Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over‐expression. Here we present data showing that short‐interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell‐lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN‐negative SH‐SY5Y NB cell‐line, or in two immortalized human fibroblast cell‐lines. Immunoblotting of NB cell‐lines shows that high PRMT5 expression is strongly associated with MYCN‐amplification  and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN‐amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN‐overexpressing cells, including the SHEP‐21N cell‐line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN‐associated cell‐death in SHEP‐21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post‐transcriptional level. Reciprocal co‐immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK‐N‐BE(2)C and NGP cell lines. By using liquid chromatography – tandem mass spectrometry (LC‐MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post‐translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small‐molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene. The development of NB is believed to be contingent on defective differentiation of neural crest cells, although the precise cell of origin remains unknown. Several genetic markers are found in poor prognosis NB, including loss of chromosome 1p, loss of 11q, gain of 17q and, most notably, amplification of the tal NBs) . Importatal NBs) . MYCN istal NBs)  and Polytal NBs) , the lattal NBs) . Genes rr (NGFR) , and allMYCN transcription  can trigger depletion of MYCN protein in NB cell‐lines and in the murine model of NB, leading to NB growth inhibition and tumour regression. MYCN destabilisation is not contingent on Aurora kinase A enzymatic activity, but rather on its conformation and ability to protect MYCN from FBXW7‐mediated degradation . Anothercription .ST7 (suppression of tumorigenicity 7)  known to catalyse repressive epigenetic modifications in a variety of cancers, specifically protein arginine methyltransferase 5 (PRMT5). PRMT5 is active in chromatin remodelling complexes, and catalyses symmetric dimethylation of the histone 3 tail at arginine 8 (H3R8) and histone 4 arginine 3 (H4R3), and is one of two “type II” arginine methyltransferases, the other being PRMT7 . PRMT5 oicity 7)  and geneicity 7) . PRMT5 aicity 7)  and the icity 7) . As wellicity 7) . Other picity 7) , NF‐κB (icity 7)  and E2F‐icity 7) .In this report we provide evidence showing that PRMT5 is involved in poor prognosis NB, and is a key post‐translational regulator of the MYCN oncoprotein. PRMT5 inhibition may therefore represent a novel alternative for treatment of NB and other cancers driven by the MYCN oncoprotein.22.1http://www.cogcell.org), Manfred Schwab (German Cancer Research Center), Robert Ross, (Fordham University), Carmel McConville, (University of Birmingham), Pramila Ramani (University of Bristol). Cells were cultured in Dulbecco's modified eagle's medium (DMEM):F12‐HAM (Sigma) supplemented with 10% foetal bovine serum (PAA Cell Culture), 200 mM l‐Glutamine, 100 mM penicillin, 0.1 mg/mL streptomycin (Sigma) and 1% (v/v) non‐essential amino acids (Life technologies). SHEP‐Tet21N cells were cultured in RPMI 1640 supplemented with 10% tetracycline‐free foetal bovine serum (Life technologies) with tetracycline added to 1 μg/ml. Human fibroblast lines were made from primary human fibroblasts from the Coriell Institute Cell Repository stably transfected with human telomerase reverse transcriptase , and were grown in DMEM with 10% foetal bovine serum. All cells were mycoplasma free, and cultured as adherent monolayers at 37 °C with 5% CO2.Neuroblastoma cell lines used in this study were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) or kindly provided by The Children's Oncology group (For short‐interfering RNA (siRNA) treatments, exponentially growing cells were reverse‐transfected using Lipofectamine 2000 (Life technologies) at a final concentration of 25–50 nM according to the manufacturer's instructions. After 48–72 h, cells were lysed for protein in Cell signalling lysis buffer (New England Biolabs), or for RNA in QIAzol (QIAGEN). Total RNA was DNAsed and purified using the miRNeasy Mini Kit (QIAGEN). Details of siRNAs (Sigma) are given in Cell counts were done in quadruplicate and repeated at least twice using a Countess cell‐counter (Invitrogen). Photographs were taken with a Leica microscope camera using Spot Advanced software.2.2To assess protein stability, cells were treated with 50 μg/ml cycloheximide (Sigma) following siRNA transfection for 72 h. At the given timepoints, cells were washed in ice‐cold PBS and prepared for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) as below. For inhibition of apoptosis, siRNA transfected cells were treated with 10 μM caspase inhibitor Q‐VD‐OPh  24 h after knockdown.2.3® Protein G Immunoprecipitation Kit, Life Sciences Technology). Briefly, for each immunoprecipitation 8 μg antibody was conjugated to 50 µL Protein‐G magnetic beads (Millipore). After preclearing with normal mouse or rabbit IgG, 500 μg protein lysate was incubated overnight with the antibody‐coated beads at 4 °C with rotation. Beads were washed twice in IP lysis buffer and once in PBS and boiled in 1× Laemmli sample buffer  glycerol, 10% (w/v) SDS , 5% (v/v) 2‐Mercaptoethanol (Sigma) before SDS‐PAGE.Cells were lysed in Cell Signalling Lysis buffer complemented with Complete Mini Protein inhibitors (Roche), 5 mM dithiothreitol and 10% (v/v) glycerol, and sonicated using a Bioruptor (Diagenode). The co‐immunoprecipitations were carried out according to the manufacturer's protocol  was performed as previously described . Details2.6For analysis of DNA content, cells were harvested, rinsed in phosphate‐buffered saline (PBS), permeabilised in ice cold 70% ethanol, and stored at 4 °C overnight. Before fluorescence‐activated cell‐sorting (FACs), cells were washed with PBS, treated with 100 μg/ml RNase A (Qiagen) and then stained with 50 μg/ml propidium iodide (Sigma) for 30 min in the dark at 37 °C. DNA content of propidium iodide stained cells was measured on a LSRFortessa X‐20 (BD Biosciences) and data analysed using ModFit LT (Verity Software House). All assays were conducted on triplicate samples unless otherwise stated.2.7® Ruby (Invitrogen) staining and visualised using a Typhoon 9400 variable mode imager . Proteins equal or above the MYCN band were excised and subjected to in‐gel tryptic digestion using a Pro‐Gest automated digestion unit (Digilab UK). The resulting peptides were fractionated using a Dionex UltiMate 3000 nano HPLC system in line with an LTQ‐Orbitrap Velos mass spectrometer (Thermo Scientific). In brief, peptides in 1% (v/v) formic acid were injected onto an Acclaim PepMap C18 nano‐trap column (Dionex). After washing with 0.5% (v/v) acetonitrile 0.1% (v/v) formic acid peptides were resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse phase analytical column (Dionex) over a 150 min organic gradient, using 7 gradient segments  with a flow rate of 300 nl min−1. Solvent A was 0.1% formic acid and Solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionized by nano‐electrospray ionization at 2.1 kV using a stainless steel emitter with an internal diameter of 30 μm (Thermo Scientific) and a capillary temperature of 250 °C. Tandem mass spectra were acquired using an LTQ‐Orbitrap Velos mass spectrometer controlled by Xcalibur 2.1 software (Thermo Scientific) and operated in data‐dependent acquisition mode. The Orbitrap was set to analyse the survey scans at 60,000 resolution (at m/z 400) in the mass range m/z 300–2000 and the top twenty multiply charged ions in each duty cycle selected for MS/MS in the LTQ linear ion trap. Charge state filtering, where unassigned precursor ions were not selected for fragmentation, and dynamic exclusion  were used. Fragmentation conditions in the LTQ were as follows: normalized collision energy, 40%; activation q, 0.25; activation time 10 ms; and minimum ion selection intensity, 500 counts.MYCN was immunoprecipitated as described above from control and PRMT5 siRNA‐treated SK‐N‐BE(2)C cells. Prior to harvest, cells were treated with 20 μg/ml MG132 for 2 h to circumvent possible proteosomal degradation of unmethylated MYCN. Protein was fractionated by SDS‐PAGE using SYPROThe raw data files were processed and converted into mgf text files using Proteome Discoverer software v1.2 (Thermo Scientific). The mgf files were submitted to the Mascot search algorithm  and searched against the UniProt human database , using the following settings: peptide precursor mass tolerance was set at 10 ppm, and MS/MS tolerance was set at 0.8 Da. Search criteria included carbamidomethylation of cysteine (+57.0214) as a fixed modification and oxidation of methionine (+15.9949) and methylation of arginine  and lysines  as variable modifications. Searches were performed using trypsin as the enzyme with one missed cleavage. The reverse database search option was enabled and all peptide data was filtered to satisfy false discovery rate (FDR) of 5%. Any modified peptides which were identified by Mascot were manually verified by examining the raw MS/MS data.2.8t‐test and Mann–Whitney tests were used for statistical analysis.Gel images were quantified using Image J software, and Student's 33.1We evaluated the effect of short‐interfering RNA (siRNA) – mediated PRMT5 depletion on the SK‐N‐BE(2)C cell‐line, representative of the poor prognosis subset of NB, together with two other HMTs known to be involved in cancer. Knockdown of CARM1 (PRMT4), EZH2 and PRMT5 all induced some cell death in SK‐N‐BE(2)C cells, but PRMT5 depletion triggered a significantly greater effect with approximately double the cell death compared to the negative control siRNA, and substantially greater death than either CARM1 or EZH2 knockdowns. Treatment of knockdowns with the caspase inhibitor Q‐VD‐OPh rescued cell death, and apoptosis was further confirmed by immunoblotting for poly(ADP‐ribose) polymerase‐1 (PARP‐1) cleavage A–C. PRMT3.2MYCN amplification and high MYCN protein expression, whereas SH‐SY5Y cells do not, suggesting a relation between PRMT5 and MYCN expression. Examination of PRMT5 expression in NB cell‐lines confirmed that MYCN over‐expression was associated with higher PRMT5 protein levels   and with poor overall prognosis (P = 2.2e−08)  (Both SK‐N‐BE(2)C and NGP cells are known to have  U test) A–B. At t2.2e−08) . Notably1.7e−06) , alludin1.7e−06)  and modi1.7e−06) .MYCN amplification. In poorly differentiated neuroblastomas lacking MYCN amplification, 57% (n = 14) showed predominant nuclear staining whereas 43% showed equal or more staining in the cytoplasm. In striking contrast, poorly differentiated MYCN‐amplified tumours showed intense nuclear PRMT5 staining together with weaker cytoplasmic staining. In total, 89% (n = 9) poorly differentiated MYCN‐amplified neuroblastomas showed predominant nuclear staining, whereas only 