A systematic study of mechanical properties using nanoindentation of epoxy resin with gyroid- and diamond-structures after modification revealed significant enhancement in energy dissipation, with the values of 0.36 ± 0.02 nJ (gyroid) and 0.43 ± 0.03 nJ (diamond), respectively, when compared to intrinsic epoxy resin (approximately 0.02 ± 0.002 nJ) with brittle characteristics. This enhanced property is attributed to the synergic effect of the deliberate structure with well-ordered nanonetwork texture and the toughening of BCP-based modifiers at the molecular level. In addition to the deliberate structural effect from the nanonetwork texture, the BCP modifier composed of epoxy-philic hard segment and epoxy-phobic soft segment led to dispersed soft-segment domains in the nanonetwork-structured epoxy matrix with superior interfacial strength for the enhancement of applied energy dissipation.Herein, this work aims to fabricate well-ordered nanonetwork epoxy resin modified with poly(butyl acrylate)- Epoxy resin is one of the most versatile thermosets due to its processability, cost-effectiveness, and superior mechanical performance, giving excellent characteristics for industrial applications. The existence of multiple oxiranes or epoxy groups in the molecular structure of epoxy resin can provide high crosslinking density for superior mechanical strength, but the excessive crosslinking of epoxy rings in the molecular structure results in the brittle or glassy characteristics, and thus limits their engineering performance . As a reg) [A well-dispersed rubber modifier without any molecular level interactions can significantly reduce the brittle characteristics of epoxy resin by lowering the crosslinking density, which enhances their toughness by enabling the internal cavitation of well-bounded rubber particles . Howeverg) . Recent g) ,10 at whg) . The BCPg) ,12. Mostg) ,14,15, wg) ,17,18,19Moreover, nanonetwork materials have been reported for their enhanced mechanical properties due to their network structure in the nanoscale, where the nanosize can be the secondary aspect of the mechanical property . Howeverb-poly(methyl methacrylate) (PBA-b-PMMA) modified epoxy resin with well-ordered nanonetwork structures for the enhancement of energy dissipation capability using the self-assembled BCP, followed by templated polymerization. As shown in b-poly(dimethylsiloxane) can be fabricated by solution casting using selective solvents. Selective etching of the PDMS block from PS-b-PDMS can be acquired using hydrofluoric (HF) acid to give nanoporous PS with gyroid- and diamond-structured nanochannels as templates for polymerization of modified epoxy resin. The examination of the mechanical energy dissipation of these two distinct nanonetwork epoxy resins after the modification with PBA-b-PMMA has been carried out by using nanoindentation; the PBA-b-PMMA BCP can be self-assembled into spherical nanosized micelle in the epoxy matrix which acts as a toughening agent for the formation of soft domains. The synergic effect of the deliberate structuring in nanoscale and the toughening of the BCP-based modifier on a molecular level can significantly contribute to the energy dissipation capability of nanonetwork epoxy resins as compared to intrinsic brittle epoxy resin.Herein, this work aims to demonstrate the fabrication of poly(butyl acrylate)-b-PDMS was discussed previously [The detailed synthesis procedure for PS-eviously ,35. Epoxb-PDMS used in this study was 86,000 g/mol  and chloroform (Sigma Aldrich) (10 wt% concentration), in a vial with a controlled solvent evaporation rate. After drying, the samples were further dried at 60 °C in a vacuum oven. The formation of network phases from the self-assembly of lamellae-forming PS-b-PDMS indicates that a double gyroid structure can be formed using toluene for solution casting while a double diamond structure can be formed using chloroform as a solvent. Moreover, the affinity of PS towards the PS-selective solvents induces a reduction in the PDMS volume fraction causing the flat interfaces (lamellar morphology) to generate network or cylindrical structures. For toluene , the difference in the solubility parameters  indicates