By doing so, Daple serves two opposing roles; earlier during oncogenesis it suppresses neoplastic transformation and tumor growth, but later it triggers epithelial-to-mesenchymal-transition (EMT). We have identified and characterized two isoforms of the human Daple gene. While both isoforms cooperatively suppress tumor growth via their GBA motif, only the full-length transcript triggers EMT and invasion. Both isoforms are suppressed during colon cancer progression, and their reduced expression carries additive prognostic significance. These findings provide insights into the opposing roles of Daple during cancer progression and define the G-protein regulatory GBA motif as one of the minimal modules essential for Daple’s role as a tumor suppressor.Previously, Aznar  Daple, a multimodular signal transducer and a cytosolic protein, was originally discovered as a Dishevelled (Dvl)-binding protein that regulates Wnt signaling3. Subsequent work showed that Daple directly binds ligand-activated FZDs, and serves as a guanine-nucleotide exchange factor (GEF) that activates the G-protein, Gαi1. Binding to the G-protein is mediated via Daple’s C-terminally located Gα-binding and activating (GBA) motif; such binding triggers non-canonical activation of Gαi1. Binding to FZD is also brought on via a C-terminally located stretch distal to the GBA motif. Upon ligand stimulation, Daple dissociates from Dvl, binds to and displaces Dvl from FZDs, and assembles a Daple:Gαi complexes on the receptor. How Daple:Dvl complexes are disassembled was unknown until recently, when we demonstrated that phosphorylation of Daple’s PDZ-binding motif (PBM) by both receptor and non-receptor tyrosine kinases can trigger this change4. Disassembly of Daple:Dvl complexes and formation of FZD:Daple:Gαi complexes facilitates the activation of Gαi near ligand-activated Wnt receptors. Daple activates Gαi within the FZD:Daple:Gαi complexes; such non-canonical activation of Gαi by Daple-GBA suppresses cAMP, whereas released ‘free’ Gβγ heterodimers enhance Rac1 and PI3K-Akt signals1. Although Daple-dependent enhancement of non-canonical Wnt signals can suppress tumor growth1, it can also fuel EMT, trigger cancer cell migration and invasion5 and drive metastasis6. Furthermore, elevated expression of Daple-GBA in circulating tumor cells prognosticates a poor outcome7. In doing so, Daple serves a dual role during oncogenic progression—a tumor suppressor early during oncogenesis and driver for metastatic invasion later.We previously defined a novel paradigm in Wnt signaling in which Wnt receptors, Frizzled (FZDs), activate G-proteins and trigger non-canonical Wnt signaling via Daple/CCDC88CHere we identified a novel Daple isoform (Daple-V2) which corresponds only to the C-terminal region of full length Daple . We demonstrate that Daple-V2 possesses all the functional domains to bind Dvl, FZD7 and Gαi and represents the smallest autonomous Daple unit able to inhibit the β-catenin/TCF/LEF pathway and suppress tumor cell growth. We also demonstrate that both isoforms have different subcellular distribution, and unlike Daple-fl, Daple-V2 does not enhance tumor cell invasiveness and may, therefore, serve as a more effective tumor suppressor and a better prognostic marker than Daple-fl.8, we asked if Daple’s seemingly opposite and bi-faceted roles in cancers may be due, in part, to the functions of varying isoforms of the same protein. Because prior work by others and us have demonstrated the importance of the PBM and GBA modules within the C-terminus of Daple in the regulation of Wnt signaling, we narrowed our analysis to transcripts encoding Daple’s C-terminus.We noted that there are 6 other transcript variants catalogued in Ensembl and/or UniProt databases: V2 (552aa), V3 (506aa), V4 (478aa), V5 (96aa), V6 (88aa) and V7 (234aa)  which adds a sequence comprised of 5 amino acids (MSVLS) on the N-terminus of the isoforms. Upon inspection of the V2 and V3 variant, we noticed that both transcripts contained the same transcript ID but are different version of the same transcript , while V4 has neither property on its 5′UTR  between Daple-fl, V2, and V4 revealed that the full-length transcript contains a putative consensus kozak sequence (high strength of prediction), with two notable features – a G on the +4 and an A on the −3UTR Fig. . In summTo determine how the expression of Daple isoforms change during normal to cancer progression, we analyzed the relative transcript expression of each Daple isoform in normal and tumor tissues annotated in the TCGA (The Cancer Genome Atlas) data portal using the web server- ISOexpresso. This server facilitates expression-based isoform-level analysis. Such analysis revealed that the cumulative levels of Daple transcripts are reduced from a median of 23.14 to 15.6 between normal colon and colorectal tumor tissues, respectively Fig. , howeverWhen we expanded this analysis to include all 9,690 tumors and 732 normal controls of 30 cancer types annotated in the TCGA data portal, Daple-V2 still remained the most dominant isoform across all tissue types  and several bands around the corresponding molecular weight of Daple-V2 (~70 kDa) are depleted in shDaple cells compared to shControl . Next we asked if Daple-V2 is expressed in cells (endogenous pool). Because all transcripts sharing the C-terminal GBA and PDZ-binding motifs share an identical 3′UTR Fig. , we utilrol Fig. . Becauserol Fig. .Figure 2). Furthermore, we confirmed the protein expression of Daple-fl and Daple-V2 .Next, we checked the expression of mRNA and protein of Daple-fl and Daple-V2 in normal adult colon. We analyzed mRNA levels of both Daple-fl and Daple-V2 in 14 colons samples by qPCR; both isoforms were detected Fig. . Further-V2 Fig.  by analy1. We also generated V2 constructs with previously defined specific mutants of Daple that lack the important motifs, GBA [Daple-V2-FA (F194A)] and PBM [Daple-V2- ΔPBM (deleted from 549–552aa)] and used these mutants to further decipher the properties of Daple-V2. As expected, we confirmed by GST pulldown assays that myc-Daple-V2 interacts with Gαi3, PDZ domain of Dvl and the cytoplasmic tail of FZD7 .These findings indicate that Daple-V2 represents the smallest autonomous Daple unit possessing the key biochemical features of the C-terminus of Daple-fl previously described1.We noted that the V2 isoform possesses the C-terminal region of the full length Daple protein and hence, contains a GBA, a frizzled binding domain, and a PDZ Binding motif (PBM) Fig. . To studZD7 Fig. .These fi11. Therefore, we next asked if the two isoforms of Daple differs in its subcellular localization. As expected, transfection of mCherry tagged Daple-fl into HeLa cells revealed that the full-length protein localizes to a pericentriolar compartment, as determined by their proximity to the centrosomal marker γ-tubulin  in localizing Daple to the pericentriolar recycling compartmentlin Fig. . By contlin Fig. . Immunoblin Fig. . Others nol Fig. . These r1. Because Daple-V2 possesses a GBA motif, and because the motif is required for binding G-proteins, we asked if this motif is functional in cells. Using DLD1 colon cancer cells stably expressing 7-TGP , an eGFP expressing Wnt activity reporter construct1, or parental DLD1 cells ; Daple-V2-WT, but not Daple-V2-FA, inhibited eGFP expression . Such growth suppressive effect required an intact GBA motif because, compared to Daple-V2-WT, expression of the GBA-deficient F194A  mutant not only failed to inhibit cell colony formation, but also enhanced anchorage-dependent growth .We found that Daple-V2 and Daple-fl have two similarities and one dissimilarity. First similarity was that both Daple-fl and Daple-V2 antagonize the β-catenin/TCF/LEF pathway Fig. ; Daple-Vion Fig. , indication Fig. . Second ion Fig. . Such grwth Fig. .. Furthermore, in 3-D matrigel invasion assays using the transformed NIH3T3 cells, as done previously1, enhanced invasion, as determined by the area of invasion was detected exclusively in the presence of Daple-fl-WT, but not in cells expressing Daple-V2 or Daple-fl-FA, indicating that only Daple-fl can trigger cell invasion , which is in keeping with our findings that Daple-V2 does not contribute to EMT or higher invasiveness.The dissimilarity between Daple-fl and Daple-V2 was observed in whether they used their GBA motifs to trigger EMT. Compared to cells expressing the GBA-deficient FA mutants of Daple-V2 or Daple-fl, those expressing Daple-fl WT had significantly higher expression of Lox-L3 and Vimentin, two genes commonly associated with epithelial-mesenchymal transition (EMT) ion Fig. . We founig. Fig. , which i14. We found that the treatment of DLD1 cells with low-dose Aspirin reduces both mRNA and protein for Daple-fl . The ratio of Daple-V2:fl was increased exclusively in those cell lines which were previously shown to be most sensitive to the growth suppressive action of Aspirin15, and share a common driver oncogene, PIK3CA  by half-fl Fig. , which h-fl Fig. , which h-fl Fig. , we founig. Fig. . The rat3CA Fig. . Whether, and that their expression may be differentially regulated by tumor cells to gain advantages in growth and invasiveness. Findings also reveal that Aspirin-mediated growth suppression is associated with an increase in the Daple-V2:Daple-fl ratio, and that such a change occurs exclusively in CRC cells harboring a constitutively activated PI3K-Akt pathway.Taken together, these findings demonstrate that compared to Daple-fl, Daple-V2 may serve as a more effective inhibitor of the β-Catenin-dependent canonical Wnt pathway and tumor growth in colonies, but it does not enhance EMT or invasion Fig. , and tha1]. In both assays we found that co-expression of Daple-fl and Daple-V2 isoforms suppressed cell growth and proliferation more effectively than compared to either isoform alone , and that high levels of expression carry a worse prognosis. We asked if and how the expression of Daple-V2 changes during cancer progression in the colon and what, if any, may the prognostic impact of such changes. Once again, we found several similarities and one dissimilarity. Analysis by qPCR in 12 paired colorectal tumors and their adjacent normal tissue showed that, much like Daple-fl, the expression of Daple-V2 is decreased in CRCs . When we analyzed a cohort of patients with Duke’s Stage II CRCs, we found that tumors that express low Daple-V2 had a significantly higher incidence of oncogenic K-ras mutation ; no such correlation was seen in the case of Daple-fl. Tumors that express low Daple-fl  have a higher frequency of progression to distant metastases. Kaplan-Meier analyses revealed that Daple-V2 is a better prognosticator than Daple-fl 25 (Table We previously showed that Daple-fl is downregulated earlier during cancer initiation (at the stage of polyp to cancer conversion), and that its expression at high levels carries a good prognosis in CRCsRCs Fig. . When weion Fig. ; Table 1-fl Fig.  or low D-fl Fig.  or low lig. Fig.  have a h7; high Daple is associated with higher tumor recurrence at distant sites and poorer survival. Here we asked how each isoform contributed to the prognostic impact using the same cohort. We found that Daple-fl,but not Daple-V2, expression is increased in disseminated tumor cells compare to healthy subjects . When we assessed their prognostic impact on survival, we found that although high expression of each isoform correlated with worse PFS , only Daple-fl levels carried a prognostic impact for DSS and overall survival (OS) . These findings are in keeping with our prior observations that pro-invasive and pro-EMT signatures are triggered exclusively by Daple-fl, but not Daple-V2  CRCs is associated with poorer prognosis compared to those with low Daple in CTCs-V2 Fig. .From these results we deduce that both Daple-fl and Daple-V2 cooperatively suppress tumor cell proliferation, that both transcripts are reduced during adenoma-to-carcinoma progression, and that the two isoforms have an additive prognostic impact, in that their reduced expression in tumors carries a poor prognosis. However, the two