Here, this work screens the mevalonate metabolic pathway enzyme PMVK to stabilize β‐catenin signaling using a human genome‐wide library. On the one hand, PMVK‐produced MVA‐5PP competitively binds to CKIα to prevent β‐catenin Ser45 phosphorylation and degradation. On the other hand, PMVK functions as a protein kinase to directly phosphorylate β‐catenin Ser184 to increase its protein nuclear localization. This synergistic effect of PMVK and MVA‐5PP together promotes β‐catenin signaling. In addition, PMVK deletion impairs mouse embryonic development and causes embryonic lethal. PMVK deficiency in liver tissue alleviates DEN/CCl4‐induced hepatocarcinogenesis. Finally, the small molecule inhibitor of PMVK, PMVKi5, is developed and PMVKi5 inhibits carcinogenesis of liver and colorectal tissues. These findings reveal a non‐canonical function of a key metabolic enzyme PMVK and a novel link between the mevalonate pathway and β‐catenin signaling in carcinogenesis providing a new target for clinical cancer therapy. β‐catenin via the PMVK‐generated intermediate MVA‐5PP. MVA‐5PP blocks β‐catenin phosphorylation at Ser45 by inhibiting the CKIα kinase. This stabilizes β‐catenin by preventing its cytoplasmic sequestration and proteolysis. Concurrently, PMVK also phosphorylates β‐catenin at Ser184, which facilitates its nuclear translocation. PMVK thus represents a novel therapeutic target in cancers that rely on aberrant β‐catenin signaling.The mevalonate pathway kinase PMVK stabilizes  Metabolic reprogramming, considered one of the hallmarks of cancer, can involve changes in lipid metabolism, which supplies membrane components needed to support tumor cell proliferation. The targeting of lipid metabolism has thus been examined as a promising treatment option.Hepatocellular carcinoma (HCC) is the second leading cause of cancer‐related deaths worldwide.1 Alth Mevalonate, which is produced from acetoacetyl‐CoA, is converted to sterol isoprenoids such as cholesterol, which is an indispensable precursor of bile acids, lipoproteins, and steroid hormones. Mevalonate is also used in the synthesis of other hydrophobic molecules including nonsterol isoprenoids. The role of these mevalonate pathways in tumorigenesis and progression has been extensively described. In addition to the final metabolite cholesterol, mevalonate kinase promotes tumor growth by stabilizing mutant P53 protein levels through the metabolite mevalonate 5‐phosphate (MVA‐5P). Geranylgeranyl diphosphate (GGPP) and farnesyl pyrophosphate (FPP) can be used as geranylgeranylation and farnesylation substrates in post‐translational modifications of proteins generically known as protein prenylation. Such post‐translational modifications are required for the proper sub‐cellular targeting and activation of many oncogenic proteins, including some members of the RAS family.The mevalonate anabolic pathway provides metabolites for multiple cellular processes in eukaryotes, archaea, and bacteria.5 MevaPhosphomevalonate kinase (PMVK) catalyzes the cation‐dependent reaction of MVA‐5P with ATP to form mevalonate 5‐diphosphate (MVA‐5PP) and ADP and represents a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis and protein prenylation. Intermediates of this network play important roles in the post‐translational modification of numerous proteins involved in inter‐ and intra‐cellular signaling. Although the mevalonate pathway has been implicated in various aspects of cancer development and progression, the role(s) of PMVK remains unknown.9β‐catenin signaling pathway regulates embryonic development, hepatobiliary development and homeostasis, and the repair response to liver injury. Abnormal Wnt‐β‐catenin signaling is also closely related to the development of many cancers. In the absence of active WNT signaling, cytoplasmic β‐catenin is rapidly degraded by the “APC complex” that includes the adenomatous polyposis coli (APC) tumor suppressor protein, Axin, glycogen synthase kinase (GSK)‐3β, casein kinase Iα (CKIα) and the E3‐ubiquitin ligase β‐TrCP. β‐catenin's phosphorylation by GSK‐3 and CKIα within the APC Complex facilitates β‐TrCP‐mediated β‐catenin ubiquitination and proteasomal degradation. Genetic alteration in the core components of Wnt/β‐catenin signaling, most notably β‐catenin itself, lead to WNT‐independent signaling, which can drive tumorigenesis. Mutations of the CTNNB1 gene, which encodes β‐catenin occur in most cases of HCC. Wnt‐β‐catenin activation can increase fatty acid utilization to promote tumor progression and mutant forms of β‐catenin can increase glutamine levels in tumor cells to drive anaplerotic oxidative metabolism. The accumulation of cholesterol leads to reduced expression of squalene epoxidase which disrupts the GSK3β and p53 complexes, ultimately activating β‐catenin signaling. However, the cross‐talk mechanism between lipid metabolism and Wnt‐β‐catenin pathway are still poorly understood.The highly conserved Wnt‐r injury.10 Abnβ‐catenin inhibitors in Huh7 cells. This identified PMVK as an enzyme that can stabilize β‐catenin protein. Mechanistically, PMVK‐generated MVA‐5PP inhibits CKIα‐mediated β‐catenin phosphorylation at Ser45. This stabilizes the protein by preventing its proteolytic degradation. Additionally, PMVK directly phosphorylates β‐catenin at an alternate site, Ser184 to facilitate its nuclear localization. Finally, we describe a novel small molecule inhibitor of PMVK that attenuates β‐catenin signaling and potentially represents a novel therapeutics. Collectively, our study reveals a novel, phosphorylation‐dependent mechanism by which the key lipid‐metabolizing enzyme PMVK regulates Wnt‐β‐catenin signaling and the ensuing oncogenicity of this otherwise highly regulated pathway.We describe here a genome‐wide Crispr/Cas9 screen to identify genes that are responsible for imparting resistance to 22.1β‐catenin signaling pathway itself in HCC, we instead used a human genome‐scale CRISPR‐Cas9 knockout (GeCKO) v2 library to identify genes that support the pathway. We chose the CTNNB1 gene wild‐type (WT) HCC cell line Huh7 to avoid the abnormal activation of β‐catenin signaling caused by the gene mutations. Preliminary screening showed that the IC50 of Huh7 cells for compound XAV‐939, a tankyrase1/2 inhibitor stabilizing of both Axin1 and Axin2, which indirectly promotes the degradation of β‐catenin   overlapping with metabolite interconversion enzyme, while the screened candidate genes should also be up‐regulated in HCC to fit the role of an oncogene  experiments with the endogenous proteins indicated that PMVK interacts directly with in Figure3A. Subsβ‐catenin phosphorylation was responsible for stabilizing β‐catenin. To this end, we generated β‐catenin β‐catenin WT. This further suggested that PMVK‐mediated phosphorylation of β‐catenin at Ser184 promotes its nuclear localization    to confirm binding of MVA‐5PP to the K46, Y64, and D157 sites of CKIα, with much less binding being seen with a mutant CKIα   catalyzes the fourth step of mevalonate biosynthesis involving the cation‐dependent reaction of MVA‐5PP with ATP to form isopentenyl diphosphate and ADP to release a carbon dioxide and a phosphate group, which binds to MVA‐5PP as a positive control  Figure . In vitrβ Figure . Finally To evaluate whether this can promote β‐catenin p‐Ser184 and β‐catenin signaling, we treated HCC cells with methyl‐β‐cyclodextrin (M‐β‐CD) which depletes cholesterol. Subsequent immunoblotting showed that M‐β‐CD treatment partially decreased the levels total and p‐S184 β‐catenin. This suggested that β‐catenin S184 phosphorylation is dependent on certain minimal levels of MVA‐5PP  via AAV8 in WT mice. Our results showed that overexpression of PMVK, β‐catenin WT or S184D significantly promoted HCC growth  mice were constructed , c‐Myc and Cyclin D1 were significantly upregulated relative to matched normal liver ‐[3‐[methyl]‐4‐methoxyphenyl]methylideneamino]‐3,4,5‐trihydroxybenzamide) from a library of 1.7 million ChemDiv small molecule compounds   every 2 days for a total of 14 doses  mouse model  cells, in which the destruction complex is non‐functional. This suggested that, by phosphorylating the critical Ser184 residue, PMVK activates β‐catenin via a destruction complex‐independent mechanism. In the HCC mouse model, overexpression of β‐catenin p‐Ser184 strongly promoted tumor growth. Although stabilizing β‐catenin indirectly promotes its nuclear localization, the actual mechanism by which β‐catenin Ser184 phosphorylation accomplishes this requires further clarification. Regardless of the actual means by which this is accomplished, β‐catenin Ser184 phosphorylation serves as a new and novel marker for β‐catenin pathway activation.Interestingly, PMVK knockdown also impaired Wnt3a‐activated β‐catenin Ser184 is dependent on its classical metabolic enzyme function. Because PMVK mutants cannot affect the interaction of β‐catenin with the destruction complex, this implies that the downstream metabolites of PMVK may cooperate with mutations in the destruction complex or in β‐catenin itself to further stabilize the protein, promote its nuclear localization and enhance it's signaling. Interestingly, the PMVK metabolite, MVA‐5PP similarly inhibited the interaction of β‐catenin with the destruction complex with similar consequences. We found that the mechanistic underpinnings of this activity involves the binding of MVA‐5PP to CKIα. Genetic knockdown of MVD also increased β‐catenin protein level. This suggests that PMVK stabilization of β‐catenin is dependent on its metabolite MVA‐5PP rather than on the mevalonate pathway metabolite isopentenyl diphosphate or other intermediates. In the developing chicken liver, MVK and PMVK remain highly active for the first 5 days, while the activity of MVD is low. This suggests that increased MVA‐5PP may be elevated in a way that satisfies the strong dependency on β‐catenin signaling during early development. Collectively, we uncovered a non‐canonical function of MVA‐5PP by which it and the PMVK enzyme that generates it, play synergistic roles in regulating the β‐catenin signaling pathway.PMVK phosphorylation of D is low.32 Thiβ‐catenin p‐Ser184 correlated with total β‐catenin and its downstream signals. This indicates that PMVK and β‐catenin p‐Ser184 protein levels are potentially useful as prognostic markers in HCC. Finally PMVK inhibitors represent a novel targeted therapy directed against a previously unsuspected target that drives HCC progression and may be useful in CRC as well. We also evaluated other inhibitors of the mevalonate pathway on PMVK‐regulated β‐catenin signaling. Simvastatin showed the same effect as PMVKi5 on β‐catenin, due to the fact that statins block the mevalonate pathway at its source . Zoledronic acid instead promotes β‐catenin protein levels, the target of zoledronic acid is the inhibition of farnesyl pyrophosphate synthesis which may indirectly contribute to the accumulation of upstream metabolites, similar to knockdown of MVD  have loxP sites flanking exons 2 and 5 of the PMVK gene. Alb‐Cre mice were kindly provided by Prof. Yong Liu (Wuhan University). To generate PMVKfl/flAlbcre mice, PMVKfl/fl mice were crossed with Alb‐Cre mice. All mouse genotype identification primer sequences used are shown in Table All animal studies were approved by the Animal Care Committee of Wuhan University (No. WQ20210130). 