selectivity towards PS domains, giving rise to double gyroid. Furthermore, chloroform  enables the formation of different network phases such as double diamond and double primitive (DP), as already reported in the literature [b-PDMS using HF solution (HF/H2O/methanol = 0.5/1/1 by volume). After the complete removal of the PDMS followed by washing with water and methanol, well-ordered nanoporous PS templates with gyroid- and diamond-structures with the corresponding nanochannels  for templated polymerization can be obtained.The lamellae-forming PS-terature . Note thterature . Accordib-PMMA  with 5% wt% of well soluble PBA-6  at an accelerating voltage of 200 kV by mass thickness contrast.Bright-field (BF) transmission electron microscopy (TEM) imaging was used to determine the pore-filling of epoxy resin in the PS template using JEOL JEM-2100 LaBField emission scanning electron microscopy (FESEM) observation was performed at an accelerating voltage of 5 keV on a JEOL JSM-7401F (Akishima). The samples were collected on a silicon wafer and sputter-coated with platinum at approximately 2 to 3 nm.q*(4π sin θ)/λ, where 2θ is the scattering angle).National Synchrotron Radiation Research Center (NSRRC) with synchrotron X-ray beamline X27C was used to study the SAXS where the wavelength of the beam was 0.155 nm. The two-dimensional SAXS pattern was obtained using the MAR CCD X-ray detector , at which the (1D) linear profile was obtained by integration of the 2D pattern. The scattering angle of the SAXS pattern was calibrated using silver behenate with the first order scattering vector rE was determined from the P h curve, using the Sneddon formula for spherical indenter frictionless punch.Hysitron Ti950 triboindenter  was used to perform the nanoindentation tests using a spherical indenter with a 2 µm diameter. The indentation measurements were conducted on a microtome film sample with 5 µm thickness in a silicon wafer as a substrate at room temperature. The load–displacement curve was recorded at the same rate of loading and unloading (60 μN/s) with a maximum load of 500 μN applied. In the nanoindentation tests, the load–displacement data were recorded continuously, while the tip was driven into the composite materials, and then smoothly removed. The load–displacement (L-D) curves were then used to calculate the mechanical energy dissipation of the fabricated materials at the same rate of loading and unloading (60 μN/s). For nanoindentation, the mechanical properties of reduced elastic modulus and hardness can be calculated from the load–displacement curve (P-h) based on the widely used Oliver-Pharr model. In the present study, the reduced elastic modulus rE is the reduced elastic modulus (indentation modulus) which represents the elastic deformation that occurs in both the sample and indenter tip. S is stiffness. tA represents the projected contact area. Note that the deformation in the diamond indenter tip is negligible. As a result, the reduced elastic modulus (indentation modulus) is a representative value for the discussion with respect to mechanical performance.Here, b-PMMA to brittle material such as epoxy resin starts from the basic characterization of the modifier (PBA-b-PMMA) (The detailed information about the sample is described in g) of the soft PBA segment is approximately −35 °C, whereas the hard PMMA shows Tg at 87 °C  type of epoxy resin to achieve the desired toughness. The hard segment (PMMA) was expected to provide the molecular level association with epoxy resin in the nanoscale. Moreover, the Tg of the soft segment (PBA) was reaffirmed by DMA analysis, as shown in b-PMMA modifier to the epoxy resin, the feasibility of reaction-induced phase separation was evaluated using TEM analysis of the dispersed PBA-b-PMMA in the epoxy matrix. As shown in b-PMMA can be self-assembled into a spherical micelle structure dispersed in the epoxy matrix. The samples were vapor stained with 0.5 wt% OsO4 aqueous solutions for 24 h at ambient temperature to provide adequate contrast between the two segments. In b-PMMA domains act as an impact modifier due to the compatibility between epoxy and PMMA, based on their interfacial strength. Namely, the incompatible PBA