isoforms differ in their ability to trigger EMT and invasion; Daple-fl, but not Daple-V2 can support that.. In cancers, ~60% expressed Daple, but ~40% did not.Next we asked if suppression of Daple-fl and Daple-V2 transcripts during adenoma-to-cancer progression is also associated with reduced Daple proteins. Using an antibody raised against the C-terminus of Daple, which is identical between Daple-fl and Daple-V2, we confirmed that total Daple is expressed in the normal colon and in early and intermediate adenomas, but it is suppressed in advanced adenomas with villous features or high-grade dysplasia Fig. . In cancTaken together, our findings support the following model Fig. . While bThe major discovery we report here is the identification and characterization of a novel physiologic isoform of Daple, Daple-V2, which appears to contain the minimal modules that enable Daple to antagonize canonical β-Catenin-dependent Wnt signals and inhibit tumor cell growth and proliferation. Both isoforms are reduced during polyp-to-cancer progression in the colon. Compared to Daple-fl, Daple-V2 appears to be a more potent tumor suppressor and a better prognostic marker in primary tumors.1, that Daple-V2 cannot trigger EMT despite a functionally intact GBA suggests that these functions not only require the coupling of Daple’s C-terminus to G-proteins, but also the functions of its N-terminal HOOK and coiled-coil domains, e.g., phosphoinositide (PI3P) binding and localization to the pericentriolar recycling endosomes11, or binding to the dynein-dynactin motor complex26.What Daple-V2 lacks is the ability to trigger EMT and invasion, which is a feature unique to Daple-fl. Consistent with these findings, we also found that although both isoforms collaboratively suppress tumor cell growth, and have an additive prognostic impact, only Daple-fl is increased in invasive margins and in CTCs disseminated during metastatic progression. Because we previously showed that a functional GBA motif is essential for Daple-fl to trigger EMT and invasion28 and studies on cultured cells29 have confirmed that CRCs/cells that harbor PIK3CA mutation are most sensitive to the growth-suppressive action of Aspirin. Based on the properties of each isoform, we conclude that such a tradeoff is expected to overall maximize the desirable tumor-suppressive effect of Daple without the undesirable pro-EMT and pro-invasive effects. In fact, a decrease in the levels of Daple-V2 with a concomitant increase in Daple-fl transcripts in the circulating blood has recently been shown to correlate with increased stemness markers, an aggressive disease course and a shorter relapse-free survival in patients with melanoma30.Although it remains unknown how cells modulate the expression of each Daple isoform, our studies provided precious clues. When exposed to the chemoprevention drug, Aspirin, CRC cells that are sensitive to its growth-suppressive action were able to suppress Daple-fl but concomitantly increased Daple-V2; this effect was observed only in CRC cell lines that harbor the PIK3CA mutation. Prior clinical trials in humansDespite the insights gained, a lot remains unknown. For example, it is possible that the mRNA for Daple-V2 isoform could have cellular functions independently of protein expression. Similarly, the numerous other splice variants, including those not interrogated here, may contribute to a rich complex of Daple biology in cancer.In conclusion, we have revealed one of perhaps many ways how expression of Daple and its functions are regulated in the colon. Our findings that Daple isoforms can be pharmacologically manipulated to selectively augment its tumor-suppressive functions, while suppressing its pro-metastatic functions raises hope that therapeutic strategies could be tailored to meet such goals.3.Unless otherwise indicated, all reagents were of analytical grade and obtained from Sigma-Aldrich. Cell culture media were purchased from Invitrogen. All restriction endonucleases and Escherichia coli strain DH5α were purchased from New England Biolabs. E. coli strain BL21 (DE3) and phalloidin-Texas Red were purchased from Invitrogen. Genejuice transfection reagent was from Novagen. PfuUltra DNA polymerase was purchased from Stratagene. Recombinant Goat anti-rabbit and goat anti-mouse Alexa Fluor 680 or IRDye 800 F(ab′)2 used for immunoblotting were from Li-Cor Biosciences. Mouse anti-α-tubulin and anti-actin were obtained from Sigma; anti-Myc was obtained from Covance, and anti-GFP from Santa Cruz Biotechnology. Rabbit anti–pan-Gβ (M-14), and anti-Gαi3 were obtained from Santa Cruz Biotechnology. Anti-Daple antibodies were generated in collaboration with Millipore using the C-terminus of Daple (1660–2028 aa) as an immunogen. WNT proteins were purified from 6 liters of CHO CM. CM was complemented with 1% Triton X-100 (vol/vol), 20 mM Tris-Cl pH 7.5, and 0.01% NaN11. The subsequent site-directed mutagenesis and truncated constructs (myc-Daple full length F1675A (FA) and deleted from 2025–2028 aa (ΔPBM) were carried out on this template using Quick Change as per manufacturer’s protocol. Cloning of N-terminally tagged myc-Daple-V2 was carried out by PCR cloning directly from HeLa cDNA using primers containing the unique 5′ region (MSVLS) in Daple V2 sequence (corresponding to UniProtKB - Q9P219-2) and being inserted into myc-pcDNA 3.1 (+) between Kpn-1/EcoR1. To clone mCherry Daple-V2, Daple-V2 was PCR amplified from HeLa cDNA with primers flanked with recombination site for Gateway cloning system (Invitrogen), as previously described11. Daple-V2-FA (F194A) and Daple-V2- ΔPBM (deleted from 549–552aa) were also directly cloned out from pcDNA3.1(+)-Daple-fl-FA and pcDNA3.1(+)-daple-fl- ΔPBM. Cloning of rat Gα-proteins into pGEX-4T-1 GST-Gαi3 has been described previously35. GST-tagged FZD7-CT construct36 was a generous gift from Ryoji Yao . GST-Dvl2-PDZ was from Raymond Habas .Cloning of N-terminally tagged myc-Daple-fl and mCherry Daple-fl was carried out as described previouslyE. coli strain BL21 (DE3) (Invitrogen) and purified as described previously33. Briefly, bacterial cultures were induced overnight at 25 °C with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Pelleted bacteria from 1 L of culture were re-suspended in 10 ml GST-lysis buffer  for 90 min at room temperature as described before46. Lysates (~250 µg) of Cos7 cells expressing appropriate myc-Daple-V2 constructs were added to each tube, and binding reactions were carried out for 4 h at 4 °C with constant tumbling in binding buffer . Beads were washed (4x) with 1 mL of wash buffer  and boiled in Laemmli’s sample buffer. Immunoblot quantification was performed by infrared imaging following the manufacturer’s protocols using an Odyssey imaging system (Li-Cor Biosciences).Purified GST alone, GST-Gαi3 or GST-PDZ (5 µg) were immobilized on glutathione-Sepharose beads and incubated with binding buffer [50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.4% (v:v) Nonidet P-40, 10 mM MgCl1. Next morning, GST-FZD7-CT immobilized on glutathione beads were washed and subsequently incubated with His-tagged Daple-CT or Gαi3 proteins at 4 °C with constant tumbling. Washes and immunoblotting were performed as previously.GST-FZD7-CT construct was immobilized on glutathione-Sepharose beads directly from bacterial lysates by overnight incubation at 4 °C with constant tumbling as described before47. Stable cells lines expressing this reporter were generated by lentiviral transduction and subsequent selection using standard procedures. Lentiviral infection and selection were performed according to standard procedures. Briefly, 10 cm plates DLD1 cells at 70% confluency were incubated with media containing 8 µg/mL polybrene and 10 µl of lentivirus for 6 h. After 24 hours post infection, selection of puromycin-resistant clones was initiated by adding the antibiotic at 2 µg/ml final concentration. The resultant DLD1-7TGP stable cells were subsequently transfected with various myc-Daple V2 constructs and selected for G418 resistance as described earlier in methods. The DLD1-7TGP cells stably expressing myc-Daple were incubated overnight at 0.2% FBS, analyzed by fluorescence microscopy, and photographed prior to lysis. Whole cell lysates samples were then boiled in Laemmli’s sample buffer and GFP protein expression was monitored by immunoblotting.These assays were carried out using the well-established reporter 7xTcf-eGFP(7TGP)2 incubator. At various times cells were trypsin released and manually counted with a haemocytometer. Results are means ± SEM (n = 3). For cell viability assays, cells were seeded into 96-well plates at 2 × 105 cells/ml and cultured in DMEM/Ham’s F12 media supplemented with 2% FBS for 1–5 days. Cell growth was documented every 24 hours via a colorimetric assay using a 3--2,5-diphenyltetrazolium bromide (MTT) assay (Sigma). Absorbance values were collected at 490 nm using a SpectraMax 190 microplate reader . Control samples were treated with vehicle (0.1% DMSO or ethanol in DMEM/Ham’s F12 culture media). In each individual experiment, proliferation was determined in triplicate, and the overall experiment was repeated at least three times.Two complementary methods were used. For cell counting assays, cells were seeded at 1 × 104 per well in Ham’s F12 media with 10% (v/v) FBS and grown at 37 °C in a humidified 5% CO2 at 37 °C for ~2 weeks in 0.2% FBS growth media. Colonies were then stained with 0.005% crystal violet for 1 h. The remaining DLD1 cells were lysed and analyzed by WB to confirm Daple construct expression. Each experiment was analyzed in triplicate.Anchorage-dependent growth was monitored on solid (plastic) surface. Approximately ~1000 DLD1 cells stably expressing various Daple constructs were plated in 6-well plates and incubated in 5% CONIH3T3 cell invasion assay in 3D culture was performed according to the manufacturer’s protocol . Briefly, NIH3T3 cells (3000 cells) transfected with empty vector (control) or myc-Daple constructs were incubated first in the Spheroid Formation extracellular matrix (ECM) containing 0.2% FBS for 3 days. Invasion matrix was then added and layered on top with media containing FBS. Serum-triggered cell invasion was photographed under light microscope every day for 10 days and fresh media (FBS concentration is increased each time in order to maintain a gradient) was replenished every 48 hours. Photographs were analyzed and pseudocolored by Image J to reflect cell density.48. Total mRNA was extracted by standard procedures , after histology guided sample selection to ensure a tumor cell content of >50%, and transcribed to cDNA as described in detail earlier, for expression analysis of DAPLE48.These studies abide by the Declaration of Helsinki principles.The ethics committee of the Klinikumrechts der Isar, Munich, Germany, approved collection of the patient samples . All samples were obtained after prior informed written consent. Tumor tissue from 173 patients with histopathologically confirmed stage II (AJCC/UICC) colon cancer who underwent complete surgical resection (R0) between 1987 and 2006 was obtained, by a pathologist immediately after surgical resection. Specimens were transferred into liquid nitrogen and stored at −80 °C until further processing. None of the patients received neoadjuvant treatment. No metachronous tumors were found in the colon or rectum. As reported previously in detail, clinical data and post-operative follow-up was collected for all patients; moreover, DNA was isolated for KRAS and BRAF mutation analysis, as well as microsatellite instability testingTotal RNA was isolated using an RNeasy kit (QIAGEN) as per the manufacturers’ protocol. First-strand cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen), followed by ribonuclease H treatment (Invitrogen) prior to performing quantitative real-time PCR. A standard curve, to quantify mRNA copy number, was constructed using larger PCR products (~700 bp) that included the target sequence used in qPCR. Reactions omitting reverse transcriptase were performed in each experiment as negative controls. Reactions were