4‐week‐old female BALB/c nude mice were purchased from Gempharmatech . PMVKV (mm3) = a × b × (a + b)/2, where a is the length and b is the width.For xenograft experiments, tumor growth was monitored every 3 days for a total period of 30 days. Tumor volumes were calculated by the equation fl/fl and PMVKfl/flAlbcre male mice were intraperitoneally administrated 25 mg kg−1 of N‐Nitrosodiethylamine (DEN) (Sigma) at 2 weeks after birth. From 4 to 15 weeks, mice were intraperitoneally injected with 10% CCl4 (5 mL kg−1) weekly. After 28 weeks, livers were examined. For induction of CRCs, C57BL/6J mice were intraperitoneally administrated AOM (12.5 mg kg−1) at 8 weeks after birth. From 9, 12, and 15 weeks, 2.5% DSS was added to the drinking water of mice for 1 week, after 25 weeks, the colons were examined.For induction of HCCs, C57BL/6J, PMVKLittermate mice were randomly divided into control and various treatment groups including: control, PMVKi5 prevention, and PMVKi5 treatment. All cohorts were maintained as blinded cohorts following these treatments. Twelve T225 culture flasks were prepared for library virus generation according to the HEK293 culture conditions described above. Each flask was transfected with 20 µg of library plasmid (10 µg each of libraries A and B), 10 µg of pMD2.G and 15 µg of psPAX2 were transfected using 100 µL of Lipofectamine 2000 and 200 µL of Plus Reagent. The medium was replaced after 24 h and the cell supernatants were collected at 48 and 72 h of transfection and passed through 0.45 µm filters. The packed lentiviral library was then used to infect at least 5 × 107 Huh7 cells in the presence of 1 µg mL−1 polybrene, ensuring a multiplicity of infection (MOI) of 0.3 so that most cells receive only one stably integrated RNA guide. 24 h after infection, puromycin was added to select for stably transduced cells. Allow the cell count to expand to >8 × 107, ensuring that the initial sieve cell count is a library coverage multiple >300×. Each group of 4 × 107 cells were treated with vector and XAV393 (30 µm), respectively, and cells were passaged every 2 days for a total of 14 days of drug treatment. All cells were collected and genomic DNA was extracted, enriched for fragments containing sgRNA sequences using amplification primers  and sequenced on an Illumina NovaSeq 6000 following a second round of PCR reactions with the addition of splice primers and indexT sequences used to identify different samples. The number of sgRNAs of XAV‐939 and vehicle was identified using the MAGeCK algorithm and analyzed using DESEq2 to obtain significantly enriched or depleted sgRNAs.Amplification of the sgRNA library followed the description of Feng Zhang et al.35 Tweβ‐catenin, and β‐catenin p‐Ser184. Images were obtained on a Leica Aperio VERSA 8 microscope.Liver sections or mouse embryos were fixed in 10% formalin and embedded in paraffin according to standard protocols. Liver sections or mouse embryo were sectioned and stained with H&E, Ki67, PMVK,  Briefly, HEK293 cells were transfected with the indicated vectors. 48 h later, they were fixed in 4% paraformaldehyde for 15 min, followed by 0.2% Triton X‐100 permeabilization for 5 min and 3% BSA blocking for 1 h. After incubation with primary antibody against FLAG (1:200) overnight in 4 °C, the cells were washed well with PBST and incubated with ABflo 488‐conjugated goat anti‐rabbit (1:500) for 1 h at room temperature. After additional extensive washing, the cells were labeled with DAPI (5 µg mL−1) for 5 min at room temperature. Fluorescence imaging was performed with a LSM800 confocal laser scanning microscope.Immunofluorescence analysis was performed as previously described.34 Brim Tris (pH 7.4), 150 mm NaCl, 1% NP‐40, 0.5% Sodium deoxycholate, 0.1% SDS, 5 mm EDTA, 10 mg mL−1 aprotinin, 10 mg mL−1 leupeptin, and 1 mm PMSF]. Sepharose beads were washed three times with 1 mL lysis buffer containing 0.1% NP‐40. Co‐IP and immunoblot analysis were performed as described.Transfected cells were lysed in 1 mL lysis buffer [50 mescribed.36β‐actin was used as an internal control.Total RNA was isolated using Trizol followed by DNase treatment. Reverse transcription was performed with a cDNA Synthesis Kit and qPCR were performed using SYBR Green master mix using standard protocols. All qPCR primer sequences used are shown in Table −1) for the indicated times before collection for immunoblot analysis.After achieving ≈70% confluence, cells in 12‐well plates were transfected with the indicated plasmids using Lipofectamine 2000. 36 h later, the cells were treated with cycloheximide  and cultured in DMEM supplemented with 10% FBS. After 3–7 days of incubation, MTT was added and cells were incubated for 4 h at 37 °C. At the indicated time, this was replaced with 200 µL of DMSO. Absorbance was then read at 570 nm using an ELX800 absorbance microplate reader.For MTT assays, cells were seeded into 96‐well plates (10β‐catenin‐His and PMVK‐His were prepared and purified from Rossita. 1 µg GST‐β‐catenin‐His protein was incubated with 1 µg PMVK‐His in a 50 µL kinase reaction buffer (20 mm Tris‐HCl (pH 7.5), 10 mm MgCl2, 50 mm KCl, 30 µm ATP) at 37 °C for 0.5 h. Reactions were stopped by the addition of SDS sample buffer, and samples were then heated for 5 min at 95 °C. Phosphorylation of β‐catenin Ser184 was identified by mass spectrometry and immunoblot with β‐catenin p‐Ser184 antibody that was generated by ABclonal by immunizing rabbits with a S184‐monophosphorylated peptide (KKEA(S‐pho)RHA) conjugated with keyhole limpet hemocyanin.Recombinant GST‐µm MG132 for 6 h. They were then lysed in RIPA lysis buffer as described above. The samples were boiled for 10 min in SDS sample buffer and analyzed by immunoblot with the indicated antibodies.36 h after transfection, cells were treated with 20 Kd of binding of MAV‐5PP to CKIα‐GFP protein and PMVKi5 to PMVK‐GFP protein. All samples to be tested were first solubilized in RIPA buffer. The CKIα‐GFP or PMVK‐GFP was added to each dilution and incubated at room temperature for 5 min. The samples were loaded into silica capillaries . The measurements were performed at 25 °C using 60% LED power and medium MST power. Data were analyzed with Nano‐Temper Analysis software, v.2.3.A Monolith NT.115 instrument  was used to measure the Serum cholesterol, triglyceride, ALT, and AST were measured using colorimetric analysis kits . All experimental steps were performed according to the manufacturer's instructions. All procedures that involved human HCC samples collection were approved by the ethics committee of Wuhan University (No.2022030) and the Union Hospital in Wuhan, China and conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Informed written consent of all participants was signed by all individuals or their families and obtained at the Union Hospital in Wuhan, China. The diagnoses of all samples were confirmed by histological review.Human HCC samples were used as previously described.34 All Finally, the top 5 candidate small molecules were selected for cellular experiments. ChemBioDraw Ultra 17.0 was used to draw the structure of MVA‐5PP, the protein structures of the destruction complexes were all from the PDB database , and were docked using Discovery Studio.The PMVK‐based crystal structure (PDB: 3CH4) was used for computer‐based virtual docking of 1.7 million small molecules from the ChemDiv database. Hydrogenation and energy optimization of database molecules relied on Schrodinger's Ligprep program. Pre‐processing of protein structures used Schrodinger's Protein Preparation program. Next, the HTVS, SP, and XP docking procedures of Schrodinger's Glide module were used to cascade docking of the database.37 Finm HEPES (pH = 8.0), 50 mm KCl, 3 mm MgCl2, 200 µm ATP, 40 µm MVA‐5P, and 5 µm PMVK, which was incubated at 37° for 5 min. Fluorescence intensity was measured by a luminometer. All experimental steps follow the manufacturer's instructions.A Luminescent Kinase Assay Kit was used to quantify enzymatic activity of PMVK by quantifying ATP consumption. The kinase reaction system consisted of 50 mhttps://www.pantherdb.org). HCC datasets were downloaded from The Cancer Genome Atlas (TCGA) data portal (https://www.tcgadata.nci.nih.gov). PMVK mRNA levels were analyzed from TCGA and NCBI GEO databases (https://www.ncbi.nlm.nih.gov). Protein crystal structures were downloaded from PDB databases (https://www.pdbus.org). Small molecules were downloaded from the ChemDiv database (https://www.chemdiv.com).High‐throughput sequencing results for protein class analysis and pathway enrichment analysis were analyzed in the websites (t‐test was used for comparisons between two groups. One‐way ANOVA was used for multiple comparisons in patient populations. The correlation coefficient (r) and p values were obtained from Pearson correlation analysis, Pearson's r was calculated by GraphPad Prism 8. GraphPad Prism 8 was used for statistical calculations. p < 0.05 was considered to be statistically significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant. The data analyses were performed using unbiased software programs or algorithms. All experiments were repeated with a minimum of three independent repeats unless otherwise noted. Each experiment was repeated independently with similar results.Data are presented as mean ± SD or mean ± SEM. Log‐rank tests were used for survival analyses. Student's The authors declare no conflict of interest.Z.C., F.W., and Y.L. designed the study; Z.C. performed most of the experiments; Y.T., M.L., and M.W. constructed plasmids; X.‐J.Z., Z.C., and F.W. performed to analyze sequencing and clinical data; J.Z., C.T., and X.‐Y.Z. performed some animal experiments. All authors discussed the results. Z.C., E.P., F.W., and Y.L. wrote the manuscript with comments from all authors.Supporting InformationClick here for additional data file.


