10664] (in FALCP); Var, la Garde-Freinet, 366 m, N43.31597°, E6.47534°, emerged 28.ii.2013, ex Dryocosmuskuriphilus on Castaneasativa (N. Borowiec & M. Thaon) (1 ♂) [2013CYN448/10663] (in FALPC); Var, Porquerolles, 13 m, N42.99534°, E6.2044°, ex Bactroceraoleae on Oleaeuropaea  (2 ♀) ; Var, Puget-Ville, 143 m, N43.26728°, E6.10671°, ex Bactroceraoleae on Oleaeuropaea  (1 ♂)  (in GDPC). ITALY: Liguria, Bussana-Vecchia, N43.84026°, E7.82905°, 02.i.2012, emerged 24.ii.2012, ex Myopitesstylata on Dittrichiaviscosa (E. Spagnol) (2 ♀)  (in MNHN); (1 ♀) [GDEL4122/10194] (in FALPC) same data except collected 25.i.2011 and emerged iv.2011; (1 ♀) [FAL1415/10481] (in CBGP) same data except collected 06.vi.2012 and emerged 07.vi.2012; Sardinia, Province Oristano, N39.70041°, E8.739690°, 20.x.2012, sweeping on Pistacialentiscus (L. Brancaccio & M. Thaon) (1 ♀) [FAL1508/10405] (in CNC); Sardinia, Province Oristano, N39.70041°, E8.739690°, 20.x.2012, emerged 24.x.2012, ex Megastigmuspistaciae on Pistacialentiscus (L. Brancaccio & M. Thaon) (1 ♀) [FAL1513/10407] (in CNC).Taxon classificationAnimaliaHymenopteraEupelmidaeDelvare & Gibsonhttp://zoobank.org/27BFFF36-E3EE-4B94-AC0B-D4AB07836E1BEupelmus(Eupelmus)janstaiHolotype ♀. CZECH REPUBLIC: Břeclav district, Pavlov, 48.86750°N, 16.65416°E, sweeping on Tiliaplatyphyllos, 03.vii.2010 (G. Delvare) [GDEL4046/10032] (in MNHG). Paratypes. Moravia, Vranov riv., Dyje, 48.89472°N, 15.81250°E, 13.viii.1991, riparian forest (L. Masner) (2 ♀) (in CNC).Taxon classificationAnimaliaHymenopteraEupelmidaeAl khatib & Fusuhttp://zoobank.org/F83B5381-0448-4EE3-B42F-F9AD7749F964Eupelmus(Eupelmus)longicalvusPageBreakHolotype ♀. SWEDEN: Gotlands, Go, Gotlands kommun, Roleks, grazed calcareous pine forest. 57°32.207'N, 18°20.273'E, 16.vii–02.viii.2004, Trap ID 28, Coll. event 1458 (SMTP) [LF.ma.SW 02/10429] (in NHRS). Paratypes. FRANCE: Alpes-Maritimes, La Bollène-Vésubie, 1700 m, N43.96778°, E7.38111°, 19.vii.2009 (G. Delvare) (2 ♀)  (in GDPC); Aveyron, Peyreleau, 850 m, N44.17528°, E3.23750°, 22.vi.2009 (G. Delvare) (1 ♀) [GDEL4194/10604] (in GDPC); Hautes-Alpes, Mont-Dauphin, 1869 m, N44.68972°, E6.6786°, 18.viii.2008 (G. Delvare) (1 ♀) [GDEL4199/10609] (in FALPC). ITALY: Friuli Venezia Giulia, Giulia, Chiusaforte, 1450 m, N46.40527°, E13.4450°, 12.vii.2008 (G. Delvare) (1 ♀) [GDEL4038/10019] (in GDPC); Friuli Venezia Giulia, Giulia, Chiusaforte, 1380 m, N46.39944°, E13.45944°, 12.vii.2008 (G. Delvare) (1 ♀) [GDEL4191/10603] (in FALPC). SWEDEN: Södermanland, Sö, Södertälje kommun, Tullgarns näs, Rävsalaviken, mixed forest next to pasture, N58.955217°, E17.607550°, 03.vii/19.viii.2004, Trap ID 30, Coll. event ID 1055 (SMTP) (4 ♀ not sequenced & 1 ♀ sequenced) [LF.ma.SW 01/10428]; same data but 16.vi/17.vii.2005 and coll. event ID 1717 (5 ♀); same data but 17.vii/08.ix.2005 and coll. event ID 1718 (4 ♀ not sequenced & 1 ♀ sequenced) [LF.ma.SW 03/10430] (in NHRS and AICF); Södermanland, Sö, Huddinge kommun, Pine forest with garbage, N59.1765333°, E17.9938500°, 13.vii/10 viii 2004, Trap ID 5, Coll. event ID 766 (SMTP) (1 ♀not sequenced) (AICF).Taxon classificationAnimaliaHymenopteraEupelmidaeDelvarehttp://zoobank.org/B0EC22D2-129B-4A37-BF5C-FAE526CA3439Eupelmus(Eupelmus)minozonusHolotype ♀. HUNGARY: Veszprém, Hegyesd, 175 m a.s.l, 46.93333°N, 