The therapeutic strategies for advanced gastric cancer (GC) remain unsatisfying and limited. Therefore, it is still imperative to fully elucidate the mechanisms underlying GC aggressive progression. The prognostic value and biological functions of low density lipoprotein receptor class A domain containing protein 2 (LDLRAD2) in GC have never been studied yet. We found that LDLRAD2 expression was significantly upregulated in GC and closely correlated with poor prognosis in GC patients. Functionally, LDLRAD2 promoted epithelial-mesenchymal transition, migration and invasion, and metastasis of GC cells. Mechanistically, LDLRAD2 interacted with and inhibited Axin1 from binding to cytoplasmic β-catenin, which facilitated the nuclear translocation of β-catenin, thereby activating Wnt/β-catenin pathway. Inhibition of β-catenin activity markedly abolished LDLRAD2-induced migration, invasion and metastasis. Together, these results suggested that LDLRAD2 contributed to invasion and metastasis of GC through activating Wnt/β-catenin pathway. LDLRAD2/ Wnt/β-catenin axis may be a potential therapeutic target for GC treatment. Gastric cancer (GC), a lethal disease, prevails worldwide . In chinDuring epithelial-to-mesenchymal transition (EMT) process, epithelial cells lose their apical-basal polarity and cell-cell adhesion, ultimately transforming into the invasive mesenchymal cells . EMT is LDLRAD2 (Low density lipoprotein receptor class A domain containing 2) gene, located on chromosome 1p36.12, is mainly expressed on cellular membrane. To date, the expression status, prognostic value and biological functions of LDLRAD2 in GC has never been investigated. In this study, we found that LDLRAD2 expression was highly expressed in GC tissues and cell lines, which was significantly related to unfavorable prognosis in GC patients. Additionally, it was observed that LDLRAD2 promoted epithelial-mesenchymal transition, in vitro migration and invasion, and in vivo metastasis of GC cells. Mechanistically, LDLRAD2-induced GC progression was dependent on the activation of Wnt/β-catenin pathway. Collectively, these findings revealed that LDLRAD2 facilitated migration, invasion and metastasis of GC by activating Wnt/β-catenin/EMT axis, suggesting that LDLRAD2/ Wnt/β-catenin axis may serve as a potential therapeutic target for GC treatment.To explore the expression status and prognostic value of LDLRAD2 in GC, dataset downloaded from The Cancer Genome Atlas (TCGA). We found that mRNA expression of LDLRAD2 was significantly upregulated in GC samples compared with normal samples  and it wAs the results above shown, MGC-803 and MKN-28 cell lines have the lowest LDLRAD2 expression, while BGC-823 cell line has a relatively high LDLRAD2 expression . Hence, Based on the role of LDLRAD2 in facilitating migration and invasion, we further explored whether LDLRAD2 could promote GC metastasis in vivo. We injected LDLRAD2-overexpresion MGC-803 cell and LDLRAD2-silence BGC-823 cell, as well as their corresponding vector/scramble control cells into nude mice by tail vein, separately, to explore the role of LDLRAD2 in GC metastasis in vivo. Four weeks later, the mice were sacrificed and the lung metastatic nodules were counted. Remarkably, the number of lung metastatic nodules from mice injected with LDLRAD2-overexpresion MGC-803 cell was more than that from control mice, while the number of lung metastatic nodules from mice injected with LDLRAD2-silenced BGC-823 cell was less than that from control mice, suggesting that LDLRAD2 could promote GC metastasis in vivo .To explore the potential molecular mechanism underlying the accelerative effect of LDLRAD2 on the migration, invasion and metastasis of GC, firstly we performed gene-set enrichment analysis (GSEA) to screen the LDLRAD2-associated signaling pathway. As shown in Then we further tried to elucidate the mechanism responsible for the LDLRAD2-induced activation of Wnt/β-catenin signaling pathway in GC cells. Nuclear translocation of β-catenin, is a typical hallmark of activated Wnt/β-catenin signaling pathway. Degradation of cytoplasmic β-catenin is a pivotal mechanism of inhibiting the nuclear translocation of cytoplasmic β-catenin, which negatively regulates Wnt/β-catenin signaling pathway. The destruction complex (DC) consisting of the tumor suppressor protein Axin1, APC, β-catenin and two constitutively active serine-threonine kinases (CK1α/δ and GSK3α/β) has been demonstrated to participate in the degradation of cytoplasmic β-catenin. Of these components, Axin1 plays a crucial role in maintaining the DC activity of degrading cytoplasmic β-catenin. Therefore, we hypothesized that LDLRAD2 might activate Wnt/β-catenin signaling pathway by targeting Axin1. Strikingly, co-immunoprecipitation assay showed that LDLRAD2 could bind to Axin1 . FurtherWe further explored whether Wnt/β-catenin signaling pathway was involved in the effect of LDLRAD2 on migration, invasion and metastasis of GC cells. We observed that KYA1797, a Wnt/β-catenin signaling inhibitor, could significantly mitigate the accelerative effect of LDLRAD2 overexpression on the invasion of MGC803 cells , while ΔThe tumorigenesis of GC is a complex process that involves aberrant expressions of multiple genes . In receThe EMT, a highly conserved cellular program, can induce epithelial cells to lose their cell polarity and cell-cell adhesion, by which epithelial cells transform into mesenchymal cells with migratory and invasive properties , 16. EMTNumerous cancer-associated signaling pathways are involved in tumor invasion and metastasis –35. To iNuclear translocation of cytoplasmic β-catenin that increases the expression of nuclear β-catenin is a prerequisite event for the activation of Wnt/β-catenin pathway , 36. HenIn summary, our findings demonstrate that high LDLRAD2 expression strongly correlates tumor aggressiveness and poor prognosis, and LDLRAD2 activates Wnt/β-catenin pathway by interacting with Axin1 to EMT, invasion and metastasis of GC cells. In conclusion, these findings indicate that LDLRAD2/Wnt/β-catenin axis may be a potential therapeutic target for GC patients.A total of 180 GC patients, who received radical gastrectomy and D2 lymphadenectomy followed with postoperative chemotherapy from April 2010 to June 2012, were histopathologically and clinically diagnosed at Lanzhou University Second Hospital. All tumor tissues were confirmed as GC using hematoxylin and eosin (H&E) staining after surgical resection. The clinicopathologic staging of the patients were determined according to the American Joint Committee on cancer criteria. Normal GC tissues were obtained from a standard distance (2 cm) from the margin of resected neoplastic tissues of patients with GC cancer and confirmed by pathological evaluation. All the patients did not receive any anti-cancer therapy, such as chemotherapy and radiotherapy before surgical treatment. All patients were followed up for at least 5 years. This study was approved by the Institute Research Ethics Committee of Lanzhou University Second Hospital and the approved study protocol number is 2019A-100. Written informed consent was signed by each patient. We performed all experiments using these samples strictly based on relevant regulations and laws. The clinical parameters of GC patients are summarized in http://gepia.cancer-pku.cn/). In this study, we analyzed TCGA database to compare the mRNA levels of LDLRAD2 between in 29 GC tissues and in 29 normal tissues (TCGA-STAD). Additionally, OS and DFS analyses of GC patients in the TCGA-STAD database were performed using the online tool GEPIA and the results were downloaded from the website. To explore the association of LDLRAD2 with cancer-associated signaling pathways, Global mRNA expression profiles of a subset of TCGA GC specimens were subjected to Gene set enrichment analysis (GSEA). The GSEA was fulfilled using GSEA software version 3.0. GSEA-GO analysis was performed using GSEA against c6.oncogenic signatures. gmt. The normalized enrichment score is the primary statistic for examining gene set enrichment results.The Cancer Genome Atlas (TCGA) database is an online tool Gene Expression Profiling Interactive Analysis (GEPIA) . All cell lines were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium with supplementation of 10% fetal bovine serum(FBS)  and appropriate amounts of penicillin (100U/ml) and streptomycin (100mg/ml). Cells were incubated in a humidified atmosphere of 5% COThe LDLRAD2 ORF sequence was amplified and cloned into LV003-IRES-EGFP . Lentiviruses were produced by co-transfecting the constructed plasmid with the packaging plasmids psPAX2 and pMD2.G  into 293T cells using Lipofectamine 2000  for approximately 72 hours. Next, we collected culture supernatants for filtration and concentration, and then we used the concentrate to infect MGC-803 and MKN28 cells. After 48 hours, the infected cells were selected by 2 μg/mL puromycin , and successful establishment was validated using western blotting analysis.Two small interfering RNA (siRNA) duplexes targeting LDLRAD2 were bought from RiboB . Then, we designed two shRNAs targeting LDLRAD2 based on the above siRNAs. BGC-823 cells were chosen to establish LDLRAD2-silenced GC cells.Transwell Matrigel invasion assay was performed as previously described. After incubation for 24 hours, cells invading to the lower chambers were fixed, stained, photographed and quantified by counting them in five random 200× magnification fields. As to wound healing assay, we first starved cells seeded in six-well plates for 12 hours to leave them in cell cycle synchronization, and then scratched the confluent monolayer of cells with sterile 200-ml pipette tips for artificially creating wounds. The wound healing process was observed and photographed under 100× magnification at indicated time points.6 cells with MGC803-Vector, MGC803-LDLRAD2, BGC823-Scramble, BGC823-LDLRAD2-Ri1 or BGC823-LDLRAD2-Ri2 were injected intravenously through tail vein into nude mice. All mice were sacrificed for excision of lungs 6 weeks after injection. The surface metastatic sites on lung surface were counted. Animal protocols were approved by the Institutional Animal Care and Use Committee of Lanzhou University Second Hospital. Moreover, all animal experiments were also performed based on the Declaration of Helsinki and the guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.To explore the role of LDLRAD2 in regulating GC metastasis, we evenly divided 25 BALB/c nude mice into 5 groups in a random manner. 2 × 10Cells were seeded in triplicate in 24-well plates. After 24 hours, indicated plasmids (TOP flash or FOP flash) plus 1 ng pRL-TK Renilla plasmid were transfected into the cells using Lipofectamine 3000 Reagent (Life Technologies). 