Here, we show that cochleae lacking β-Catenin transcriptional function lost IPCs and displayed extranumerary OPCs, indicating its requirement for establishing IPC identity. Overexpression of β-Catenin induced proliferation within IPCs but not ectopic IPCs. Single-cell transcriptomes of supporting cells lacking β-Catenin transcriptional function show a loss of the IPC and gain of OPC signatures. Finally, targeted deletion of β-Catenin in IPCs also led to the loss of IPC identity, indicating a cell autonomous role of β-Catenin in establishing IPC identity. As IPCs have the capacity to regenerate sensory hair cells in the postnatal cochlea, our results will aid in future IPC-based hair cell regeneration strategies.The mammalian cochlea is composed of sensory hair cells as well as multiple different types of non-sensory supporting cells. Pillar cells are one type of supporting cell that form the tunnel of Corti and include two morphologically and functionally distinct subtypes: inner pillar cells (IPCs) and outer pillar cells (OPCs). The processes of specification and differentiation of inner versus outer pillar cells are still unclear. Here, we show that β-Catenin is required for establishing IPC identity in the mammalian cochlea. To differentiate the transcriptional and adhesion roles of β-Catenin in establishing IPC identity, we examined two different models of  The organ of Corti, the hearing apparatus, requires a precise arrangement of various cell types including hair cells (required for sound reception) and supporting cells (supporting hair cell function) for proper function. β-Catenin is an essential component of the Wnt/β-Catenin signaling pathway, which is required for normal development of different cell types. Here, we show that β-Catenin is required for differentiation of the supporting cell subtype inner pillar cells. Loss of transcriptional activity of β-Catenin caused loss of inner pillar cells and gain of outer pillar cell-like cells. In addition, we show that β-Catenin works in a cell autonomous manner within pillar cells to establish inner pillar cell identity. These findings advance our understanding of the mechanisms involved in cell identity establishment during cochlear development and thereby will guide regenerative efforts to repair damaged cells within the organ of Corti. The sensory epithelium of the mammalian cochlea (the organ of Corti) consists of multiple unique cell types arranged in a highly ordered pattern that is necessary for proper auditory function. Cells in this organ are categorized into sensory hair cells and non-sensory supporting cells. Hair cells are arranged into 4 rows: three rows of outer hair cells and one row of inner hair cells. Supporting cells consist of multiple cell types including : several rows of Hensen’s cells, 3 rows of Deiters’ cells (DCs) that underlie the outer hair cells, one row of outer pillar cells (OPCs), one row of inner pillar cells (IPCs) and few rows of inner phalangeal cells that support inner hair cells  resulted in loss of NGFR (p75) expression in the IPCs [β-Catenin deletion, via its cell adhesion role, disrupts the boundaries between medial and lateral compartments of the developing cochlea [β-Catenin deletion alleles in this study: (1) the β-Cateninfl allele that has a deletion from exon 2 (which contains the ATG translation start site) to exon 6, generating a β-Catenin null allele that deletes both transcriptional and structural functions [β-Catenindm allele which retains cell adhesion function but deletes transcriptional function by point mutating Aspartic Acid of 164 to Alanine (D164A) and deleting the C-terminus [β-Catenin deletion models, to determine the role of β-Catenin in pillar cell fate determination. Through genetic modification of β-Catenin gene, lineage tracing, and single cell RNA sequencing, we showed that that β-Catenin transcriptional activity within supporting cells is required to establish IPC identity during cochlear development.There is growing evidence that Wnt/β-Catenin signaling pathway is essential for mammalian cochlear development. In the developing cochlea, canonical Wnt/β-Catenin signaling reporter activity becomes restricted to medial supporting cells including pillar cells and inner phalangeal cells . In two the IPCs ,34. Addiunctions , and (2)terminus . In thisβ-Cateninfl allele to eliminate both the transcriptional and cell adhesion functions of β-Catenin, and showed that Sox2-Cre- and Fgf20-Cre-mediated deletions of β-Catenin caused hair cell and supporting cell disorganization throughout the cochlear duct. Additionally, p75 expression (IPC marker) was downregulated or absent. To further investigate the role of β-Catenin in the development of cochlear supporting cells, we utilized an additional Cre driver, Emx2-Cre, which is active in cochlear epithelial cells by E12.5. Emx2-Cre has a broader activity domain and higher efficiency compared to the Sox2-Cre and Fgf20-Cre models, as well as higher animal survival rates [β-Cateninfl/fl; Emx2Cre/+) and littermate controls  at E18.5 showed hair cell disorganization in all cochlear turns, disrupted boundaries between inner and outer hair cells (as indicated by E-cadherin staining), without a significant change in hair cell density , and gain of OPCs (CD44+ cells) across all cochlear turns   . Most ec p<0.05) . To inveferation . Altogetβ-Catenin conditional mutant cochleae is due to the deletion of β-Catenin cell adhesion or transcriptional function, we generated and compared 2 different mouse models: β-Cateninfl/fl; Fgf20Cre/+  to delete both β-Catenin cell adhesion and transcriptional functionalities, and β-Catenindm/fl; Fgf20Cre/+  to delete β-Catenin transcriptional activity while retaining the cell-cell adhesion function [β-Catenindm/dm mice exhibit early embryonic lethality [Fgf20Cre mice to delete one copy of β-Cateninfl allele, while the other allele is β-Catenindm . β-Cateninfl/+; Fgf20Cre/+ was used as the control for both cKO and dm-cKO models. Both models have demonstrated high efficiency of β-Catenin deletion from the cochlear supporting cells [To test whether the phenotype observed in function . Since hethality , we utilng cells . Postnatβ-Catenin in prosensory cells around the time of pillar cell specification, we utilized an inducible Cre allele (Lgr5CreERT2/+) that is active in the Sox2+ supporting cells of the cochlear duct [RosatdTomato/+ allele that expresses tdTomato upon Cre recombinase activity to trace Cre activity [β-Catenin deletion at later time points compared to Fgf20-Cre line. We generated pregnant female mice carrying β-Cateninfl/fl; Lgr5CreERT2/+; RosatdTomato/+ embryos (lacking both β-Catenin functions) and β-Cateninfl/dm; Lgr5CreERT2/+; RosatdTomato/+ embryos (lacking β-Catenin transcription but retaining cell adhesion function). Pregnant females were induced with tamoxifen (250 mg/kg) once at E14.5, before pillar cell differentiation. Analysis of the cochleae from control mice at E18.5 for the recombination efficiency within the region of IPCs showed efficient recombination ranging from 75% to 82% in both models (β-Catenin deletion using cochlear immunostaining against β-Catenin 48 hours post induction (E16.5) . On the other hand, β-Catenin staining was present in the transcription deletion model , as β-Catenin cell adhesion function is expected to be preserved in this model. Analysis of β-Catenin deficient cochlea showed loss of p75 staining from many recombined (tdTomato+) cells within the region of IPCs in both conditional deletion models when compared to controls  . The majcontrols . In compcontrols   allele that generates constitutively active β-Catenin under the control of Cre recombinase [Fgfr3iCreER/+ inducible Cre model which is activated in supporting cells upon induction [β-Cateninfl(ex3)/+; Fgfr3iCreER/+) and littermate controls . Pregnant mice were induced with tamoxifen at E14.5 and cochleae were collected and analyzed at E18.5. Constitutively active β-Catenin cochlea exhibited multiple foci of p75+ cell clusters within the IPC region, yet no ectopic IPC formation was found outside the IPC region /+; Fgfr3iCreER/+; RosatdTomato/+ embryos and littermate controls . We induced recombination in pregnant females with tamoxifen at E14.5, injected EdU (proliferation marker) at E18.5, then collected embryos 2 hours later (β-Cateninfl(ex3)/+; Fgfr3iCreER/+; RosatdTomato/+ cochlea, EdU marked some of the tdTomato+ IPCs (12.9% (base), 13.1 (middle) and 3.2% (apex)), whereas none were marked in control cochlea  P0 pups and littermate controls . We pooled both cochleae of one control pup at P0 and analyzed 2,830 cells with 58,718 mean reads per cell, and a sequence saturation of 73.5%. Around 87% of the reads were mapped to the mouse genome and a total of 18,757 genes were detected. We analyzed control sample data using unsupervised graph-based clustering in Loupe Browser v.6 (10X genomics) to cluster similar transcriptome signatures from control cochlear cells. We were able to identify unique transcriptomic signatures belonging to specific cell populations based on previously published markers. Hair cells markers such as Barhl1, Pou4f3, Pvalb were enriched in hair cell clusters [Lfng, Fgfr3 and Prox1 [To understand the molecular identity of the β-Catenin-deficient supporting cells within the IPC region, we utilized single cell RNA (scRNA) sequencing of cochlear duct cells from dm-cKO  expression (rd row of Deiters’ cells (DC3) clustered separately from the 1st and 2nd rows of Deiters’ cells (DC1/2). OPC cells (CD44+ cells) were located within the DC1/2 cluster, indicating that OPCs’ molecular signature is similar to the DC1/2 signature at P0  was lower in dm-cKO (38.9%) compared to control (54%) cochleae, confirming the loss of IPC signatures in dm-cKO cochleae , CD44+ cells (OPCs) and p75-negative, CD44-negative cells (Deiters’ cells) to the total Prox1+ cells in dm-cKO and control cochleae . IPCs recochleae .Igfbpl1, p75, Npy and Cryab, while 44% of upregulated genes in dm-cKO belong to the DC3 signature, including Srp9, Frmd4a, Hnrnpu and Mgst3. These results suggest a loss of the IPC signature upon deletion of β-Catenin transcriptional activity. For the top DEGs, please refer to We next analyzed differentially expressed genes (DEGs) in Prox1+ cells in control and dm-cKO cochleae  and crosfzd4 