22% of a group including ganglioneuromas, ganglioneuroblastomas and differentiating neuroblastoma (n = 9) showed equivalent or increased nuclear staining compared to cytoplasmic staining. Together these expression analyses strongly suggest a PRMT5‐MYCN oncogenic axis in NB.We further examined PRMT5 protein expression in primary NB tumour sections by immunohistochemistry. As shown in ST7 and RBL2 genes might also be targeted by PRMT5/MYCN in NB. These genes are established PRMT5‐silenced TSGs in other cancers (NTRK and NGFR (NGFR mRNA was markedly increased (over 3‐fold) with PRMT5 knockdown in NGP cells, with little effect being apparent in SK‐N‐BE(2)C cells C cells . Thus PR3.3MYCN amplification. As shown in MYCN transcript levels were only moderately decreased accompanying PRMT5 knockdown C, NGP and Kelly cell‐lines, all of which have nockdown , suggestP < 0.0.005) whereas cells without induced MYCN did not show a significant increase in cell death. Immunoblotting also showed an increase in cleaved PARP indicative of apoptosis accompanying PRMT5 knockdown in the induced cells.We also evaluated PRMT5 knockdown in the SHEP‐Tet21N cell‐line, which has inducible MYCN expression following removal of tetracycline B–D. PRMTIn order to assess PRMT5 influence on post‐translational MYCN stability, we conducted cycloheximide chase assays on PRMT5 siRNA transfected cells. As shown in P < 0.005), concomitant with an increase of cells in S‐phase. A similar effect on S‐phase has been demonstrated for c‐myc in medulloblastoma cells C cells, as such an interaction may serve to stabilise MYCN and provide initial evidence that MYCN might be a substrate for the arginine methyltransferase activity of PRMT5. As shown in Lastly, we assessed whether MYCN protein may be methylated by PRMT5 using liquid chromatography – tandem mass spectrometry (LC‐MS/MS) analysis of immunoprecipitated MYCN protein from SK‐N‐BE(2)C cells before and after PRMT5 knockdown. This analysis gave us >53% coverage of MYCN, and identified several potential sites of arginine monomethylation and dimethylation on the MYCN protein . By insp4NGFR reactivation by PRMT5 knockdown also suggest that PRMT5 is a co‐operative co‐repressor for MYCN‐mediated gene regulation. This is also supported by the report that the gene encoding MEP50/WDR77, which is a key component necessary for PRMT5 enzymatic activity, is positively regulated by MYCN  High PRMT5 mRNA expression correlates with MYCN amplification (MYCN‐amp) in NB. Data analysed is from Valentijn et al., 2012, using the R2 microarray analysis and visualization platform (http://r2.amc.nl). Tumours with no MYCN amplification (MYCN‐un) show lower PRMT5 mRNA expression. Kaplan Meier curve showing high PRMT5 mRNA expression correlates with poor prognosis. The Bonferroni corrected probability (Bonf P) is shown. (B) Kaplan Meier curve showing high PRMT5 mRNA expression correlates with poor prognosis in MYCN‐unamplified tumours.Click here for additional data file.Supplementary Figure S3 For PRMT5 immunohistochemistry controls, we used skeletal muscle which is negative for PRMT5 (left) and normal prostate where PRMT5 expression has been reported to be strong in the nucleus of the epithelial cells  (right). Weak staining in the blood vessels is indicated (ves), contrasting with the skeletal muscle (ske). Note the strong nuclear staining in prostate epithelial cells (epi) contrasting with the myoepithelium (myo).Click here for additional data file.Supplementary Figure S4 (A) Gene expression changes accompanying PRMT5 and MYCN knockdowns in NB cell‐lines. (B) Quantitative reverse transcriptase PCR analysis of MYCN expression after PRMT5 knockdown.Click here for additional data file.Supplementary Figure S5 MYCN protein sequence and peptide coverage by LC‐MS/MS is shown by shading and the number of peptides detected. Arginines predicted to be mono‐ and/or dimethylated are marked by red boxes, whereas the arginine predicted to be monomethylated is indicated by a stippled red box.Click here for additional data file.


In this study, Cha et al. show that the Wnt/β-catenin signaling pathway is the link between fluid flow and lymphatic vascular morphogenesis. They provide a molecular and structural framework to study mammalian lymphatic vasculature by demonstrating that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling. Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/β-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of β-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/β-catenin signaling in LECs. In turn, Wnt/β-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking β-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling and, in turn, FOXC2. The lymphatic vasculature collects and returns interstitial fluid to the blood. The mammalian lymphatic vasculature is composed of lymphatic endothelial cells (LECs) that originate predominantly from embryonic veins, although additional sources might exist . LEC proLEC progenitors are specified in a subpopulation of venous ECs by the activation of PROX1 expression. Most of the LEC progenitors migrate out of the vein to form the lymph sacs. However, a subset of LEC progenitors stays on the veins to form four LVVs through the up-regulation of genes in addition to PROX1, such as FOXC2, GATA2, integrin α9 (ITGA9), and ITGA5 . Next, lProx1+/− pups die soon after birth with severe edema. The surviving Prox1+/− pups have LVs; however, LVs are absent in those that die soon after birth . Mutations in FOXC2 are associated with human lymphedema. The lymphatic plexuses of Foxc2−/− embryos are mispatterned and abnormally covered with mural cells, do not undergo proper maturation, and lack LVs and LVVs  . In vitrmph flow . TherefoIntriguingly, OSS is not sufficient to promote PROX1 expression in LECs . Additio+ LEC progenitors on the vein are H2B-EGFP+ at embryonic day 11.0 (E11.0) . However, LECs outside the vein are only rarely labeled. At E12.0 and E14.5, a subpopulation of LVV-forming ECs (LVV-ECs) express nuclear H2B-EGFP . To analyze LVs, we performed whole-mount IHC of the mesentery of E16.5–E18.5 TCF/LEF-H2B-EGFP embryos. We identified modest H2B-EGFP expression in LECs at all stages . However, H2B-EGFP expression is consistently stronger in the LV-forming ECs (LV-ECs) at the three obvious stages of LV formation; i.e., up-regulation of PROX1 expression, formation of the circular sheath, and a mature valve.To determine whether Wnt/β-catenin signaling is active in the mouse lymphatic vasculature, we used transgenic TCF/LEF-H2B-EGFP reporter mice . In thes+ cells in the entire lymphatic vasculature (data not shown). Therefore, we used additional approaches to assess Wnt/β-catenin signaling activity in the valves. We performed in situ hybridization for Axin2, a well-characterized target of the Wnt/β-catenin signaling pathway, in numerous contexts. As a positive control, we evaluated cardiac semilunar valves, which display activated Wnt/β-catenin signaling  using Lyve1-Cre mice . LVs and VVs that develop at later time points are also labeled by Lyve1-Cre. We bred Lyve1-Cre;Ctnnb1f+/ mice with Ctnnb1f+/ mice and failed to obtain any surviving Lyve1-Cre;Ctnnb1f/f  pups from >200 pups analyzed at postnatal day 0 (P0). Instead, we observed several dead, cyanotic, and edematous Lyve1-Cre;Ctnnb1LOF pups in the cages, suggesting perinatal lethality. We collected E14.5, E16.5, and E18.5 Lyve1-Cre;Ctnnb1LOF embryos and determined that they had severe edema . Occasionally, some blood was observed in the peripheral skin of the mutant embryos . We found that the lymph sacs of these embryos were severely dilated, resulting in the constriction of the surrounding veins .To elucidate the importance of the Wnt/β-catenin signaling pathway in valve development, we first conditionally deleted Cre mice . Lineagehigh FOXC2high GATA2high LVV-ECs are first observed at E12.0 . In scanning electron microscopy (SEM) images of control embryos, LVV-ECs could be seen delaminating from the walls of the vein and loosely aggregating with each other; however, these cells are absent in Lyve1-Cre;Ctnnb1LOF embryos . These results demonstrate that β-catenin is necessary for the differentiation of LVV-ECs. Consistently, IHC revealed that PROX1high FOXC2high GATA2high LVV-ECs are present in E14.5 control embryos but absent in their Lyve1-Cre;Ctnnb1LOF littermates . At E17.5, LV rudiments are more clearly visible in control embryos and are not observed in mutants . By E18.5, oval-shaped lymphangions are clearly distinguishable in control mesenteries, and LVs with PROX1high cells are seen. In contrast, the lymphatic vessels of E18.5 Lyve1-Cre;Ctnnb1LOF embryos are dilated, and LV-ECs are only rarely observed .LV formation occurs in a stepwise manner starting at E16.5 with the up-regulation of PROX1, FOXC2, and GATA2 in a subset of cells within the lymphatic vessels . We dete+ FOXC2+ cells on the downstream side of cardiac valves (Lyve1-Cre mice (data not shown); therefore, we used previously reported Prox1Cre+/ mice to conditionally delete Ctnnb1 in these cells. Prox1Cre+/ mice are heterozygous for Prox1 and lack LVVs, LVs, and VVs . Hence, Prox1Cre+/ will specifically delete Ctnnb1 in the PROX1+ ECs of the cardiac valves. Importantly, the well-characterized roles of β-catenin during cardiac cushion formation are unperturbed .Combined, these results conclusively show that β-catenin is necessary for the differentiation of all types of PROX1Lyve1-Cre;Ctnnb1LOF littermates express uniformly high levels of PROX1 and VEGFR3  cell coverage. In contrast, the lymphatic vessels of Lyve1-Cre;Ctnnb1LOF littermates are dilated well-short of the midline and are abnormally covered with α-SMA+ mural cells . Tip cells located at the forefront of vessels dictate vascular patterning with their numerous filopodia, which sense the environment and determine growth and branching. We found that the number of filopodia per tip cell and the length of filopodia are significantly reduced in Lyve1-Cre;Ctnnb1LOF embryos . In contrast, the lymphatic vessels of Lyve1-Cre;Ctnnb1LOF littermates are dilated with bulbous architecture . These abnormal lymphatic vessels have migrated to only the middle of the heart .We also analyzed the lymphatic vessels of the heart. Lymphatic vessels start migrating from the atrial end of the heart at around E14.5 (data not shown). In E17.5 control embryos, lymphatic vessels are seen running from the atrium until the ventricular tip of the hearts . In control embryos, PROX1 and FOXC2 are expressed within the lymphatic vasculature in a heterogeneous manner. A modest expression of these molecules is observed in most of the lymphatic vasculature, including the tip cells . In contrast, PROX1 is up-regulated in the immature collecting lymphatic vessels. A modest down-regulation of FOXC2 expression is observed in the immature collecting lymphatic vessels of Lyve1-Cre;Ctnnb1LOF embryos . These results demonstrate that Wnt/β-catenin signaling