48 hours after transfection, Dual-Luciferase Reporter Assay (Promega) was performed according to the manufacturer’s instructions, as previously described .Protein was electrophoretically separated by 10% SDS-PAGE and transferred to PVDF membranes . The membranes were blocked for 2 hours with 5% skim milk in TBST at room temperature, and then incubated with specific primary antibodies  overnight at 4 °C. After incubated with primary antibodies, the membranes were washed with TBST for three times, incubated with rabbit or mouse radish peroxidase-coupled secondary antibodies for 2 hours at room temperature, and then washed with TBST for three times. Antibody binding was detected by the enhanced chemiluminescence . Immunofluorescence staining assay was performed using anti-β-catenin (dilution 1:200) and FITC-conjugated goat anti-Rabbit secondary antibody . Images were recorded with a fluorescence microscope.Samples were lysed with Trizol reagent , then total RNA was isolated based on the manufacturer’s instructions. The extracted RNA was re-suspended in RNase-free water, and 1 μg RNA from each sample was used for cDNA synthesis primed with random hexamers. Then we used the synthesized cDNAs for Quantitative RT-PCR. All Quantitative RT-PCR reactions were fulfilled using LightCycler 480 (Roche). The fold change in expression was calculated using the 2-ΔΔCt method, in which with the GAPDH mRNA was used as an internal control. PCR amplification was performed using the SYBR Green PCR master mix Kit . The primer sequences were as follows: GAPDH forward, 5′-GACTCATGACCACAGTCCATGC-3′, GAPDH reverse, 5′ AGAGGCAGGGATGATGTTCTG-3′ MYC forward, 5′- GGCTCCTGGCAAAAGGTCA-3′, MYC reverse, 5′-CTGCGTAGTTGTGCTGATGT-3′ TWIST forward, 5′-TCCATTTTCTCCTTCTCTGGAA-3′, TWIST reverse, 5′-GTCCGCGTCCCACTAGC-3′ MMP7 forward, 5′-GAGTGAGCTACAGTGGGAACA-3′, MMP7 reverse, 5′ CTATGACGCGGGAGTTTAACA T-3′ VEGF forward, 5′-CTACCTCCACCATGCC AAGT-3′, VEGF reverse, 5′-AGCTGCGCTGATAGA CATCC-3′.Cells were transfected with various constructs and washed with PBS after 48hours, then lysed in an NP-40-containing lysis buffer supplemented with protease inhibitor cocktail (Roche), and then immunoprecipitated, as instructed by the protocol of a standard method, as previously described , with thImmunohistochemistry assays were performed on the paraffin-embedded GC samples, using the following primary antibodies: anti-LDLRAD2  and anti-b-catenin . Three pathologists assessed the staining intensity and percent of each section independently, and graded it according to the criteria as described below. The staining intensity score was evaluated as: 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. The staining area score was evaluated as: 0 = 0%, 1 = 1-25%, 2 = 26-50%, 3 = 51-75%, and 4 = 76-100%. The scores of staining intensity and staining area were multiplied to produce the final score. The final staining scores ≤ 3 were regarded as low LDLRAD2 or β-catenin expression and the scores ≥ 4 were regarded as high expression.Data analysis was performed by using the SPSS 19.0 software . Comparisons between any two groups were conducted using Student’s t-test (two-sided). To assess the association of LDLRAD2 expression with clinic-pathologic parameters of GC patients, they were divided into high and low LDLRAD2 expression groups according to the median value of LDLRAD2 expression. The clinic-pathologic parameters of GC patients between high and low LDLRAD2 expression was compared using chi-squared test. Kaplan-Meier method was used to establish survival curves for OS and DFS. The differences in survival of patients between high and low LDLRAD2 expression were assessed by log-rank test. P < 0.05 was regarded as statistically significance.Supplementary Figures


Hepatoblastoma (HB) is the most common form of primary liver malignancy found in pediatric populations. HB is considered to be clonal and arises from hepatoblasts, or embryonic liver progenitor cells. These less differentiated tumor-initiating progenitor cells, or cancer stem cells (CSCs), may contribute to tumor recurrence and resistance to therapies, and have high metastatic abilities. Phenotypic heterogeneity, undesired genetic and epigenetic alterations, and dysregulated signaling pathways provide CSCs with a survival advantage over current therapies. The molecular and cellular basis of HB and the mechanism of CSC induction are not fully understood. The Wnt/beta-catenin pathway is one of the major developmental pathways and is believed to play an important role in the pathogenesis of HB and CSC formation. This review summarizes the cellular and molecular characteristics of HB with a specific emphasis on CSCs and Wnt/beta-catenin signaling. Hepatoblastoma (HB) is the primary liver cancer found in children generally under three years of age . The terIGF-2), delta-like 1 homolog (DLK1), transforming growth factor beta 1 (TGF beta-1), mitogen-inducible gene 6 (MIG-6), and non-coding RNA MALAT1 are induced in HB but not in HCC [HB and HCC (hepatocellular carcinoma) are common liver malignancies that affect different age groups. Even though there are similarities in their tumorigenic characteristics, recent studies have shown that HB differs from HCC not only in its origin, but also in various molecular signatures associated with each malignancy . Global t in HCC . On the t in HCC . The FXRt in HCC .CTNNB1 gene, which encodes beta-catenin, is the most frequently mutating gene in exon 3 in HB; consequently, an adenomatous polyposis coli (APC) germline mutation in HB has been associated with a familial adenomatous polyposis history. Genomic analysis has revealed a CTNNB1 mutation rate of ≈68% in HB [The presence of various fetal and embryonic liver progenitor markers strengthens the hypothesis that HB originates from transformed hepatoblasts. HB tumors are categorized into two distinct subtypes: C1 and C2 . The C1 8% in HB ,20,21,22AFP, TACSTD1, DLG7, CDC2, BUB1, AURKB, IGF2, DLK1, PEG3, PEG10, BEX1, MEG3, NDN, BIRC5, NPM1, and HDAC2, and the activation of Myc signaling have been associated with the HB C2 subtype, which has embryonal characteristics and is enriched with CSCs [GLUL, RHBG, CYP2E1, and CYP1A1. It is also important to note that different sets of Wnt/beta-catenin-targeted genes are overexpressed in the two HB subtypes [NFE2L2 , which encodes a transcription factor involved in the antioxidant response pathway [NRF2 mutations are correlated with the activation of nicotinamide adenine dinucleotide phosphate dehydrogenase quinone 1 (NQO1), which has high expression levels in HB and has been found to be associated with invasive and metastatic C2 subtype HB tumors [Overexpression of genes, such as ith CSCs . On the subtypes . The HB subtypes . Another pathway . NRF2 muB tumors . ComerfoB tumors . This moAdditionally, cytoplasmic activation/proliferation-associated protein-2 (CAPRIN2), a known oncogene, and tumor suppressors, such as speckle-type POZ protein (SPOP), olfactory receptor-511 (OR5I1), and cell division cycle 20B (CDC20B), have been found to influence HB cell growth. CAPRIN2 is known to activate Wnt signaling via low-density lipoprotein receptor-related protein (LRP) 5/6 phosphorylation , and gaiHB differs in its developmental origin from HCC in adults. HB occurs in the very early stages of life, while HCC frequently develops on a background of chronic liver diseases. Studies have demonstrated that HB tumors consist of heterogenous populations of stem/progenitor cells ,13,28. TCSCs express numerous progenitor/stem cell-specific genes and share many characteristics with normal stem cells. HB tumors have been found to express progenitor markers, such as clusters of differentiation 133 (CD133), EpCAM , CD44, CD24, CD90, oval cell antigen-6 (OV6), and aldehyde dehydrogenase1 (ALDH1) ,30. CD13EpCAM is another potential CSC marker in the liver. In a normal liver, EpCAM is expressed in the bile duct cells at low levels. EpCAM is a transmembrane protein induced by Wnt/beta-catenin signaling and expressed in a variety of progenitor and stem cells , includiCD90 and CD44 have also emerged as markers of liver CSCs. Yang et al. found that the expression of CD90 correlated with tumorigenic potential in liver cancer cell lines. The presence of CD90-positive and CD44-positive cells was found to correlate with tumor metastasis. Blockade of CD44 by a neutralizing antibody induced apoptosis of CD90-positive cells and prevented tumor formation in mice .Epithelial–mesenchymal transition (EMT) is a cellular reprograming process whereby epithelial cells undergo transformation into cells with mesenchymal characteristics. In EMT-activated cells, intercellular junction proteins, such as E-cadherin, are downregulated, while mesenchymal-related proteins, such as N-cadherin, fibronectin, and vimentin, are upregulated. When primary tumor cells undergo EMT, polarized cells become highly migratory and are capable of invasion, leading to distant metastatic colonization ,51. SeveCTNNB1 or mutations prevent beta-catenin phosphorylation, and thus its degradation leads to an increase in both cytoplasmic and nuclear beta-catenin protein levels, which results in the constitutive activation of the Wnt/beta-catenin pathway  are a family of small, endogenous, non-coding RNAs, approximately 19–25 nucleotides in length. MicroRNAs regulate key signaling pathways in the pathogenesis of hepatoblastoma and are known to regulate CSCs ,74. HB tTargeting CSCs is challenging due to their high degree of heterogeneity and the intrinsic survival mechanisms that they develop over time to bypass apoptotic signals. At present, conventional therapies, such as chemotherapy, radiotherapy, and immunotherapy, can effectively suppress the growth of differentiated tumor cells; however, they potentially lag behind in inhibiting CSC proliferation . AlthougCTNNB1 [Several basic and clinical studies have demonstrated the critical role for Wnt/beta-catenin signaling in HB. The greatest challenge in targeting Wnt/beta-catenin signaling is its complexity, as it consists of several components regulated by protein post-translational modifications at various levels, as well as extensive signaling crosstalk with other cell-signaling pathways. Targeting nuclear beta-catenin and/or its nuclear regulators responsible for the transcription of downstream Wnt/beta-catenin genes could be a plausible approach to target CSCs. Beta-catenin interaction with cyclic adenosine 3′,5′-monophosphate response element-binding protein (CBP) promotes stem cell renewal, while interaction with P300 enhances progenitor cell differentiation. Therefore, targeting the beta-catenin-CBP interaction is a promising strategy, and small-molecule drugs, such as ICG-001, have been found to effectively target cancer stem cells ,86,87,88CTNNB1 . ActivatCTNNB1 . Based oEven though several cancer therapeutic approaches are available at present, tumor recurrence is a growing challenge in modern medicine. Recent evidences strongly suggest that CSCs that resist current therapies are the basis of cancer recurrence. Better understanding of the ways in which they originate from normal progenitor/stem or hepatoblast cells, their intrinsic self-renewal and survival mechanisms, and differential drug-metabolizing abilities compared to normal and differentiated cancer cells is critical for developing CSC-targeting therapies.