17.52278°E, 27.vi.2010, sweeping Quercuscerris (G. Delvare) [GDEL4030/10010] (in MNHG). Paratypes. Same data as holotype (4 ♀) .Taxon classificationAnimaliaHymenopteraEupelmidaeDelvarehttp://zoobank.org/23647401-C103-4AD7-ABA5-C41EC6126087Eupelmus(Eupelmus)opacusHolotype ♀. SWEDEN: Östergötland, Ög, Ödeshögs kommun, Omberg, Stocklycke äng, lime meadow, 58°18.452'N, 14°37.859'E, 23.viii/16.ix.2005, Trap ID 13, Coll. event 1648 (SMTP) [LF.ur.SW 02/10460] (in NHRS). Paratype. GREECE: Kerkini Lake N. Park, Kerkini, Krousia Mts site, Malaise tr., 06.vi–12.vii.2007, 41°11’32.4”N, 23°03’59.5”E, 190 m a.s.l., Leg. Gordon Ramel (1 ♀) [LF.ur.GR 01/10459] (in AICF).PageBreakTaxon classificationAnimaliaHymenopteraEupelmidaeAl khatibhttp://zoobank.org/111A405E-470F-481A-A43D-663460CEC078Eupelmus(Eupelmus)pistaciaeHolotype ♀. FRANCE: Hérault, Cazevieille, 230 m a.s.l, 43.75222°N, 3.77000°E, 28.x.2011, emerged v.2012, ex Megastigmuspistaciae on Pistaciaterebinthus (G. Delvare) [GDEL4027/10507] (in MNHG). Paratypes. FRANCE: Hérault, same data as holotype (6 ♀)  (3 ♂) ; Hérault, Viols-le-Fort, 200 m, N43.74583°, E3.70389°, 28.x.2009, emerged v. 2010, ex Megastigmuspistaciae on Pistaciaterebinthus (G. Delvare) (1 ♀) [GDEL4022/3704bis] (in GDPC).Taxon classificationAnimaliaHymenopteraEupelmidaeDelvarehttp://zoobank.org/3565E30B-92D9-4DC4-BB94-7224D6BA3D22Eupelmus(Eupelmus)priotoniHolotype ♀. FRANCE: Aveyron, Sauclières, 700 m a.s.l, Lit de la Virenque, 43.96389°N, 3.35583°E, 15.vi.2011 (G. Delvare) [GDEL4051/10038] (in MNHG).Eupelmuspriotoni in Eupelmus species described in this paper, the upper surface of the costal cell on the fore wing has only one row of setae on the apical half and not 3-4 rows as previously written.Unfortunately, a mistake was included in the original description of Taxon classificationAnimaliaHymenopteraEupelmidaeFusu & Al khatibhttp://zoobank.org/1F354047-F041-4CD6-B1DA-1D53815B9B7CEupelmus(Eupelmus)purpuricollisHolotype ♀. GREECE: Kerkini lake nr Neo Petritsi; Malaise trap, Midway Site, 30.vi–06.vii.2008, 41°18’49.8”N, 23°16’35.6”E, 750 m a.s.l., Leg. Gordon Ramel, [LF.ur.GR 02/10650] (in AICF). Paratypes. GREECE: same data as for holotype but 09–13.vii.2008 (1 ♀ not sequenced). Kerkini Lake N. Park, Kerkini, Krousia Mts site, Malaise tr., 11–17.vii.2007, 41°11’32.4”N, 23°03’59.5”E, 190 m a.s.l., Leg. Gordon Ramel, (1♀) [LF.ur.GR 05/10653] (in AICF); same data but 18–24.vii.2007 (1♀) [LF.ur.GR 03/10651] (in GDPC).PageBreakTaxon classificationAnimaliaHymenopteraEupelmidaeAl khatibhttp://zoobank.org/CCD5B87E-9C81-456A-9DD8-F737AE6AC2F3Eupelmus(Eupelmus)simizonusHolotype ♀. FRANCE: Ardèche, Les Vans, 175 m a.s.l., Lit du Granzon, 44.38722°N, 4.15444°E, 15.vii.2012, sweeping on Quercuspubescens, (G. Delvare) [GDEL4142/10297] (in MNHG).Taxon classificationAnimaliaHymenopteraEupelmidaeDelvarehttp://zoobank.org/3001FBF7-A5AF-4D65-A274-6673F34264F1Eupelmus(Eupelmus)tremulaeHolotype ♀. CZECH REPUBLIC, Jindóichùv Hradec, Veselí nad Lužnicí, 1 km E of Charles University field station (Ruda), 422 m a.s.l, 49.15296°N, 14.70646°E, ex Harmandia sp. (Cecidomyiidae) on Populustremula, 05.vi.2007, adult emergence 13.vi.2007 (P. Jansta) [PJ07003_1_1/10570] (in MNHG). Paratypes. Same data (1 ♀) [PJ07003_1_1/10569] (in MNHN) (1 ♂) [PJ07003_1_1/10571] (in MNHN).