then run on a real-time PCR system . Gene expression was detected with SYBR green/Taqman assay (Invitrogen), and relative gene expression was determined by normalizing to GAPDH using the comparative ΔCt/Relative standard curve method.Primer and probe sequences are as listed below.Probes and primers used in verifying Daple isoforms rms Fig. Primer 1: ACCTGCCAAGAAAGAAGGPrimer 2: ACCTGCAGTGGCAATGGCProbes and primers used in Taqman assays: for human tumor/tissue and CTC samplesGAPDH:hGAPDH-fwd: 5′-CAGTTGTAGGCAAGCTGCGA-3′hGAPDH-rev: 5′-TATGACAGGCCCGAAGCTTCT-3′hGAPDH-probe: 5′-CCAAGCCTGAGGGCAAGGCTATAATAGATGAAT-3′hGapdh-standard fwd: 5′-GCT GTG ACA TCA GGG CAA T-3′hGapdh-standard rev: 5′-GGC GGT GGT GGC TTT ATT T-3′Daple-fl:hDaple-fl-fwd: 5′-CGGGACCTCACCAAGCAA-3′hDaple-fl-rev: 5′-CTGCTGAGCTGCTGGCTCTT-3′hDaple-fl-probe: 5′-CAACTCTGAGGGAGGACCTGGTGCTC-3′hDaple-fl-Standard-fwd: 5′-GGATGCAGTCTTGGACGATAG-3′hDaple-fl-Standard-rev: 5′-CTTCTTTCATGGCTAGTGTTGTTT-3′Daple-V2:hDaple-V2-fwd: 5′-GGAGCCTCAGGATATACGTGCA-3′hDaple-V2-rev: 5′-TCAAGGCTGCCTCTGTGTGG-3′hDaple-V2-probe: 5′-CAGGATGTCCGTACTAAGCCCTGGGGATC-3′hDaple-V2 Standard-fwd: 5′-CACTCCCTGGACCATTTCTT-3′hDaple-V2 Standard-rev: 5′-CTGTAGTGGTGGCTGAAGTT-3′Primers used in SYBR-green assays: for cell-based analysesLox-3:hq-LOXL3 fwd: 5′-ATGGGTGCTATCCACCTGAG-3′hq-LOXL3 rev: 5′-GAGTCGGATCCTGGTCTCTG-3′Axin-2:hAxin-2-fwd: 5′-GAGTGGACTTGTGCCGACTTCA-3′hAxin-2-rev: 5′-GGTGGCTGGTGCAAAGACATAG-3′Vimentin:hVim-fwd: 5′-AAGAGAACTTTGCCGTTGAA-3′hVim-rev: 5′-GTGATGCTGAGAAGTTTCGT-3′SFRP-1:hSFRP-1-fwd: 5′-GAGTTTGCACTGAGGATGAAAA-3′hSFRP-1-rev: 5′-GCTTCTTCTTCTTGGGGACA-3′GADPH:hGADPH q-fwd2: 5′-TCA GTT GTA GGC AAG CTG CGA CGT-3′hGADPH q-rev2: 5′-AAGCCAGAGGCTGGTACCTAGAAC-3DAPLE:hDaple-fl-fwd: 5′-TGA CAT GGA GAC CCT GAA GGC TGA-3′hDaple fl-rev2: 5′-TTTCATGCGGGCCTCACTGCTGA-3′hDaple V2-Fwd: 5′-GTT GTC ACA CTC CCT GGA CCA TTT C-3′hDaple V2-Rev: 5′-GCTTTGGTTTTAGATCCCCAGGGC-3′Formalin-fixed paraffin embedded (FFPE) normal, polyp and cancer tissues used in this study were obtained from patients undergoing routine colonoscopies and provided by the section of Gastroenterology, VA San Diego Healthcare System. The protocol was approved by the Human Research Protection Program Institutional Review Board . These studies abide by the Declaration of Helsinki principles.Colon specimens of known histologic type were analyzed by IHC using a previously-validated anti-Daple-CT rabbit polyclonal antibody raised against Daple (1660–2028 aa) . Briefly, formalin-fixed, paraffin-embedded tissue sections of 4 µm thickness were cut and placed on glass slides coated with 3-aminopropyl triethoxysilane, followed by deparaffinization and hydration. Heat-induced epitope retrieval was performed using citrate buffer (pH 6) in a pressure cooker. Tissue sections were incubated with 3% hydrogen peroxidase for 15 min to block endogenous peroxidase activity, followed by incubation with primary antibodies overnight in a humidified chamber at 4 °C. Immunostaining was visualized with a labeled streptavidin-biotin using 3,3′-diaminobenzidine as a chromogen and counterstained with hematoxylin. All the samples were first quantitatively analyzed and scored based on 2 independent criteria. First, the intensity of staining was scored on a scale of 0 to 3, where 0 = no staining, 1 = light brown, 2 = brown, and 3 = dark brown. Second, the percentage of the cells that stained positive in the tumor area was scored on a scale of 0 to 4, where 0 = 0, 1 = ≤10%, 2 = 11–50%, 3 = 51–75%, and 4 = >75%. Subsequently, each tumor sample was assigned a final score, which is the product of its (intensity of staining) × (% cells that stained positive). Tumors were categorized as negative when their final score was <3 and as positive when their final score was ≥3.t test. The associations between the expression level of Daple isoforms and K-ras mutation status and metastatic status of disease were investigated by Fisher’s exact test. Parametric Pearson’s correlation and nonparametric Spearman’s correlation analysis were used to assess the relationship between the expression of Daple isoforms and patients’ age, tumor size, differentiation and grading, the level of Ki67-Index, Osteopontin, SASH1, MACC1 and CEA. In order to derive optimal cut-off values of gene expression levels, maximally selected log-rank statistics performed by Cutoff Finder (R Software version 2.15.0.)49 were used. Time-dependent survival probabilities were estimated with the Kaplan-Meier method using the log-rank test. All statistical tests were performed two-sided, and p- values less than 0.05 were considered to be statistically significant.Statistical evaluation was performed using GraphPad Prism 5 software. Unless stated otherwise, statistical significance was determined using Student’s Supplementary Figures and Legends


TULA-2 acts as a negative regulator of CLEC-2 signaling by dephosphorylating Syk on Y346 and restraining subsequent Syk-mediated signaling.Platelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors such as glycoprotein VI, the protease-activated receptors, and C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a HemITAM (hem-immunoreceptor tyrosine activation motif)-bearing receptor that binds podoplanin and signals through spleen tyrosine kinase (Syk). T-cell ubiquitin ligand-2 (TULA-2) is a protein tyrosine phosphatase that is highly expressed in platelets and targets phosphorylated Y352 of Syk. We wanted to determine whether TULA-2 regulates Syk phosphorylation and activity downstream of CLEC-2. To that end, we used TULA-2 knockout mice and wild-type (WT) littermate controls. We found that TULA-2 deficiency enhances the aggregation and secretion response following stimulation with an excitatory CLEC-2 antibody or the CLEC-2 agonist rhodocytin. Consistently, Syk phosphorylation of Y346 is enhanced, as well as phosphorylation of the downstream signaling molecule PLCγ2, in TULA-2 knockout platelets treated with either CLEC-2 antibody or rhodocytin, compared with WT control platelets. Furthermore, the kinetics of Syk phosphorylation, as well as that of PLCγ2 and SLP-76, is enhanced in TULA-2 knockout platelets treated with 2.5-μg/mL CLEC-2 antibody compared with WT platelets. Similarly, thromboxane production was enhanced, in both amount and kinetics, in TULA-2 Release of secondary mediators such as TXA and adenosine diphosphate reinforces the original excitatory signal and recruits new platelets to the growing thrombus. Just as this process is essential to maintain hemostasis, a similar process is necessary to insure lymphatic and blood vessel separation.Platelets are the primary mediators of thrombosis and hemostasis. They are small anucleate cells that exist in a quiescent state, and in a discoid shape. Platelets respond to vascular damage by binding to von Willebrand factor and collagen, which initiates an intracellular signaling cascade that culminates in shape change, production of thromboxane AXX(L/I)). The binding of podoplanin to CLEC-2 initiates a signaling cascade that involves tyrosine phosphorylation of the hemITAM via Src-family kinases (SFKs), and subsequent spleen tyrosine kinase (Syk) phosphorylation.Lymphatic endothelial cells differentiate from venous endothelial cells and, unlike venous and arterial endothelial cells, they express podoplanin, which is the only known physiologic ligand for C-type lectin-like receptor 2 (CLEC-2).Platelets, along with lymphatic endothelial cells, are responsible for maintaining separation of blood and lymph. Deletion of either SH2 domain–containing leukocyte protein of 76 kDa (SLP-76) or Syk  in mice resulted in embryonic lethality due to disrupted separation of blood and lymph.The CLEC-2 and podoplanin are also important for several other physiological and pathological processes. There is evidence in the literature that CLEC-2 plays a role in thrombosis and hemostasis, probably in conjunction with glycoprotein VI (GPVI) as deletion of both CLEC-2 and GPVI in mice causes bleeding and reduced arterial thrombus formation.Syk is a crucial mediator of signaling initiated by podoplanin engagement of CLEC-2. Syk is phosphorylated on several tyrosine residues following activation of CLEC-2, just as it is following activation of GPVI, which contains two ITAM motifs.Syk is a target of the protein tyrosine phosphatase T-cell ubiquitin ligand-2 (TULA-2).All reagents were purchased from Thermo Fischer Scientific unless otherwise noted. Rhodocytin was isolated as previously described.TULA-2 knockout mice were described previously.2, 0.42-mM NaH2PO4, 10-mM HEPES, and 0.2-U/mL apyrase, pH 7.4), and platelets were counted using a Hemavet 950FS blood cell counter  and adjusted to a final concentration of 1.5 × 108cells/mL.Mouse blood was collected and processed as previously described.All aggregation and secretion experiments were performed using a lumi-aggregometer (Chrono-log) at 37°C under stirring conditions. Platelets (250 μL) were stimulated with rhodocytin, CLEC-2 antibody, or CLEC-2 antibody plus DAR-IgG and aggregation was measured via light transmission. ATP secretion was measured simultaneously using Chrono-lume (a luciferine/luciferase reagent).−/−and wild-type (WT) mice were heated to 37°C without stirring. The platelets were then stimulated with 2.5 μg/mL of CLEC-2 antibody and allowed to incubate at 37°C. The reaction was stopped by snap freezing. Samples were collected every 30 seconds for 6 minutes. Thromboxane B2 was measured by enzyme-linked immunosorbent assay (ELISA), using a kit from Enzo Life Sciences  as per the manufacturer's instructions.Washed murine platelets (50 μL) isolated from TULA-24and washed with water prior to solubilization in sample buffer. Platelet protein samples were then boiled for 5 minutes. Proteins were resolved via SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with Odyssey blocking buffer and incubated overnight with primary antibodies against a protein of interest. After washing, the membranes were incubated with the appropriate secondary antibodies for 1 hour at room temperature. The membranes were then washed and imaged using a Li-Cor Odyssey infrared imaging system.Western blotting was performed as described previously.t-test. Significance was determined byp < 0.05. All bar graphs represent mean ± SEM for that dataset.All statistical comparisons were calculated using Student'sRhodocytin is a snake venom protein that binds and activates the CLEC-2 receptor.−/−platelets compared with WT control platelets  samples. The time points are presented in−/−samples and the time to reach that point was recorded. That time was used to collect the complementary point from WT platelets. For instance, if point 1 (the beginning of shape change) was reached in 30 seconds using TULA-2−/−platelets, then the complementary WT sample was collected 30 seconds after agonist stimulation. We also collected a sample when maximal aggregation was reached, which we termed “full.” Following SDS-PAGE of the samples collected using the previously described method, it was clear that Syk Y346 and Y519/520 were phosphorylated earlier and to a greater extent in TULA-2−/−platelets than WT platelets after stimulation with a CLEC-2-activating antibody  and SLP-76 are both phosphorylated downstream of Syk. Therefore, we analyzed the phosphorylation of PLCγ2 and SLP-76 using the same methodology described above. We observed that PLCγ2 was phosphorylated earlier and to a greater extent at both Y759 and Y1217 in TULA-2ylation  followiylation ,D. AdIn this report, we demonstrate that TULA-2 is a negative regulator of signaling initiated by the hemITAM-containing CLEC-2 receptor. The regulation of CLEC-2-induced signaling may be important for several physiological processes such as separation of blood and lymph, thrombosis and hemostasis, and tumor cell metastasis.−/−mouse platelets compared with WT control platelets when either GPVI or FcγRIIA were stimulated. This suggests that TULA-2 acts as an important regulator of Syk phosphorylation when Syk is activated by either a hemITAM or ITAM-containing receptor. Upon ligand engagement of a hemITAM or ITAM-containing receptor, SFKs phosphorylate the tyrosine within the YXX(L/I) motif (one tyrosine per hemITAM and 2 per ITAM).