With an increase in the EHTPB content, the tensile strength (σm) of the modified epoxy resin decreased and the breaking elongation (εb) increased gradually. The initial decomposition temperature (T5%) and glass transition temperature (Tg) of the modified epoxy resin decreased with an increase in the EHTPB content. The modified epoxy resin had a rough fractured surface and the interface was blurred, presenting a ductile fracture.Epoxy resin (EP) was toughened by epoxidized hydroxy-terminated polybutadiene (EHTPB), with the corresponding modified epoxy resin being prepared. In this paper, the microstructure of EHTPB-modified epoxy resin was characterized, while the influence of different contents of EHTPB on curing kinetics, mechanical properties, morphology, thermal properties, dynamic thermomechanical (DMA) properties and crosslink density of the modified epoxy resin were also discussed. The results showed that the EHTPB-modified epoxy resin was successfully prepared and cured completely. The activation energy ( Epoxy resin (EP) is a material with excellent properties, which is widely used in semiconductor equipment manufacturing ,2,3, adhThe addition of flexible rubber components to EP  is a common method for improving the brittleness of epoxy resin, especially liquid rubbers, such as carboxy-terminated butadiene acrylonitrile (CTBN) rubbers, amino-terminated butadiene acrylonitrile (ATBN) rubbers, hydroxy-terminated butadiene acrylonitrile (HTBN) rubbers, vinyl-terminated butadiene acrylonitrile (VTBN) rubbers, epoxy-terminated butadiene acrylonitrile (ETBN) rubbers and hydroxy-terminated polybutadiene (HTPB). Kargarzadeh  studied Epoxidized hydroxy-terminated polybutadiene (EHTPB) is a novel polymer obtained by partial double bond epoxidation of the hydroxy-terminated polybutadiene (HTPB) ,20. SincIn this paper, a polyurethane prepolymer was firstly synthesized by epoxidized hydroxy-terminated polybutadiene (EHTPB) and toluene diisocyanate (TDI), before the prepolymer was introduced into the epoxy resin. The microstructure of EHTPB-modified epoxy resin was characterized by FTIR. The effects of different contents of EHTPB on the curing kinetics, mechanical properties, morphology, thermal properties, dynamic thermomechanical properties and crosslink density of EHTPB-modified epoxy resin were discussed. Epoxy resin E-51 (EP) , epoxidized hydroxy-terminated polybutadiene (EHTPB) , toluene diisocyanate (TDI) , polyamine (A60) , ethyl acetate , diisooctyl sebacate (DOS)  were dried by standard methods . DibutylThe preparation principle is shown in 1 (–NCO (from TDI)/–OH (from EHTPB) mole ratio is 2) was carried out at 75 °C for 3 h. After this, the ethyl acetate solution of epoxy resin E-51 was added to the flask and the reaction was continued for 2.5 h to obtain compound 2. The mixture was cooled to room temperature. A constant amount of curing agent A60 was added to the cooled prepolymer solution, which was stirred for 10 min. The solution was transferred to a polytetrafluoroethylene mold and the solvent was removed at room temperature under a vacuum, with the sample film obtained after vacuum curing. The EHTPB-modified epoxy resin containing 5–20 wt % EHTPB was prepared according to the procedure shown in T-12, which was 1 wt % of the total mass of EHTPB and TDI, was added to a three-neck flask containing ethyl acetate. After mixing, a certain amount of EHTPB was added. After this was dissolved completely, TDI was added dropwise. The pre-polymerization of compound −1. Potassium bromide (KBr) and the samples were grinded, before being pressed in the IR pellet die at 40 psi pressure, before the pellet was placed near the window of FTIR spectrophotometer. The changes in the structure of the polymer at the molecular level were studied from the recorded spectra using a computerized recorder. Fourier-transform infrared (FTIR) spectra of the prepared samples were recorded on an infrared spectrophotometer  in the wavelength range of 500–4000 cmThe curing behavior of EHTPB/EP system and the corresponding curing kinetics parameters were studied by dynamic differential scanning calorimetry (DSC) . The samples were subjected to scanning rates of 5 K/min, 10 K/min, 15 K/min and 20 K/min in the test temperature range of 50–300 °C with a nitrogen flow rate of 40 mL/min. aE) and reaction order (n). The activation energy was an important parameter to determine whether the curing reaction could be carried out. The curing mechanism could be deduced by the reaction order.The curing kinetics parameters of the system were studied by the Kissinger method, the Ozawa method and the Crane method ,23,24, saE could generally be obtained by the Kissinger Equation (1):β is the heating rate, pT is the temperature, R is the ideal gas constant of 8.314 J/(mol·K), aE (J/mol) is the activation energy and A is the pre-exponential factor. aE could be also obtained by the Ozawa Equation by Gaussian fitting of ln(β/pT2) − 1/pT.n could be obtained by the Crane Equation (2):Reaction order pT could be neglected since a/nRE was much larger than 2pT and thus, the Crane Equation was simplified to Equation (3):In the Crane Equation, 2β −1/pT and aE (the average number of aE obtained by the Kissinger Equation and the Ozawa Equation) into the Crane Equation.Reaction order could be obtained by Gaussian fitting of ln The mechanical properties were conducted on the AGS-J Electronic Universal Testing Machine  according to the Chinese National Standard GB/T 528-1998. The micrographs were obtained on a scanning electron microscopy (SEM) . The fracture surface of the stretched EP samples after tensile testing and the fracture surface of the unstretched EP samples under liquid nitrogen conditions before tensile testing were observed respectively to determine the morphology of the fracture surface.The thermal property of the modified epoxy resin was investigated by thermal gravimetric analyzer (TGA)  at the heating rate of 10 K/min from 30 °C to 600 °C with a nitrogen flow rate of 40 mL/min.Dynamic thermomechanical analysis (DMA)  of the modified epoxy resin was carried out in shearing mode with the frequency of 10 Hz, the temperature ramp of 3 K/min and scanning range from −50 °C to 200 °C.The samples of the modified epoxy resin were cut into 10 mm × 5 mm × 2 mm uniform specimens. These specimens were weighed and immersed in pyridine at room temperature for about 5–7 days. After this, the specimens were removed from the solvent, blotted and weighed in a stoppered weighing bottle. eν was the crosslink density, ν2 was the volume fraction of the elastomer phase in the swollen elastomer, ν was the molar volume of the solvent, χ was the interaction parameter between the elastomer and the solvent, f was the functionality of the elastomeric network, bρ was the density of the elastomer and cM was the relative average molecular weight of the elastomer.The crosslink density of the modified epoxy resin was obtained using the equilibrium volume swelling method, according to the Flory–Rehner Equation (4) ,26:(4)νeν2 from Equation (4) can be calculated according to Equation (5):pV was the polymer volume and sV was the volume of solvent in the swollen elastomer.The χ between the elastomer and the solvent was determined by Equation (6):pδ and sδ were the solubility parameters of the elastomer and the solvent, respectively, R was the ideal gas constant, T was the absolute temperature and ν was the volume of the solvent in the swollen elastomer. The solubility parameter pδ of the elastomer could be determined by the swelling method [The interaction parameter g method .−1 was due to the stretch vibration of N–H, while the band located at 3073 cm−1 and 2918 cm−1 was attributed to the stretch vibration of the unsaturated –CH and saturated –CH, respectively. The characteristic absorption band of –NCO appeared around 2267 cm−1. The band located at 1733 cm−1 represented the stretch vibration of C=O, the band located at 1596 cm−1 was due to the benzene ring and the absorption at 1072 cm−1 was attributed to the stretch vibration of C–O–C. The carbamate characteristic absorption bands located at 3309 cm−1, 1733 cm−1 and 1532 cm−1 indicated that the hydroxy groups reacted with the isocyanate groups to form the polyurethane prepolymer containing carbamate groups [e groups .−1 disappeared, indicating that the isocyanate groups in the polyurethane prepolymer reacted with the secondary hydroxy groups on the epoxy resin to form an epoxy resin–polyurethane graft copolymer.For the spectrum of the EHTPB/EP system before curing (curve B), the –NCO characteristic absorption at 2267 cm−1 attributed to the epoxy groups of the EHTPB and the EP [For the spectrum of the EHTPB/EP system after curing (curve C), the characteristic absorption band at 913 cmd the EP  disappeaaE values obtained by the Ozawa Equation were similar to those calculated by the Kissinger Equation.aE(avg) was 1.37. This might be due to the same addition content of the T-12 catalyst, which affected the activation energy primarily. The curing reaction order was determined by the chemical reaction type and the effects of each reaction in the reaction process, which subsequently reflects the change of the curing reaction mechanism. As seen from The activation energy of the curing reaction was an important parameter reflecting the reactivity of the curing system, which determined whether the curing reaction could proceed successfully. It can be seen from The mechanical properties of the modified epoxy resin with different w(EHTPB)/w(EP) ratios are shown in To analyze the overall performance of the modified epoxy resin, the stress–strain curves were obtained by testing data directly, which is shown in As shown in The micrographs of the modified epoxy resin were obtained by SEM, which are shown in As shown in As the polyurethane prepolymer was grafted to the epoxy resin, the whole system introduced more polyurethane flexible segments. Thus, the flexibility of the entire network structure was improved. When subjected to an external tensile load, the flexible segment itself would yield deformation, which hindered proliferation of microcracks, so the fracture surface showed obvious ductile fracture characteristics. At the same time, with an increase in the EHTPB content, the number of entanglements increased and the damage to this network structure required more stress or energy. Thus, the system exhibited toughness enhancement.One of the main advantages of epoxy resin is its good thermal properties. The general method of characterizing the thermal property of the material is to use thermal gravimetric analysis (TGA), where the thermal decomposition temperature is the most important parameter. The TG and DTG curves of the modified epoxy resin are shown in The TG and DTG curves of the modified epoxy resin with different w(EHTPB)/w(EP) ratios were analyzed and the corresponding thermal decomposition temperatures were obtained, which are shown in max was at about 240 °C, which was due to the decomposition of the carbamate [max was approximately at 360–370 °C, which was due to the chemical bond breaking of epoxy resin backbone [max was at about 440 °C, which was due to the EHTPB polyurethane backbone chemical bond breaking [5% of the modified epoxy resin was lower than that of the neat epoxy resin. The initial decomposition temperature was gradually reduced with an increase in the EHTPB content, which might be attributed to the thermal stability of the polyurethane being lower than that of the epoxy resin.The TG and DTG curves of the neat epoxy resin showed one decomposition stage. The initial decomposition temperature was 307 °C and the temperature at the maximum weight loss rate was 369 °C . After aarbamate ; the secg, especially when differential scanning calorimetry (DSC) testing is not appropriate due to the glass transition temperature being hard to determine [Dynamic thermomechanical analysis (DMA) is a common method for determining the glass transition temperature Tetermine .Storage modulus (E') curves are shown in g and tan δ max decreased with an increase in the EHTPB content. This may possibly be due to the polyurethane prepolymer pendant scaffolds having played an internal plasticizing effect in the modified epoxy resin, resulting in a decrease in Tg. Furthermore, the decrease in Tg could also be caused by a decreasing crosslink density [g. We proposed a concept of the ratio of epoxy reaction sites, which was the product of molecular weight and epoxy value. The EHTPB had a higher epoxy reaction sites ratio (3.48 mol/mol) compared to E51 (2.00 mol/mol) by calculation. These reaction sites made the EHTPB curing by-product molecular chain segments rigid and confined the movement of chains. Therefore, a Tg peak of the EHTPB curing by-product appeared at 120.2 °C.From  density ,40. It wmax decreased.The value of tan δ reflected the relationship between the stress and deformation of the material. When the deformation fell behind the stress, the material would lag behind and the corresponding energy would be consumed due to the hysteresis ,42. Withc and the crosslink density of the modified epoxy resin were calculated by the Flory–Rehner Equation. The final results are shown in The modified epoxy resin with different EHTPB/EP ratios were tested in pyridine, while the crosslink molecular weight M−4 to 2.12 × 10−4 mol/g. This was due to the epoxy value of epoxy resin E-51 (5.10 mmol/g) being greater than that of the EHTPB (3.26 mmol/g). Therefore, compared to the same quality of several modified epoxy resin, the total epoxy groups amount of the modified epoxy resin decreased with an increase in the EHTPB content. Thus, the modified epoxy resin crosslink network reduced, reducing the crosslink density, while the molecular weight became larger, allowing more solvent to enter the modified epoxy resin network and causing it to swell until equilibrium.It could be seen from −4 mol/cm3 to 2.12 × 10−4 mol/cm3.The modified epoxy resin with different EHTPB content was successfully prepared. During the curing process, the activation energy of the curing process decreased due to the self-catalysis effect and the addition of T-12, while the curing reaction mechanism did not change. With an increase in the EHTPB content, the tensile strength of the modified epoxy resin decreased from 31.83 MPa to 6.20 MPa and the breaking elongation increased from 21.43% to 118.76%. Compared with the neat epoxy resin, the fracture surface of the modified epoxy resin toughened with EHTPB was rough and the interface was blurred, presenting a ductile fracture. For the fracture behaviors under liquid nitrogen conditions, the modified epoxy resin toughened with EHTPB presented a phase separation, showing island structures. Furthermore, the thermal stability and the glass transition temperature were reduced. The equilibrium volume swelling method showed that the crosslink density decreased from 2.83 × 10In short, the addition of an appropriate amount of EHTPB could significantly enhance the toughness of epoxy resin. The preparation of the modified epoxy resin had the characteristics of low curing time and low curing temperature. Therefore, this is an ideal surface coating matrix material, which would attract the attention of researchers worldwide.