block dispersed in the epoxy resin matrix can be characterized as an epoxy-phobic core and the compatible PMMA block as an epoxy-philic shell that led to the reinforcement of the interfacial strength. Additionally, as shown in b-PMMA reduces the Tg to 83 °C; note that the reduction in Tg with respect to the addition of a modifier was negligible. After the gradual addition of PBA-b-PMMA up to 20% w/w, there is an obvious reduction in the Tg to 73 °C. Following the platform technology developed in our laboratory, the fabrication of well-ordered nanonetwork epoxy modified with PBA-b-PMMA can be successfully achieved using templated polymerization. By taking advantage of the strong segregation strength between PS and PDMS, gyroid-structured PS-b-PDMS can be fabricated from lamellae-forming BCP using toluene as a selective solvent [b-PDMS used in this study. b-PDMS; a typical projection of a trigonal planar gyroid phase can be observed. The 1D SAXS results at the relative q values of √6, √8, √16, √22, √38, and √52 (Iad). By using chloroform as a selective solvent, a typical projection of a double diamond phase with a tetrapod-like pattern can be identified by TEM analysis  with an additional slight deformation peak (denoted by red arrow). The appearance of an extra peak at a relative q value of √2 can be observed for PS/epoxy nanocomposite in q values of √2, √3, √4, √6, √8, and √10 are identified for the PS template and PS/epoxy nanocomposites. Consequently, a characteristic reflection of the nanoporous epoxy at the relative q value of √3 can be found in b-PMMA. Based on the unloading curve, the reduced elastic modulus (rE) of 4.2 GPa was calculated for intrinsic epoxy resin by the Oliver-Pharr model [b-PMMA shows approximately 90% of plastic deformation behavior with minor retracting of the unloading curve  times of the non-structured, intrinsic epoxy resin). The enhancement of the energy dissipation explicitly indicates the synergic effect of the deliberate structuring of network texture in the nanoscale and the toughening of self-assembled modifiers (BCPs) in the epoxy matrix at the molecular level.As shown in rr model ; note thng curve . The rede curing  due to t µN load . By contnloading ; both gynloading , compareb-PMMA, can be successfully fabricated by templated polymerization, using PS templates. The periodic structured templates were acquired from PS-b-PDMS self-assembled samples, followed by preferential removal of PDMS through HF etching. The incorporation of PBA-b-PMMA in the matrix of epoxy resin imparts self-assembled spherical micelles. The PBA core serves as an energy absorbing soft domain due to the core–shell characteristics with reinforced interfacial strength from the association of PMMA in the matrix of epoxy resin. By taking advantage of the well-ordered nanonetwork structure, a further enhancement on plasticity can be achieved. The synergic effect of the deliberate structuring of nanonetwork texture and the toughening of the BCP-based modifier, thus, presented outstanding enhancement of plastic energy dissipation for gyroid and diamond-structured resin.In conclusion, well-ordered nanonetwork-structured epoxy resin with gyroid  and diamond (tetrapod) structures modified with a BCP based modifier, PBA-


To screen potential β-catenin-associated genes involved in OSCC, the intersection of these genes in the STRING and IMEx databases was assessed using differential expression genes (DEG) from public microarrays, and 22 were further selected to construct a β-catenin-protein interaction network. The top 14 hub genes (node degree > 10) within the network were selected. Pearson's correlation analysis showed that β-catenin expression correlated positively with the expression of 11 genes, including AR, BIRC5, CDK6, DKK1, GSK3B, MET, MITF, PARD3, RUVBL1, SLC9A3R1, and SMAD7. A heat map of overall hub gene survival was created, and elevated expression of DKK1 and RUVBL1 was associated with poor survival using the Mantel-Cox test. To identify the function of RUVBL1, colony formation assay, transwell assay, and western blotting revealed that knock-down of RUVBL1 by siRNA decreased H157 and Cal-27 cell proliferation and metastasis by inhibiting β-catenin signaling. These findings suggest that RUVBL1 may serve as a diagnostic and prognostic biomarker for OSCC, as well as a therapeutic target, and may help to uncover additional molecular mechanisms of β-catenin-driven OSCC tumorigenesis.Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of head and neck squamous cell carcinoma (HNSCC), but the causes and molecular mechanisms remain unclear. The wingless-integrated/ Aberrant activation of WNT/β-catenin signaling is found in many types of tumors and plays an intrinsic role in cancer cell proliferation, differentiation, metastasis, and other functions [Oral squamous cell carcinoma (OSCC) is the most frequent malignant tumor of head and neck squamous cell carcinoma (HNSCC) . Incidenunctions .β-catenin signaling is considered the most important signaling pathway in OSCC [β-catenin signaling pathway have been identified as therapeutic targets and diagnostic biomarkers of OSCC [β-catenin [β-catenin are closely correlated with differentiation, invasion, and poor prognosis [β-catenin (78.3%) in OSCC correlates with histologic grade [β-catenin staining is a potential prognostic marker for ovarian cancer in univariate analysis, while cytoplasmic and membranous β-catenin staining is not [β-catenin expression in the nucleus [β-catenin and the related gene expression and clinicopathological features of OSCC patients in China.WNT/ in OSCC . Several of OSCC , includi of OSCC , Msi2 [5-catenin . Alteratrognosis , 8. In aic grade . Other sg is not . The epig is not . OSCC ti nucleus , 12. Thuβ-catenin-associated genes (BCAGs) was developed. A flowchart of the analysis pipeline is shown in Figure β-catenin-protein interaction network was constructed, and several hub genes were screened out based on differential expression of genes (DEGs), nodes were connected within the network, and the outcome of overall survival (OS) was measured. RUVBL1, also known as Pontin protein, was associated with low survival rates. Therefore, it was postulated that RUVBL1 may serve as a potential diagnostic and prognostic biomarker, as well as a therapeutic target against OSCC, and may help to define the molecular mechanisms of β-catenin-induced tumorigenesis in OSCC.In the present study, an integrated method to screen n = 164 and adjacent nontumor tissue n = 45), GSE37991 , and GSE31056 (OSCC n = 21 and adjacent nontumor tissue n = 24) were obtained and downloaded from the NCBI Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) using the R package, “GEOquery.” R packages “limma” and “ggstatsplot” were used to annotate all probes and produce a differential expression matrix. After data preprocessing, p < 0.05 and ∣log2 FC | ≥1  were defined as the threshold to screen differential expression genes (DEGs). Principal component analysis (PCA) was also performed using “prcomp” in R studio.OSCC microarray datasets of GSE30784 (OSCC β-catenin-associated protein (BCAP) interaction network was constructed through STRING and IMEx database by Networkanalyst . Venn plot (http://bioinfogp.cnb.csic.es/tools/venny/index.html) was visualized by identifying genes that intersected among DEGs and genes from STRING and IMEx. The selected DEGs were further used to construct a β-catenin-associated protein interaction network using Networkanalyst and visualized by Cytoscape [β-catenin. Hub gene expression was also shown by box plot using GSE datasets. Correlation analysis between β-catenin and hub gene expression was analyzed by Pearson's correlation analysis using HNSC data from the Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/) database. A p value < 0.05 was considered significant. BCAGs within the PPI network were annotated and analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome pathway database, and Gene Ontology (GO) with default parameters through Metascape (https://metascape.org/gp/index.html#/main/step1). p values< 0.05 and FDR < 1 were considered significant.To identify BCAGs in OSCC, ytoscape . After tp < 0.05 was considered significant.The effects of hub genes on OS were analyzed using the Mantel-Cox test and shown in a heat map created by GEPIA. The Kaplan-Meier curves were used to analyze the survival outcome of HNSCC patients with