regulates the expression of FOXC2, PROX1, and CX37 in LECs.Gap junction molecule Connexin37 (CX37) is important for valve formation and also plays an incompletely understood role in lymphatic vascular morphogenesis . CX37 isLyve1-Cre;Ctnnb1LOF embryos are most similar to Foxc2−/− embryos. Specifically, both β-catenin and FOXC2 are required for the formation of LVs, LVVs, and VVs and proper lymphatic vessel maturation. To further delineate the functional relationship between β-catenin and FOXC2, we analyzed the lymphatic vascular plexus of Foxc2−/− embryos. We found that the lymphatic vessels in the peripheral skin of Foxc2−/− embryos do not reach the dorsal midline and are dilated . These phenotypes are strikingly similar to Foxc2−/− and Lyve1-Cre;Ctnnb1LOF embryos. Consistent with this observation, FOXC2 expression is down-regulated in the LECs of Clec2−/− embryos  (FOXC2 regulatory element is significantly enhanced in Bio-treated LECs (FOXC2 in a region devoid of H3 epigenetic modifications.The sequence preference of TCF/LEF transcription factors is well characterized . By usinte (TSS) B. Accordte (TSS) C. To detted LECs D. In conPROX1, which is a known target of the Wnt/β-catenin signaling pathway, in other contexts such as colon cancer cells and neural stem cells . These results show that Wnt/β-catenin signaling directly regulates PROX1 and FOXC2 expression in LECs.Our ChIP assay revealed that the association of β-catenin with the regulatory elements upstream of ted LECs E. As a pThe defects in the tip cells and filopodia of mice lacking FOXC2 or β-catenin suggested that these molecules might be necessary for the migration of LECs. We evaluated this possibility by using an in vitro wound healing assay in which a scratch is introduced within a plate of confluent LECs and the time for cells to migrate and close the “wound” is evaluated. We found that the Wnt/β-catenin antagonist iCRT3 significantly delayed wound closure, suggesting an inhibition of LEC migration A,B,E. AnIn order to determine whether FOXC2 could compensate for the loss of Wnt/β-catenin signaling in cell migration, we repeated the scratch assay using LECs that are infected with retroviral particles to overexpress FOXC2 . We deteGOF.We next wanted to determine whether FOXC2 could compensate for the loss of Wnt/β-catenin signaling in vivo. We generated a transgenic mouse line that conditionally over-expresses FOXC2 using a previously reported approach . In thisLyve1-Cre;Cttnb1LOF;FOXC2GOF embryos and found that they exhibit reduced edema compared with Lyve1-Cre;Cttnb1LOF embryos . The diameter of the lymphatic vessels and their migration pattern are comparable in control and Lyve1-Cre;FOXC2GOF embryos . Further analysis revealed that the morphological defects in the lymphatic vessels of E16.5 Lyve1-Cre;Ctnnb1LOF embryos—namely, the dilation, growth retardation , and abnormal recruitment of mural cells by the lymphatic capillaries —are completely rescued by overexpression of FOXC2. We also determined that the lymphatic vessels in the mesenteries of Lyve1-Cre;Ctnnb1LOF;FOXC2GOF embryos have a normal morphology . Therefore, FOXC2 is unable to compensate for the loss of β-catenin during LVV and VV development. Additional targets of Wnt/β-catenin signaling, such as PROX1, CX37, and GATA2, are likely necessary for the differentiation of VECs.LVVs and VVs are present in E16.5 wild-type and Lyve1-Cre;FOXC2GOF embryos . A similar result was obtained when the experiments were performed using 24-well plates with a shear stress of 0.09 dynes/cm2 (data not shown). Despite the apparent difference in the shear stress values, we were able to precisely recapitulate the results of The pioneering work by OSS was originally proposed to be critical for LV development . AlthougOur results show that OSS promotes Wnt/β-catenin signaling and FOXC2 expression, both of which are necessary in turn for valve formation and lymphatic vascular maturation. While FOXC2 compensates for the loss of β-catenin in lymphatic vessels, other targets of Wnt/β-catenin signaling appear to be necessary for valve formation. What are the factors that modulate the LEC response to OSS and determine whether they differentiate into valves or mature lymphatic vessels? We speculate that the OSS response could be regulated by whether PROX1 is up-regulated at a specific site. Accordingly, OSS and OSS+PROX1 will promote lymphatic vascular maturation and valve formation, respectively.In the future, it will be important to determine the OSS-specific effects on LECs. Valve-forming cells undergo complex migratory events such as delamination, realignment with respect to flow, condensation, and elongation to form valves . LymphatSupplemental Fig. 12). Based on the published literature, we discuss a few alternate mechanisms that may be involved in sensing and translating OSS into Wnt/β-catenin signals.Despite its elegance, a definitive proof for the accuracy of the “OSS model” is still missing. Identifying the mechanosensory molecules in the lymphatic vasculature will bring us a step closer to testing this model in vivo. Primary cilia function as mechanosensors in many cell types . Using SIntegrins are well-known mechanosensors in numerous contexts . IntegriFrizzled receptors might function as mechanosensors, as reported recently . AccordiIn tumor cells, mechanical strain activates a receptor tyrosine kinase (Ret) that phosphorylates and promotes the nuclear translocation of β-catenin by inhibiting catenin–cadherin interaction at cell junctions . VEGFR2 Mutations in the mechanically activated ion channel PIEZO1 are associated with human lymphedema . Hence, Lyve1-Cre in mice did not reveal any obvious defects in LEC progenitor specification. Conditional deletion of β-catenin using Tie2-Cre in the entire vascular network resulted in embryonic death at E12.5, as reported previously , goat anti-human PROX1 (R&D Systems), sheep anti-mouse FOXC2 (R&D Systems), goat anti-mouse VEGFR3 (R&D Systems), goat anti-mouse LYVE1 (R&D Systems), rat anti-mouse CD31 (BD Pharmingen), goat anti-mouse GATA2 (R&D Systems), rabbit anti-total β-catenin and rabbit anti-active β-catenin antibodies , goat anti-mouse NRP2 (R&D Systems), chicken anti-GFP (Abcam), rat anti-mouse Endomucin (eBioscience), rabbit anti-CX37 (Invitrogen), and Cy3-conjugated monoclonal anti-α-SMA (Sigma-Aldrich). The following secondary antibodies were used: Cy3-conjugated donkey anti-rabbit, Cy3-conjugated donkey anti-sheep, and Cy5-conjugated donkey anti-rat . Alexa 488-conjugated donkey anti-goat, Alexa 488-conjugated goat anti-chicken, and Alexa 488-conjugated donkey anti-rat were purchased from Life Technologies.Primary antibodies used for Western blotting were as follows: mouse anti- β-Actin (Sigma), mouse anti-mouse β-catenin (BD Pharmingen), rabbit anti-active β-catenin , goat anti-human PROX1 (R&D Systems), sheep anti-mouse FOXC2 (R&D Systems), and goat anti-mouse GATA2 (R&D Systems). The following HRP-conjugated secondary antibodies from Santa Cruz Biotechnology were used: goat anti-mouse IgG, goat anti-rabbit IgG, donkey anti-goat IgG, and donkey anti-sheep IgG.IHC on sections was done according to our detailed protocols that we published recently . Whole-mAxin2 probe will be provided on request.In situ hybridization and coimmunohistochemistry experiments were performed as described previously . InformaCells were lysed in lysis buffer . Concentration of proteins was measured using Bradford reagent (Bio-Rad). Western blot was carried out according to standard protocols.SEM was done according to our previous protocol .Total RNA was isolated from cells using Trizol reagent (Invitrogen) according to the manufacturer's protocol. cDNA was synthesized from total RNA using the SuperScript III first strand synthesis system (Invitrogen) following the manufacturer's instructions. Quantitative PCR (qPCR) was performed using SYBR Green PCR master mix reagent (Bio-Rad) in a CFX96 real-time system (Bio-Rad). Primer sequences will be provided on request. The threshold cycle (Ct) value for each gene was normalized to the Ct value for β-actin.6 to 10 × 106 human primary LECs (Lonza) as described previously , scratch wounds were made at the center of the well using a sterile 1000-µL pipette tip. Loose cells were removed by a PBS wash, and DMSO-containing (Sigma) or iCRT3-containing (Sigma) medium was added. Images were recorded at 2 for 48 h. The rocker went through an angle of 48o in ∼2 sec. One milliliter of medium was used for the 24-well plates, and 6 mL of medium was used for the six-well plates. In both the cases, the amount of medium was sufficient to cover the center of the wells at all times, although the medium did lose contact with the sides of the wells at high angles of oscillation.Human LECs were cultured to confluency in six-well plates and exposed to OSS using a test tube rocker  with a preset frequency (18 rpm). The entire setup was kept inside a sterile humidified incubator with 5% COL is the length of the dish along the direction of motion of the rocker, x is the distance of the point of interest from a side of the dish, h0 is the height of the fluid surface when the dish is horizontal, θ is the angle of the rocker, θmax is the maximum angle of the rocker, θ0 is the “critical flip angle” (defined as the angle at which the fluid just leaves contact with the bottom of the dish), t is the time, and T is the time period of the sinusoidal motion of the rocker.An analytical formulation for a rectangular Petri dish was presented in max of 48o and T of ∼2 sec. The dish had an L of ∼3.48 cm for the six-well plates and 1.56 cm for the 24-well plates. The volume of fluid used was 6 mL and 1 mL, and the h0 was ∼0.63 cm for the six-well plates and ∼0.53 cm for the 24-well plates, respectively. With these values, the maximum shear stress is calculated to be ∼0.3 dynes/cm2 for the six-well plates and ∼0.09 dynes/cm2 for the 24-well plates at the center of the wells.The aforementioned analytical formulation was adapted for the present scenario, since the lubrication approximation holds for the small volume of fluid used, and the region of interest is the center of the wells, which was always covered with fluid. For the medium, µ is considered as the same as that of water, which is 1 centiPoise. The rocker had a θ