In amyotrophic lateral sclerosis (ALS), loss of motoneuron function leads to weakness and, ultimately, respiratory failure and death. Regardless of the initial pathogenic factors, motoneuron loss follows a specific pattern: the largest α-motoneurons die before smaller α-motoneurons, and γ-motoneurons are spared. In this article, we examine how homeostatic responses to this orderly progression could lead to local microcircuit dysfunction that in turn propagates motoneuron dysfunction and death. We first review motoneuron diversity and the principle of α-γ coactivation and then discuss two specific spinal motoneuron microcircuits: those involving proprioceptive afferents and those involving Renshaw cells. Next, we propose that the overall homeostatic response of the nervous system is aimed at maintaining force output. Thus motoneuron degeneration would lead to an increase in inputs to motoneurons, and, because of the pattern of neuronal degeneration, would result in an imbalance in local microcircuit activity that would overwhelm initial homeostatic responses. We suggest that this activity would ultimately lead to excitotoxicity of motoneurons, which would hasten the progression of disease. Finally, we propose that should this be the case, new therapies targeted toward microcircuit dysfunction could slow the course of ALS. Many diverse provinces of the central nervous system are involved in the production of movement, and, through their interconnections, the coordination of activity of circuits in these regions leads to organized behavior. Microcircuits within and between many regions of the cerebral cortex, basal ganglia, cerebellum, brain stem, and spinal cord each play a role in movement, whether as selection circuits, command neurons, organization circuits, or the final common path leading to muscle contraction. These circuits are remarkably adaptive: movement is well controlled in a multitude of environmental conditions. To do so, multiple modalities of sensory input are involved in the moment-to-moment adjustments of motor output to ensure appropriate coordination and function of these disparate motor circuits.There are homeostatic processes in neurons and circuits that ensure that the output of neurons, in the form of trains of action potentials, is maintained within a specific range  needed fHomeostatic mechanisms also play important roles in maintaining movement following damage to the nervous system. For example, after spinal cord injury, spinal circuits can regain activity needed for locomotor function . SimilarConversely, there are situations in which circuit homeostasis may be maladaptive. For example, following spinal cord injury, adaptations including changes in MN serotonin receptors  and/or cIn this review, we ask whether maladaptive plasticity of motor circuits can contribute to progression of neurodegenerative diseases. We focus on ALS, describing two fundamental spinal circuits involving MNs. We do not suggest that circuit dysfunction is causative of ALS, but rather propose that ALS-induced changes in these circuits disrupt normal homeostatic mechanisms and may thus accelerate the progression of MN degeneration. We present an hypothesis whereby maladaptive plasticity of MN circuits leads to excessive glutamate receptor activation, excitotoxicity, and hence further MN dysfunction and, ultimately, death. We therefore suggest that the development of strategies to target microcircuits involved in these maladaptive processes could slow the progression of disease.ALS was described by Charcot in the 19th century . ALS is The underlying causes of ALS are not clear and are not explored in this review. Various cellular and molecular hypotheses have been proposed to explain MN death, e.g., aggregation of toxic proteins, defects in RNA metabolism, or disrupted axonal transport  or spinal cord. The time of onset varies considerably, although it is most commonly diagnosed between the 6th and 8th decades of life, and the speed of progression is quite variable  are affected to a greater extent than those of the medial motor column (MMC). Furthermore, sacral MNs that innervate external anal and urethral sphincter muscles (Onuf’s nucleus) are spared . In the There is also selective vulnerability within motor pools, the populations of MNs that innervate a single muscle. α-MNs, those that innervate extrafusal muscle fibers responsible for force production, are vulnerable, whereas γ-MNs, those that innervate the contractile elements of muscle spindles to regulate proprioceptive feedback, are resistant to degeneration . FurtherGiven these differences in MN vulnerability, we next explore local microcircuits involving different types of MNs. Spinal MNs, termed the “final common path” for movement by c), muscle sensory feedback evolved, with the development of muscle spindles and associated afferent fibers to relay stretch length and velocity data back to the central nervous system . Spindles developed contractile elements such that their tension could be regulated during muscle shortening. Early in vertebrate evolution, spindle contractile elements were innervated by the same MNs (β-MNs) that innervated extrafusal fibers (b). About 30% of most MN pools are composed of γ-MNs, which overlap in size with small α-MNs, have simpler dendritic branching patterns  conveying feedback related to length. Dynamic γ-MNs innervate nuclear bag1 fibers and regulate the dynamic sensitivity of primary endings (Ia sensory afferents) conveying information on lengthening velocity . Although there is some variability, in many rhythmic movements α- and γ (both static and dynamic)-MNs are coactivated , but not to γ-MNs  and weakest on type FF MNs (e)  .There is evidence that group II afferents also project directly to homonymous MNs, but these connections are weak . ContrarNot all motor pools receive direct spindle afferent input. In the spinal respiratory motor columns, for example, despite the presence of a few spindles in the diaphragm , there iMonosynaptic connectivity between proprioceptive afferents and MNs has been less well studied in ALS-resistant motor pools. The external urethral and anal sphincters have few, if any, spindles , includig). A single Renshaw cell is excited by axon collaterals from several α-MNs, which can be from different motor pools . In addition to projecting to the MNs that excite them (i).Renshaw cells have several targets , but in ite them , Renshawite them , and, asite them , althougite them  Fig. 1BBh. In adIt is not clear whether the effects of Renshaw inhibition differ on different MN types. Initially, it was thought that Renshaw inhibition was weighted toward tonically active MNs  . On the The functional role of Renshaw inhibition is not clear, with many hypotheses having been raised . For exaExamination of the evolution and distribution of Renshaw cells has failed to unearth a unifying hypothesis of their function. Although about one-half of frog lumbar MNs have recurrent axon collaterals , they doWhereas it is clear that Renshaw cells are not involved in circuits for all vulnerable MNs, Renshaw cell circuits have not been identified in circuits of ALS-resistant motor pools. Motor axon collaterals have been observed from neurons in Onuf’s nucleus , but RenIn summary, Renshaw cells are activated by axon collaterals of α-MNs, in particular those in proximal motor pools that are vulnerable in ALS, and in turn inhibit α- and γ-MNs in homonymous and synergist MN pools.In ALS, the underlying neurodegenerative process, regardless of how it starts, ultimately leads to death of α-MNs. FF MNs degenerate first, followed by FR MNs, whereas S MNs are well preserved until late stages of the disease . ClearlyThe underlying assumption of this hypothesis is that MNs are susceptible to excitotoxic cell death  γ-MNs are not affected in ALS (2) during most movement, α-MNs and γ-MNs are for the most part coactivated  1) changes at NMJs, including changes in synaptic transmission at the NMJ (2) changes in MN physiology, including increased excitability or increased glutamate receptors to produce higher rates of firing in response to a given input  (3) changes in circuits, including increased synaptic input to drive MNs to higher firing rates.We will assume that the first step in the degenerative process is α-MN dysfunction , so the  the NMJ  and/or c the NMJ ; 2) chanN death) ; and/or 1).Given that initial dysfunction lies in the high force-producing activity of FF MNs, it is likely that homeostatic responses at the NMJ alone would be insufficient to compensate for this loss, and the homeostatic response of the nervous system would be to increase the firing rates of MNs. Although this could be accomplished in a cell autonomous process, e.g., by increasing membrane voltage-gated calcium channels  or incre3), such that intrafusal fibers would contract out of proportion to extrafusal fibers (2 and 4). Increased attention to the task secondary to any perceived weakness may also increase γ-MN activity (5). This could initially assist the homeostatic response, compensating for the reduction in motor pool output via “servo-assistance” (6) and will thus contribute to excitotoxicity  but before changes in motor axons in another (TDP43A315T)  . This re43A315T) C, whethe7), which would reduce Renshaw pool activity. Although this would diminish α-MN inhibition (8) and thus could aid in a homeostatic compensatory process leading toward normalization of muscle contraction, it would also diminish γ-MN inhibition (9). That is, the loss of Renshaw cell activation by α-MNs would lead to a further imbalance of α-γ output (2 and 4), leading to a relative increase in spindle contraction (5). This in turn would lead to an increase in spindle afferent activity (6) and, as above, contribute to excitotoxic cell death . At these presymptomatic stages, there is evidence of axonal sprouting of Renshaw cells leading to transient upregulation of glycinergic synapses on MNs , they reveal neither afferent nor γ-MN activity in the intact animal . Reflex One clinical sign that would be seen with this maladaptive plasticity would be spasticity. ALS patients with supraspinal disease tend to have a greater degree of spasticity than those with predominantly spinal disease . The mecIn summary, we propose that the homeostatic response to reduced neuromuscular activity following early MN dysfunction would be for the nervous system to increase inputs to α-MNs. With ongoing loss of α-MN function, these increased inputs would lead to a shift in the α-γ balance, leading to increased afferent input to α-MNs. Concomitantly, in some motor pools there would be a reduction in Renshaw cell inhibition resulting from reduced MN excitation of Renshaw cells. The changes to both Ia-MN and MN-Renshaw cell circuits would initially be homeostatic but would ultimately lead to excitotoxic death of MNs. Furthermore, we note that the increased inputs to MNs will affect more than a single motor pool [consider muscle synergies, e.g., If this hypothesis is correct, then therapies that target this microcircuit dysfunction should slow the progression of disease B. MicrocThere is no evidence in humans that such therapies might be effective, and we do not yet have the knowledge or methods to specifically target these microcircuits, but there is some recent evidence provided by animal models of ALS. In two models, the progression of disease was slowed concomitant with a reduction in γ-MN activity. In the first, muscle spindles were targeted, and their degeneration was associated with a loss of γ-MNs . In the https://clinicaltrials.gov/ct2/show/NCT03306277 for spinal muscular atrophy). As we learn more about gene expression profiles in mature MNs or Renshaw cells, for example, promoters for these genes could be used in viral approaches to drive expression confined to these populations. For example, introduction of potassium channels to γ-MNs or inhibitory “designer receptors exclusively activated by designer drugs” (DREADDs) could be expressed to reduce activity . Perhaps the advantage of a DREADD approach would be that dosage could be titrated and the therapeutic window thus defined .No conflicts of interest, financial or otherwise, are declared by the authors.R.M.B. conceived and designed research; C.L. prepared figures; R.M.B. and C.L. drafted manuscript; R.M.B. and C.L. edited and revised manuscript; R.M.B. and C.L. approved final version of manuscript.