relatively high or low expression of selected hub genes on GEPIA. Log-rank μm thick sections were cut and mounted on silanized slides. After several steps of deparaffinizing, rehydrating, and blocking with endogenous peroxidase, the sections were rinsed with 0.01 M PBS and incubated in blocking buffer  for 30 min. The slides were then exposed to 1 : 100 primary anti-RUVBL1 antibody at 4°C overnight. After further incubation with the secondary HRP-antibody  for 30 min at 37°C, the slides were washed five times with PBS. The chromogenic reaction was performed using freshly prepared diaminobenzidine (DAB) for 10 min and Harris hematoxylin. Hamamatsu NanoZoomer RS Digital Pathology System image analysis software was used to analyze the pathological images. Two clinical pathologists independently examined and evaluated all IHC staining results.Tissue microarray of OSCC (Cat # TC0224) including 50 OSCC samples and ten normal oral tissue samples was purchased from the Auragene Bioscience Company . RUVBL1 protein levels were detected by tissue microarray using anti-RUVBL1 antibody  followed by an SV-DAB two-step kit . Several serial 4 The OSCC cell line, H157 (Cat # 3153C0001000000260), was obtained from Kunming Cell Bank of Chinese Academy of Sciences and cultured in DMEM/F12 medium with 10% fetal bovine serum (FBS). Small interfering RNA (siRNA) was purchased from GenePharma , and the sequences are listed in Table The Cal-27 cell line was purchased from the Cell Resource Center, Chinese Academy of Medical Sciences, and cultured in DMEM-H medium with 10% FBS. The H157 and Cal-27 cell lines were seeded in culture insert (ibidi) with DMEM medium containing 5% FBS and grown to 90% confluence to evaluate the migration ability. The culture inserts were removed before the straight scratch in each culture insert was made. After washing twice with 1×PBS, the cells were transfected with siRUVBL1 or siNC for 24 h, respectively, as described above. The cells that migrated to the scratched area were examined under a microscope after 48 h.μm pores, Costar, USA, Cat #. 3422). After transfecting the cells with siRNA, the H157 cells were seeded in the upper chamber with Matrigel matrix (BD Biosciences) and serum-free DMEM/F12 medium. The lower matched 6-well plates contained DMEM/F12 medium supplemented with 10% FBS. After a 48 h incubation, the H157 and Cal-27 cells that had invaded the lower membrane surface of the upper chamber were stained with crystal violet (0.05%), scanned, and examined by microscope.A cell invasion assay was used to assess the invasive ability of the H157 and Cal-27 cells in a transwell chamber , anti-E-cadherin , or anti-β-actin antibodies . Immunoreactive proteins were detected by chemiluminescence, and the relative intensity of the gray band was calculated using ImageJ.After two independent treatments with siRUVBL1 or siNC, the H157 and Cal-27 cells were incubated and lysed with RIPA buffer. The total protein was isolated, and the concentrations were determined using the bicinchoninic acid (BCA) method . Standard western blotting was performed, and after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred from the gel onto polyvinylidene fluoride (PVDF) membranes. PVDF membranes were blocked using blocking solution  and incubated with anti-Χ2 was the chi-square value. ∗A value of p < 0.05 was considered significant.RUVBL1 expression in each group of clinical traits was compared by chi-square test using SPSS version 17.0 . β-catenin signaling, a BCAP interaction network was constructed, and 170 and 331 genes from STRING and IMEx, respectively, were screened out  showed that normal and OSCC samples could be clearly separated . Differeened out . Twenty-ened out . These g network . Based o network .