−/−platelets following stimulation of CLEC-2.In terms of Syk phosphorylation, our data agree with published data describing the effect of TULA-2 on Syk downstream of two ITAM-containing receptors, FcγRIIA and GPVI.−/−platelets stimulated with a CLEC-2 antibody, but also is detected earlier than similarly treated WT platelets. Furthermore, Syk phosphorylation continues to persist, and even increases throughout the aggregation process, which does not occur in time course experiments when platelets are stimulated with agonists to GPVI.−/−platelets. Interestingly, Syk Y346 and Y519/520 phosphorylation decreases from that point forward, even in TULA-2−/−platelets.Our data show that Syk phosphorylation not only is greater in TULA-2The reason for this discrepancy could be that a burst of Syk phosphorylation occurs following GPVI stimulation due to the ITAM having two YXX(L/I) motifs, which would allow Syk to bind immediately upon GPVI activation, thus resulting in phosphorylation of a large portion of total Syk. From that point, any alterations in Syk phosphorylation could be due to phosphatase activity. The absence of a phosphatase such as TULA-2 would delay the reduction in Syk phosphorylation, at least at Y346. Conversely, time may be required for CLEC-2 to cluster and present two hemITAM domains for Syk to bind (one for each tandem SH2 domain) and autophosphorylate, which would delay Syk phosphorylation. The absence of a phosphatase such as TULA-2 to remove the initial phosphorylation on the small amount of Syk that is phosphorylated initially may give the impression that Syk is phosphorylated at an earlier time point when, in fact, it is not dephosphorylated. Furthermore, CLEC-2 clustering due to the CLEC-2 antibody may continue throughout the aggregation process leading to greater Syk phosphorylation as the experiment continues. The lack of TULA-2 would only enhance the increase in Syk phosphorylation, which is exactly what we observed.It is also possible that the phosphatases that function downstream of GPVI are different from those that function downstream of CLEC-2. The data reported for Syk phosphorylation downstream of GPVI in the absence of TULA-2 suggest that there are phosphatases other than TULA-2 that dephosphorylate Syk, as Syk phosphorylation is reduced over time. Our data suggest that TULA-2 could be the main initial phosphatase that acts on Syk downstream of CLEC-2, since Syk phosphorylation continues to increase over time in the absence of TULA-2.q.qand G12/13, can augment Syk phosphorylation when CLEC-2 is likewise stimulated. In this report we demonstrated that thromboxane production is greatly enhanced in TULA-2−/−platelets stimulated with a CLEC-2 antibody compared with WT control platelets. Therefore, it is possible that the enhanced Syk phosphorylation observed downstream of the CLEC-2 receptor in TULA-2−/−platelets is partly due to feedback from the TP receptor.We have previously detailed an important crosstalk mechanism that occurs between CLEC-2 and GWe demonstrate in this report that TULA-2 acts as a negative regulator of CLEC-2-mediated signaling by dephosphorylating its target substrate, Syk. TULA-2 is one of several phosphatases, such as SHP-1, SHP-2, SHIP-1, and CD148, that control ITAM and hemITAM-mediated signaling in platelets.


Rheumatoid arthritis (RA) is an autoimmune disease that affects the synovial cavity of joints, and its pathogenesis is associated with an increased expression of pro-inflammatory cytokines, namely tumour necrosis factor-alpha (TNF-α). It has been clinically shown to have an adequate response to systemic administration of TNF-α inhibitors, although with many shortcomings. To overcome such limitations, the immobilization of a TNF-α antibody on a nanofibrous substrate to promote a localized action is herein proposed. By using this approach, the antibody has its maximum therapeutic efficacy and a prolonged therapeutic benefit, avoiding the systemic side-effects associated with conventional biological agents’ therapies. To technically achieve such a purpose, the surface of electrospun nanofibers is initially activated and functionalized, allowing TNF-α antibody immobilization at a maximum concentration of 6 µg/mL. Experimental results evidence that the biofunctionalized nanofibrous substrate is effective in achieving a sustained capture of soluble TNF-α over time. Moreover, cell biology assays demonstrate that this system has no deleterious effect over human articular chondrocytes metabolism and activity. Therefore, the developed TNF-capturing system may represent a potential therapeutic approach for the local management of severely affected joints. Rheumatoid arthritis (RA) is an autoimmune disease that affects primarily the synovial cavity tissues on the small diarthrodial joints of the hands and feet , leadingThe disease pathophysiology involves several interconnected mechanisms. Specific antibodies for immunoglobulin G (IgG) mediate the autoimmune process, as well as the imbalanced expression of the pro-inflammatory cytokine’s profile and its functionality ,12,13,14From the autoimmune disease (AD) subsets that entail the multiple inflammatory cascades, one of the most significant players is the TNF-α, particularly the over-expression of the TNF-α ,13,17. TThe increasing knowledge on RA pathogenesis stimulated the development of different therapeutic modalities aiming to avoid joint destruction, minimize the symptomatic profile, and enhance physical function . These oConsidering the significant involvement of TNF-α in RA, as well as the rising evidences that this cytokine heads the pro-inflammatory cytokine cascade, it becomes a significant therapeutic target ,32. SpecFurthermore, some attention has been given to drug carriers, in order to maintain effective concentration levels in plasma for extended periods . ExampleConsidering the shortcomings of these treatments, there is a need for innovative approaches that circumvent the aforementioned adverse side effects. Therefore, the main objective of this work was to develop an implantable system capable of capturing excessive TNF-alpha present in intra-articular cavities of an RA patient. For that, we immobilized a neutralizing TNF-α antibody at the surface of a polymeric substrate . This strategy takes advantage of specific interactions between the antibody and the antigen, where the antibody (TNF-α antibody) binds to the antigen (TNF-α) avoiding/limiting its harmful pro-inflammatory effects. After the system´s assembly and proved ability to capture TNF-α secreted by activated monocyte-derived macrophages, the cytotoxicity of the biofunctionalized system was tested with human articular chondrocytes. Indeed, the cytocompatibility and chondrogenic differentiation potential of bare electrospun nanofibers were previously reported by us ,46,47, cN,N-dimethylformamide (DMF), 1,6-hexamethylenediamine (HMD), Ellmans reagent (DTNB), 2-iminiothiolane (2IT), 2-(N-morpholino)-ethanesulfonic acid (MES hydrate), phosphate buffered saline (PBS), N-hydroxysuccinimide (NHS), and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich  and kept at room temperature (RT), with the exception of 2IT which was kept at 4 °C and EDC which was kept at −20 °C. 4-(dimethylamino) pyridine (DMAP) was purchased from Millipore  and kept at RT. Anti-TNF-α antibody [B-C7] was purchased from Abcam  and kept at 4 °C until further use. Alexa Fluor® 488 goat anti-mouse rabbit IgG (H + L) and TNF-α human ELISA kit were purchased from Life Technologies  and kept at 4 °C until further use.Polycaprolactone , chloroform, w/v) PCL dissolved in an organic solvent mixture of chloroform and dimethylformamide (7:3 ratio). This PCL solution was electrospun by applying a voltage of 12 kV, a needle tip-to-ground collector distance of 20 cm and a flow rate of 1 mL/h. After the complete processing of 1 mL of PCL solution, the NFMs were left to dry for 1 day at RT inside a chemical hood, in order to completely evaporate the solvents. All further tests described were performed in samples of 10-mm squares.Nanofiber meshes (NFMs) were prepared as described elsewhere ,49. Brie®, ProCleaner 220, Bioforce Nanoscience, Salt Lake City, UT, USA). Afterwards, the amine groups (–NH2) were inserted by immersion of the irradiated NFM in a 1 M hexanediamine (HMD) solution during 1 hour at 37 °C.The surface functionalization of electrospun NFMs was performed as described elsewhere ,49. Brie−2) molar absorption coefficient, the value of 14,151 M−1 cm−1 was used [Quantification of amine groups present in each NFM was performed as described elsewhere ,51. For was used ,51.N-morpholino)-ethanesulfonic acid (MES) buffer with 0.9% (w/w) NaCl, followed by pH adjustment to 4.7 [The TNF-α antibody was immobilized at the surface of activated and functionalized electrospun NFMs. To determine the substrate’s maximum immobilization capacity, a wide range of primary antibody concentrations were considered (from 0 to 12 µg/mL). The TNF-α antibody was firstly activated by a 15-minute incubation with a solution of EDC/NHS (50 mM EDC and 200 mM NHS), dissolved in a 0.1 M 2-(t to 4.7 .® 488 solution diluted in PBS (1:200 ratio). As a negative control, NFMs with no antibody immobilized (plotted as 0 µg/mL) were used. The fluorescence of the supernatant was determined in triplicate, using an excitation of 495/20 nm and an emission of 519/20 nm, and used to quantify the amount of antibody effectively immobilized.The functionalized NFMs were incubated with 200 μL of the activated TNF-α antibody overnight at 4 °C. Biofunctionalized NFMs were washed three times with PBS. A 3% bovine serum albumin (BSA) solution was used as a blocking step. To determine the degree of TNF-α antibody immobilization, biofunctionalized NFMs were incubated with an Alexa FluorSEM was used to analyse the morphology of biofunctionalized NFMs. Briefly, the biofunctionalized NFMs were sputter-coated with a thin layer (9–12 nm) of gold/palladium (Cressington 208 HR) and then analysed by SEM . Micrographs were recorded at 15 kV with magnifications ranging from 100 to 2000 times.A fluorescence microscope  was used to analyze the spatial distribution of the TNF-α antibody at the surface of electrospun NFMs. The TNF-α antibody was linked by a secondary antibody with green fluorescence (Alexa Fluor™ 488). Photographs were recorded at magnifications of 50, 200 and 400 times.A human monocytic cell line THP-1 was maintained in RPMI 1640 media supplemented with 2 mM L-glutamine, 100 µg/mL of penicillin, 100 μg/mL of streptomycin, 10 mM HEPES, and 10% fetal bovine serum  . For the induction of cell differentiation, cells (106 per mL) were seeded in cRPMI with 100 nM phorbol 12-myristate-13-acetate (PMA) for 24 h. After incubation, non-attached cells were removed by aspiration, and the adherent cells were washed three times with cRPMI. To ensure reversion of cells to a resting macrophage phenotype before stimulation, cells were incubated for an additional 48 h in cRPMI without PMA. For stimulation and retrieval of conditioned media, cells were further incubated for 4 h with 100 ng/mL of lipopolysaccharide (LPS) in fresh cRPMI and the supernatants were collected and stored at −80 °C. Production of TNF-α was assessed in the conditioned media by commercial ELISA . Four systems were tested at least in quadruplicates, in two independent assays: (1) the biofunctionalized NFM (with immobilized TNF-α antibody); (2) a positive control with the soluble TNF-α antibody; (3) a negative control with the UV-O activated NFM; and (4) the conditioned cell culture medium alone. Each system was placed in sterile Eppendorf tubes and 2 mL of monocyte-derived macrophage conditioned medium was added individually, and kept at 37 °C in agitation in an orbital shaker at 120 rpm. Initially, a 3 days assay was performed with a 1 ng/mL TNF-α concentration from monocyte-derived macrophage conditioned medium. A specimen was collected at the following time points  without conditioned medium