Advances in computational psychometrics and mathematical methods have been gaining a significant role in both medicine and psychology over these past years. The mainstream in psychometrics is moving towards ever greater use of computational and mathematical modeling techniques. Such techniques are critical in the emerging fields of affective and wearable computing, where new biomedical instruments available both in the laboratory and in the field are allowing for deeper understanding of human psychology. These experimental methods offer new opportunities but also new challenges in data interpretation and analysis.This special issue has two foci, namely, to feature works that (a) advance scientific knowledge in the area of computational psychometrics and (b) explore deep investigated methods, techniques, and instruments for the assessment of cognitive, emotional, and medical  as well as mental health at the cutting edge of current technology.There have recently been an increasing number of research initiatives that utilize computational technologies in order to support patients in maintaining or regaining a healthy mental state. Computational psychometrics and related tools have been exploited for assessing, measuring, and defining new methods for an effective and focused psychological intervention.It is of utmost importance to provide people with higher quality of life and also to shift a part of monitoring tasks from therapists and caregivers to unobtrusive technological systems. Efforts have started with Internet-based self-help therapies, but recently systems make an increasing use of computational psychometrics, including ambient intelligence, pervasive computing, smart phones, and sensor systems. Their common goal is to provide effective solutions for maintaining and improving mental health and related assessment.This special issue received many articles, accepting for publication five exciting contributions to the field.P. Cipresso et al. describe a promising approach for managing data from the interaction of two communicating individuals by collecting multiple electrophysiological signals and eye movements with computational methods. D. Cardone et al. introduced a promising thermal infrared imaging-based tool for the computational assessment of human autonomic nervous activity and psychophysiological states in a contactless and noninvasive way. I. A. C. Giglioli et al. presented a systematic review in the novel field of augmented reality for the assessment and treatment of psychological disorders, highlighting 13 selected articles after an initial screening of 784 articles emerging from the scientific databases. Last but not least, two articles in the exciting field of virtual reality are published: C. Wilson and A. Soranzo review some current uses for VR environments when examining visual perception and discuss limitations or questions that can arise; and S. Segkouli et al. define a more robust cognitive prediction model, accurately fitted to human data to be used for more reliable interface evaluation through simulation on the basis of virtual models of users with mild cognitive impairment (MCI).We hope that this special issue will foster wider discussion for these exciting themes in computational psychometrics.Pietro CipressoPietro CipressoAleksandar MaticAleksandar MaticDimitris GiakoumisDimitris GiakoumisYuri OstrovskyYuri Ostrovsky


This study addresses how tension between chromosomes signals their proper alignment for segregation during cell division. Marston and colleagues show that budding yeast shugoshin dissociates from the pericentromere reversibly in response to tension. The antagonistic activities of the kinetochore-associated Bub1 kinase and the shugoshin-bound phosphatase PP2A-Rts1 underlie a tension-dependent circuitry that enables shugoshin removal upon sister kinetochore biorientation. The findings expose shugoshin as the pivotal sensor of tension between sister kinetochores. During mitosis and meiosis, sister chromatid cohesion resists the pulling forces of microtubules, enabling the generation of tension at kinetochores upon chromosome biorientation. How tension is read to signal the bioriented state remains unclear. Shugoshins form a pericentromeric platform that integrates multiple functions to ensure proper chromosome biorientation. Here we show that budding yeast shugoshin Sgo1 dissociates from the pericentromere reversibly in response to tension. The antagonistic activities of the kinetochore-associated Bub1 kinase and the Sgo1-bound phosphatase protein phosphatase 2A (PP2A)-Rts1 underlie a tension-dependent circuitry that enables Sgo1 removal upon sister kinetochore biorientation. Sgo1 dissociation from the pericentromere triggers dissociation of condensin and Aurora B from the centromere, thereby stabilizing the bioriented state. Conversely, forcing sister kinetochores to be under tension during meiosis I leads to premature Sgo1 removal and precocious loss of pericentromeric cohesion. Overall, we show that the pivotal role of shugoshin is to build a platform at the pericentromere that attracts activities that respond to the absence of tension between sister kinetochores. Disassembly of this platform in response to intersister kinetochore tension signals the bioriented state. Therefore, tension sensing by shugoshin is a central mechanism by which the bioriented state is read. For accurate dissemination of the genome, chromosomes are first duplicated during S phase of the cell cycle to generate identical sister chromatids that are held together by cohesion. During mitosis, to ensure that each daughter cell receives one copy of each chromosome after cohesion is lost, sister kinetochores must attach to microtubules from opposite poles. Cohesion resists the pulling force of microtubules, resulting in the generation of tension at sister kinetochores. Sister kinetochore tension is critical in enabling biorientation to be sensed, thereby allowing chromosome segregation to proceed. Although central to the segregation process, the underlying mechanism by which this state of tension is read is not known.The conserved shugoshin family of proteins has been implicated in the sensing of intersister kinetochore tension . ShugoshIn addition to protecting pericentromeric cohesin during meiosis and mammalian mitosis, shugoshins play a conserved role in promoting biorientation of sister chromatids . BiorienAlthough the ability to discriminate between tension-generating and tension-less attachments is the key to achieving chromosome biorientation , it is nInterestingly, in both mitosis and meiosis, shugoshin plays its critical roles at pericentromeres only when sister kinetochores are not under tension. This suggests that shugoshins may govern the tension-sensing process. Indeed, shugoshin undergoes a tension-dependent relocation from the inner centromere to the kinetochores in spermatocytes, oocytes, and human somatic cells . In humaCDC20 was placed under the control of the methionine-repressible promoter pMET3 (pMET3-CDC20) to induce metaphase arrest by addition of methionine. Cells carrying SGO1-6HA and pMET3-CDC20 were released from G1 into medium containing methionine and either nocodazole (to depolymerize microtubules) or DMSO (as a control). In cells that were not treated with nocodazole, Sgo1 first appeared as a bright dot within the nucleus, likely representing the pericentromere  and SGO1-GFP from a G1 arrest and imaged them at 15-min intervals as they progressed into a metaphase arrest induced by CDC20 depletion . It is unlikely that tension causes removal of core kinetochore proteins and tethered TetR-GFP from the centromere, as no such change was observed by microscopy . Addition of nocodazole to cells already arrested in metaphase led to Sgo1 accumulation at the centromere, indicating that Sgo1 removal under tension is reversible . We conclude that Sgo1 associates with the pericentromere only in the absence of microtubules and that this reduction can be readily observed by ChIP.With this in mind, we used ChIP to analyze Sgo1 association with the pericentromere in cells arrested in metaphase in the presence and absence of microtubules. Cells carrying ipl1-as5) sensitive to the ATP analog NAPP1, which results in syntelic attachments (both sister kinetochores attached to microtubules from the same pole)  using a version (me pole) . Althougith Sgo1 , this fiachments . In stu2codazole . The obsBUB1-GFP and MTW1-tdTomato to correlate Bub1 disappearance with sister kinetochore separation  system  to condicdc55Δ cells arrested in metaphase without microtubules, deletion of RTS1 led to an approximately fourfold increase in pericentromeric Sgo1, although total cellular levels remained unchanged , regulatory (B), and catalytic (C) subunit . In budded , accumulated to high levels on the pericentromere in metaphase-arrested cells lacking microtubules, and decreased in the presence of tension . We conclude that association with PP2A-Rts1 negatively regulates the pericentromeric localization of Sgo1.To determine whether direct association with PP2A is important for controlling the pericentromeric levels of Sgo1, we analyzed the ith PP2A . Similar tension . The perociation . Furtherrts1Δ or sgo1-3A cells. Wild-type and rts1Δ cells carrying SGO1-yeGFP and MTW1-tdTomato were released from a G1 arrest and imaged at 15-min intervals. We simultaneously analyzed a strain in which Sgo1-3A was tagged with GFP (SGO1-3A-yeGFP) and that also carried MTW1-tdTomato  and a Sgo1-bound phosphatase (PP2A-Rts1) control Sgo1 localization in the pericentromere . Bub1 isBUB1 in cells where H2A-S121D is the only source of H2A and measured Sgo1 levels at the pericentromere in metaphase-arrested cells in both the presence and absence of spindle tension. Next, we considered the possibility that H2A-S121 phosphorylation is not the only way that Bub1 promotes Sgo1 localization to the pericentromere. We deleted RTS1. If dephosphorylation of H2A contributes to Sgo1 removal, we would anticipate higher levels of Sgo1 at the pericentromere in the H2A-S121D mutant cells, but this is not the case , condensin (BRN1-6HA), or CPC  components were arrested in metaphase by treatment with nocodazole, and the levels of the tagged proteins at CEN4 were measured by ChIP-qPCR. Subsequently, we treated half the culture with NAA (to induce Sgo1 degradation), while the other half received no treatment  with or without nocodazole and in either the presence  or absence  of doxycycline. We first confirmed that tethered Sgo1-TetR-GFP remained bound to tetO repeats as they separate under tension. In metaphase-arrested cells with tetO repeats adjacent to CEN4, similar levels of Sgo1-TetR-GFP associated with a site close to CEN4 in the presence and absence of nocodazole  . While this indicates that mono-orientation is defective in mam1Δ cells, the fraction of cells with separated CEN5-GFP foci is much lower than expected if sister kinetochores on chromosome V were bioriented in all cells. We reduced the number of ways that kinetochores could stably attach to microtubules in metaphase I by deleting SPO11, the endonuclease required for the initiation of meiotic recombination, thereby abolishing chiasmata .During meiosis I, sister kinetochores must be mono-oriented (attached to microtubules from the same pole), and therefore the biorientation of sister kinetochores is suppressed. Inactivation of the monopolin complex, which is required for kinetochore mono-orientation, does not abolish the protection of pericentromeric cohesion during meiosis I, which has led to the idea that a lack of tension between sister kinetochores during