and klf4), MAPK signaling pathway genes , and few transcription factors . Enriched gene ontologies within upregulated genes included multiple genes involved in cell differentiation   was downregulated in dm-cKO supporting cells  ,45. With Prrc2b) . To vali2b)  was restricted to IPCs and glial cells in the cochlea at P0 . Analysis of P1 cochlea from this conditional knockout showed reduced levels of β-Catenin in the majority of IPCs  did not show statistically significant changes, a trend of reduction in p75 expression and occasional ectopic CD44+ cells were observed  along with multiple Cre drivers, we show that lack of β-Catenin causes cells within the IPC region to lose IPC signature genes  and gain OPC genes (such as CD44 and Tuba1b)  indicatiUsing a constitutively active β-Catenin model, we tested whether β-Catenin is sufficient for inducing IPC fate. Data show no ectopic IPCs outside their respective cochlear domain, but that some IPCs proliferated to create clusters of IPCs . This da-Catenin transcriptional deletion model to confirm the loss of IPC identity and to identify targets downstream β-Catenin during supporting cell development. Single cell transcriptomic analysis at P0 showed that supporting cells lacking the β-Catenin transcriptional function failed to establish the unique IPC molecular signature as identified in the controls. Although we utilized one biological sample per condition for the scRNA sequencing experiment, we have validated a subset of DEGs using immunostaining and in situ hybridization to support IPC identity loss. Analyzing supporting cells from the β-Catenin transcriptional deletion model, we observed a significant loss of IPC specific genes and gain of other supporting cells-specific genes including Deiters’ and/or OPC marker genes. Downregulated genes in β-Catenin deficient supporting cells did not show canonical Wnt target genes such as Axin2, Lgr5 or Sp5, which may be due to the late time point of the scRNA sequencing sample (P0) relative to the time of IPC specification and differentiation (around E15) [β-Catenin deletion models at earlier time points (E15.5–16.5). Nevertheless, P0 single cell transcriptome analysis shows the ultimate impact of β-Catenin deletion on pillar cell fates.In addition to using p75 and CD44 as markers for IPCs and OPCs, respectively ,51, we aund E15) –25. A vaβ-Catenin deletion to IPCs to determine its cell autonomous role in IPC fate establishment. IPC-specific conditional β-Catenin deletion resulted in a similar phenotype of IPC to OPC marker conversion within IPCs. Notably, this phenotype was more evident in the apex than in the middle and basal turns. We suggest that pillar cell specification follow a basal to apical gradient similar to hair cell differentiation. Basal cells may have already established IPC identity at the time of Cre-mediated β-Catenin deletion. This may explain why we found fewer p75 to CD44 conversions in the basal and middle turns compared to the apex. Nevertheless, the occurrence of converted cells and CD44/p75 double positive cells in all turns in the cochlea suggests a cell autonomous role of β-Catenin in IPC identity establishment.Finally, we limited Wnt5a and Wnt7a are expressed in cochlear duct at E14.5 [Fzd1 and Fzd2, are expressed within the supporting cells during development  is required for IPC fate? It has been shown that at E14.5 ,53 and piewed in ) and mayOne of the most significant applications of Wnt/β-Catenin signaling role in IPC fate is for hair cell regeneration. Activation of Wnt signaling within IPC was sufficient to drive proliferation, as shown in this study. Since proliferation is a prerequisite for regeneration after damage in avian cochlea , activatβ-Catenin complete deletion models versus β-Catenin transcriptional deletion models .Emx2Cre [β-Cateninfl [Fgf20Cre [β-Catenindm [Lgr5CreERT2 [RosatdTomato [β-Cateninfl(ex3) [Fgfr3iCreER [NpyCre [Emx2Cre , β-Catenateninfl , Fgf20CrFgf20Cre , β-Catenatenindm , Lgr5Cre5CreERT2 , RosatdTtdTomato , β-Catennfl(ex3) , Fgfr3iCr3iCreER , and Npy [NpyCre  were preFor whole mount immunostaining, embryos from different time points and groups were collected, inner ears were dissected in cold 1X Phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) overnight at 4°C then washed with 1X PBS three times. Samples were blocked in 1X PBS containing 0.5% Triton and 5% normal donkey serum. Samples were then incubated in primary antibody overnight at 4°C, then washed with 1X PBS three times. Samples were incubated with a secondary antibody for 2 hours at room temperature then washed, mounted in 95% glycerol or DAPI mounting medium, cover-slipped, and imaged using a Zeiss LSM 700 confocal microscope. For cryosection immunostaining, PFA-fixed samples were washed in 10%, 20%, and 30% sucrose solutions for 1 hour each at 4°C, then embedded in optimum cutting temperature medium (OCT), frozen on dry ice, and stored at −80°C until further processing. OCT-embedded samples were sectioned horizontally at 10μm thickness using a cryostat, then sections were mounted and left to dry at room temperature overnight. Primary antibodies/stains used: Phalloidin , Prox1 , Myo6 , Myo7a , CD44 , p75 , E-cadherin , Sox2 , Npy , β-catenin , and Ki67 .Pregnant females were injected with Ethynyl deoxyuridine (EdU) at 400μg/g body weight two hours ahead of collecting the embryos. Embryos were collected and prepared for cryosectioning. Staining for incorporated EdU along was performed according to Click-iT EdU Cell Proliferation Kit (Invitrogen C10340) manufacturer protocol. Stained sections were then imaged on a Zeiss LSM 700 confocal microscope.https://umgear.org/index.html?multigene_plots=0&layout_id=d4505add&gene_symbol_exact_match=1&gene_symbol=sox2A P0 pup was euthanized, and cochleae were dissected in sterile cold HBSS, then placed in Dispase (1U/mL) in DMEM/F-12 (Stem cell Technologies) for 15 min at 37°C. Cochlear ducts from both sides were dissected and pooled into a single tube, incubated in 0.25% trypsin-EDTA for 15 min at 37°C with gentle trituration every 5 min, followed by trypsin inactivation by adding an equal volume of DMEM/F-12. Dissociated cells were then passed through a 30μm strainer, pelleted at 300g and then resuspended in 100μl of cold PBS supplemented with 1% fetal bovine serum . Single cells were captured and lysed, and mRNAs were reverse transcribed into cDNAs using a 10X Genomics Chromium Controller at University of Nebraska Medical Center Sequencing core. cDNA libraries were prepared using Chromium Single Cell 3′ Reagents according to the manufacturer’s instructions. Libraries were sequenced on an Illumina NextSeq. Sequences were aligned to the Ensembl mouse MM10 assembly using Cell Ranger 2.1.1 analysis software (10× Genomics). Processing of the Cell Ranger output data was done with Loupe Browser v.6 (10X Genomics). Gene expression-based clustering information, including t-SNE projections and differential gene expression were done utilizing Loupe Browser v.6. The raw data from the scRNA sequencing is deposited in gEAR database and can be accessed through the following link: Cryosections (10μm) were used for RNA FISH analysis following the manufacturer’s protocol  was used to adjust for multiple comparisons using two-stage step-up. For ScRNA sequencing data analysis, Cell Ranger 2.1.1 analysis software (10X Genomics) was used.Data analysis was done using Image J software Version 1.53n . To measS1 Figβ-Catenin deletion  versus control showing scarce proliferating cells within the prosensory domain (PD) in both conditions. Most proliferating cells are located within the greater epithelial ridge (GER) (B) Quantification of Ki67/Sox2 double-positive cells within the prosensory domain per 100μm. Bar on graph is mean±SE. Scale bar = 50μm.(A) Immunostaining of whole mount E14.5 cochlear epithelium for the proliferation marker Ki67 (green) and the prosensory marker Sox2 (red) from (TIF)Click here for additional data file.S2 Figβ-Cateninfl/fl; Lgr5CreERT2/+; RosatdTomato/+ cochlea (full deletion) compared to control 48 hours post induction with tamoxifen (E14.5), showing staining for β-Catenin (white) along with tdTomato fluorescence (red) in cells with Lgr5CreERT2 recombination. The majority of IPCs along with other supporting cells shows loss of β-Catenin staining (yellow arrow heads), but a few supporting cells still show some β-Catenin expression (green arrow heads). (B) Immunostaining from β-Cateninfl/dm; Lgr5CreERT2/+; RosatdTomato/+ cochlea  showing β-Catenin expression that is similar to controls. Scale bar = 100μm.(A) Immunostaining of the basal turn whole mount E16.5 cochlear epithelium from (TIF)Click here for additional data file.S3 Figβ-Cateninfl/+; Fgf20Cre/+) showing 8 different cell clusters (color-coded) identified based on known markers per population. (B) t-SNE plots representing Prox1+ expression levels within the supporting cell population in P0 control cochlear cells. (C) Differentially expressed genes (DEGs) within each identified cluster from (A) showing level of enrichment of each gene within each cluster .(A) t-SNE plot representing graph-based clustering of cochlear cells from P0 control ((TIF)Click here for additional data file.S4 Fig(A-B) Graphs representing enriched gene ontologies within differentially expressed genes in dm-cKO versus control Prox1+ cells .(TIF)Click here for additional data file.S5 Figβ-Cateninfl/+; NpyCre/+; RosatdTomato/+ cochlea showing p75 (green), tdTomato (red) and CD44 (blue) showing restricted Cre activity within IPCs. (B) Immunostaining of whole mount P1 cochlear epithelium from IPC-specific complete β-Catenin deletion  cochlea showing β-Catenin (green), tdTomato (red) showing efficient β-Catenin deletion within the IPCs. (C) Immunostaining of whole mount P1 cochlear epithelium from IPC-specific transcriptional β-Catenin deletion  cochlea showing p75 (green) and CD44 (red) showing examples for p75/Cd44 double-positive cells in IPC region (blue arrowhead) and Cd44+ ectopic cells (white arrowhead). Scale bar = 100μm unless otherwise specified.(A) Immunostaining of whole mount P0 cochlear epithelium from (TIF)Click here for additional data file.S1 Table(CSV)Click here for additional data file.S2 Table(CSV)Click here for additional data file.S3 Table(CSV)Click here for additional data file.