β-catenin signaling were enriched in cancer-related pathways. Some genes were involved in vital signaling pathways, such as cell morphogenesis required for differentiation that occurs via Wnt signaling. Other hub genes are critical for cancer cell survival and growth, playing an important role in epithelial and glial cell development, cell division, and microtubule-based processes.To characterize the molecular function of node genes involved in the BCAP network, hub genes were annotated on Metascape . The resβ-catenin expression (p < 0.05) (BIRC5 and SLC9A3R1 correlated weakly (R < 0.2), AR, CDH2, CDK6, DKK1, MET, PARD3, and RUVBL1 correlated moderately (R < 0.3), GSK3B, MITF, and SMAD7A associated strongly , and ERBB2 and ERBB3 were not associated with β-catenin expression . These findings suggest that aberrant expression of β-catenin correlates with expression of the 12 hub genes. CDH2 expression did not change between the normal and OSCC tissue. Eleven genes were used in the following survival analysis except for ERBB2, ERBB3, and CDH2.Using GSE37991, six overexpressed and seven downregulated hub genes were found in OSCC versus adjacent nontumor epithelial tissue . Two oth < 0.05) . BIRC5 aSMD7, SLC9A3R1, RUVBL1, PARD3, MITF, MET, GSK3B, DKK1, CDK6, BIRC5, and AR, were used to assess the effect on overall survival using the Mantel-Cox test in GEPIA. An OS heat map showed that log10 HR values of DKK1 and RUVBL1 were higher than 0.2   . These rRUVBL1 functions as a component of several chromatin-remodeling complexes and plays an essential role in transcriptional regulation, DNA damage repair, and telomerase activity [β-catenin, overexpression of RUVBL1 was found in the GSE37991 dataset of OSCC. To further identify the results, a tissue microarray of OSCC samples was used to analyze RUVBL1 expression. Compared to normal oral tissue, RUVBL1 expression was primarily higher in the cytoplasm and nucleus of OSCC (n = 25) and low (n = 25) groups. A relationship analysis was conducted by chi-square, and RUVBL1 expression was correlated with histological grade , but the suppression of RUVBL1 by siRNA delayed the growth and migration of the H157 cells and Cal-27 cells (#p < 0.05)   and vehicle control . The res Figures . In the  Figures . Since R Figures . Moreoveβ-catenin signaling was shown to play a vital role in the tumorigenesis of malignant tumors which promoted an investigation into whether β-catenin was expressed in the nucleus of OSCC cells [p < 0.001) [β-catenin was expressed in 35.9% of adjacent and 44.4% of “distant” OSCC samples [β-catenin signaling to promote cell proliferation and migration in OSCC. Evidence indicates that targeting β-catenin using specific inhibitors and siRNA abrogated β-catenin-induced malignant growth of OSCC and enhanced sensitivity to chemotherapeutics [While surgical and chemotherapy strategies to treat OSCC have improved significantly, the diagnosis and prognosis of OSCC patients require further investigation. Numerous studies have indicated that cancer cell mutations can promote cell proliferation and metastasis in several cancer types, including OSCC , 16. WntCC cells . Indeed, samples . Dysregu samples  and miR- samples , lncRNA  samples , FTH1P3  samples , CCAT1 [ samples , and MEG samples  activateapeutics –28.β-catenin signaling in OSCC, screening for β-catenin-related proteins that can serve as novel biomarkers is essential to better understand this disease. Based on the differential expression of genes in OSCC and β-catenin-associated genes in the β-catenin-protein network, 22 genes were selected from the intersection of DEGs and β-catenin-associated genes in the STRING and IMEx databases. These 22 potential β-catenin-associated genes were used to construct a β-catenin-protein network, and the top 14 genes (node degree > 10) were chosen. Signaling pathway enrichment analysis indicated that many hub genes were enriched in cancer-related pathways and essential signaling pathways related to cell growth, survival, and differentiation. Pearson's correlation analysis was performed, and the results showed that 12 hub genes containing AR, BIRC5, CDH2, CDK6, DKK1, GSK3B, MET, MITF, PARD3, RUVBL1, SLC9A3R1, and SMAD7 were significantly related to β-catenin expression, except ERBB2 and ERBB3. Eleven genes were used to analyze overall survival except for ERBB2, ERBB3, and CDH2.Given the aberrant activation of p < 0.05). The Kaplan-Meier curve also showed that high expression of DKK1 and RUVBL1 was associated with poor survival outcomes. Many studies have shown that DKK1 plays various roles in Wnt signaling during several cancers [DKK1 is frequently upregulated in OSCC-derived cell lines