Big data offer a great opportunity for nature-based recreation (NbR) mapping and evaluation. However, it is important to determine when and how it is appropriate to use this resource. We used Scotland as a case study to validate the use of data from Flickr as an indicator of NbR on a national scale and at several regional spatial and temporal resolutions. We compared Flickr photographs to visitor statistics in the Cairngorms National Park (CNP) and determined whether temporal variability in photo counts could be explained by known annual estimates of CNP visitor numbers. We then used a unique recent national survey of nature recreation in Scotland to determine whether the spatial distribution of Flickr photos could be explained by known spatial variability in nature use. Following this validation work, we used Flickr data to identify hotspots of wildlife watching in Scotland and investigated how they changed between 2005 and 2015. We found that spatial and temporal patterns in Flickr count are explained by measures of visitation obtained through surveys and that this relationship is reliable down to a 10 Km scale resolution. Our findings have implications for planning and management of NbR as they suggest that photographs uploaded on Flickr reflect patterns of NbR at spatial and temporal scales that are relevant for ecosystem management. Nature-based recreation (NbR) is a key cultural ecosystem service provided by nature and it represents a big component of global recreation . NbR is The widespread use of the Internet and the popularity of smartphones and social media websites offer the opportunity to use the data generated by their billions of users ,13. GeotHowever, following promising results from the first study , the useThis paper presents a rare case study in which the patterns observed in social media data were validated against survey data over time and at a relatively fine spatial scale. Wildlife watching contributes roughly £127 million per year to the Scottish economy , generatAll the data used in this study are publicly available and were collected following terms and conditions of the data providers. The data are anonymised and we did not collect any personal information. All the R code necessary to reproduce this study is available on GitHub. See Data Accessibility statement for the links to the repositories.We downloaded data from the Flickr Application Programming Interface (API) to create two datasets. The first dataset was a monthly time series of Flickr photographs taken in the CNP between 2009 and 2014 and it was compared to time series of visitor numbers provided by the CNP authority and to a time series of popularity of Flickr. The second dataset contained the number of Flickr photographs depicting wildlife that were taken on the coast of Scotland between October 2014 and October 2015 and it was compared to a spatial dataset of wildlife watching from the Scottish marine recreation and tourism survey . In the CNP STEAM visitor days—The CNP visitation dataset contained the number of visitor days to the park (a person spending at least a portion of a day at the CNP) per month from 2009 to 2014. These visitor days were estimated by the STEAM model [AM model , which uAM model .CNP Flickr visitor days—Data from Flickr were collected through the Flickr API [https://data.gov.uk) was used to select only the photographs taken inside the boundaries of the park. We downloaded the following metadata associated with the photographs: photograph and user ID, the date when the photograph was taken and the geographic coordinates of where it was taken. To avoid bias coming from having a small number of very active users, we used the combination of user ID and date to delete multiple photographs from the same user on the same day, thus retaining only the first photograph taken every day by each user. By counting the number of photographs retained in each month we then obtained the monthly number of Flickr visitor days in the CNP (a person taking at least one photograph a day in the CNP).ickr API  and R [3ickr API , using tickr API  version ickr API  version ickr API  version Flickr active users–To quantify changes in the popularity of Flickr over the years, we used the number of active users (i.e. users posting content on Flickr). We downloaded a publicly available global dataset of 100 million media objects uploaded on Flickr from 2004 to April 2014 [ril 2014  and calcCoastal survey visitor days—The dataset from the Scottish marine recreation and tourism survey [m survey  containeThis dataset, as most survey data, has some limitations and weaknesses. The survey was sent to a number of relevant organisations , advertised through press releases to key media organisations, user groups, industry organisations and on social media (Facebook and Twitter). As a result of this dissemination strategy, the survey was not based on a random sample of the Scottish population, therefore it is likely to have a bias towards a proportion of the population that is highly active in outdoor coastal activities. A total of 933 people provided spatial information on the places they visited to take part in wildlife watching activities, however, the data is not likely to be fully comprehensive. There was a small number of responses from remote parts of Scotland, which means that the spatial information for areas such as the Western Isles and Shetland is likely to be partial.Coastal Flickr visitor days—We used keywords  to query the Flickr API and select all the geotagged photographs of the main groups of wildlife sought by wildlife watchers in Scotland and taken between October 2014 and October 2015. We spatially restricted the query to the Flickr API by providing Scotland’s WOEID (Where On Earth IDentifier), which is a unique 32-bit reference identifier assigned by Yahoo! that identifies any feature on Earth. We downloaded the same metadata as for the CNP Flickr visitor days, but, in addition, we downloaded the user tags, which we used to eliminate photographs that were not relevant (such as photographs of statues or paintings and photographs taken in zoos). The tags were examined and a list of keywords for non-relevant photographs was compiled, then following a method similar to that used in [ used in  to selecIn order to test whether the temporal patterns shown by the CNP Flickr visitor days and the CNP STEAM visitor days time series were similar, we used Wavelets Analysis (WA) . WA decoTo exclude the possibility that the time series of CNP Flickr visitor days was influenced by the changes in the popularity of Flickr, we tested whether the number of Flickr active users had an effect on CNP Flickr visitor days. We fitted three Generalised Estimating Equations (GEE) with autoregressive correlation structures and the following formulas:Due to the strong seasonal trend in both the CNP STEAM visitor days and the Flickr active users, we checked for collinearity between these two variables. We used the function vifcor in the R package usdm . Given aWe compared the spatial distribution of wildlife watchers obtained from Flickr  and the one obtained from the survey  at three different spatial scales: 5 km, 10 km and 20 km. We fitted Generalised Linear Models (GLM) to the three datasets using the number of Coastal Flickr visitor days in each cell as the response variable and the number of Coastal survey visitor days in each cell as explanatory variable. The data contained a high number of zeros, so we fitted a GLM with a binomial error distribution to the presence/absence of Coastal Flickr visitor days in a cell and a GLM with a negative binomial error distribution to the number of Coastal Flickr visitor days present. This allowed us to test two hypotheses: 1) the probability of finding at least one photograph by one user on Flickr is higher for areas with higher Coastal survey visitor days 2) the number of users posting photographs on Flickr is higher for areas with higher Coastal survey visitor days. Since densely populated areas tend to have a higher average number of Flickr users  taken in the CNP between 2009 and 2014 and uploaded on Flickr.The query to Flickr API returned 92,229 results  for photographs with the word “bird” in tags, title or description taken in Scotland between 2005 and 2015. From the search with the word “seal” we obtained 7212 photographs  and from the search with the word “dolphin” and the word “whale” we obtained 5994 photographs .The power spectra of the Flickr and CNP STEAM time series were very similar, with significant 12-month cycles throughout the 5-year period . The higher the number of visitors captured by the survey, the higher the probability of finding photographs on Flickr taken in that area.The probability of finding a Flickr photograph taken in a certain cell was higher in cells with higher Coastal survey visitor days . This waSE = 0.01, Z = 8.1, p-value < 0.001; 10 km: estimate = 0.02, SE = 0.001, Z = 3.4, p-value < 0.001; 5 km: estimate = 0.01, SE = 0.006, Z = 1.7, p-value > 0.05). The number of Flickr users taking a photograph in a cell was significantly higher in cells with higher number of visitors captured by the survey, but only at a 10 and 20 km resolution.We found a significant correspondence between the number of Coastal Flickr visitor days and the number of Coastal survey visitor days at 10 km and 20 km resolution but not at 5 km  and harbour seals (Phoca vitulina) [The seal hotspot maps  revealeditulina) . This maitulina) ,68. The The whale and dolphin watching density maps  showed aData from social media still presents some limitations that need to be acknowledged, some of which also apply to traditional sampling methods ,71. TherIt is also important to consider shifts in the popularity of Flickr as a possible source of bias. Our analysis indicated that, in our study period (between 2009 and 2014), changes in Flickr popularity did not influence the time series of photographs taken in an NbR destination  in each month from January 2009 to April 2014. Figure B. Distance between multiple photographs by the same user. Distribution of the distance (in meters) between multiple photographs taken by each user on the same day. Figure C. Flickr visitor days. Spatial distribution of Flickr visitor days (FVD). Figure D. Variograms. Directional variograms of residuals of binomial GLM at the 5km resolution. Each panel shows semivariance in residuals in different directions: from North (panel 0) to North West (panel 320). Figure E. Time series wavelet power spectra. Wavelet power spectra of the two time series: a) CNP authority and b) Flickr time series. Colour code from dark blue  to red . White contour lines indicate significance. The shaded area near the edges in the graphs is the cone of influence, and indicates the range of the graph where the results are not reliable because of edge effects. Figure F. Survey visitor days. Spatial distribution of coastal survey visitor days. Figure G. Flickr visitor days normalised. Spatial distribution of coastal Flickr visitor days normalised by population size. Figure H. Bird watching density maps. Each panel represents the density of Flickr visitor days in a different year, from 2005 to 2015. The blue dots on the maps are the data. Different colours represent different density levels, from low (yellow) to high (red). Table A. QIC of the three alternative models.(DOCX)Click here for additional data file.