N-glycans on LRP6, thereby enhancing trafficking of LRP6 to the plasma membrane and promoting Wnt/β-catenin signalling. Our findings further highlight the importance of LRP6 as a regulatory hub in Wnt signalling and provide one of the few examples of how a specific glycosyltransferase appears to selectively target a signalling pathway component to alter cellular signalling events.Reception of Wnt signals by cells is predominantly mediated by Frizzled receptors in conjunction with a co-receptor, the latter being LRP6 or LRP5 for the Wnt/β-catenin signalling pathway. It is important that cells maintain precise control of receptor activation events in order to properly regulate Wnt/β-catenin signalling as aberrant signalling can result in disease in humans. Phosphorylation of the intracellular domain (ICD) of LRP6 is well known to regulate Wntβ-catenin signalling; however, less is known for regulatory post-translational modification events within the extracellular domain (ECD). Using a cell culture-based expression screen for functional regulators of LRP6, we identified a glycosyltransferase, B3GnT2-like, from a teleost fish (medaka) cDNA library, that modifies LRP6 and regulates Wnt/β-catenin signalling. We provide both gain-of-function and loss-of-function evidence that the single human homolog, B3GnT2, promotes extension of polylactosamine chains at multiple  The Wnt pathway is an evolutionarily conserved, highly regulated cellular signalling network comprising 19 secreted Wnt proteins (ligands), 10 Frizzled proteins  and a variety of co-receptors that regulate embryonic development and tissue homeostasis ,3. Wnt sLRP5/6 are type I single-span transmembrane proteins with a large extracellular domain (ECD) harbouring four YWTD β-propeller (BP) domains, each linked to an epidermal growth factor-like (EGF-like) domain and three LDLA (low-density lipoprotein receptor type A) domains . The fouN-glycans to proteins in the ER starts with transfer of an immature, mannose-rich, dolichol-linked oligosaccharide. ALG8 is one of the enzymes involved in the biosynthesis of the dolichol-linked oligosaccharide precursor and has it been implicated in the regulation of Wnt/β-catenin signalling and LRP6 glycosylation . I. IN-glyc) enzyme b. When b) enzyme b. These N-glycans attached to LRP6, which enhances its cell surface localisation and therefore its ability to transduce Wnt/β-catenin signals. To address the requirement of B3GnT2 for Wnt/LRP6 signalling, we generated B3GnT2−/− HEK293T cell lines using a lentiviral-based CRISPR/Cas9 gene editing approach designed to remove the entire B3GnT2 coding region  B3GnT2-like cDNA identified in this study may have arisen from a Teleost genome duplication event around 350 million years ago that generated extra copies of fish genes rocesses . It is arocesses . In compOur loss-of-function data show that B3GnT2 is required for normal levels of LRP6/Wnt signalling and the reduced LRP6 signalling seen in cells lacking B3GnT2 is only restored to normal levels upon reintroduction of B3GnT2. Nevertheless, significant LRP6 signalling remains in cells lacking B3GnT2. This likely reflects a non-essential role of B3GnT2 for LRP6 activity and points more to its role as a regulatory factor. However, we show that different members of the B3GnT family appear to modify LRP6 and we cannot rule out functional redundancy by other members that may compensate for the absence of B3GnT2. It remains unclear why overexpression of LRP6 in the B3GnT2−/− cell line results in a slightly increased amount of LRP6 compared to wild-type cells; however, it displays reduced signalling. The distribution of LRP6 in different cellular compartments plays a crucial role in its activity: for example, it has been reported that lipid-raft-located LRP6 forms a complex with Wnt and Frizzled proteins and undergoes caveolin-mediated endocytosis to activate Wnt/β-catenin signalling, while LRP6 in nonlipid rafts is internalised with clathrin to inhibit Wnt/β-catenin signalling ,67. A reN-glycans present on these sites may promote maturation of LRP6 in the ER/Golgi through their ability to aid interactions with glycosyltransferases or LRP6 chaperones such as MESD. LRP6 is known to require N-glycosylation for trafficking and membrane localisation [N-glycan at N859 results in increased levels of polylactosamine, suggesting that lack of an N-glycan here may promote the extension of polylactosamine chains on other N-glycans. This means that N-glycans present at N859 may inhibit the function and/or processing of nearby N-glycans on LRP6. The fact that the LRP6 N859Q mutant shows both enhancement of basal LRP6 activity  of LRP6 alter the bending angle of the ectodomain which consequently affects its signalling activity [N-glycan sites cooperate to regulate the conformation and activity of LRP6 using different LRP6 N-glycosylation site mutant combinations. Although our MS analysis detected modification at only a few of the N-glycan attached to LRP6, such as N81 and Asn433, it is highly likely, considering the difficulty in detecting such modifications, that other N-glycans on the ECD of LRP6 are also modified by B3GnT2. Our LEL lectin blot analysis as well as analysis of LRP6 glycosylation mutants support this.We have identified two LRP6 glycosylation sites, N81 and N692, that are essential for the maturation of LRP6 during its biogenesis. lisation ,26, and lisation . Interesactivity D and a sactivity E suggestactivity . It willConsidering B3GnT2 has recently been reported to modify several ligands and receptors that alter T-cell activation via interaction with cancer cells , there mN-glycans attached to many membrane proteins and we have demonstrated that B3GnT2, in particular, modifies multiple LRP6 N-glycans to promote Wnt/β-catenin signalling. This is a significant finding that will have an impact on our understanding of cellular signalling in general, but also for research on disease states, stem cell maintenance, and cellular aging, all of which are known to be controlled to a large part through Wnt signalling.This study had identified glycosylation of LRP6, a co-receptor for Wnt, as a new regulatory mechanism for Wnt/β-catenin signalling. The B3GnT family of glycosyltransferases plays an important role in the extension of polylactosamine on the antenna of complex


A novel dimension splitting method is proposed for the efficient numerical simulation of a biochemotaxis model, which is a coupled system of chemotaxis–fluid equations and incompressible Navier–Stokes equations. A second-order pressure correction method is employed to decouple the velocity and pressure for the Navier–Stokes equations. Then, the alternating direction implicit scheme is used to solve the velocity equation, and the operator with dimension splitting effect is used instead of the traditional elliptic operator to solve the pressure equation. For the chemotactic equation, the operator splitting method and extrapolation technique are used to solve oxygen and cell density to achieve second-order time accuracy. The proposed dimension splitting method splits the two-dimensional problem into a one-dimensional problem by splitting the spatial derivative, which reduces the computation and storage costs. Finally, through interesting experiments, we show the evolution of the cell plume shape during the descent process. The effect of changing specific parameters on the velocity and plume shape during the descent process is also studied. Biochemotaxis models are used to describe oxidation patterns of some cells in nature. These cells tend to inhabit viscous fluids. The cells and chemical attractants are moved about with the fluid. The gravity produced by the cell aggregation then has an impact on the fluid’s flow. Therefore, the biochemotaxis equation is usually coupled to the incompressible Navier–Stokes (NS) equation that governs fluid motion. The aim of this study is to develop an effective second-order dimension splitting scheme for the chemotaxis-Navier–Stokes equations proposed by ,2. This L and We now introduce the dimensionless chemotaxis-Navier–Stokes equation and define the dimensionless parameters. The equation is defined in terms of non-negative parameters, namely As shown above, the chemotaxis-Navier–Stokes system is a non-linear, coupled system with multiple variables. To efficiently solve this system, it is important to decouple the equations. Additionally, a fine-mesh discretization is necessary for achieving high-resolution results in the two-dimensional case. However, it may result in high storage requirements and computational complexity. Thus, achieving high resolution while maintaining stability and computational efficiency remains an important research field.In response to numerical instabilities and maintaining algorithmic accuracy, researchers have developed many effective numerical methods. In , a high-In response to the huge storage and computational effort required for two-dimensional discretization, the dimension splitting method is developed. The key is to split the operator according to the direction of the spatial derivative. An early dimension splitting method was the alternate direction implicit (ADI) method, which relied heavily on the finite difference method ,9. OperaOur innovation is to establish a fast and low-cost numerical method for the chemotaxis-Navier–Stokes equation by using the dimension splitting method. First, a second-order pressure correction scheme with splitting effect is used to decouple the NS equation. The key is to use the operator pressure , so thatpressure ,15.The rest of the paper is organized as follows. In In this section, we present the dimension splitting scheme of the chemotaxis-Navier–Stokes model and the second-order central difference scheme in the MAC grid configuration.We give a second-order pressure-stabilization perturbation scheme for the NS equation in :(6)∂u∂t+eference ).c, and q at Steps 1–5 as follows.Let Step 1:The ADI scheme for updating the velocity field:Step 2:The dimension splitting scheme for updating the pressure Step 3:c based on the first-order operator splitting method:The dimension splitting scheme for solving the oxygen concentration given by  correspoStep 4:q based on the first-order operator splitting method:The dimension splitting scheme for solving the cell density given by  correspoStep 5:Step 3 and Step 4, the time step Since the numerical solutions Then, we get second-order solutions by adopting the extrapolation When applying the standard finite difference scheme directly in a standard Cartesian grid for the incompressible Navier–Stokes equation, the resulting numerical pressure is unstable. To address this issue, we use the second-order central difference scheme in the MAC grid configuration to provide a fully discrete scheme for the dimension splitting method above. In the MAC scheme , the unkx and y directions, respectively. For intermediate nodes, we can define as:p in the decoupled velocity equation, we define:p, q, and c at the cell center, we let Set the computational domain as The full discretization for the chemotaxis-Navier–Stokes system can be summarized with the following five steps.Step 1:problems –23)::v1n can problems –26): Core (TM) i7-10510u CPU (2.30 GHz) laptop. To evaluate computational efficiency, we compare the time required to solve the chemotaxis-Navier–Stokes equation using dimension splitting and non-dimension splitting schemes. The dimension splitting scheme adopts the spatiotemporal second-order scheme proposed in To numerically simulate the biochemotaxis phenomenon, we ran a number of computational tests on the dimensionless chemotaxis-Navier–Stokes model. Mixed boundary conditions  are consq, the second column is chemoattractant concentration c, and the third column is the fluid velocity field In all subsequent numerical result graphs shown, the first column is cell density In this subsection, we investigate how altering the parameter In this part, we examine what happens to the cell plume as l set to . CompariThe initial density of cells in this paragraph is adjusted to ent with . The simA fast dimension splitting scheme for chemotactic-Navier–Stokes systems is proposed. This method combines the pressure correction method with the dimension splitting effect, the ADI method, the operator splitting method, and the extrapolation method to achieve a fast second-order solution. The method decomposes the two-dimensional model into a series of one-dimensional subproblems, thus reducing computational complexity and storage space. The effectiveness and practicability of this method are demonstrated by numerical experiments simulating chemotaxis. Future research will focus on the following directions: (1) the dimension splitting schemes for three-dimensional chemotactic-Navier–Stokes systems: the method proposed in this article can be appropriately modified for solving the coupling system of the incompressible NS equation and the second-order parabolic equation. For example, the effective numerical results of the Allen–Cahn model of two-phase incompressible fluid, the surfactant model of binary fluid, etc., are what we will present in the future. (2) Due to the complexity and nonlinearity of the model, its stability analysis is a challenging problem, which will continue to be solved in the future. (3) The rapid numerical simulation of biochemotaxis in complex regions has more important practical application value. How to establish the dimensional splitting scheme of complex regions is the focus of our follow-up research.