meiosis I is not important for the maintenance of Sgo1 . Howeverr pCLB2) . In wildhiasmata . In spo1mam1Δ spo11Δ cells gave us the opportunity to test how tension across sister kinetochores influences Sgo1 association with the pericentromere during meiosis I. Wild-type, spo11Δ, mam1Δ, and spo11Δ mam1Δ cells were arrested in metaphase I by depletion of CDC20 (pCLB2-CDC20), and the area occupied by Sgo1-GFP was measured in live cells directly after kinetochore clusters became bilobed . Interestingly, centromeric Sgo1 levels were highest in spo11Δ cells in which both intersister tension and interhomolog tension are abolished , perhaps representing the average of a population of cells that includes attachments that lack tension as well as those that generate intersister tension . The idea that Sgo1 levels at the pericentromere are sensitive to all types of tension at kinetochores was confirmed by treatment of wild-type and spo11Δ metaphase I-arrested cells with the microtubule-destabilizing drug benomyl, which resulted in thin metaphase I spindles and increased levels of Sgo1 at the pericentromere . Together, these findings indicate that the pericentromeric levels of Sgo1 are responsive to spindle tension also during meiosis I and that Sgo1 levels at the pericentromere are lowest when sister kinetochores are bioriented.The increased sister kinetochore biorientation of  bilobed . Althoug1Δ cells . Therefo tension . Express of Sgo1 . Similarspo11Δ mam1Δ cells implied that cohesin may not be efficiently protected in these cells. Consistent with this idea, spo11Δ mam1Δ cells undergo a single meiotic division in which sister chromatids separate to opposite poles  was reduced in spo11Δ mam1Δ cells . We confirmed these observations in live single cells progressing through meiosis by examining Rec8-GFP localization immediately after Pds1-tdTomato (securin) degradation in cells that also carried Mtw1-tdTomato (to label kinetochores)  . Intereseiosis I . Since oo signal . These mmam1Δ and spo11Δ mam1Δ mutants results in the segregation of sister chromosomes to opposite poles in meiosis I, we filmed cells carrying CEN5-GFP foci on one homolog together with Pds1-tdTomato and the spindle pole body marker Cnm67-3mCherry (CEN5-GFP segregated away from each other (CEN5-GFP foci separated to >2 μm) directly following Pds1 degradation in meiosis I. Separation of sister CEN5-GFP foci to a distance of <2 μm suggests that sister kinetochores are bioriented, but pericentromeric cohesion is retained. As reported previously for mam1Δ mutants (CEN5-GFP separated only a short distance (<2 μm), indicating sister kinetochore biorientation without loss of cohesion, and this phenotype was also apparent in 40% of spo11Δ mam1Δ cells . We showed that PP2A-Rts1 negatively regulates Sgo1 levels at the centromere. We propose that Sgo1-bound PP2A, and possibly other phosphatases too, promote dephosphorylation of as yet unknown chromatin-associated substrates, the phosphorylation of which is required for Sgo1 association with the pericentromere. In the absence of tension, Sgo1 remains pericentromere-bound because of the proximity of the kinetochore-bound kinase Bub1. Spindle tension leads to the spatial separation of Bub1 from the chromatin, leading to the reversal of phosphorylation of its chromatin-bound substrates by PP2A-Rts1, releasing Sgo1. Eventually, upon stable biorientation, Bub1 kinase itself dissociates from its Spc105/Spc7/KNL1 receptor in the kinetochore due to reversal of Mps1-dependent phosphorylation by PP1, which also binds to Spc105/Spc7/KNL1 . We specSCC1-6HA was described in CLB3 promoter with pCUP1; and generate null alleles  was a kind gift from Dr. F. van Leeuwen (Netherlands Cancer Institute). Plasmids AMp920 (H2A-S121D) and AMp921 (H2A-S121A) were generated by site-directed mutagenesis of pER1 using a QuikChange II XL kit (Agilent Technologies).All yeast strains were derivatives of W303 or SK1 and are listed in Supplemental Table S1.  alleles . SGO1-ye tagging . Auxin-i tagging . pMET3-C112xtetO  to generpMET3-CDC20 were arrested in G1 in synthetic medium lacking methionine (SC/−Met/D) with α factor (4 or 5 mg/mL). Cells were then washed with rich medium lacking glucose (YEP) and released into rich medium containing 8 mM methionine (YPDA/Met). Methionine was readded to 4 mM every hour. To achieve a metaphase arrest in the absence of microtubules, 15 μg/mL nocodazole was added immediately after release into YPDA/Met and readded to 7.5 μg/mL every hour. To inhibit Ipl1-as5, 1 NA-PP1 was added to a final concentration of 50 mM. The stu2-277 allele was inactivated by shifting to 37°C. Doxycycline was used at 5 μg/mL. Meiosis was performed as described in pGAL-NDT80 and GAL4-ER, prophase release was induced by addition of β-estradiol to 1 mM .CENV-GFP), and 0.2 sec (red channel). ONIX software was used to control the microfluidics system, and SoftWoRx software was used for the control of the DeltaVision microscopy system and taking images. Image analysis was performed using Image-Pro and ImageJ programs, and final images were assembled using Adobe Photoshop. A custom-written plug in for Image J was used to generate V plots. Line scans were manually drawn across Mtw1-tdTomato kinetochore foci/focus of 100 single cell images. The center point between the two brightest pixels was chosen as a reference for alignment, and line scans were ordered according to their length. Details are available on request.For live-cell imaging, the ONIX microfluidic perfusion platform by CellASIC was used within a heated chamber set to 30°C, with the exception of the experiment shown in For fluorescence intensity measurements of kinetochore/microtubule-associated Sgo1-GFP signal, we used the “box in box” method described in


Such an MPA provides tailored permittivity and permeability to approach the impedance of free space for minimizing reflectance and a great imaginary part of the refractive index for reducing transmittance and finally results in high absorbance. Both experimental measurement and numerical simulation are in a good agreement. The flexible, ultrathin and double-sided MPA significantly differs from traditional quarter-wavelength absorbers and other single-sided perfect absorbers, paving a way toward practical THz applications in thermal emission, sensing and imaging, communications, stealth technique, and even energy harvesting.We design and demonstrate a flexible, ultrathin and double-sided metamaterial perfect absorber (MPA) at 2.39 terahertz (THz), which enables excellent light absorbance under incidences from two opposite sides. Herein, the MPA is fabricated on a λ J. Padilla More recently, an alternative design termed as metasurfaces has been reported to absorb light8911121417r) of 2.75 × (1 + 0.12i) and the thickness of 12 μm, about λ0/10.1-thick. As for the metallic pattern of the double-sided MPA, it is worth mentioning that it is not achievable by simply copying the front pattern to replace the back-reflector in cases of conventional MPAs232 . In addition, we only consider the one eighth of the metallic pattern in the unit cell to create a four-fold symmetric pattern by mirroring the generated patterns three times to achieve the polarization-insensitive performance. Then, we randomized a value between 0 and 1 to determine whether or not there exists metal on a pixel (see Method for more details). By repeating this random process on each pixel, we could obtain the metallic patterns capable of double-sided absorbance. Note that the stochastic design process could be the very first step of the genetic algorithm, thus possessing a potential to further optimize the performance of the MPA through targeted goals in a genetic algorithm252627CST Microwave StudioTM, to numerically calculate the corresponding reflectance, transmittance and absorbance. In simulation, we choose build-in material properties of gold with conductivity of 4.56 × 107 S/m, which is sufficient for simulation within terahertz regionThe double-sided MPA is comprised of a metal-insulator-metal (MIM) structure. For the insulating substrate, we utilized a flexible polyethylene terephthalate (PET) thin film with the electric permittivity . Finally, a flexible, ultrathin, optically transparent and double-sided MPA is presented in the middle and lower parts of For the sample fabrication, we applied an ultraviolet (UV) lithography technique and an electron-gun deposition process, and then a lift-off technique to realizing the designed metallic pattern. Since both of the rotation and translation alignment between the two metallic patterns of the double-sided MPA are a request to ensure the functionality of the structure, we also employed an alignment mark to provide 2-μm accuracy. The fabricated structure is comprised of 10-nm-thick titanium (as an adhesion layer) and 200-nm-thick gold films, and its unit cell is shown in the upper part of Next, we characterized the performance of the fabricated MPA by a micro-Fourier transform infrared (μ-FTIR) spectrometer (Vertex 70V) in the frequency range from 2.2 to 2.5 THz. Notice that we conducted the reflectance measurement at 11° instead of normal incidence, and in this case, there appear two different conditions of TE and TM incidences. As shown in 0/10.1, where λ0 is the free space wavelength. In fact, we could further reduce the thickness of the double-sided MPA once we obtain a thinner substrate in stock  is divided into 20 × 20 square pixels and then whether there would appear a metallic pattern on the front and back sides of each pixel or not depends on a randomized number between 0 to 1. Once the random number is smaller than 0.5, there would not appear metal on that pixel; otherwise, there would exist metallic squares on both sides. Further, in order to achieve a polarization-independent pattern, we only encode one eighth of total 400 pixels and generate a four-fold symmetric pattern by mirroring the one-eighth pattern three times. Thus, by repeating the random process on the one-eighth pixels 55 times, we can easily achieve a stochastic designed double-sided MPA whose performance is further determined in the aid of the commercial electromagnetic solver of the finite integration method.Designed photo-mask of metamaterial pattern from stochastic processAttached 12-μm-thick PET on Si substrateConducted UV photolithographyDeposited 10 nm-thick titanium and 200 nm-thick goldProceeded Lift-off processFlipped the 12 μm-thick PET with first patternRepeated step1–6 for the second pattern with tolerable alignment on the other sideMicro-Fourier transform infrared (μ-FTIR) spectrometer (Vertex 70V)Light source: Mercury lamp−1Spectral range: 100–10 cm−1Spectral resolution: 1 cmAperture size: 8 mmScan velocity: 1.6 kHzHow to cite this article: Huang, T.-Y. et al. Experimental realization of ultrathin, double-sided metamaterial perfect absorber at terahertz gap through stochastic design process. Sci. Rep.5, 18605; doi: 10.1038/srep18605 (2015).