Acute myeloid leukaemia (AML) is a heterogeneous clonal malignancy of hematopoietic progenitor cells. The Wnt pathway and its downstream targets are tightly regulated by β-catenin. We recently discovered a new protein, FLYWCH1, which can directly bind nuclear β-catenin. Herein, we studied the FLYWCH1/β-catenin pathway in AML cells using qRT-PCR, Western blot, and immunofluorescence assays. In addition, the stemness activity and cell cycle were analysed by the colony-forming unit (CFU) using methylcellulose-based and Propidium iodide/flow cytometry assays. We found that FLYWCH1 mRNA and protein were differentially expressed in the AML cell lines. C-Myc, cyclin D1, and c-Jun expression decreased in the presence of higher FLYWCH1 expression, and vice versa. There appeared to be the loss of FLYWCH1 expression in dividing cells. The sub-G0 phase was prolonged and shortened in the low and high FLYWCH1 expression cell lines, respectively. The G0/G1 arrest correlated with FLYWCH1-expression, and these cell lines also formed colonies, whereas the low FLYWCH1 expression cell lines could not. Thus, FLYWCH1 functions as a negative regulator of the Wnt/β-catenin pathway in AML.  Acute myeloid leukaemia (AML) is a malignancy characterized by uncontrolled proliferation and failure in stem cell lineage differentiation, causing the accumulation of non-functional cells called myeloblasts . AML hasMYC, c-JUN, and cyclin D1 (CCND1) [The Wnt/β-catenin pathway plays a critical role in hematopoietic stem cell (HSC) self-renewal, and its impairment might also affect LSCs and AML cells. Wnt pathway activation leads to the accumulation of β-catenin in the cytoplasm and its translocation to the nucleus, where it replaces the Groucho proteins . It inte (CCND1) . β-caten (CCND1) ,8,9, and (CCND1) . Recentl (CCND1) ,12,13,14 (CCND1) ,15. GiveFLYWCH1 gene. Interestingly, the majority of identified transcription regulators associated with β-catenin, for example; KLF4 [Drosophila modifier of the mdg4 protein [Caenorhabditis elegans: PEB-1 [FLYWCH1 gene has been mapped on chromosome 16, and contains five FLYWCH-type zinc finger motifs that are highly conserved between mammals. The role of FLYWCH is unclear; however, based on our recent study, FLYWCH1 antagonizes β-catenin/TCF4 signalling during cell polarity/migration in colorectal cancer [Identifying proteins that regulate nuclear β-catenin, particularly in AML, is necessary for controlling the aberrant Wnt/β-catenin pathway. Apart from TCF4, the involvement of several other DNA-binding transcription factors with β-catenin is reported in the literature ,17,18,19le; KLF4 , and Glile; KLF4  belong tle; KLF4 ,26. The  protein ,28. In as: PEB-1  and FLYWs: PEB-1 . The huml cancer . AccordiFLYWCH1 mRNA expression and the specificity of designed primers, in nine AML cell lines using RT-PCR. The RT-PCR data confirmed that FLYWCH1 primers render a single product   assays using MethoCult H4230 methylcellulose-based medium . Howevern = 16) F, but noFLYWCH1 mRNA and compared with the expressions of c-MYC and c-JUN proto-oncogenes in human primary AML samples. Notably, qRT-PCR analysis of twelve cases indicated a relatively low expression of FLYWCH1 mRNA in all AML patients  promotes tumour progression and initiation. Recent data suggest that Wnt/β-catenin signalling is linked with AML pathogenesis ,13,37,38Our results suggest that there is differential expression of FLYWCH1 at both transcriptional and translational level. However, the AML cell lines expressed different levels of FLYWCH1 mRNA and protein . This diIn addition, the data suggest that the differential expression of FLYWCH1 correlates with cell cycle phase transition, as we observed the loss of FLYWCH1 expression in dividing cells or in cells immediately after division A,B. The APC mutation. In contrast, other investigators have suggested that LSCs tolerate higher Wnt levels [Further functional analysis of FLYWCH1 was performed through the stemness activity assay. The OCI-AML3 cells had low colony formation efficiency, whereas the KG1a cells were notable for not forming colonies at all C. Familit levels . Howevert levels . Such cet levels ,45.Mechanistic understanding of the link between these effects and FLYWCH1/β-catenin require further investigation. We speculate that the observed differences in different levels of growth activator are related to oncogenes as their target genes. For example, c-Myc is a proto-oncogene that is correlated with the control of cellular proliferation, stemness, and programmed cell death ,47. KarnMany laboratories are examining potential inhibition strategies for Wnt/β-catenin signalling in a variety of diseases, including cancer. However, despite these efforts, therapeutic agents specifically targeting the Wnt pathway have not been introduced in the clinic. In the present study, we identify for the first time FLYWCH1 as a potential negative regulator of the downstream targets of the Wnt/β-catenin pathway in AML cells. Hence, FLYWCH1 might be an intrinsic inhibitory factor of the oncogenic nuclear β-catenin in AML cells.L-glutamine , and 1% penicillin/streptomycin . The M07e and KG1a cell lines were cultured as above, but supplemented with 20% fetal calf serum and 10 ng/mL granulocyte-macrophage colony–stimulating factor . All experiments were performed when cells were in the stationary phase. The MOLM-13, M0-7e, and OCI-AML3 myeloid leukaemia cell lines were obtained from the German Collection of Microorganisms and Cell Cultures . U937, HL-60, and KG-1a cell lines were from the European Collection of Animal Cell Cultures . The MV4.11 and TF1A cell lines were from the American Tissue Culture Collection . M0-91 were a kind gift from Dr. Joseph M. Scandura  . The humTaq polymerase  and SYBR Green master mix  were used to perform PCR and quantitative RT-PCR (qRT-PCR), respectively. The comparative CT method was applied for quantification of gene expression. β-actin was used as endogenous controls, unless otherwise stated. The following primers were used:Total RNA was extracted from the AML cell lines using TRIzol  according to the manufacturer’s instructions. Total RNA (2 µg) was used to synthesize complementary DNA using a PrimeScript RT reagent kit  following the manufacturer’s instructions. FLYWCH1-Fwd: 5′-CTGGATGCAGCCCCTCAGT-3’ FLYWCH1-Rev: 5-′TTGGCGGCACTTCCAGTAC-3’β-actin-Fwd: 5′-GCGCGGCTACAGCTTCA-3’β-actin-Rev: 5′-CTTAATGTCACGCACGATTTCC-3’CXXC4-FWD: 5’-ATGCACCACCGAAACGACTC-3’CXXC4-Rev: 5’-GCAGTGTTCAGGGGATAAGGT-3’FGF-4-FWD: 5’-TATGGCTCGCCCTTCTTCAC-3’FGF-4-Rev: 5′-TCGGTTCCCCTTCTTGGTCTTC-3’PPARD-Fwd: 5′-AAGAGGAGGAGAAAGAGGAAG-3’PPARD-Rev: 5′-TGAACACCGTAGTGGAAGC-3’c-Jun-Fwd: 5′-ACCCCAAGATCCTGAAACAG-3’c-Jun-Rev: 5′-ATCAGGCGCTCCAGCTCG-3’LGR5-Fwd: 5′-GACAACAGCAGTATGGACG-3’LGR5-Rev: 5′-GCATTACAAGTAAGTGCCAG-3’C-MYC-Fwd: 5′-GGAGACACCGCCCACCA-3’C-MYC-Rev: 5′-GCGCTGCGTAGTTGTGCTG-3’Radio-immunoprecipitation assay (RIPA) buffer  freshly supplemented with a 1:100 cocktail of phosphatase and protease inhibitors  was used for protein extraction. The protein was quantified using the Bradford assay . Equal amounts of protein were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to a hydrophobic polyvinylidene difluoride membrane . Blots were blocked using 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) and 0.1% Tween-20  for 1 hour at room temperature. Then, the blots were incubated at 4 °C with anti-primary antibody in 5% BSA–TBS-T overnight. The antibodies used were those against: cyclin D1 , c-Myc , c-Jun , and β-actin . To detect the protein bands, fluorescent secondary antibodies (1: 5000 dilution) were measured using an Odyssey Fc Dual-Mode Imaging System (LI-COR Biosciences).4) suspended in 100 µL of phosphate-buffered saline (PBS) were plated on slides after cytospin at 800 rpm for 3 mins (Thermo Fisher Scientific). The cells were fixed in 4% paraformaldehyde, blocked with 3% BSA, and washed in 3% BSA and 0.1% phosphate-buffered saline (PBS)–Tween-20. The cells were probed overnight at 4 °C with primary antibody targeting FLYWCH1 , followed by secondary Alexa flour labelled antibody  and DAPI  staining, and mounted with ProLong Diamond Antifade Mountant with DAPI . Images were captured using a Leica fluorescent microscope.Immunofluorescence was carried out following standard procedures . BrieflyFLYWCH1 gene is composed of 2151 nucleotides encoding a protein with 716aa residues called FLYWCH1. The IMAGE-clone plasmid DNA then digested with several restriction-enzymes and cloned into different vectors, while fused with the eGFP in pLVX-Puro backbone vector, as previously described [https://www.abmgood.com/FLYWCH1-shRNA-Lentivectors-i008000.html) as previously described [The IMAGE-clone of human FLYWCH1-cDNA purchased from Geneservice . The human escribed . The screscribed . Human EK293T cells maintained in RPMI supplemented with 10% FBS. Cells transiently transfected using Lipofectamine-2000  and processed for virus production, as described . KnockedThe AML cells were washed in cold PBS, fixed in ice-cold 70% ethanol, and stained for 15 min at room temperature with propidium iodide  and RNase  . The cel3) were seeded in triplicate in 24-well plates in a semi-solid matrix  and incubated for 14 days according to the manufacturer’s protocol. The colonies were monitored microscopically every 2 days, identified and counted on day 14, and images were captured using a phase-contrast microscope.Cells (1 × 10Blood or bone marrow samples were obtained at diagnosis after written consent from adult patients with AML (excluding M3 patients). National and Local Research Ethics Committees approved the use of these samples . Mononuclear cells were isolated using a standard density gradient centrifugation method with histopaque and were cryopreserved in liquid nitrogen. RNA was prepared from AML blasts using QIAamp RNA kits with DNase treatment according to the manufacturers’ instructions . Up to 2 μg of RNA was used in a reverse transcription reaction with MMLV reverse transcriptase  and random primers .t-test using Microsoft Excel. Significance testing was performed using SPSS version 15. Mean ± SD  values are shown.Statistical analyses were performed by the two-tailed Student’s Differential expression of FLYWCH1 appears to be an important determining factor to balance the β-catenin/Tcf activity within AML cells. There seem to be at least two different stages at which dysregulation of Wnt/β-catenin signalling plays a role in leukemogenesis. First, at the initiation phase of the disease, elevated Wnt signalling could be the crucial factor to progress from a pre-LSC into an LSC (as for AML), as has been recently discovered for T-ALL (T-cell acute lymphoblastic leukaemia) where the absence of the Wnt-nuclear factor Tcf1 appears to predispose to T-ALL development. However, for CML (chronic myeloid leukaemia), ectopically activated Wnt signalling is also essential. Hence, depending on the differentiation status of the cell and or the location of the leukaemia-initiating cells in its microenvironment, Wnt/β-catenin signalling plays different roles in leukemogenesis. Thus, a dysregulated FLYWCH1/β-catenin signalling can contribute to leukemogenesis, both on the differentiation and leukemic status of the cell. The data warrant further investigation of FLYWCH1/β-catenin signalling as a critical component in all hematologic malignancies including AML pathogenesis.