Synechococcus PCC 7002 (S.7002) and Synechococcus elongatus PCC 7942 (S.7942), for the photosynthetic production of four chemically-different high-value terpenes: two monoterpenes limonene and pinene, and two sesquiterpenes bisabolene and farnesene. We showed, for the first time, that S.7002 and S.7942 can produce farnesene and bisabolene, respectively. Both cyanobacteria produced farnesene (S.7942 produced more efficiently than S.7002) more efficiently than the other tested terpenes . S.7002 produced limonene more efficiently than bisabolene, whereas S.7942 produced bisabolene more efficiently than limonene. These findings suggest that S.7942 is better suited to produce sesquiterpenes than monoterpenes. Interestingly, higher levels of terpenes were produced by S.7942 and S.7002 expressing a terpene-synthase gene from both an RSF1010-derived replicating plasmid and a neutral chromosomal site, as compared to either the plasmid alone or the chromosome alone. These results suggest that in both cyanobacteria, the production of terpenes is more limited by the activity of terpene synthases than the abundance of terpene precursors. Finally, higher levels of terpenes were produced by S.7002 growing on urea (a frequent pollutant) as compared to nitrate or ammonium, the standard nitrogen sources for cyanobacteria.We have performed the first comparative analysis of the potential of two physiologically-diverse model cyanobacteria,  Many high-value terpenes are exploited by cosmetic, food, pharmaceutical, and/or biofuel industries, including bisabolene, farnesene, limonene, and pinene, the focus of the present study [Terpenes are a large family of chemicals with the formula  such as limonene and pinene. The subsequent addition of one IPP unit on GPP forms the farnesyl pyrophosphate  precursor of sesquiterpenes (C15H24), such as bisabolene and farnesene (All terpenes are derived from the five-carbons (C5) building blocks, isopentenyl pyrophosphate (IPP), and dimethylallyl pyrophosphate (DMAPP) that are condensed to form the geranyl pyrophosphate  precursor of monoterpenes  and Synechococcus elongatus PCC 7942 (S.7942) are attractive for this purpose, in having good genetic tools and interesting physiological differences  cyanobacteria iew, see ) which miew, see . In contiew, see . S.7002 iew, see .Two proof-of-concept studies showed that S.7002 can be engineered for the photosynthetic production of bisabolene and limonene  or pinenIn this study, we have performed the first comparative analysis of the potential of the two physiologically-diverse model cyanobacteria, S.7002 and S.7942, for the photosynthetic production of four chemically-different high-value terpenes. Practically, we have employed the same genetic strategy to engineer S.7002 and S.7942 for the photoproduction of two monoterpenes, limonene (cyclic molecule) and pinene (bicyclic), and two sesquiterpenes, bisabolene (cyclic) and farnesene (linear), in order to rationally compare the potential of these two model cyanobacteria for the production of diverse terpenes. Each terpene synthase gene was expressed from the same strong promoter propagated by the same autonomously replicating plasmid vector in both S.7002 and S.7942. We showed for the first time that S.7002 and S.7942 can produce farnesene and bisabolene, respectively. Both S.7002 and S.7942 produced farnesene and pinene with the best (farnesene) and the lowest (pinene) efficiencies. Furthermore, S.7002 produced limonene better than bisabolene, whereas S.7942 produced bisabolene more efficiently than limonene. We also showed for the first time that S.7002 cultivated on urea (a frequent pollutant) produces terpenes more efficiently than cultures growing on either nitrate or ammonium (standard but more expensive nitrogen sources for cyanobacteria). Finally, we showed in both S.7002 and S.7942 that increasing the copy number of the terpene synthase-encoding gene increases the level of terpene production, suggesting that terpene production is more limited by the activity of terpene synthases rather than the abundance of terpene precursors. E. coli strains used for gene manipulations  50 μg·mL−1, streptomycin (Sm) 25 μg·mL−1 or spectinomycin (Sp) 75 μg·mL−1.−2·s−1) and agitation  in liquid mineral medium: MM, i.e., BG11 [2CO3 [−1) for S.7002 [−1 for both S.7002 and S.7942, and/or Sm 50 μg·mL−1 and Sp 50 μg·mL−1 for S.7002 and Sm 5 μg·mL−1 and Sp 5 μg·mL−1 for S.7942. For some experiments carried out with S.7002, the standard nitrogen source  was replaced by urea or ammonium. Nitrate-grown cells were washed twice and resuspended in nitrogen-free media that were supplemented with either urea or nitrate, as indicated. Growth in liquid cultures was monitored by regular measurements of optical density at 750 nm (OD750) using a spectrophotometer (Jenway 6700). S.7942 and S.7002 were grown at 30 °C, under continuous white light , or A+ medium containing 4 μg·L−1 B12 vitamin, 3 g·L−1 sodium thiosulfate, Km 50 μg·mL−1 or both Sp 50 μg·mL−1 and Sm 50 μg·mL−1 (S.7002), which were solidified with 1% (S.7942) or 1.5% (S.7002) Bacto Agar (Difco). The pC-derived replicative plasmids for high-level expression of terpene synthase genes  were intescribed ,22. CellThe presence of the terpene-synthase-encoding DNA cassette propagated in pC-derived plasmids or a neutral chromosomal site was verified by PCR and DNA sequencing  using appropriate oligonucleotide primers .S.7002 and S.7942 strains engineered for terpene production were photoautotrophically grown in the presence of selective antibiotics in 250 mL erlenmeyers containing 50 mL cell suspensions overlaid with 20% (vol/vol) dodecane  to trap terpenes ,20,23. AAbies grandis E-α-bisabolene synthase (bs), (ii) the Picea abies α-farnesene synthase (fs), (iii) the Mentha spicata 4S-limonene synthase gene (ls) and (iv) the Pinus taeda (+)-α-pinene synthase gene (ps). Each codon-adapted terpene synthase gene was cloned downstream of the strong lambda-phage pR-promoter and associated cro ribosome-binding site of the autonomously replicating RSF1010-derived [R/SpR clones were selected and analyzed by PCR and DNA sequencing  [Malus domestica Borkh or Picea abies (we used only the Picea abies enzyme) expressed from the strong pTrc promoter (we used the strong lambda-phage pR promoter). However, the multiple experimental differences between these reports and our own study make their comparison difficult. This explains why we employed the same genetic engineering strategy to explore the capability of various model cyanobacteria to photosynthetically produce various high-value terpenes. The level of farnesene production by S.7942 (~1.5 mg·Lrements) ,12) who Synechocystis PCC 6803 (hereafter S.6803) [−1 after 21 days, respectively, −1 after 21 days, [The level of farnesene production driven by the pCFS plasmid in S.7942 and S.7002 could be really compared to the level driven by the same pCFS plasmid we previously reported in  S.6803) , another21 days, , Figure −1 after 21 days, respectively, The photosynthetic production of bisabolene by the S.7002 and S.7942 strains propagating the pCBS plasmid, or the empty pC plasmid, was measured over a 21-day period of cultures B. GC-MS −1 after four days; ~0.5 mg·L−1 after 21 days) is lower than the previously reported level -α-bisabolene synthase gene expressed from the cpc phycocyanin promoter (we used the lambda-phage pR promoter) into a neutral chromosomal site (we used a replicative plasmid). The important differences between the previous report and our study make their comparison difficult.The production of bisabolene by S.7002 (~0.1 mg·Lsurement ) observe−1 after 21 days, respectively, as seen in −1 after 21 days, [The presently-observed levels of bisabolene production driven by the pCBS plasmid in S.7002 and S.7942 . Tog−1 after 14 days and ~1.7 mg·L−1 after 21 days, −1 after 14 days, The photosynthetic production of limonene by the S.7002 and S.7942 strains propagating the pCLS plasmid, or the empty pC plasmid, was monitored for 21 days C. Dodeca−1 after four days, −1 after four days, but no subsequent measurement [Mentha spicata limonene synthase gene expressed from the cpc phycocyanin promoter (we used the lambda-phage pR promoter). The presently-observed production of limonene by S.7002 (~0.3 mg·Lsurement ) observe−1 after seven days, −1 after seven days, but no subsequent measurement [Mentha spicata limonene synthase gene expressed from the psbA promoter (we used the lambda-phage pR promoter). Similarly, the presently-observed production of limonene by S.7942 (~0.06 mg·Lsurement ) observeThe multiple experimental differences between the previous reports and our study make it difficult to ascribe the difference in limonene production to one or several parameters: growth conditions, nature of the promoter expressing the limonene synthase gene, and/or of the cloning vehicle propagating it.−1 after 21 days, −1 after 21 days [The presently-observed level of limonene production driven by the pCLS plasmid in S.7002  is lower than the previously reported level  expressed from the cpc promoter (we used the lambda-phage pR promoter). As both the pAQ1 and pC vectors, on the one hand, and the cpc and pR promoters, on the other hand, function well in cyanobacteria [Abies grandis is more efficient than the native Pinus taeda enzyme we have used in this study  observeix days, ) a Abiesbacteria , we assuis study D, and ouis study .R-LS recombinant gene (LS expressed from the strong pR promoter) in the NS7002 neutral chromosomal site  often uy Gibson   in the NSI7942 neutral chromosomal site  a site y Gibson  , ammonium  or urea (CO(NH2)2, 6 mM). In the case of urea (and nitrate as a control), we tested both protocols in which nitrogen is supplied either all at once, at the beginning of the culture, or provided gradually as successive doses during cultivation as was required by the other cyanobacteria Cyanothece PCC 7425 and Synechocystis PCC 6803 [Cyanothece PCC 7425 and S.6803 produced terpene less efficiently (C.7425) or similarly (S.6803) when cultivated on urea as compared to nitrate [In the view of reducing the costs of future industrial production of terpenes, eventually coupled with wastewater treatment, we have tested the influence of urea  on cellPCC 6803 ,23. The  nitrate ,23.Synechococcus PCC 7002 (a marine host) and Synechococcus elongatus PCC 7942 (a freshwater host), to photosynthetically produce chemically-diverse terpenes of high-value . For this purpose, each terpene synthase gene was expressed from the same strong promoter and propagated by the same RSF1010-derived replicative plasmid vector. We showed, for the first time, that S.7002 and S.7942 can be engineered for the photoproduction of farnesene and bisabolene, respectively. S.7942 and S.7002 (in this order) produced farnesene more efficiently than the other tested terpenes, especially pinene, the weakest produced terpene, similar to what we previously observed in the other model cyanobacterium Synechocystis PCC 6803 (S.6803) [Pinus taeda pinene synthase does not work well in cyanobacteria. Furthermore, S.7002 produced limonene more efficiently than bisabolene, whereas S.7942 produced bisabolene more efficiently than limonene. We showed for the first time that S.7002 can produce terpenes when growing on urea (a frequent pollutant) as the sole nitrogen source, similar to what we observed for S.6803 [Synechococcus elongatus UTEX 2973, can be used for chemical production from urea because they both lack all urea transport and catabolism enzymes [Synechocystis PCC 6803 [We have performed the first rational and comparative analysis of the ability of two physiologically-diverse model unicellular cyanobacteria, (S.6803) . These fr S.6803 . In the PCC 6803 . These rPCC 6803 . FinallyPCC 6803 . We foun