Drug administration via the transdermal route is an evolving field that provides an alternative to oral and parenteral routes of therapy. Several microneedle (MN) based approaches have been developed. Among these, coated MNs  are a minimally invasive method to deliver drugs and vaccines through the skin. In this review, we describe several processes to coat MNs. These include dip coating, gas jet drying, spray coating, electrohydrodynamic atomisation (EHDA) based processes and piezoelectric inkjet printing. Examples of process mechanisms, conditions and tested formulations are provided. As these processes are independent techniques, modifications to facilitate MN coatings are elucidated. In summary, the outcomes and potential value for each technique provides opportunities to overcome formulation or dosage form limitations. While there are significant developments in solid degradable MNs, coated MNs (through the various techniques described) have potential to be utilized in personalized drug delivery via controlled deposition onto MN templates. Transdermal drug delivery  offers an attractive alternative to oral and parenteral routes of drug administration. These methods have provided significant contributions towards pharmaceutical (emerging therapies) applications . Such administration routes have a distinct advantage in overcoming the first pass effect of the liver, which can prematurely metabolize active drugs . Most trSeveral enhancement approaches have been utilized in order to increase skin permeability, ranging from chemical/lipid penetration enhancers to non-cavitation ultrasound, thermal ablation, iontophoresis, sonophoresis, microdermabrasion, electroporation, cavitational ultrasound and microneedles (MNs) ,7,8,9,10In the last twenty years, MNs have been proposed as pain-free systems (significantly less painful than a 26-gauge hypodermic needle)  with higMNs have the advantages of delivering small quantities of high-potency medication through the skin to minimize the pain factor  and alloThe application of MNs is limited due to their size and material properties. MN tips are prone to breakage and subsequent embedment within the skin if prepared from materials with poor mechanical properties ,23. FurtThis short review focuses on MNs as transdermal drug delivery systems. The review provides an overview of the advantages and disadvantages of MN systems. Moreover, examples of different MN structures, coating methods and coating formulations are highlighted. Specifically, emerging and existing MN coating methods are summarized in a table  that detMNs have been developed in ways that enable them to share advantages of both hypodermic needles and transdermal patches to deliver drugs through the skin at therapeutically desirable quantities ,7,8,9,10MNs are significantly different from hypodermic needles based on their length and the pore size they generate (ranging from sub-microns to millimeters). Generally, MN patches or substrates possess similar basic design elements such as an ordered array of MNs ranging from a few to a few hundred in number. MNs are prepared from various materials and manufactured in a plethora of shapes and sizes ,9,10,11.co-glycolic acid) (PLGA) polymer). These degrade safely in the skin while providing a sustained release of drug [The design of MNs and the way in which the drug or formulation is incorporated to target the skin varies. There are four main MN designs. The first is an array of solid MNs (with no drug) which is used to penetrate into the skin, increasing skin permeability for the intended drug . The second type is degradable MNs, prepared using biodegradable polymers  on pain compared to a 26-gage hypodermal needle. All MNs investigated were 5% to 40% less painful than the selected hypodermic needle. The thickness, width and tip angle had no significant impact on pain. However, decreasing the number and length of the MNs decreased the pain significantly. In addition, increasing the number of MNs 10-fold resulted in a two-fold increase in pain (by score). They also concluded that a three-fold increase in MN length resulted in a seven-fold increase in pain (by score) [Gill y score) .Coated MNs are attractive devices for drug delivery through the skin. Previously, MNs were coated by immersing patches in a liquid solution for several hours to ensure a full coat onto their surface. However, this simple procedure has the drawback of drug wastage and loss, variable coating thickness of active onto MNs and thus inaccuracy in drug dosage . DiffereThe dip coating process is the simplest procedure to coat MNs. MNs are first dipped into the formulation and then withdrawn. This step produces a liquid film on MNs. The liquid layer is then allowed to dry to form a solid film coating. The dip coating method has been utilized to deliver hydrophilic and hydrophobic drugs. Several biomolecules have been coated onto MNs using this technique, e.g., proteins, viruses and DNA, for rapid transdermal delivery .MW = 270.8 Da) is a hydrophobic drug which is used as a local anesthetic agent. Usually, the administration of lidocaine to the patient is either topically  or parentally . Here, the molten dip coating process was used to develop uniform lidocaine coated MNs for transdermal delivery. Polyethylene glycol (PEG) was used as a hydrophilic matrix with a lidocaine base to create the solid dispersion. Drug stability was achieved even at elevated temperatures of ~130 °C. The mass fraction of lidocaine in the drug dispersion had an impact on the PEG-lidocaine molten solution, as decreasing the mass fraction of lidocaine increased the solution viscosity. Compared to the conventional 1 h application of topical cream , the in vitro dissolution studies of PEG-lidocaine coated MNs in porcine skin demonstrated a significant increase in lidocaine delivery within 3 min [Ma and Gill, have reported one of the earliest attempts at coating a hydrophobic drug onto MNs using molten dip coating. Lidocaine (in 3 min .w/w). rhGH MN patches were stable for 6 months at 40 °C. Compared to commercial subcutaneous Norditropin injection (rhGH), rhGH coated MNs were found to provide a similar absolute bioavailability. This suggests rhGH MN patches have potential to replace rhGH injections in the pharmaceutical market due to reduced pain benefits and ease of administration [Human growth hormone (191 amino acids) is a peptide which is important for growth, cell regeneration and reproduction in humans. Recombinant human growth hormone (rhGH) was successfully dip coated onto titanium MNs for transdermal delivery  of low viscosity carboxymethyl cellulose sodium salt and 0.5% (w/v) Lutrol F-68 NF). Here, MN shafts were coated without contaminating the base. Coated materials dissolved (to liquid form) within 20 s in porcine cadaver skin with complete delivery into the skin [The dip coating method works by submerging MNs into a drug solution and could result in a non-uniform coating . Surfacethe skin .et al., to overcome the addressed problem, especially for very small (<90 micron length) and very closely (~20.000 cm−2) spaced MNs. Solid silicon microprojections were sputter coated with a thin layer of gold. The whole length of the microprojections was coated with a (6–8 µL) solution which possessed ideal surface tension and viscosity properties. The coating solution contained methylcellulose (works by increasing the viscosity and decreasing the surface tension of the coating solution at the same time), Quil-A (has benefits of serving as a surfactant to reduce the surface tension and works as a vaccine immune-stimulatory adjuvant) and selected concentrations of model active drugs (vaccines or fluorescent dyes). Formulations were applied and were modified using (6–8 m/s) a gas jet. The viscosity of the coated layer (5 µm thick) onto the microprojections increased rapidly, allowing the coated material to dry rather than relocate on the base substrate. This was followed by a fast gas jet (10 m/s) at an incident angle of 20° to remove all excess coating solution. The uniformly dried coating remained intact during skin penetration and the model drugs were released in 3 min within wet skin [The slow drying process associated with the dip coating approach is limited practically, especially for curved MNs. While the drug coating solution is still wet on the MN surface, the solution has potential to move  and relocate off the MN surface, reducing and varying the desired dose. Even the multi-dip coating approach is potentially problematic as a thick multi-layer  coating wet skin .et al., have improved vaccine delivery efficiency by using gas jet coated MNs, by focusing on depositing actives on MN tips rather than whole MNs. This was performed by increasing the gas jet incident angle to 70°, removing the patch edge and rotating the patches during the coating process to ensure uniformity. The delivery efficiency of vaccines was increased from 7.3% ± 1.1% to 17.8% ± 1.5% (for incident angle 20°) simply by removing the patch edge. Delivery efficiency increased from 17.8% ± 1.5% (for incident angle 20°) to 32.5% ± 3.9% (for incident angle 70°) based on the incident angle with continuous rotating of patches [Vaccine delivery using MNs has merits over diffusion (the movement of drug from an area of high drug concentration to low concentration) delivery, biolistic MN delivery and electroporation delivery approaches. Vaccines comprise large molecular weight actives which has limited their MN based deployment. Typically, transdermal delivery favors small drug molecules (<500 Da) to pass through the stratum corneum. Chen  patches .The spray coating process is similar to conventional coating approaches  to achieve millimetre thicknesses. The micron sized design of MNs  requires a coating thickness in and below the micron range (particles size < 280 μm). Spray coating of microparticles onto MNs undergoes three steps. Firstly atomization, which is the generation of formulated microdroplets from the spray coater. Secondly, the deposition and adherence of droplets onto the surface of MNs. Finally, the coalescence of droplets on the substrate to form an intact film coating .et al. [w/v CMC solution had a higher surface tension than 5% w/v CMC and both HPMC solutions (5% and 12% w/v). Solutions with high polymer concentrations  demonstrated greater viscosities . These parameters adversely affected film-coating coalescence as the resulting film had a combination of small patches of coating and large uncoated areas. The addition of a surfactant (Tween 80) in the case of CMC solutions was necessary to assist coalescence of the sprayed droplets onto the silicon surface [The spray coating process was first developed by McGrath et al. . The con surface .Spray coated solid MN patches were also used for transcutaneous delivery of live recombinant adenovirus (rADV) and modified vaccinia virus Ankara (MVA) vectors as vaccines. Viruses with suitable sugar-based formulations were spray coated in a dry form at the shaft of each silicon MN rather than the inter-needle space. A uniform coating pattern with an effective preservation of the virus’s activity was successfully delivered into the skin. The potency of recombinant virus vaccine coated onto MNs patches to induce antibody response, transcutaneous infection and induced antibody (or CD8+ T cell) response was equivalent to the response induced by transdermal injection of the same vaccine .etc. The EHDA system can generate both particles (electrospraying) and fibers (electrospinning).The electrohydrodynamic atomisation (EHDA) process has been developed to generate near uniform micro- and nano-meter scaled architectures in one step. The principle process was described by Grace and Marijnissen (1994). Here, atomized droplets are produced by an electrically imposed moving liquid  that jet through a capillary nozzle exit and are subsequently collected over a ground electrode positioned below the nozzle tip . The liqet al., presented a multi-structural MN coating approach employing EHDA principles. Stainless steel MNs (600–900 µm height) were attached to the ground electrode. The coating solution (containing 5 wt % of selectable molecular weights of polyvinylpyrrolidone (PVP) dissolved in methanol:ethanol (50:50) and fluorescein dye ) was infused into a three needle co-axial device which was connected to a high voltage supply. The MNs were coated with particles (100 nm to 3 µm) and fibres (400 nm to 1 µm) under controlled processing parameters, flow rate (~5–15 µL/min) and applied voltage (6–19 kV) at room temperature. This coating process can assist in advancing transdermal drug delivery [Using the EHDA process, controlled particle and fibre coating thickness is achievable. Khan delivery . This isdelivery ,39,43. IThere are three main types of EHDA processes: single needle (in which the formulation is injected into a single nozzle by a single precision syringe pump), coaxial EHDA (this system uses two or more immiscible liquids which are fed through separately enveloped nozzles) and multiplexed EHDA . One of the benefits of EHDA using a coaxial system is protection of the sensitive drug from direct exposure to the biological environment . This isCertain parameters need to be considered when using EHDA process: flow rate, applied voltage and the distance between the nozzle and the collecting platform . These fIn addition to processing parameters, material properties also impact resulting particles/fibres as they significantly affect the jet stability. The viscosity, surface tension, electrical conductivity and density are all major factors to be considered before processing . The mosThe inkjet printing approach is a valuable engineering apparatus that enables controlled distribution and accurate arrangement of fine liquid droplets (1–100 picoliters) onto a substrate  before solidification ,50. UnliPiezoelectric (piezo) inkjet printing technology is the most acknowledged industrial inkjet printing process. In this approach, a piezoelectric crystal (ceramic actuator) undergoes distortion by the effect of an electric field that creates a pressure pulse in the ink chamber forcing drops to eject from the nozzle. The droplet size is correlated with the nozzle dimensions .et al., fabricated biodegradable polyglycolic acid MNs coated with voriconazole  using piezoelectric inkjet printing. This system was compared with unmodified and vehicle modified MNs against different micro-organisms . Voriconazole-polyglycolic acid MNs showed antifungal activity against Candida albicans while other devices were ineffective. The method was identified as a useful application of piezoelectric inkjet printing for drug loading onto MNs for poorly soluble pharmacological agents [® AN 169 BF ) using piezoelectric inkjet printing technology. Dimethyl sulfoxide was used as a solvent to enhance the antifungal drug penetration. Miconazole-loaded Gantrez® AN 169 BF MNs exhibited antifungal activity against Candida albicans [Combination of MNs (as drug delivery systems) with piezoelectric inkjet printing (as precise drug coating technology) permits an advanced approach in the pharmaceutical arena. Boehm l agents . Micronaalbicans .® coatings  were uniform, reproducible and printable onto metallic MNs using the piezoelectric inkjet printing approach. The release profile depended on drug solubility. Hydrophilic 5-FU showed a rapid release profile compared to water insoluble CRC and CPT. However, varying antiproliferative action was observed for the three anticancer agents. Antiproliferative activity was concentration dependent, and at low concentrations (15 µg/mL) no antiproliferative activity was observed but observation increased with drug dose. This was dependent on drug potency as 7% and 9.4% viability was observed for 7 µg/mL (CRC) and 200µg/ml (CPT), respectively, sufficient to trigger antiproliferative activity. 5-FU was least potent with 20% cell viability at 400 µg/mL [Three anticancer agents with varying solubilities (5-fluororacil (5-FU), curcumin (CRC) and cisplatin (CPT)) were utilized for transdermal delivery using MNs. At various drug-polymer ratios, anticancer agents with soluplus00 µg/mL .et al., used this combination of two prototyping methods to fabricate biodegradable acid Gantrez AN-139 anhydride copolymer MNs containing quantum dots. Deposition of quantum dots was confirmed in deeper layers of the skin (>200 μm depth from surface) after successful penetration into the stratum corneum. This valuable combination enables the development of sensors and theranostic devices (combination of detecting and drug delivery in one system) [Piezoelectric inkjet printing technology was also combined with visible light dynamic mask micro-stereolithography-micromolding as a new approach of engineering polymeric MNs. Beohm  system) .Microneedle based delivery of therapeutics is a rapidly evolving area in the pharmaceutical remit, and within the approach of microneedle based delivery, several sub-classes exist. Coated MNs offer advantages over existing routes of parenteral drug delivery including pain-free and self-administration options (when compared to injections). Coated MN systems also possess benefits over other MN types, including the ability to pattern MN surfaces and control the dose (based on the coating formulation) in a facile manner. They also allow the retention of microstructures within the coated system and allow formulation to be located specifically onto MN templates. The coating of MNs can be achieved using several techniques, including emerging process such as EHDA technology, printing and gas jet drying. In particular, these current advances provide opportunities for developing advanced drug delivery systems for personalized and tailored transdermal medicines.