replacement. Afterwards, a 15-day assay was conducted, where a sample was collected daily and recharged with the same volume of recovered cell culture medium each day, which had a TNF-α concentration of 500 pg/mL. This concentration was chosen due to being closer to TNF-α levels in active RA patients (76.1 ± 103.2 pg/mL) , consideFor the quantification of TNF-α in the conditioned medium at each time point, sandwich TNF-α ELISA  was performed according to the manufacturer. Absorbance was read at 450 nm on a microplate reader . A standard curve was built with concentrations ranging from 0 to 1000 pg/ml. The final concentrations were calculated by subtracting the value of each sample  to the conditioned culture medium baseline , and the average was plotted for each testing condition along time.Cartilage tissue consists of only one cell type, the chondrocyte, embedded in a dense extracellular matrix (ECM). To evaluate the cytotoxicity of the developed biofunctionalized nanofibrous substrates, human articular chondrocytes (hAC) isolated from knee cartilage samples, collected from arthroplasties surgeries biopsies, were used. These cells were chosen as they were isolated from diseased joints cartilage. Samples were collected under the cooperation agreement between Centro Hospitalar do Alto Ave, Guimarães, Portugal, and the 3B’s Research Group, after informed donor consent. Briefly, cells were isolated by enzymatic digestion, according to the protocol described previously .l-alanyl-l-glutamine (Sigma), Non Essential Aminoacids (Sigma) 10,000 units/ml penicillin, 10,000 μg/mL streptomycin, and 10% foetal calf serum was the basis for the expansion and differentiation medium used herein (Basic medium). Basic medium was supplemented with 10 ng/mL of bFGF for hAC expansion. Basic medium was further supplemented with 1 mg/mL of insulin and 1 mg/mL of ascorbic acid, when hAC were seeded onto the PCL NFM (differentiation medium). These supplements enhanced extracellular matrix (ECM) deposition by the cultured hACs.Dulbecco’s modified Eagle’s medium , containing 10 mM Hepes buffer , Human articular chondrocytes where used at passage 4. Expansion medium was changed every 2 days until the cells reached a confluence of 90%. The cells were harvested and seeded onto both the activated NFM, as well the NFM with the TNF-α antibody immobilized using the differentiation medium.Electrospun PCL NFMs were sterilized by UV-O treatment. All the surface functionalization and TNF-α antibody immobilization steps were performed in sterile conditions. For the biofunctionalized NFMs, antibody immobilization was performed overnight, and after the BSA blocking step, cell seeding was performed. Activated and biofunctionalized electrospun NFMs were used as controls.2 over 4 hours. After cell attachment to the NFMs, 1 mL of expansion medium was added to each well. Afterwards, the medium was changed into differentiation medium. The seeding was performed in three independent experiments. For each independent experiment, constructs were cultured for 1, 14, 21 and 28 days under static conditions and collected at each time point for quantification of cell viability, DNA, total protein analysis, as well as GAGs deposition. Cell morphology was evaluated by SEM.Confluent hACs were detached from the culture flask using trypsin, counted on a hemocytometer and seeded at a density of 200.000 cells/NFM. Seeding was performed using the droplet method, using a 50 µL drop of cell suspension on all sample groups in 24-well plates. The plates were placed at 37 °C and 5% CO® dsDNA Reagent and Kit , according to the manufacturer´s instructions. Triplicates of each condition collected at 1, 14, 21 and 28 days were evaluated. The specimens were collected, 1 mL of sterile distilled water was added, and then samples were stored at −80 °C until further analysis. Prior quantification, the samples were defrosted and sonicated for 15 min. To extrapolate the DNA values for each sample, a set of standards were prepared with concentrations ranging from 0 to 1.5 μg/mL. The fluorescence of each sample was measured on an opaque 96-well plate using an excitation of 485/20 nm and an emission of 528/20 nm, using a microplate reader .DNA quantification was assessed using Quant-iT™ PicoGreenTotal protein was performed using Micro BCA™ Protein Assay Kit  according to the manufacturer´s instructions. Triplicates of each condition collected at 1, 14, 21, and 28 days were evaluated. The samples were collected as described in N-acetyl cysteine (Sigma Aldrich) at concentrations of 0.05% and 0.096%, respectively, to 50 mL of digestion buffer (200 mM of phosphate buffer containing 1 mM EDTA (Sigma Aldrich), pH 6.8). Each specimen was incubated with 600 μL of digestion buffer, overnight at 60 °C. After a 10-min centrifugation at 1300 rpm, the supernatant was collected. Dimethymethylene Blue (DMB) stock solution was prepared dissolving 16 mg of DMB powder in 900 mL of distilled water containing 3.04 g of glycine and 2.73 g of NaCl. pH was adjusted to 3.0 with HCl and the volume adjusted to 1 L. The solution was stored at RT covered by aluminum foil. Chondroitin sulphate  solution was prepared in water in a 5 mg/mL stock solution and kept refrigerated. Standards were prepared from serial dilutions of this solution. Three samples per condition were considered per time point, and the absorbance of each sample was measured at 530 nm using a microplate reader.GAG quantification was performed using papain digestion. Triplicates of each condition collected at each previously established time point were tested. The samples were collected and stored in eppendorf tubes at −80 °C until further analysis. Digestion solution was prepared by adding papain  and Samples were collected at the end of the experiment and processed for histology. Samples were then fixed in 10% neutral-buffered formalin and then kept at 4 °C until the staining procedures. Alcian Blue staining was performed by rinsing the samples in 3% acetic acid, keeping them in 1% alcian blue solution  for 1 h. After that, the stain was poured off and sections were washed with water, let to dry, and then rinsed in absolute alcohol, cleared in xylene, and mounted in Entellan rapid .Statistical analysis of values related with antibody immobilization, TNF-α capturing profiles and biological studies was performed using IBM SPSS software . Firstly, a Shapiro-Wilk test was used to ascertain the assumption of data normality. For antibody immobilization, protein capture and biological assays, the results showed that the data was not following a normal distribution. P values lower than 0.01 were considered statistically significant in the analysis of the results. A Kruskal-Wallis test was also performed for the comparison of more than two independent groups of samples for one variable.The primary antibody (anti-TNF-α antibody) immobilization at the surface of electrospun nanofiber meshes (NFMs) was performed in a wide range of concentrations (0–12 µg/mL) to determine the maximum antibody immobilization capacity. The fluorescence of the unbound secondary antibody was measured and the values were plotted against the various primary antibody concentrations . As thisp < 0.01). Concentrations of 6 μg/mL and 8 μg/mL exhibited significantly lower values of fluorescence intensity when compared to all lower concentrations (p < 0.001). Moreover, these two concentration values did not display differences between themselves. Finally, when the antibody was immobilized at a concentration of 12 μg/mL, the fluorescence intensity of secondary antibody did not present significant differences when compared to the fluorescence intensity of NFMs with primary antibody immobilized at concentrations above 2 µg/mL (p < 0.01). Therefore, primary antibody immobilization at the surface of the nanofibrous substrate reached a maximum concentration of 6 µg/mL, since no statistically significant differences were found between the concentrations above. This value is within the range of concentrations systemically administrated in clinical practice using Infliximab  [The statistical analysis showed that the immobilized primary antibody, with concentrations above 4 μg/mL, displayed significantly lower values of fluorescence intensity than 0 μg/mL (dy mass) .® secondary antibody was not immobilized at the surface of activated and functionalized NFMs (data not shown), confirming the specific bond between the primary and secondary antibodies.After the optimization of the anti-TNF-α antibody immobilization, its spatial distribution at the surface of electrospun NFMs was evaluated by fluorescence microscopy . The fluAntibody immobilization was achieved by the amine groups inserted at the nanofibers’ surface, which provides binding sites. Consequently, the availability of this functional group influences directly the primary antibody immobilization. To confirm the success of aminolysis functionalization, as well as to determine if the binding points were enough for primary antibody immobilization, quantification of available amine groups was performed. For this, SH groups were inserted at the surface of the aminolysis-treated NFMs and determined by the Ellman’s reagent method . Table 1The ultimate goal of this work was to engineer a system that could capture TNF-α. Accordingly, after the primary antibody immobilization optimization, the TNF-α capturing capacity of the biofunctionalized substrate was assessed. A TNF-α rich conditioned culture medium from stimulated monocyte-derived macrophages was used over two distinct time intervals: 3 days representing the half-life of the anti-TNF-α antibody and 15 days representing the time interval of anti-TNF-α antibody administration in the clinic. Four conditions were tested and compared: (1) the biofunctionalized NFM (with primary antibody); (2) a positive control with the soluble form of the anti-TNF-α; (3) a negative control with UV-O activated NFM; and (4) the conditioned culture medium alone. The quantification of TNF-α levels from the conditioned culture medium (condition 4) was used as a baseline, eliminating the quantification of degraded TNF-α but not captured. Therefore, to guarantee the values’ accuracy and reproducibility between experiments, the values herein presented do not correspond to the ones quantified by ELISA, but just to the concentration of the TNF-α captured.p < 0.01) (p < 0.01). In addition, no significant differences were found between the sAb and the control condition UV-O activated NFM (NFM).The proposed system comprising anti-TNF-α immobilized at the surface of NFMs (NFM + Ab) was found to capture soluble TNF-α in significant amounts after 8 hours of incubation when compared to the UV-O activated NFM (NFM) ( < 0.01) . This obThe initial efficiency of the activated NFM over the TNF-α levels at early time points might be justified by the presence of free binding sites  was performed. Experimental results show that the sAb only has a biological effect for the first 3 days, since from the fourth time point on, it does not present significant differences when compared to the UV-O activated NFM . The NFMThese results confirm the initial hypothesis that the immobilization of the primary antibody at the NFMs’ surface had a longer effect than the soluble antibody. This longer effect might be supported by the increased stability that the antibody immobilization provides . MoreoveAfter the successful assembly of the TNF-capturing system, we then tested it in contact with arthritic joint cells. Therefore, the toxicity of the biofunctionalized NFMs was assessed by culturing them with human articular chondrocytes (hACs). These cells were isolated from diseased knee arthroplasties, thus presenting a phenotype that is associated with RA and osteoarthritis pathologies, where inflammation was present. Different biological assays were conducted to assess the hACs morphology, proliferation, total protein, and glycosaminoglycans (GAGs) synthesis. hACs were cultured at the surface of three different substrates: (1) anti-TNF-α antibody immobilized at the surface of NFMs (NFM + Ab), (2) UV-O activated