Mounting evidence has proposed the importance of the Wnt/β-catenin pathway and tripartite motif 31 (TRIM31) in certain malignancies. Our research aimed to clarify the correlation between aberrant TRIM31 expression and the Wnt/β-catenin pathway during gastric cancer (GC) oncogenesis and development. TRIM31 was drastically elevated in GC tissues and was closely associated with aggressive clinical outcomes and poor prognosis. Moreover, TRIM31 downregulation attenuated GC cell proliferation and invasion in vitro. Mechanistically, TRIM31 could bind and ubiquitinate Axin1 protein, thereby facilitating the activation of the Wnt/β-catenin pathway. Additionally, Axin1 knockdown partially abrogated the inhibitory effects on the proliferative, invasive and migratory abilities of GC cells induced by TRIM31 silencing. Furthermore, TRIM31 was negatively correlated with Axin1 protein expression in GC tissues. In summary, we revealed a new TRIM31-Axin1-Wnt/β-catenin axis that contributed greatly to the progression of GC, and targeting this regulatory axis may represent an effective treatment for GC patients. The overall survival of patients with GC is poor due to its high recurrence and metastasis rates4. Hence, it is of importance to elucidate the mechanisms and pathological processes underlying GC tumorigenesis and development.Gastric cancer (GC) is a major malignant cancer and contributes to a heavy social burden globally owing to its increasing prevalence5. When the Wnt/β-catenin pathway is stimulated, intracellular β-catenin rapidly accumulates, and translocates into the nucleus to facilitate cyclin D1 and c-Myc transcription, resulting in cell proliferation and metastasis7. While the Wnt signaling pathway is absent, the accumulated β-catenin is phosphorylated by the destructive complex (APC-Axin1/2-GSK-3β)7. Next, the cytoplasmic β-catenin will be ubiquitinated for the degradation through the ubiquitin–proteasome system. As its dysregulation contributes greatly to several cancers, including GC, further studies are needed to investigate the complicated mechanisms underlying the activation of Wnt/β-catenin pathway.The Wnt/β-catenin signaling pathway has been shown to play a fundamental role in multiple biological and pathological processes, including human cancers10. Some studies have demonstrated that TRIM31 is involved in tumor growth and metastasis in nasopharyngeal carcinoma, glioblastoma, pancreatic and colorectal cancer14. TRIM31 promotes liver cancer progression by degrading the tuberous sclerosis complex (TSC) 1 and TSC2 complex and activating the mammalian target of rapamycin complex1 signaling pathway15. Other dysregulated signaling pathways are responsible for TRIM31 functions including NF-κB and Akt pathways, which are tightly associated with cancer cell proliferation and invasion16. More interestingly, TRIM31 overexpression has been reported in early GC tissues18. Additionally, TRIM31 is also as a positive activator of the Wnt/β-catenin pathway in acute myeloid leukemia19. However, the exact correlation between the Wnt/β-catenin pathway and TRIM31 in GC remains unknown.Tripartite motif 31 (TRIM31), a member of the TRIM protein family, is an E3 ubiquitin ligase and serves as an important regulator of carcinogenesis and the progression of several cancers owing to its role in promoting substrate degradation and activating signal transduction through ubiquitin modificationsThe aim of our study is to unveil the correlation between TRIM31 and Wnt/β-catenin signaling in GC tumorigenesis and progression. Our data show that TRIM31 overexpression activates the Wnt/β-catenin pathway by promoting the ubiquitination and degradation of Axin1 protein, and thereby providing novel insights into the treatments for GC patients.20  and GEPIA221  online tools. As illustrated in Fig. To determine TRIM31 expression in various cancers, preliminary screening was conducted via TIMERAs TRIM31 was predicted to correlate with GC tumorigenesis and progression, we further examined the expression levels and clinical significance of TRIM31 in GC specimens. In total, 170 paraffin-embedded GC tissue samples were collected for IHC staining. As shown in Fig. We conducted qRT-PCR, CCK-8, colony-forming, transwell and wound healing assays to assess the biological functions of TRIM31 in GC cells. qRT-PCR assays were performed to confirm the efficiency of shRNAs targeting TRIM31 , MMP2/9, p53-AMPK axis, NF-κB and PI3K/Akt pathwaysIn summary, this study revealed a novel correlation between TRIM31 and the Wnt/β-catenin pathway. TRIM31 associated with and destabilized Axin1 protein to orchestrate the Wnt/β-catenin pathway, resulting in the tumorigenesis and progression of GC.We use TIMER database to detect TRIM31 expression levels in multiple cancers. Then GEPIA2 database was utilized for TRIM31 expression in gastric cancerous and normal tissue from TCGA database or TCGA and GTE) databases. Next, we downloaded the gastric cancerous and corresponding non-cancerous tissues from TCGA online tool, which were dealt with R software version 4.2.1 for statistical analysis and visualization.We retrospectively collected 170 paraffin-embedded GC tissues from the Department of Pathology at the Third Affiliated Hospital of Nanchang University from 2017 to 2020. Demographics of patients from which the samples were obtained were as follows: 101 (59.4%) were aged < 65 years and 69 (40.6%) were aged ≥ 65 years, and 75 (44.1%) and 95 (55.9%) were female and male, respectively. None of the enrolled patients received treatment before surgery. The clinicopathological features of these 170 patients were sorted and analyzed by two pathologists  were from the American Type Culture Collection (Manassas) and the Shanghai Institute of Cell Biology (China). All cell lines were raised in RPMI-1640  or Dulbecco’s Modified Eagle Medium (DMEM) supplied with 10–15% fetal bovine serum  in 37 °C at 5% COTotal proteins of GC cells and tissues were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer  with 1% protease inhibitor cocktail . Nuclear protein was extracted using Nuclei Isolation Kit  according to the manufacturer’s instructions. All the proteins were separated using gradient SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with TBST solution containing 5% skim milk powder at 25 °C. Due to different experimental purposes, the blots were cut prior to hybridization with antibodies. After incubation with primary antibodies at 4 °C overnight, the membranes were washed with TBST for 5 min each time, followed by incubation with secondary antibodies for 60 min. Finally, the protein bands were visualized using the chemiluminescence kit  and be analyzed through mage J software.All primary antibodies used in this study were as follows: TRIM31 , β-catenin , Histone H3 , c-Myc , GSK-3β , Axin1 , Axin2 , cyclinD1 , β-actin  and GAPDH . β-actin and GAPDH were used as negative controls.41. To each well, 10 μL CCK-8 reagent was added at 0, 24, 48, 72, or 96 h, and the plate was cultured at 37 °C for 1 h. The absorbance of these cells was evaluated at 450 nm according to the manufacturer’s instructions.Briefly, 5000 GC cells were seeded into a 96-well plate before transfection, as previously reported23. The immunohistochemical evaluation of TRIM31 expression was conducted independently by two experienced pathologists. We multiplied the staining levels and positive ranges to calculate the staining values of the score index (SI) according to the previously described criteria25. High TRIM31 expression was defined as SI ≥ 6, and low TRIM31 expression levels were the rest of specimens.The clinical specimens were stained using IHC assays as described previously42. The SYBR® Green kit  was employed to detect gene expression using the Step-One Plus System. The gene primer sequences were listed in detail in Table −ΔΔCT method was used to determine the relative expression level of mRNA with GAPDH as an internal control.Total RNA was first collected from cells or tissues using TRIzol reagent , and then reversely transcribed to complementary DNA via the PrimeScript kit  according to the manufacturer's protocol22. The colonies were fixed with 4% formaldehyde for approximately 30 min and stained with Giemsa for 20 min. ImageJ was used to count the number of colonies. These assays were conducted at least thrice.Approximately 500 indicated cells were placed in six-well plates with the complete medium and cultured until colonies were visible, as previously described5 cells per well were implanted into a six-well plate until fully confluent, as described previously30. The cell layers were scratched using 20 µL sterile pipette tips. The plates were then washed with PBS and cultured in a medium with 5% FBS. Typical images were captured at 0 and 48 h to measure the wound distance after scratching to evaluate the wound closure percentage. Closure rate (100%) = [wound distance (0 h) − wound distance (48 h)]/wound distance (0 h) × 100%. The experiments were performed at least twice.Approximately 4.0 × 103 cells were suspended in a serum-free medium. Matrigel gel BD (Biosciences) was used in the chambers  GC cells were seeded into the upper chambers, and the lower chambers contained 500 μL of medium with 15% FBS as a chemical attractant. After incubation for indicated time points, the migrated and invasive cells were fixed and stained according to a previously published method28. Invasive cells were counted and photographed in at least five randomly-selected microscopic fields. The assays were performed in triplicate.First, the transfected cells were trypsinized, and 3 × 1025.SiRNA targeting Axin1 and shRNAs targeting TRIM31, along with the corresponding control siRNA and scramble shRNA, were both bought from GenePharma . The siRNA and shRNA sequences were as follows: Axin1 siRNA (5′-GGACAUGGAUGAGGACGAUTT-3′), TRIM31 shRNA-1 (5′-TATGATGGACTCATGCCTTGC-3′), and TRIM31 shRNA-2 (5′- TTCCCGTCAAAGGAAGTTTGG-3′). The Myc-TRIM31 plasmid was constructed by subcloning the PCR-amplified human TRIM31 coding sequence into the 2 × pcDNA3.1 vector, while Axin1 plasmids were subcloned into the pcDNA3.1 vector with an HA tag. The detailed gene primer sequences were listed in Supplementary Table 43. Designated cells were harvested after transfection for 36 h, and treated with the CHX reagent (100 μg/ml) at the indicated time points. Lysates were assessed using western blotting. Half-time decay curves were drawn using the GraphPad Prism software.CHX assays were performed to evaluate the protein stability of Axin1 as previously describedHGC-27 cells were seeded into 10 cm plate and transfected with scramble shRNA or TRIM31 shRNAs. Upon 48 h, the cells were treated with MG132 for 6 h before harvested. Then indicated cell were lysed by RIPA buffer, and immunoprecipitated with Ni–NTA beads. The ubiquitination levels of Axin1 protein were detected using western blotting assays.To investigate the exogenous interaction of Myc-TRIM31 and HA-Axin1, 800–1000 μg of total proteins were mixed with anti-HA beads  at 4 °C for 2 h. The beads were washed with IP lysis buffer for more than 3 times and denatured with SDS-PAGE protein loading buffer. Bound proteins were detected by western blotting analysis with anti-Myc  or anti-HA  antibodies described in the figure legends.Biological assays were independently conducted at least twice. All data were displayed as the mean ± standard deviation. The Kaplan–Meier plotter was used to plot and calculate the survival rate via the log-rank test. The analyses of ROC and time-dependent ROC were made using the R software (version 4.2.1) package pROC (version 1.17.0.1). T-test or one-way analysis of variance (ANOVA) was used for statistical analyses of two or more groups. Chi-square tests were utilized to analyze the correlation between TRIM31 expression and clinicopathological parameters of GC patients. The Pearson correlation test was used for examining correlation analysis of TRIM31 and Axin1 expression in GC tissues. Statistical analysis was performed using R or SPSS software  with *P < 0.05 and **P < 0.01 as statistically significant.The informed consents were obtained from all participants and the acquisition of human tissues used in our study was approved by the Ethics Committee of the Third Affiliated Hospital of Nanchang University (No: KY2022021). This study conforms to the provisions of the Declaration of Helsinki.Supplementary Information 1.Supplementary Information 2.