We have recently demonstrated that ZnPP diminishes β-catenin protein expression in cancer cells. The present study examined the cellular mechanisms that mediate ZnPP’s suppression of β-catenin expression. We demonstrate that ZnPP induces a rapid degradation of the β-catenin protein in cancer cells, which is accompanied by a significant inhibition of proteasome activity, suggesting that proteasome degradation does not directly account for the suppression. The possibility that ZnPP induces β-catenin exportation was rejected by the observation that there was no detectable β-catenin protein in the conditioned medium after ZnPP treatment of cancer cells. Further experimentation demonstrated that ZnPP induces lysosome membrane permeabilization, which was reversed by pretreatment with a protein transportation inhibitor cocktail containing Brefeldin A (BFA) and Monensin. More significantly, pretreatment of cancer cells with BFA and Monensin attenuated the ZnPP-induced suppression of β-catenin expression in a concentration- and time-dependent manner, indicating that the lysosome protein degradation pathway is likely involved in the ZnPP-induced suppression of β-catenin expression. Whether there is cross-talk between the ubiquitin-proteasome system and the lysosome pathway that may account for ZnPP-induced β-catenin protein degradation is currently unknown. These findings provide a novel mechanism of ZnPP’s anticancer action and reveal a potential new strategy for targeting the β-catenin Wnt signaling pathway for cancer therapy.Zinc protoporphyrin (ZnPP) has been found to have anticancer activity both  Due to ZnPP’s structural similarity to that of free heme, an established substrate of the antioxidant enzyme heme oxygenase-1 (HO-1), ZnPP acts as a competitive inhibitor for HO-1 enzymatic activity [ in vivo –5 and it in vivo . In our  in vivo .Because β-catenin is a key player in the canonical Wnt signaling pathway, which is a well appreciated target pathway for cancer therapy , the sigIn the present study, we have explored the cellular mechanisms of ZnPP-induced suppression of β-catenin expression in human cancer cells. The rapid and dramatic nature of the ZnPP-induced suppression of β-catenin protein expression strongly suggests that this suppression is primarily due to β-catenin protein degradation. β-catenin protein levels are well controlled by the β-catenin destruction complex that is tightly coupled to the ubiquitin-proteasome system . It is tThe β-catenin, phospho-β-catenin (Ser33/37/Thr41) and K48 (lysine 48)-linkage specific polyubiquitin antibodies were from Cell Signaling Technology, Inc. . MG132 and Brefeldin A/Monensin cocktail were from Cayman Chemical . Suc-LLVY-AMC was from Anaspec . Z-ARR-AMC and Z-LLE-AMC were from Millipore . Other fluorescent probes were from Life Technologies . The Corning Spin-X concentrators (6 mL) and monensin sodium salt was from VWR International LLC . The β-actin antibody and other chemical reagents were analytic grade and obtained from Sigma-Aldrich .2. All cells were sub-cultivated twice a week and applied to the various experiments as described in the results section.The A2780 cell line (human ovarian cancer) was a kind gift from Dr. Stephen Howell . The DU145 cell line (human prostate cancer) and MDA-MB-231 cell line (human breast cancer) were purchased from American Type Culture Collection . A2780 cells were cultivated in RPMI 1640 medium, and DU145 and MDA-MB-231 cells were cultivated in DMEM medium. Both RPMI 1640 and DMEM mediums were supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cells were routinely grown in a 75-mm flask at 37°C in a humidified environment containing 5% COZnPP and SnPP were purchased from Frontier Scientific, Inc. . The manufacturer’s advice and a previous report  were folProtein expression was analyzed by Western blot as we previously described ,13. CellCo-immunoprecipitation (co-IP) using β-catenin antibody was performed as described previously . In shorLysosome permeability was analyzed by fluorescence microscopy using the Operetta High Content Imaging System from PerkinElmer . A2780 cells were plated in Cell Carrier-96 plate from PerkinElmer  at a density of 10,000 cells per well. Forty-eight hours after plating, the cells were treated with ZnPP or SnPP or pre-treated with Brefeldin A/Monensin (21.1 μM / 4 μM) cocktail for 4 hours followed by treatment with ZnPP. The medium was then replaced with fresh medium containing 2.5 μM Acridine Orange . After 30 minutes incubation the cells were washed three times with HBSS and viewed under the Operetta. Lysosome permeability was measured using the AO staining . AO was 6 cells by incubating at room temperature for 30 minutes and vortexing every 10 minutes. The lysates were then centrifuged and supernatant collected. A total of 10 μg of protein for each sample was incubated with 20 µM fluorogenic substrate  in 100 μl assay buffer  at 37°C for 2 hours. After incubation, the fluorescence was read at 380 nm excitation and at 460 nm emission using Molecular Devices Fmax fluorescent microplate reader Proteasome activities were measured as previously reported . In brieThe ZnPP-induced suppression of β-catenin expression is accompanied by an inhibition of proteasome activity. We have previously reported that ZnPP suppresses β-catenin protein expression in human cancer cells . This wa50 for ZnPP’s inhibition of proteasome activity was determined to be 6.2 μM in A2780 cells and 4.2 μM in MDA-MB-231 cells (ZnPP’s inhibition of proteasome activity was further confirmed by direct measurement of the 20S proteasome chymotryptic activity, which was analyzed using the fluorophore linked peptide Suc-LLVY-AMC . The euk31 cells . ZnPP, b31 cells . Note thThese results indicate that ZnPP-induced β-catenin protein degradation is accompanied by a significant suppression of ubiquitin-proteasome activity, suggesting that proteasome degradation does not directly account for ZnPP-induced suppression of β-catenin expression.ZnPP does not promote β-catenin protein exportation. A recent report demonstrated that the β-catenin protein is secreted in exosomes from HEK293 cells, leading to a significant suppression of the canonical Wnt signaling pathway . To deteThe lysosome pathway is likely involved in ZnPP-induced β-catenin protein degradation. We have previously shown that lysosomal enzymes can be released upon enhanced lysosome membrane permeability, leading to cleavage of cellular proteins . To deteBFA is known to block intracellular transportation of lysosomal enzymes  and MoneThe present study was designed to explore the potential cellular mechanisms that mediate ZnPP-induced suppression of β-catenin expression using cancer cell model systems. The most interesting finding from this study is that ZnPP-induced β-catenin protein degradation is accompanied by a significant inhibition of the ubiquitin-proteasome degradation pathway; and lysosome-mediated protein degradation seems to mediate this event. These results further elucidate the cellular mechanisms of ZnPP’s anticancer activity and indicate a potential new strategy in targeting the β-catenin Wnt signaling pathway for cancer therapy.β-catenin protein levels are well controlled by phosphorylation and ubiquitin-proteasome degradation . TherefoThe possibility that ZnPP might induce β-catenin protein exportation through exosomes  thereby We have recently reported that zinc ionophores enhance lysosome membrane permeability leading to the release of lysosomal enzymes and cleavage of cellular proteins . In the In summary, we have explored cellular mechanisms that mediate the ZnPP-induced suppression of β-catenin expression in cancer cells. Our results indicate that the lysosome degradation pathway is likely involved in ZnPP’s suppression of β-catenin expression and that this process is accompanied by an inhibition of proteasome activity. These findings provide a novel cellular mechanism of ZnPP’s anticancer activity and implicate a new strategy for targeting the canonical Wnt signaling pathway.