NFM (NFM) and (3) tissue culture polystyrene (TCPs) as positive control. The influence of the anti-TNF-α immobilization at the surface of NFMs over hACs morphology was assessed by SEM. Micrographs  showed tp < 0.001) (p < 0.001). Taken together, these results indicate that the immobilized anti-TNF-α may inhibit chondrocytes growth by mimicking an inflammatory environment, where cells would respond by limiting their growth and proliferation. This effect is not negative, since our primary goal was not to promote an extensive cell proliferation, but merely to assess the device’s potential cell toxicity. Nevertheless, hACs keep proliferating, even if at a lower rate when compared to the controls. Indeed, IL1-β and TNF-α have been shown to inhibit the migratory potential of chondrogenic progenitor cells in osteoarthritic cartilage [Chondrocytes proliferation was also evaluated by quantifying the dsDNA concentration. Experimental results showed that the TCP’s condition displayed a significantly higher DNA concentration, for 14 and 28 days of culture, when compared to all other culture conditions (< 0.001) A. Furtheartilage . Furtherartilage , thus thp < 0.01). On the other hand, the NFM + Ab testing condition displayed a significantly lower protein concentration when compared to all other culture conditions for 28 days of culture (p < 0.01). At 14 days of hACs culture, no significant differences between conditions were found (p = 0.148). The protein synthesis in the NFM + Ab condition was lower for longer culture periods, which is possibly due to the extracellular accumulation of proteins by hACs, rather than the intracellular synthesizing rate.Quantification of total protein synthesis is presented in p = 0.186) and 14th days (p = 0.088) of the experiment. On the 21st day, the NFM + Ab testing condition displayed significantly lower GAG concentrations than control condition TCPs (p < 0.01). Moreover, on the 28th day, TCPs presented significant higher GAGs concentration than all other culture conditions (p < 0.01). Concerning the GAGs production by hACs cultured onto the NFM + Ab, the obtained results confirm the evidences found in the intracellular protein synthesis. There is a decrease in the total protein concentration values for longer time points, with a concomitant increase of GAGs production for these same periods of time, indicating that protein synthesis was being directed by the GAGs production. This increase of GAGs concentration for late time points of the experiment are in accordance to our hypothesis, as they will protect chondrocytes against the inflammatory environment.Furthermore, the impact of the anti-TNF-α antibody immobilization at the surface of NFMs on GAG production and accumulation A by hACsAs it can be observed in The present work proposes the use of nanofibrous meshes (NFM) functionalized with a neutralizing anti-TNF-α antibody, for the selective clearance of TNF-α. From a spectrum of primary antibody concentrations, 6 µg/mL was determined as the maximum immobilization capacity of a UV-O activated and aminolysis-functionalized NFM. Experimental results demonstrated the bioactivity of the developed biofunctional substrate on clearing TNF-α from conditioned culture medium of macrophages and a longer time of action when compared to the circulating/soluble antibody. Furthermore, cell biology data showed that the immobilized anti-TNF-α antibody exhibited no cytotoxicity over human articular chondrocytes and that these cells were able to maintain their metabolic functions in the presence of the TNF-capturing system. In conclusion, we herein describe an effective system for local clearance of TNF-α, which is particularly relevant in a RA scenario, but adaptable to other autoimmune inflammatory conditions.Martins A, Oliveira C, Reis RL, Neves NM. Polymeric substrates with immobilized antibodies and method of production thereof. WO/2015/166414 A1, EP 3137906 A1, US 2017/0051050 A1. Priority date 28/04/2014.


The Morris water maze (MWM) is widely used to evaluate rodent spatial learning and memory. However, current evaluation measures are not comprehensive because there is a wide distribution in the measured response. Utilizing the graph cognition hypothesis, we proposed four new deviation indices to evaluate cognitive function in the MWM that compared the optimal swim track to the actual track taken. These include the sum of the lateral deviation vectors, the sum of the offset angles, the sum of the correction vectors, and the sum of the lateral deviation vectors to the initial optimal route. We compared the four new deviation indices to the classically used escape latency measures in a vascular dementia model and demonstrated a higher consistency in the normal distribution between the vascular dementia group and the control rats. Further, the new measures displayed higher sensitivity and specificity compared to what escape latency displayed in the Monte Carlo simulation. From the receiver operating characteristic curve, the diagnostic values of the new deviation indices are higher than those of escape latency. Therefore, including these new evaluation indices in MWM experiments provided a more effective analysis of cognitive function compared to using escape latency. Many of the metrics currently used in the MWM are based on simple summaries of the rodent’s path, such as path-length, the percent of time spent away from walls, escape latency, angles of approach to the platform, and quadrant time, etc.6. These measures are convenient to observe and record, but they rarely examine the dynamic behavior of the animal while searching for the platform7, missing key parameters that could provide valuable insights into learning and memory. For example, escape latency, one of the most common parameters, was widely used in the MWM8. Reductions in escape latency suggest that the experimental subjects focused on searching strategies, but in fact, reduced escape latencies may also reflect the adoption of non-spatial strategies9. Rodents that suffer from vascular dementia swim along the wall randomly searching for escape routes until they happen to swim away from the wall and find the hidden platform. The faster they swim, the more likely they are to encounter the platform randomly. Therefore, the low escape latency in a single training does not reflect the ability of the subject to use spatial cues to find the platform.The Morris water maze (MWM) evaluates the spatial learning and memory ability of rodents8. Therefore, total distance, target quadrant activity time, searching strategies and other indices are also used as common reference indices to comprehensively evaluate the learning and memory abilities of the experimental subjects11. These parameters compensate for deficiencies in escape latency measures, but there is a clear separation between these indices and escape latency. For example, if the subject used less time (escape latency) to find the platform, the target quadrant time spent by the subject cannot support this data. In order to resolve such contradictions, Maei et al., suggested that using the P measure may reveal more efficient detection of spatial learning9.Several recent studies suggested that using escape latency to evaluate learning and memory is not comprehensiveThe new indices reported here are based upon sufficient trajectory information, and they use the accumulation of real-time deviations to quantitatively describe the dynamic processes of animal learning behavior. The aim of the present study is to explore dynamic evaluation indicators based on behavioral processes of animal learning and memory, and to make up for the lack of summative evaluation that only pays attention to the final results in a limited time. Another aim of the present study is to form a comprehensive evaluation system that combines summative and dynamic evaluation for the MWM.12, experimental subjects can determine their own location and the direction of the platform by establishing a relatively perfect cognitive map, and they can search the platform according to the optimal route. Thus, we proposed an alternative model that measures deviation indices. The new deviation indices refer to the deviation degree of the subject’s trajectory relative to the optimal route in unit time. This degree may consist of several parameters including angle, distance, or some derived index. We proposed four new deviation indices for angle and distance . Point C0 is the entry of the experimental subject, with coordinate . The curve from C0 to B is the trajectory of the experimental subject. Point Cn is any point recorded on the trajectory, with coordinate . Point n in one unit time t(s), with coordinate . Point nCn+1. The optimal route would be directly from Point Cn to B and from C0 to Point B. The direction of As shown in Fig. The lateral deviation vector length mentclass2pt{minimThe n+1CnB is the deviation angle between the motion trajectory vector and the optimal route in the unit time. The calculation method is listed below as formula (The offset angle ∠C formula .2\documen+1CnB is obtained through the inverse trigonometric function (the range of the deviation angle is 0°–180°). The Then, the offset angle ∠CThe correction vectors  formula .3\documeThe The length of the lateral deviation vectors relative to the initial optimal route  formula .4\documeThe First, we measured escape latency in the experimental (model) and control groups to calculate the normal distribution. As shown in Table 13. We first collected the experimental data from both groups  to build the database in order to establish a probability model. As shown in Fig. p > 0.05), and the true positive rates (using the t-test or the U test) were significantly higher than those of escape latency (classic index). Our results demonstrate that the new deviation indices display a higher sensitivity compared to that of the classic escape latency index through using the Monte Carlo simulation test.The Monte Carlo simulation can simulate a large number of training sessions to examine the sensitivity and specificity of each index14. The ROC curve is now widely used to evaluate and compare the diagnostic value of a test14. As shown in Fig. A ROC curve is a graphical plot that expresses the diagnostic ability of a binary classifier system as its discrimination threshold is varied. It is created by plotting the true positive rate against the false positive rate at various threshold settingsThe difference between the new deviation indices and escape latency can be evaluated by AUC. The Z test  compares the advantages and disadvantages of each index precisely. Therefore, we compared escape latency with the new indices by using the Z test. Index 1 and index 3 showed significant differences as compared to escape latency. Index 4 showed a close to a significant difference from escape latency measures. There was no significant difference between index 2 and escape latency . Our data demonstrate that the new indices better reflect the trend of continuous improvement of the experimental subjects during the training process compared to the classically used escape latency index. This reveals more stability in measurements.The index values of the subjects should decrease with increasing training times, and a stable index should reflect learningp < 0.05, Analysis of Variance by ANOVA) using escape latency measures compared to the new index measures between control and experimental groups in the 20 training sections. The new index measures detected differences in the behavior of the experimental model groups compared to the controls, while escape latency measures failed to detect differences. These results indicate that the new index measures can more precisely detect differences in learning and memory.We further compared the differences between the control and the model group by using the new deviation indices and the classic index (escape latency). As shown in Table th and 15th training results . In the 13th training , they can make an informed decision whether to enter the arm or not. According to the list hypothesis theory, the