Burkholderia mallei is a Gram-negative bacterium that causes glanders, a zoonotic disease, especially in equine populations . B. mallei usually grows slowly on most culture media, and this property makes it difficult to isolate from clinical specimens. One of the problems is that B. mallei is easily overgrown by other bacteria, especially in animal specimens collected from non-sterile sites. The aim of this study was to develop a new selective agar for the laboratory diagnosis of glanders. We formulated a new agar, named BM agar, to enrich B. mallei growth, but inhibit the growth of other bacteria and fungi based on their antimicrobial profiles. We compared the growth of B. mallei on BM with Xie’s and PC agars, the two previously described selective agars for B. mallei.B. mallei strains within 72 h: only one out of the 38 strains grew scantly after 72 h of incubation. BM agar was further tested with other Burkholderia species and various bacterial species commonly found in the nasal cavities and on the skin of horses. We have found that other Burkholderia species including B. pseudomallei and B. thailandensis can grow on BM agar, but non-Burkholderia species cannot. Furthermore, the specificities of the three selective agars were tested with or without spiking B. mallei culture into clinical specimens of non-sterile sites collected from healthy horses. The results showed that BM agar inhibited growths of fungi and other bacterial species better than PC and Xie’s agars. We have also found that growth of B. mallei on BM agar was equivalent to that on 5% horse blood agar and was significantly greater than those on the other two agars (P < 0.05).BM agar could sufficiently grow almost all of the tested B. mallei from mixed samples such as those typically collected from horses and other contaminated environments.We believe that BM agar can be used to efficiently isolate The online version of this article (10.1186/s12917-019-1874-0) contains supplementary material, which is available to authorized users. Burkholderia mallei is a Gram-negative bacterium that causes glanders, a zoonotic disease. Glanders is a significant contagious disease, particularly in equine populations , and other animals , including humans, are known to be susceptible to B. mallei infection [B. mallei [nfection . The acu. mallei . InternaB. mallei can be grown on most enriched media, including sheep blood agar and trypticase soy agar, but grows very slowly, requiring up to 72 h of incubation [B. mallei from clinical and environmental specimens because B. mallei is easily overgrown by other bacteria, especially in animal specimens collected from non-sterile sites [B. mallei is often overgrown by other bacteria even in fresh samples obtained under sterile conditions [Bacterial isolation is a gold standard method for diagnosing glanders . B. mallcubation . This prle sites , 5. Therle sites , 7. Addinditions .Pseudomonas cepacia agar (PC agar) for the isolation of B. mallei [B. mallei strains could not grow at all on PC agar and the sizes of 16 colonies, which grew on the agar were quite small (< 1 mm). Similarly, another study has reported that three selective agars, which are basically used for isolation of B. pseudomallei, could not grow 20–60% of the assayed B. mallei strains [B. mallei was reported by Xie et al. [A previous report compared four selective media and recommended . mallei . However strains . Althouge et al. , 11, theBurkholderia mallei agar: BM agar) for laboratory diagnosis of glanders and compared its bacterial selectivity and ability to grow B. mallei with previously reported media by testing with bacterial cultures and horses’ nasal and skin specimens.In this study, we developed a new selective medium  produced colonies after 72 h incubation, but there were fewer colonies on BM agar than the other strains. Most B. mallei strains formed circular, purple, smooth, and > 1 mm colonies on BM agar  for growth on all the three selective agars and blood agar at 37 °C for 72 h. Plating serial dilutions of the same three B. mallei cultures on each of the agars, we computed the average bacterial concentration of the original cultures to be 1.48 × 108, 8.03 × 107, 8.03 × 106 and 1.38 × 108 CFU/mL when using blood, Xie’s, PC, and BM agars, respectively .To assess how well BM agar can support B. mallei colonies, making it difficult to pick up B. mallei colonies. Concerning PC and BM agar, one colony of B. mallei was found on PC agar, whereas more than 10 colonies of B. mallei were observed on BM agar. The colonies were confirmed as B. mallei by the MALDI Biotyper System.Appearances of the four agars by using spiked or non-spiked specimens are shown in Fig. B. mallei from clinical specimens [B. mallei is its difficulty to isolate the bacteria from environmental samples. Investigators are often unable to isolate B. mallei from seropositive or species-specific PCR positive horses [B. mallei is mainly attributed to its slow growing character [B. mallei. For example, B. pseudomallei, a genetically closely related species to B. mallei, is often isolated on Ashdown’s agar, which uses crystal violet and gentamicin as selective agents [B. mallei growth generally fails on Ashdown’s agar [B. mallei is significantly lower than that for B. pseudomallei [B. mallei because the bacteria may or may not grow on this agar [B. mallei from contaminated clinical samples [B. mallei and its colonies formed on this agar were very small (< 1 mm) even after 72 h incubation. In addition, the effectiveness of Xie’s agar has not been compared with those of other agars. Therefore, we have developed a new selective agar “BM” to isolate B. mallei from equine specimens and compared its selectivity and growth efficiency with the previously recommended selective agars. Although non-selective agars like glycerol dextrose agar and glycerol potato agar can be used to grow B. mallei, we excluded these from our study, which focused on using selective agars to isolate B. mallei from heavily contaminated samples.Although glanders has been eradicated from many countries over the last century, several outbreaks of glanders still occurred in the equine population in Asia, Middle-East, Africa, and South America , 12. Thee horses , 16. Theharacter . The bace agents , 18. Hown’s agar  because domallei , 20. Simhis agar . The PC  samples , 9, but B. mallei [B. mallei was reportedly resistant to all the three antimicrobials  [B. mallei was reported to be intrinsically resistant to polymyxin B [B. mallei strains showed its relative susceptibility to this antimicrobial in our preliminary test. Specifically, polymyxin B of greater than 70,000 units/L could slow down the growths of B. mallei strains (data not shown). Therefore, the concentration of polymyxin B which did not affect the bacterial growths was used in this study. In addition, this view could make an impact on the difference of growth efficacy between BM agar and PC agar as described below, because PC agar contains high concentration of polymyxin B .Concerning the formulation of BM agar, glycerol is well known to enhance the growth of . mallei , and crymyxin B) , 22. TicB. mallei strains to grow within 48 h. Three of the other four strains needed up to 72 h to be easily picked up from the agar plate. The remaining one grew scantly even after 72 h, indicating that at least 72 h incubation is recommended for isolation of B. mallei by using BM agar. Recovery of the slow-growing strain might benefit from pre-enriching the clinical sample or increasing the incubation time. Other Burkholderia species tested were also grown on BM agar. Although Burkholderia species are not frequently isolated from equine specimens [Burkholderia species because most species of genus Burkholderia naturally exist in soil [B. mallei-suspected colonies from BM agar. Concerning non-Burkholderia species, BM agar showed its high specificity by inhibiting the growths of common bacteria frequently found from equine specimens. The results of comparison tests using equine nasal and skin swabs also illustrate the superiority of BM agar. BM agar can inhibit the growth of both bacteria and fungi more efficiently than PC and Xie’s agar. Several Gram-negative bacteria grew on PC agar while both Gram-positive and Gram–negative bacteria grew on Xie’s agar. Although some non-Burkholderia species, such as Herbaspirillum huttiense, were found on BM agar, its colony number was relatively low and did not complicate the isolation of B. mallei from BM agar.Our results with bacterial pure cultures showed that the BM agar could allow 34 out of 38 pecimens , 23, 24, in soil , 26. TheB. mallei, high effectiveness is also needed for isolating this pathogen because the concentration of B. mallei in clinical specimens of the infected equid is low [B. mallei colonies can be grown on BM agar. In addition to BM agar’s specificity, the agar’s high recovery rate makes it useful for isolating B. mallei from horse specimens. Therefore, focusing on combined approaches for the detection of B. mallei and B. pseudomallei, BM agar could be used as a unique selective medium that can efficiently grow these two pathogenic bacteria and inhibit the growth of non-Burkholderia species.Regarding growth efficiency of d is low , 14. OurB. mallei from horse specimens. We believe that this new agar can be used to provide sensitive and selective isolation of B. mallei for both diagnosis and future research.Our study has shown that BM agar is a novel selective agar for effectively isolating BM agar was prepared by combining 5 g of proteose peptone no.3, 10 g of tryptone pancreatic digest of casein, 0.25 g of magnesium sulfate, 1 g of sodium pyruvate, 5 g of sodium chloride, 40 mL of glycerol, 3 mg of crystal violet , 50 mg of cycloheximide (5 mL from 1% w/v cycloheximide in absolute ethanol), 15 g of agar, and distilled water 960 ml). The mixture was sterilized at 121 °C for 15 min. After cooling to 45 °C, the medium was supplemented with three antimicrobials: 16.7 mg of ticarcillin disodium salt (668 μL from 25 mg/ml stock solution), 197.8 mg of fosfomycin sodium (1520 μL from 130 mg/mL stock solution), 40,000 units of polymyxin B . The formulations of PC agar and Xie’s agar were based on previous reports 60 ml. Th. AdditioBurkholderia species and 24 isolates of non-Burkholderia species was used for evaluation of BM agar . The non-Burkholderia species, which are frequently isolated from horse specimens, were tested in this study and the collection was composed of three strains from each of the following eight bacterial species : Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Escherichia coli, Klebsiella pseumoniae, Streptococcus zooepidemicus, Streptococcus equi, methicillin-susceptible Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus.A collection of 68 isolates of Burkholderia species, one colony of each strain was picked up and subcultured onto BM agar. The plates were incubated at 37 °C for 72 h. For non-Burkholderia species, each isolate was suspended in 1 mL of saline solution (0.85% w/v) to a turbidity equivalent to a McFarland 0.5 standard (approximately 1.5 × 108 CFU/mL). Each suspension was diluted 1000-fold (approximately 1.5 × 105 CFU/mL) and a 100 μL aliquot of the dilution (approximately 1.5 × 104 CFU/100 μL) was inoculated onto the medium and spread with a plate spreader. Then, the plates were incubated at 37 °C for 72 h. The numbers of bacterial colonies were counted every 24 h.Prior to testing, each of the strains were subcultured onto blood agar or LB agar containing 4% glycerol. For A total of 34 nasal swabs and 34 skin swabs were sampled from 17 healthy horses that were kept by Equine research institute; both sides of the nasal cavity and shoulder skin were sampled from each horse. Each swab was dipped into 500 μL of distilled water, and 100 μL of the suspension was inoculated onto the three selective agars  and 5% horse blood agar. Growths of bacteria and fungi were checked after incubating at 37 °C for 72 h. Bacterial isolates from horse specimens were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry with MALDI Biotyper System , in accordance with the manufacturer’s instructions. The Biotyper software 3.1 and the MALDI Biotyper reference library version 5.0.0.0. were used in this study. The bacterial identifications were interpreted according to the criteria of the manufacturer, namely species level, identification score of ≥2.000; genus level, ≥1.700 to < 2.000; no identification, < 1.700.B. mallei strains  obtained from Gifu Type Culture Collection were used to compare growth efficiencies of B. mallei in this study. Each strain was suspended in saline solution (0.85% w/v) to a turbidity equivalent to a McFarland 0.5 standard . Ten-fold serial dilutions of the suspensions were inoculated in triplicate onto four agars  and the average colony counts of the original suspensions were separately calculated for each of the agars after incubation at 37 °C for 72 h.Three B. mallei GTC 3P0003T was suspended in 1 mL of phosphate-buffered saline (PBS) to a turbidity equivalent to a McFarland 0.5. Ten-fold serial dilutions of the suspension were prepared. One nasal swab obtained from a healthy horse was dipped into 2.5 mL of PBS. Then, 10 μL of each bacterial dilution and 200 μL of the swab suspension were mixed and 50 μL aliquots of the mixture were inoculated onto the four agars. Appearances of the agars were observed after incubation at 37 °C for 72 h. The same amount of nasal swab suspension was also inoculated onto the four agars without being spiked with B. mallei, and bacterial and fungal colonies were checked after incubation for comparison.To compare the appearances of bacteria grown on each of the four agars, p-value of < 0.05 was considered to indicate a significant difference.A Fisher’s exact test and t-test were conducted by using Excel Statistics ver. 7.0  for statistical analysis of comparisons. A Additional file 1:Table S1. Constituents of three selective agars. (XLSX 12 kb)Additional file 2:Table S2. Bacteria used in this study. (XLSX 14 kb)