The formation and attachment of new cementum is crucial for periodontium regeneration. Tissue engineering is currently explored to achieve complete, reliable and reproducible regeneration of the periodontium. The capacity of multipotency and self-renewal makes adipose tissue-deprived stem cells (ADSCs) an excellent cell source for tissue regeneration and repair. After rat ADSCs were cultured in dental follicle cell-conditioned medium (DFC-CM) supplemented with DKK-1, an inhibitor of the Wnt pathway, followed by 7 days of induction, they exhibited several phenotypic characteristics of cementoblast lineages, as indicated by upregulated expression levels of CAP, ALP, BSP and OPN mRNA, and accelerated expression of BSP and CAP proteins. The Wnt/β-catenin signaling pathway controls differentiation of stem cells by regulating the expression of target genes. Cementoblasts share phenotypical features with osteoblasts. In this study, we demonstrated that culturing ADSCs in DFC-CM supplemented with DKK-1 results in inhibition of β-catenin nuclear translocation and down-regulates TCF-4 and LEF-1 mRNA expression levels. We also found that DKK-1 could promote cementogenic differentiation of ADSCs, which was evident by the up-regulation of CAP, ALP, BSP and OPN gene expressions. On the other hand, culturing ADSCs in DFC-CM supplemented with 100 ng/mL Wnt3a, which activates the Wnt/β-catenin pathway, abrogated this effect. Taken together, our study indicates that the Wnt/β-catenin signaling pathway plays an important role in regulating cementogenic differentiation of ADSCs cultured in DFC-CM. These results raise the possibility of using ADSCs for periodontal regeneration by modifying the Wnt/β-catenin pathway. Periodontitis is one of the most common dental infectious diseases, in which inflammation extends deep into the tissues, thus damaging the periodontal tissue composed of periodont ligament, gingiva, alveolar bone and cementum covering the tooth root. In advanced cases, the destruction of periodontal ligament tissue and alveolar bone may result in tooth loss To date, adipose tissue-derived stem cells (ADSCs) have been extensively used in tissue engineering The canonical Wnt/β-catenin pathway stabilizes β-catenin; on activation of the canonical Wnt pathway, β-catenin binds to the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and mediates the transcription of Wnt target genes Wnt signaling acting on cementoblasts could inhibit cell differentiation while stimulating cell proliferation All animal experiments were approved by the Animal Care and Use Committee of Third Military Medical University and were carried out in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.Harlan Sprague–Dawley (SD) filial rats (7 days old) were used for ADSC culture. SD rats were obtained from the Laboratory Animal Research Centre of the Third Military Medical University, China, and maintained on a daily diet of Purina rodent chow in housing quarters with a light:dark cycle (12 h L∶D), regulated temperature, and sterile water.3 small pieces, then incubated in 60 units/mL type I collagenase (Gibco BRL) for 60 min at 37°C to partially dissociate adipose tissues. Single cell-derived colony cultures were obtained using the limiting dilution technique as previously described ADSCs were isolated and cultured using a previously described method 2. The isolated cells from the DF grew to confluence in about 2 weeks and remained fibroblastic in shape. No contamination of epidermal cells was observed after two passages. Once the cells reached confluence, culture medium was changed every day and collected for 3 days. The collected media were filtered through a 0.2 µm Millipore strainer and mixed with an equal volume of fresh DMEM supplemented with 10% FBS, then stored at −80°C before used as dental follicle cell-conditioned media.Rat DFCs were isolated and cultured as described previously 2 in the culture flasks, maintained and expanded in DMEM (10% FBS), and allowed to adhere overnight. The culture medium was then changed to DFC-CM, which contained either Wnt3a or DKK-1 used for the ADSC cultures. On culture day 7, the cells were harvested and subjected to assays for in vitro cementoblast-like differentiation.The primary group of cultures was treated with 100 ng/mL recombinant Wnt3a (R&D Systems) 3 cells/well) and cultured in basic medium (DMEM plus 10% FBS) for 1 day. Then, the cells were incubated with DFC-CM for another 7 days. They were then fixed in 4% paraformaldehyde for 15 min, after which the cells were incubated with β-catenin , CAP (cemetum attachment protein) or BSP  at dilutions ranging from 1∶100 to 1∶200  for 2 h and subsequently incubated with FITC(Fluorescein Isothiocyanate)- or Rhodamine-conjugated anti-rabbit or anti- goat secondary antibodies. Then, sections were counterstained with Hoechst 33342  to identify all nuclei. The image collection and superimposition were processed by DP controller  and DP manager . Isotype-matched control antibodies were used under the same conditions.ADSCs were seeded into a 24-well plate . Approximately 2–5 mg of total RNA was converted to cDNA using the Super Script First Strand Synthesis Kit (Invitrogen). The real-time PCR reactions were performed using the QuantiTect SYBR Green PCR Kit  and the Applied Biosystems 7500 Real-Time PCR Detection System. Two independent experiments were performed for each reaction in triplicates. The primers used are listed in ADSCs were harvested in a RIPA lysis buffer . After whole cell protein extracts were quantified by a BCA assay, they were separated on NuPAGE 10%–12% polyacrylamide gels, transferred to PVDF membranes , blocked in 5% BSA in TBST, and hybridized with antibodies against primary mouse β-actin/GAPDH antibody , primary rabbit β-catenin , primary rabbit GSK3β antibody , primary mouse CAP antibody , primary mouse BSP antibody , and primary rabbit OPN antibody , respectively. Beta-actin or GAPDH on the same membrane were used as a loading control. Signals were revealed after incubation with anti-rabbit or anti-mouse IgG secondary antibody (1∶2000) coupled to peroxidase by using ECL. Densitometry of Western blots was analyzed with Quantity One software and normalized to respective loading control signal on each blot.P<0.05.Data are presented as mean ± SD. Comparisons were made using a t-test or one-way ANOVA for experiments with more than three groups. All experiments were repeated three times, and representative experiments are shown. Differences were considered significant at Ex vivo expanded ADSCs expressed the cell surface molecules STRO-1 (24.7%) and CD146 (31.2%), two early mesenchymal stem-cell markers. FACS profiling also showed that ADSCs expressed CD31 (1.8%), CD34 (2.4%), CD45 (2.0%), CD90 (100%), and CD105 (96.1%)  .The pote (96.1%) . Adipoge (96.1%) .ADSCs have been reported to possess the potential to differentiate into cementoblasts Wnt/β-catenin signaling plays a critical role in bone formation and regeneration Canonical Wnt signaling appears to be capable of maintaining certain stem cell populations in a proliferative and non-differentiating state. The inhibition of canonical Wnt signaling either by deleting LEF-1 or by over-expressing the Wnt antagonist Dickkopf (DKK)1 arrests tooth morphogenesis at the late bud stage Firstly, we determined the levels of β-catenin and GSK3β (glycogen synthase kinase 3 beta) by Western blot analysis during a period of 7 d in order to investigate whether treatment of ADSCs with recombinant rat Wnt3a (100 ng/mL) influenced the Wnt signaling pathway in ADSCs on the protein level. ADSCs expressed the β-catenin protein in both the control group and the DCM-CM cultured group, and its expression was increased following Wnt3a treatment . In markIn order to investigate the ability of ADSCs to differentiate into cementoblast-like cells, immunofluorescence staining was used for determining the protein expression in ADSCs. The results showed that ADSCs cultured in DFC-CM were positive for BSP and CAP. And then we treated ADSCs with DKK-1(100 ng/mL) under DFC-CM induction conditions, we found that the number of BSP and CAP positive expression cells were increased . Further−, CD34−) but express other molecules, the combination of which is largely used for their description: CD73+, CD44+, CD105+, CD90+, CD146+ and STRO-1+It is widely accepted that periodontal defects usually do not heal spontaneously. Periodontal regeneration requires new connective tissue attachment to the root surface, a process that involves the formation of new cementum on previously exposed root surfaces, the synthesis of Sharpey's fibers and their insertion into newly formed cementum. Therefore, a variety of procedures have been advocated in order to ensure an outcome in which periodontal tissue regeneration can occur. Many studies have reported that dental stem cells can form structures resembling tooth tissue, and that dental pulp stem cells (DPSCs) and dental follicle cells have the ability to form odontoblast-like cells that produce dentine in vitro osteoblastic/cementoblastic differentiation of DPPSCs in the presence of ascorbic acid, b-glycerophosphate, and dexamethasone. This differentiation was evidenced by high alkaline phosphatase activity, osteocalcin content, and mineralized nodule formation Cementogenesis has received attention due to its importance in periodontal maintenance and regeneration. In the present study, expression of CAP as a putative cementoblast marker has been detected in cultured ADSCs stimulated with DFC-CM. In addition to osteoblastic/cementoblastic differentiation of ADSCs, the osteogenic differentiation of PDLSC and DPSC has been also reported. PDLSCs were allowed to differentiate in the presence of ascorbic acid-2-phosphate. This resulted in the expression of alkaline phosphatase, mineralization-related genes, and the formation of mineralized nodules, thus indicating osteoblastic/cementoblastic cell lineage differentiation The dental follicle (DF) is a specialized connective tissue, a loose connective tissue sac that surrounds the unerupted tooth and which plays different roles in the development of a tooth. Although some studies suggest that some of the cementoblasts may arise from Hertwig's epithelial root sheath, as well as from the DF in vivoPersistent stabilization of β-catenin in the dental mesenchyme leads to premature differentiation of odontoblasts and differentiation of cementoblasts, and induces excessive dentin and cementum formation in vitro. We found that ADSCs treated with DKK-1 displayed dramatically reduced baseline levels of β-catenin, whereas DKK-1 induced CAP, ALP, BSP, and OPN activation to promote osteoblastic/cementoblastic differentiation. Enhanced Wnt/β-catenin signaling either by Wnt overexpression or by deficiency of Wnt antagonists is associated with increased bone formation in mice and humans The aim of this research was to successfully regenerate cementum tissue. Dkk1 specifically inhibits canonical Wnt signaling by binding as a high-affinity antagonist to LRP5/6 co-receptors. Currently, we do not know whether down-regulated β-catenin levels could help to promote ADSC differentiation into cementoblast-like cells via inhibition of the n Wnt/β-catenin signaling pathway In this study, it has been suggested that the use of ADSCs could be an alternative potent source for the application of tissue engineering given their comparatively easy accessibility and multidifferentiation capacity. Our study has provided new insights into the molecular mechanisms of the Wnt/β-catenin signaling pathway, thus showing that it acts as an important regulator controlling cementoblastic differentiation of ADSCs induced in a DFC-CM microenvironment. A down-regulated Wnt/β-catenin signaling pathway could promote ADSC differentiation into cementoblast-like cells in a DFC-CM microenvironment. Future efforts should focus on developing an in-depth understanding of the regulation and functions of the Wnt pathway in the cementoblastic differentiation of MSCs, which could improve periodontal tissue regeneration.