whole training process is a series of decisions in which the subjects make micro-selections and large selections continuously in front of each arm. The subjects will also continue to make left versus right micro-selections while looking for the platform in the water maze. So, index 4 can be applied to determine the motion strategies of the experimental subjects in training under the same experimental environment  and to evaluate the mastery degree of the real-time position between the entry point and the platform.The MWM is widely used to evaluate rodent learning and memoryIn the present study, we examined the normal distribution of the data generated from the new deviation indices and escape latency, and evaluated the sensitivity and diagnostic value using the Monte Carlo simulation and ROC curve analyzations. Our results demonstrate that using the new deviation indices revealed higher consistency as compared to using the classic escape latency index. Our data suggest that the new indices might be an alternative method to evaluate the dynamic changes in animal space exploration as there is a deficiency in the simple classifications of the current searching strategies.th and 15th training. We found that the significant differences were caused mainly by two situations: (1) The experiment subjects used less time to find the platform, but they demonstrated a greater deviation from the optimal search route. This led to the shorter escape latency and higher deviation index. This might be due to the inability of the subject to construct a cognitive map between itself and the platform position. The decrease in escape latency is mainly due to the fast moving that caused an increased probability of meeting the platform. (2) The experiment subjects took a longer time to find the platform, but they demonstrated decreased deviation from the optimal search route, which led to the longer escape latency but smaller deviation index value. This was due to the fact that the experimental subjects did not recognize the relationship between the platform and the escape18. We might speculate that the water maze environment may not produce enough stimulation and force the rat to escape, which is contrary to the principle of the MWM18. In this situation, we could not evaluate their learning and memory ability through escape latency and the new deviation indices. It could be ruled out by the maximum escape latency and the smaller deviation indices.We further analyzed the results of every single training. The results showed that escape latency and deviation index clearly differed in the results of the 13th training are more reasonable. The results of the 15th training should be reconsidered after ruling out subjects who were not active in the training. In the process of training after training, if experimental subjects were not active, then escape latency and the new deviation indices cannot correctly describe their learning and memory ability. We can filter out the experimental subjects by comparing the values of escape latency and the new deviation indices to ensure the accuracy of the analysis results. Due to escape latency and the new deviation indices in the 15th training not showing significant differences, the four new indices not only could be used as a useful supplement to escape latency, but also could be combined with escape latency to eliminate the interference of some passive experimental subjects in the experiment, keeping the experimental evaluation more reasonable.Our present study suggests that the results of the new deviation indices in the 1319, because t-tests pay more attention to the value of the data than does the rank-sum (K–S) test, making the data of non-normal distribution prone to reject the correct hypothesis of H0, and then impossible to guarantee the test for homogeneity of variance and t-test facticity19. By contrary, the new deviation indices paid little attention to the training completion of the rats and never assigned the same value to the results of the subjects who had not found the platform, so the proportion of the new deviation indices’ data not conforming to the normal distribution was relatively small, which improved the test efficiency.The data on escape latency mostly did not align with the normal distribution each time because of the assignment operator of the maximum escape latency score (60 s), when applying to all subjects who failed to find the platform. Non-normal distribution will increase type I error rate to some extentThrough the Monte Carlo simulation, we found that the true positive rate (rejection rate of H0 hypothesis) of the new indices of any sample size in the t and U test was higher than that of escape latency, indicating that the sensitivity of each new index was higher than that of escape latency. Therefore, using the new deviation indices to evaluate the effect of the dementia model might be more sensitive to distinguish the behavioral differences between the model and the control group. In addition, the Monte Carlo simulation also showed that the false positive rate of both the new indices and escape latency are close to the predicted value.In the circumstance that escape latency and the new deviation indices results are paradoxical, the new deviation indices may reflect the quality of the movement process to evaluate the learning and memory ability of the experimental subjects, with less inclination to misjudge compared to how escape latency does. Therefore, only using escape latency as an index to measure the learning and memory ability may lead to errors in experimental conclusions.The ROC curve also distinctly shows the dominant position of the new deviation indices on sensitivity and specificity. Especially when the false positive rate is less than 20%, the AUC of the new deviation indices is clearly higher than that of escape latency, showing higher sensitivity. The phenomenon of escape latency ROC curve being a straight line when the values on the x axis are between 0 and 27.75% is due to the limited time window of escape latency (60 s). The false positive rate on the x axis of the ROC curve is directly crossed from 0% to 27.75% because nearly 30% of escape latency is 60 s, and this section can be observed as a straight line from the ROC curve. It can be concluded that escape latency sometimes may not ensure better specificity.The new deviation indices focus on the dynamic changes of learning and memory, rather than time. They can finely describe the motion characteristics of the experimental subject during the whole process of searching for the platform, which is helpful to observe their dynamic changes of behavior in the training process. The new deviation indices are helpful to the standardized preservation of the experimental results of the MWM and the more efficient use of the experimental data and the establishment of a database.In conclusion, the new deviation indices are not only consistent with escape latency, but also have better normal distribution, sensitivity, specificity and authenticity than escape latency alone. The new deviation indices combined with escape latency may more accurately analyze the unique behaviors and the learning and memory strategies of experimental subjects in each training. The combination of the new deviation indices and escape latency makes it possible to establish a comprehensive evaluation system, which may address the dynamic process, learning strategies, and the summative assessment. Meanwhile, a more reasonable and standardized analysis method of animal learning processes will be established with the trajectory data by developing new software.39 male Sprague-Dawley (SD) rats, weighing 200 ± 20 g, were maintained on a 12-h/12-h light/dark (7:00 am on and 7:00 pm off) cycle in a temperature- and humidity-controlled facility with food and water available ad libitum. Rats, provided by Animal Experimental Center of Qiqihar Medical University, were randomly divided into two groups . The housing environment was maintained at 23 ± 1 °C and humidity at 50 ± 10%. The light in the training condition of MWM was 10–20 lux. The water temperature in the tank was 22 ± 1 °C. All the behavioral training of the rats was completed between 7:00 am and 7:00 pm. All procedures were conducted in accordance with standards of laboratory animal care and used policy approved by the Institutional Animal Care and Use Committee of Qiqihar Medical University.20 and accelerated development of dementia22. In the dementia model group, the bilateral common carotid artery was ligated according to modified 2-VO23. The operation was done in a completely aseptic state. The rats were injected intraperitoneally with 0.5% sodium pentobarbital  according to their weight (50 mg/kg). The anesthesia level of the rats was determined by the disappearance of corneal reflex, righting reflex and toe response. The anesthesia process was smooth, and the rats were in a stable breathing condition during the operation. When the appropriate level of surgical anesthesia was reached, the rats were fixed on the operating table in the supine position, disinfecting skin, opening a 1–1.5 cm incision along the side of the neck, blunting dissection subcutaneous tissue and muscle, determining the common carotid sheath and separating the common carotid artery from nerves by a glass needle, ligating the unilateral common carotid artery permanently with surgical sutures, then using gentamicin sulfate  3–5 drops (0.1 ml/100 g) in the incision to prevent infection. The rats were recovered 20 to 30 min later after surgery. Postoperative analgesia of rats was performed when needed with injected amount of less than 20% of initial volume. After operation, the rats were placed under a warm lamp and put back into cages. In order to ensure the effectiveness of the dementia model, the other side of the common carotid artery of the rat was ligated in the same way three days later. The method for the control group was the same as the model group without ligation of the carotid artery.Double-side vascular occlusion  in the rats can cause long-term chronic cerebral hypoperfusion, leading to deterioration of spatial learning memory abilityp > 0.05), indicating that the injury of the operation did not lead to a significant difference in exercise ability between the two groups of subjects18. All rats were used for the MWM test four weeks after surgery.All subjects had no symptoms of infection. In any training, there was no significant difference in swimming speed between the two groups  used in this experiment consisted of a black stainless-steel round tank , transparent plexiglas platform (10 cm in diameter), a surrounding light-blocking curtain, and four marks of different shapes around the tank18. The platform was made of transparent material and was submerged 0.5–1 cm below the water surface and located in the center of a certain quadrant  selected randomly in four quadrants, making it invisible. The pool was filled with a depth of 40 cm water and replaced regularly. The device was placed in a dimly-lit room, and white dye was not used because of the SD rats’ white fur24. The experimenter and other possible clues remained in the same position throughout the experiment, and the room was kept quiet to avoid the stress reaction of rats25. In addition, it was necessary to clean up the feces left in the pool regularly to reduce the effect of smell information on rats26.The rat dementia model in the present study was consistent with the chronic cerebral hypoperfusion response in human aging and dementia28. In the course of formal training, they were placed into the pool from four quadrants in sequence, facing the wall; the interval of each training was more than 1 hour, and the training lasted for 5 days18. The training time was set to 60 s and recorded by an elevated video camera connected to the computer. Swimming paths were recorded from which we could figure out the value of the new deviation indices and escape latency. If the experimental subjects failed to find the platform in 60 s during the first training, they would be guided to the platform and would stay there for 10 s. Escape latency of the subjects who did not find the platform would be recorded as 60 s. All rats were dried with a towel during training intervals and placed in a warm and dry cage24.Prior to the commencement of training, all subjects were placed in the water in turn by the experimenter who did not know their grouping situation. It was made sure that they were face up to the maze wall and allowed to swim freely for 120 s to familiarize themselves with the training environment29. The probability level is P < 0.05. Using SPSS24.0, the normal distribution was conducted. The distribution of normality was tested by single sample K-S test. Using MATLAB 2017A, the Monte Carlo simulation was employed to calculate the sensitivity and false positive rate of the classic escape latency and the four new deviation indices, and the test level was set with α = 0.05, 0.01 and 0.005. MedCalc software was applied to draw ROC curves to determine the sensitivity and specificity of each index (the new deviation indices and escape latency).The experimental data included the 20 training escape latencies and the four new deviation indices: index 1, 2, 3, and 4. All behavioral data was analyzed using SPSS24.0 version statistical software package. Independent t-test and repeated measure variance analysis (ANOVA) were used to analyze the statistical differences between the two groupsSupplementary file