SPINK5 is significantly reduced in the development of esophageal cancer, and is closely related to the pathological differentiation and lymph node metastasis of esophageal cancer via bioinformatics analysis and esophageal cancer tissue array. Further studies have found that SPINK5 is closely related to Wnt/β‐catenin signaling pathway by bioinformatics analysis and western blot. In esophageal cancer cells, SPINK5 overexpression can inhibit Wnt/β‐catenin signaling pathway. Combined with LiCl or MG‐132 treatment, SPINK5 can inhibit GSK3β phosphorylation and promote β‐catenin protein degradation, thus inhibit Wnt/β‐catenin signaling pathway. In vivo study, SPINK5 overexpression can significantly inhibit the growth of esophageal cancer cells. Our study shows that SPINK5 can inhibit the proliferation, migration, and invasion of esophageal cancer cells by inhibiting Wnt/β‐catenin signaling pathway, and thus plays an important role in the development of esophageal cancer, and may serve as a treatment target of esophageal cancer.Esophageal cancer is one of the most common tumor in the world, and the morbidity rate is as high as 100/100 000 in some parts of China. Therefore, it is important and urgent to explore the pathogenesis of esophageal cancer and find new therapeutic targets for esophageal cancer. In this study, we found that a novel tumor suppressor  In 2018, there were 572 034 new cases of esophageal cancer. In addition, there were 508 585 (5.3%) patients with esophageal cancer died, which is the sixth leading cause of cancer death worldwide.SPINK) family is the largest branch of the serine protease inhibitor family.SPINK1, SPINK2, SPINK4, SPINK5, SPINK5L2, SPINK5L3, SPINK6, SPINK7, and SPINK9.SPINK 8, SPINK 10, SPINK11, and SPINK12 were found in the rat’s epididymis.SPINK family consists of the Kazal domain. A typical Kazal domain contains 3 pairs of disulfide bonds, 2 a‐helices, and 3 anti‐parallel β‐sheets.SPINK5 gene is located in the 5q32 region of the chromosome and is composed of 15 functional regions. The SPINK5 gene encodes a lymphoid epithelial‐associated inhibitor LEKTI, a serine protease inhibitor. SPINK5 is mainly related to Netherton Syndrome (NS). NS is caused by loss of expression or dysfunction of LEKTI due to mutation of SPINK5 gene.SPINK5 and human cancer. There was mRNA microarray analysis showed that SPINK5 was downregulated in esophageal squamous cell carcinoma.The serine protease inhibitor kazal type . The other tissue microarray contains 205 cases of esophageal cancer tissue which was from the tissue samples library of our laboratory. KYSE510, ECA109, and HEK293T cells were purchased from the China Center for Type Culture Collection . KYSE510 cells were cultured in RPMI1640 medium  supplemented with 10% fetal bovine serum (Gibco). ECA109 and HEK293T cells were cultured in DMEM medium  supplemented with 10% fetal bovine serum (Gibco).2.2SPINK5 overexpression plasmid was cloned into the pflag‐CMV vector by nested PCR using the CDS sequence of the SPINK5 gene (NM_001127698.1). The primers of plasmid construction could be seen in Table The SPINK5 were purchased from Genepharma Company  and contained 3 pairs of sequences, which were siRNA1 at nucleotides 1065 bp, siRNA2 at nucleotides 2046 bp, and siRNA3 at nucleotides 2910 bp. The sequences could be seen in Table The siRNAs of The antibodies in this study were used for western blot and immunohistochemistry, including SPINK5 , flag , β‐catenin , GSK3β , c‐myc , cyclin D1 , β‐actin , p‐β‐catenin , p‐GSK3β , and the goat anti‐rabbit IgG  and goat anti‐mouse IgG .SPINK5, and LV5‐NC were purchased from Genepharma Company. For lentiviral stock preparation, HEK293T cells were co‐transfected with LV5‐SPINK5 and 3 packaging plasmids . Supernatants containing packaged lentivirus were collected after 48 hours, passed through a 0.45‐µm filter and added to ECA109 cells along with 1 µg/mL polybrene (Sigma). Infected cells were selected in puromycin‐containing medium.The lentivirus vectors LV5‐2.33 per well in 96‐well plates, and the proliferation of cells in each treatment group was detected at 0h, 24h, 48h using the CCK8 detection kit. For the plate colony formation assay, the cells in the logarithmic growth phase were inoculated into the 6‐well plate at a density of 200 per well. After further culture for 1 week, the cells were stained with crystal violet solution, and the growth of the cells in each treatment group was observed. Cell counting was performed by image J software.The ability of cell proliferation was measured by CCK‐8 assay and plate colony formation assay. The procedure of the CCK8 assay was as follows. The cells in the logarithmic growth phase were seeded at a density of 1 × 102.44 cells per well. The chamber was placed in a 24‐well plate in which the upper chamber contained serum‐free cell culture medium and the lower chamber contained 10% FBS complete medium. The culture was continued for 24 hours. The medium was discarded, and stained with a crystal violet solution to observe the number of migrated cells. For cell invasion assay, Matrigel gel was uniformly applied to the upper chamber in advance, and then the cells were inoculated. The other procedures were the same as those in the cell migration experiment.Cell migration and invasion ability was measured by Transwell assay. For cell migration assay, cells in logarithmic growth phase were seeded at the upper transwell chamber insert  at a density of 2 × 102.5The TOP/FOP flash assay is a reporter gene assay used to detect Wnt/β‐catenin signaling pathway activity.2.6The cell proteins were extracted from each group of cells by RIPA lysate and sonicator, and the concentration of each group of protein samples was detected by BCA protein concentration detection kit to prepare protein samples. An equal amount of protein sample (40 μg) was added to a 10% SDS‐PAGE gel and electrophoresed at 60 and 110 V, respectively. After the electrophoresis, the protein in the SDS‐PAGE gel was transferred to the PVDF membrane at a moderate current. The PVDF membrane was then blocked with 5% skim milk for 1 hour, and the corresponding protein antibody was separately incubated overnight according to the molecular weight of the protein of interest. Then, the PVDF membrane was separately incubated with the corresponding HRP‐labeled secondary antibody, and finally observed and photographed by the chemiluminescence imaging system .2.7Immunohistochemistry was used to detect the expression level of the protein in esophageal tissue. The tissue chip was dewaxed, and after dehydration, the endogenous peroxidase in the tissue was inactivated using 1 mmol/L sodium citrate buffer (pH 6.0). After blocking with goat serum for 30 minutes, the antibodies of the protein of interest were separately incubated overnight at 4°C. The next day, the biotin‐labeled secondary antibody was incubated with the tissue chip for 1 hour at room temperature, followed by incubation with streptavidin‐labeled horseradish peroxidase for 1 hour and finally with 3, 3‐diaminobenzidine (DAB), and the depth of the color represents the expression level of the target protein. The Pannoramic MIDI automatic digital slide scanner  was used for image capture. Quantification of target protein staining was performed using IHC profiler in ImageJ.2.8SPINK5 overexpression was determined by orthotopic transplanted tumor model in nude mice. A nude mouse model of orthotopic transplanted tumor was established by subcutaneous injection of ECA109 stable cell line (1 × 108 cell/mL) in 4‐6 weeks old Balb/c nude mice . After the nude mice were sacrificed, the tumor weight and tumor volume were observed and recorded. All the animal protocols were approved by Zhang Zhongjing College of Traditional Chinese Medicine, Nanyang institute of Technology, China.The tumorigenic ability of esophageal cancer cells ECA109 after 2.9SD). Each experiment was repeated at least 3 times. The variance analysis between groups was performed using a one‐way ANOVA. Chi squared‐tests were used to detect the significance of the relationship between expression of SPINK5 and clinicopathologic features of esophageal cancer tissue microarray. P < 0.05 was considered statistically significant.The data were presented as the mean ± standard deviation  of esophageal cancer, compared with normal esophageal tissues , c‐myc, and cyclin D1 and upregulated p‐β‐catenin (S33/37) in esophageal cancer cells KYSE510 and ECA109  and upregulated the protein expression levels of β‐catenin, p‐GSK3β (S9), c‐myc and cyclin D1 , LRP5, LRP6, DVL3, LEF1, AXIN2, MYC, and cyclin D1 (CCND1) in esophageal cancer , whereas knockdown of SPINK5 upregulated the expression of p‐GSK3β (S9), suggesting that SPINK5 can affect the activity of GSK3β. LiCl is often used as an inhibitor of GSK3β which promotes GSK3β phosphorylation and inactivation.SPINK5 in esophageal cancer cells has a similar effect to LiCl and upregulates the expression level of p‐GSK3β (S9)  protein expression by LiCl treatment , but has no significant effect on the expression level of GSK3β. Furthermore, using the GSK3β inhibitor LiCl in combination with SPINK5 overexpression or knockdown, we further determined that SPINK5 can activate GSK3β and inhibit the activity of Wnt/β‐catenin signaling pathway by affecting the GSK3β/β‐catenin degradation complex.The Wnt/β‐catenin signaling pathway is a complex and precise signaling pathway in which the localization and expression level of β‐catenin protein is a key point. When the Wnt/β‐catenin signaling pathway is inactivated, CK1, AXIN, APC, GSK3β, and other proteins in the cytoplasm form a degradation complex, which promotes the ser33 and ser37 phosphorylation of β‐catenin by binding to β‐catenin protein, leading to the degradation of β‐catenin by ubiquitination.SPINK5 overexpression could reverse the effect of LiCl treatment, which confirmed that SPINK5 could indeed inhibit the proliferation and migration of esophageal cancer cells by inhibiting the activity of Wnt/β‐catenin signaling pathway. In addition, the inhibitory effect of SPINK5 overexpression on the growth of esophageal cancer cells was further verified in nude mice, suggesting that SPINK5 may be a new therapeutic target for esophageal cancer.In the process of tumor development, activation of Wnt/β‐catenin signaling pathway can upregulate cell proliferation and migration‐related target genes, such as c‐myc and cyclin D1, which promote cell proliferation and migration.SPINK5 as a tumor suppressor gene in the development of esophageal carcinoma, and further found that SPINK5 can inhibit the Wnt/β‐catenin signaling pathway to play a role in the proliferation, migration, and invasion of esophageal cancer cells. In addition, in this study, we also found that SPINK5 is mainly expressed on the cell membrane in normal esophageal tissues, while SPINK5 is mainly in the cytoplasm and nucleus in esophageal cancer tissues. In general, the position of protein determines its function. Therefore, the correlation between the transposition of SPINK5 and its function is one of the directions of future research.In conclusion, in this study, we first explored the role of None declared.TableS1Click here for additional data file.