Epoxy resin was mixed with benzoxazine resin and an aluminum trihydrate (ATH) additive to provide flame retardancy and good mechanical properties. The ATH was modified using three different silane coupling agents and then incorporated into a 60/40 epoxy/benzoxazine mixture. The effect of blending compositions and surface modification on the flame-retardant and mechanical properties of the composites was investigated by performing UL94, tensile, and single-lap shear tests. Additional measurements were conducted including thermal stability, storage modulus, and coefficient of thermal expansion (CTE) assessments. The mixtures containing more than 40 wt% benzoxazine revealed a UL94 V-1 rating with high thermal stability and low CTE. Mechanical properties including storage modulus, and tensile and shear strength, also increased in proportion to the benzoxazine content. Upon the addition of ATH to the 60/40 epoxy/benzoxazine mixture, a V-0 rating was achieved at 20 wt% ATH. The pure epoxy passed a V-0 rating by the addition of 50 wt% ATH. The lower mechanical properties at high ATH loading could have been improved by introducing a silane coupling agent to the ATH surface. The composites containing surface-modified ATH with epoxy silane revealed about three times higher tensile strength and one and a half times higher shear strength compared to the untreated ATH. The enhanced compatibility between the surface-modified ATH and the resin was confirmed by observing the fracture surface of the composites. Due to rising increasing concerns, flame-retardant adhesives are attracting enormous attention in the automotive, electronic, and construction industries. The adhesives are designed to provide thermal runaway protection and the cell-to-cell bonding of battery systems in electric vehicles ,2,3. RegThe use of flame-retardant additives is a simple and effective way of suppressing fire risk of organic polymers. Halogen-based substances reveal excellent fire resistance but generate toxic and corrosive gases during combustion, constituting a serious threat to the environment and humans alike. In the European Union (EU), halogenated flame retardants were prohibited in electronic display from March 2021 . RecentlIn the present study, the epoxy was blended with the benzoxazine in order to enhance fire resistance and mechanical properties and then the ATH was added to achieve synergistic flammability. Flammability and the mechanical properties of epoxy/benzoxazine mixtures and epoxy/benzoxazine/ATH composites have been measured in relation to blending composition. The UL94 burning test was carried out to determine the flame retardancy, while tensile and shear strength were measured to determine the material’s mechanical properties. Physical property losses at high ATH loading have been ameliorated via the surface modification of the ATH conducted using three different silane coupling agents.Epoxy, manufactured at Daicel Chemical Industries, was blended with benzoxazine  at various compositions. Pure epoxy and its mixture with benzoxazine were thermally cured in the presence of 50 wt% curing agent  and 5 wt% accelerator , respectively. Aluminum trihydrate , which has an average particle size of 3.3 μm, was used as a flame retardant. Three types of silane coupling agents, possessing epoxy , amino , and mercapto  functional groups, were used to modify the ATH surface.For surface modification, 1.0 g of coupling agent was dissolved in 200 mL ethanol and then 100 g of ATH was slowly added to the solution. The mixtures were homogenized via mechanical stirring at room temperature and heated up to 60 °C for the reaction. The product was filtered and washed with deionized water several times. Silane-treated ATH was dried at 90 °C in a convection oven for 12 hrs. No noticeable change in particle size was observed after surface modification. Uniform mixtures consisting of epoxy, benzoxazine, and ATH were obtained via a conventional three-roll mill technique  and then rotated at 100 rpm for 30 min in a vacuum bath in order to remove the residual bubbles. The three-roll mill process is commonly utilized to disperse the micron-sized particles within the resin. The mixtures were stored in a refrigerator at −40 °C prior to use. Thermal curing was carried out at 175 °C for 60 min.We conducted tensile and lap shear tests of the adhesive composites using a universal testing machine  according to ASTM D638 and ASTM D1002 standard specifications, respectively. Tensile strength was measured at a crosshead speed of 5 mm/min, while the shear strength was measured by pulling the aluminum plates (114 mm in length × 20 mm in width × 3 mm in thickness) at a rate of 1.27 mm/min and with an overlap length of 20 mm. The surface of aluminum was cleaned using ethanol prior to the application of the adhesives. The flame retardancy of cured epoxy/benzoxazine mixtures and their composites with ATH was evaluated using a UL94 vertical test, where the dimensions of the specimen were 127 mm in length × 12.7 mm in width × 3.2 mm in thickness. Thermogravimetric analysis  was used to characterize the thermal stability of epoxy/benzoxazine blends. Approximately 10 mg of sample was heated from 30 °C to 600 °C at a rate of 10 °C/min under an air environment. The coefficient of thermal expansion (CTE) of adhesives was estimated from the amount of thermal expansion versus temperature using a TMA instrument . A sample was placed on the TMA cell and heated from 20 °C to 150 °C at a heating rate of 5 °C/min. The viscoelastic behavior of the resins and their composites was probed in a sinusoidal tension mode using the Pyris Diamond DMA  technique. The cured film (200 mm in length × 6 mm in width × 0.3 mm in thickness) was heated from 25 °C to 280 °C at a heating rate of 2 °C/min in a nitrogen environment. The frequency of the dynamic mechanical measurement was fixed at 1 Hz. The fracture surface of the composites containing untreated and surface-modified ATH was observed using a scanning electron microscope (SEM) . The specimens were sputtered with silver for 90 s using a sputtering device before characterization. An accelerated voltage of 15 kV was employed. Micrographs were captured that corresponded to the cross-sectional and surface morphology.Td) at around 301 °C, which corresponds to a 5 wt% loss, and then decomposes rapidly upon further heating. The weight loss curves of the mixtures shift to higher temperatures with increases in the fraction of benzoxazine, and the residues at 550 °C are also found to increase. This indicates that the thermal stability of the blends is improved by the addition of benzoxazine. Tg), determined from a tan δ peak, slightly increases when epoxy is copolymerized with benzoxazine. The Tg of pure epoxy observed at 159 °C shifts to 50/50 epoxy/benzoxazine at 163 °C. The second peak observed above 200 °C denotes the Tg of benzoxazine. The storage modulus of neat epoxy at room temperature increases with an increase in benzoxazine content, i.e., 5.2 GPa for pure epoxy to 6.2 GPa for 70/30 epoxy/benzoxazine, as shown in 1) and CTE 2 (α2) measure below and are above a Tg of 50/50 epoxy/benzoxazine at 35 ppm/°C and 142 ppm/°C, which is much lower than the values of pure epoxy, i.e., α1 = 95 ppm/°C, α2 = 162 ppm/°C. Low CTE is advantageous in that the thermal stress generated by the thermal expansion mismatch between the adhesive and the die can be suppressed. Viscosity is one of the crucial criteria to determine the processibility of the adhesives. The viscosity of pure epoxy, showing approximately 250 mPa∙s at 5 rpm, increases substantially when benzoxazine is added. This occurs because benzoxazine enters into a glassy state at room temperature. The use of adhesives with viscosities in the range of 8000 to 30,000 mPa∙s is recommended for dispensing purposes. Considering the viscosity of the mixtures, the benzoxazine content is limited to a level below 50 wt%. The thermal and mechanical properties of epoxy/benzoxazine mixtures are summarized in The coefficient of thermal expansion (CTE) of epoxy tends to decrease when benzoxazine is added. The CTE 1  values, α1 and α2, gradually decrease with the increase in ATH content due to the low CTE of inorganic ATH (15 ppm/°C). The reduction in α2 value is more distinct, showing 141 ppm/°C for the resin and 92 ppm/°C for the composites with 40 wt% ATH. The storage modulus increases up to 8.1 GPa at 25 °C and 0.081 GPa at 250 °C when 40 wt% ATH is loaded. The viscosity of the ATH solution is measured at 2 rpm instead of 5 rpm because, at high ATP loading, it is difficult to measure the viscosity. Generally, the measurement is conducted at a rotation speed of 5 rpm when the viscosity of solution is less than 80,000 mPa∙s. The addition of ATH exponentially increases the viscosity of epoxy/benzoxazine mixtures. At 10 wt% ATH, the viscosity increases from 2278 mPa∙s to 3107 mPa∙s, and at 40 wt% the viscosity increases drastically to 11,180 mPa∙s.On the basis of thermal and mechanical behavior, the 60/40 epoxy/benzoxazine mixture is selected as the optimal composition. From The lap shear test is usually employed to measure adhesive bond strength. The composition-dependent shear strength of pure epoxy and 60/40 epoxy/benzoxazine blends is investigated. As illustrated in The flame retardancy of epoxy/benzoxazine mixtures in relation to ATH content is tabulated in We find that the incorporation of ATH into the epoxy/benzoxazine mixture as well as pure epoxy leads to a substantial reduction in both tensile and shear strength. The ATH surface is modified using three different silane coupling agents containing epoxy (EP), amino (AM), and mercapto (MC) functional groups. The thermal, mechanical, and flame-retardant behaviors of the epoxy/benzoxazine/ATH composites have been investigated via a process of varying their compositions. Compared to pure epoxy, the epoxy/benzoxazine blends exhibited a high thermal stability, tensile strength, and storage modulus, as well as a low CTE. The mixtures containing above 40 wt% benzoxazine passed a UL94 V-1 rating. The amount of benzoxazine in mixtures was limited because of low processability and low crosslinking density. The addition of ATH to pure epoxy and epoxy/benzoxazine mixtures provided a satisfactory flame retardancy. The effective amount of ATH needed to achieve a V-0 rating for pure epoxy was 50 wt%. Higher ATH loading caused a significant reduction in the tensile and shear strength of the composites. When ATH was added to the 60/40 epoxy/benzoxazine, a V-0 rating could be achieved at even 20 wt% without a significant sacrifice in the mechanical properties. It can be concluded that the combined use of ATH and benzoxazine showed noteworthy synergism in terms of flame retardancy and mechanical properties. The lowered tensile and shear strength of the composites due to the addition of ATH can be recovered by conducting surface modification with the use of a silane coupling agent. The ATH modified with epoxy silane (EP) exhibited about three times higher tensile strength and one and a half times higher shear strength as compared to untreated ATH at 60 wt% loading.