To evaluate whether low PI-RADS v2 assessment categories are effective at excluding extraprostatic extension (EPE) of prostate cancer (≥pT3a PCa).The local institutional ethics committee approved this retrospective analysis of 301 consecutive PCa patients. Patients were classified as low- or intermediate/high-risk based on clinical parameters and underwent pre-surgical multiparametric magnetic resonance imaging. A PI-RADS v2 assessment category and ESUR EPE score were assigned for each lesion by two readers working in consensus. Histopathologic analysis of the whole-mount radical prostatectomy specimen was the reference standard. Univariate and multivariate analyses were performed to evaluate the association of PI-RADS v2 assessment category with final histology ≥pT3a PCa.N = 137) and the intermediate/high-risk groups . In univariate analysis, all clinical and tumor characteristics except age were significantly associated with ≥pT3a PCa. In multivariate analysis, PI-RADS v2 assessment categories ≤ 3 had a protective effect relative to categories 4 and 5. The inclusion of ESUR EPE score improved the AUC of ≥pT3a PCa prediction . The impact of PI-RADS v2 assessment category is reflected in a nomogram derived on the basis of our cohort.For a PI-RADS v2 assessment category threshold of 3, the overall performance for ruling out  ≥pT3a PCa was 99%/98%/0.04 and was similar in both the low-risk  prostate cancer.• PI-RADS v2 assessment categories of 1 to 3 are useful for excluding ≥pT3a prostate cancer with a NPV of 98%; such patients can be considered as candidates for less invasive approaches.• The ability to exclude ≥pT3a prostate cancer may improve confidence in choosing nerve-sparing surgery or in avoiding pelvic nodal dissections, and similarly for patients undergoing radiotherapy, in adopting short-course adjuvant hormonal therapy or foregoing prophylactic nodal irradiation.The online version of this article (10.1007/s00330-019-06092-0) contains supplementary material, which is available to authorized users. The presence of extraprostatic extension (EPE) of disease in prostate cancer (PCa) patients, corresponding to a pathological stage of ≥pT3a at final histology,Multiparametric magnetic resonance imaging (mp-MRI) is an established imaging technique for PCa detection , 4 and hThe purpose of our study was to evaluate whether low PI-RADS v2 assessment categories are effective at excluding EPE (≥pT3a) of PCa.This retrospective analysis was approved by our institution’s ethics committee, who waived the requirement for a specific informed consent for the study as all patients had given separate written, informed consents for the performance of MRI, for the surgical procedures, and for the use of their clinical data for research purposes.Inclusion criteria were as follows: (a) biopsy-confirmed PCa, (b) mp-MRI, and (c) robotic-assisted radical prostatectomy (RARP) performed at our institution based on clinical-radiological indications or elective choice.The exclusion criteria were as follows: contraindications for MRI, and previous treatments or the assumption of 5a-reductase inhibitors that could affect the performance of mp-MRI or of final histology.During the period of this retrospective study (July 2012 and August 2013), 638 patients underwent mp-MRI at our institution, of whom 308 underwent RARP surgery on the basis of clinical findings and personal health management decisions. The time interval between biopsy and mp-MRI ranged from 20 to 50 days, while the time between mp-MRI and RARP ranged from 1 to 3 months.PI-RADS v2–compliant prostate mp-MRI was performed on a 1.5-T MR scanner . Anterior body (18 channel) and spinal (32 channel) phased-array coils were used without endorectal coil, providing consistently good image quality. The mp-MRI protocol , specificity (SP), positive predictive values (PPV), negative predictive values (NPV), positive likelihood ratio (LR+), and negative likelihood ratio (LR−) for predicting pathological stage ≥pT3a were calculated for the following: clinical risk group, ESUR EPE score, and PI-RADS v2 assessment categories. The diagnostic performance was also evaluated for ESUR EPE score and PI-RADS v2 assessment categories stratified by clinical risk group. For these analyses, ESUR EPE score and PI-RADS v2 assessment categories were analyzed in two classes of cancer likelihood (≤ 3 vs. 4–5), while for the univariate models, they were analyzed in three classes (1–2 vs. 3 vs. 4–5).Four unconditional logistic regression models for the association with ≥pT3a PCa were evaluated: model 1 included only the clinical risk groups, model 2 added ESUR EPE score to model 1, model 3 added the PI-RADS v2 assessment category to model 1, and model 4 included clinical risk groups, ESUR EPE score, and PI-RADS v2 assessment category. Corresponding odds ratios (ORs) and 95% confidence intervals (CI) were calculated for each model. The areas under the receiver operating characteristic (ROC) curves (AUC) of the four models were calculated and compared via the DeLong test .In addition, univariate and multivariate analyses were stratified by clinical risk groups and reported as Supplementary Material.Finally, a nomogram for the prediction of ≥pT3a PCa findings at pathology was created. Multivariable logistic regression was used to build the nomogram, considering the categorical variables: risk group, ESUR EPE score, and PI-RADS v2 assessment category. Performance of the nomogram was assessed in terms of discrimination (Harrell’s c-index), which provides an estimate of the probability that the model will correctly identify patients who had ≥pT3a.http://cran.r-project.org/).Statistical analysis was performed using the SAS software (SAS version 9.2) and R (R version 3.2.3) and its Hmisc and rms libraries , followed by pathology stages ≥pT3a, pT2a, and pT2b (Table The majority of the patients were in PI-RADS v2 assessment category 5 (62.5%), followed by categories 4 and 3; less than 2% of the patients were in category 2, and none in category 1 . The full model  produced an AUC significantly higher than all the other models .ESUR EPE score and PI-RADS v2 assessment category were seen to be significantly correlated . Our results are comparable with those of Matsuoka et al  who receAs regards ESUR EPE evaluation, our results are comparable with those of Boesen et al , whose Sp < 0.0001) and to 0.73 (p < 0.0001), respectively). Similar results were also obtained in both the multivariate analyses stratified by clinical risk groups . The combined addition of both ESUR EPE score and PI-RADS v2 assessment category to clinical risk (model 4) yielded a slight but statistically significant further increase in AUC if compared with model 2 (from 0.856 to 0.862 (p = 0.04)).Both ESUR EPE score ≤ 2 and PI-RADS v2 assessment category ≤ 3 were associated with a 96% lower risk of ≥pT3a PCa than in patients with ESUR EPE score of ≥ 4 or PI-RADS v2 assessment category ≥ 4. The inclusion of ESUR EPE score to the clinical risk alone (model 2) or PI-RADS v2 assessment category to the clinical risk alone (model 3) significantly increased the AUC  between PI-RADS v2 assessment categories and ESUR EPE score as indicators of the presence of ≥pT3a PCa. It could be expected due to the criteria that define the categories in the respective scales.We found a correlation  and could promote suitability in program of active surveillance. Conversely, the presence of PI-RADS v2 assessment categories > 3 can inform the decision to avoid a nerve-sparing approach in the site of tumor contact with capsule during radical prostatectomy, or to perform in that site intraoperative frozen sections .Numerous nomograms have been developed for the prediction of ≥pT3a, including the Partin tables and Memorial Sloan Kettering Cancer Center (MSKCC) nomograms , 25, butThe nomogram created from our cohort Fig.  had a goA key limitation of our study relates to the fact that all patients had undergone prostatectomy, of whom 54% were of intermediate/high risk with the overall prevalence of ≥pT3a PCa being 39.5%. In addition, treatment decisions were influenced by the original reporting of the mp-MRI under PI-RADS v1, and thus the cohort may be subject to possible over- or underdiagnosis associated with that reporting system. Due to patient choice to undergo surgery after positive biopsy, there is a relatively high representation of low-risk patients in our cohort (46%), but the proportion of PI-RADS v2 scores ≤ 3 patients is low (15%). Thus, our results are most applicable to a population with known prostate cancer, particularly intermediate- to high-risk disease, and cannot necessarily be extrapolated to screening in the general population, where the expected prevalence of PCa ranges from 5% at age < 30 years to 59% by age > 79 years .As well, we used consensus reporting by two radiologists to improve accuracy of PI-RADS v2 category determinations. This may limit the applicability of our results for single readers in clinical practice. Reports indicate that inter-reader reproducibility of PI-RADS v2 tends to be moderate and experience dependent , 30. We Another limitation is that in our investigation of the prediction of ≥pT3a PCa on a per-patient basis, there was no direct comparison between the regions suspected of cancer based on mp-MRI and the tumor focus detected at whole-mount histopathologic examination. The data available retrospectively did not allow analysis at the lobe level to be performed in this study; a future work evaluating PI-RADS category scores and adjacent EPE at a per lesion level would provide further insight into the local depiction of ≥pT3a PCa and should correlate the location of the suspicious lesions to the pathologic stage.Lastly, the nomogram developed based on our cohort must be validated and tested for predictive ability in a larger population of patients, and in other clinical centers.In conclusion, the addition of PI-RADS v2 assessment category to clinical risk parameters improves the prediction of ≥pT3a PCa, and thus risk stratification. In particular, PI-RADS v2 assessment categories of 1 to 3 are useful for excluding ≥pT3a PCa with a NPV of 98%. This is important for clinical practice and for appropriate patient counseling; as such patients can be considered as candidates for less invasive approaches . The ESUR EPE score should be better standardized to make full use of the available information in evaluating prostate disease. A nomogram that combines clinical and mp-MRI parameters for prediction of ≥pT3a PCa has been developed based on our cohort and requires validation in larger and different populations prior to use in clinical practice.ESM 1(DOCX 34.6 kb)