and primary OSCCs compared with normal cells, DKK1-positive patients have a low risk of regional lymph node metastasis [DKK1 and the clinical traits of OSCC.To determine whether hub BCAG expression was a prognostic indicator for OSCC, an overall survival heat map of 11 hub genes analyzed using the Mantel-Cox test showed that only two genes influenced overall survival . Pearson's relation analysis showed that RUVBL1 was positively related to β-catenin (p = 0.003) in HNSC data of TCGA using GEPIA  and vehicle control. Fewer cells were transfected with siRUVBL1 on the membrane of the upper chamber than were transfected with siNC and vehicle control, indicating that knock-down of RUVBL1 significantly decreased the invasive ability of the H157 cells. Involvement of RUVBL1 in the β-catenin signaling pathway showed that RUVBL1, β-catenin, and vimentin expression were reduced in the H157 cells. It was also found that RUVBL1 suppression by siRNA could decrease the migration and growth of the Cal-27 cells. Interestingly, E-cadherin expression was elevated in the H157 cells exposed to siRUVBL1. These results suggest that RUVBL1 knock-down inhibited the growth and metastasis of an OSCC cell line by inactivating β-catenin signaling. Recently, RUVBL1, a component of R2TP, a well-conserved molecular chaperone complex, was shown to be required for HSC-4 cell proliferation and migration [β-catenin-driven transcription. More in-depth experiments of the molecular mechanisms of RUVBL1 in OSCC require future investigation.The findings indicated that RUVBL1 was overexpressed in the cytoplasm and nucleus of OSCC cells by IHC. In addition, RUVBL1 expression was significantly related to the histological grade of OSCC (ng GEPIA . Thus, Rigration . The preβ-catenin-driven OSCC tumorigenesis.RUVBL1 expression is a favorable biomarker for predicting the overall survival of OSCC patients. In addition, knock-down of RUVBL1 in the H157 cells showed that this gene is a potential diagnostic and prognostic biomarker, as well as a target for OSCC treatment. Further investigation of RUVBL1 may reveal additional molecular mechanisms of


A commercial epoxy resin and reduced graphene oxide (rGO) were blended with a hardening reagent and then mixed with prepared BaSO4–epoxy resin (B–epoxy). The reduced graphene oxide–BaSO4–epoxy composite (rGO–B–epoxy) paste was used to coat the surfaces of Al 7205 alloy and the corrosion and thermal properties were investigated. A corrosion test in a 3.5 wt% synthetic sea water solution showed that the composite coating containing BaSO4 had the best corrosion resistance. Moreover, the rGO–B–epoxy composite showed better protection against corrosion than the epoxy alone. The rGO–B–epoxy composite with 5 wt% BaSO4 had an in-plane coefficient of thermal conductivity of approximately 165.0 W/m K, and the in-plane thermal diffusivity was 71.38 mm2/s. In standard thermal conductivity tests, all three samples had values below 40 W/m K. The rGO–B–epoxy composites showed good surface corrosion protection and in-plane thermal conductivity.The results of studies on the corrosion protectiveness and thermal conductivity of reduced graphene oxide–BaSO Graphene, as a new high-profile two-dimensional 2D) material, has shown promising application potential because of its prominent thermal ,2,3, mecD materia2 hybrid orbital form pi–pi stacking. This causes problems for the dispersion of the graphene in a solution or polymeric matrix [Although graphene should ideally be a single layer of carbon atoms for honeycomb lattices, because of its unstable thermodynamics, this single layer is likely to form multiple layers of graphene or a graphite-like material with greater stability and lower surface energy. The functional groups could help the interaction with the polymer matrix or others molecules. The reduced graphene oxide with certain surface functional groups would be formed during the chemical reduction procedures. The structure of graphene is easily formed by a layer-by-layer structure; the delocalized electrons in spc matrix ,16. It ic matrix . Of courc matrix . ReducedEpoxy resin is a thermosetting polymer that is used for high-performance applications, with properties such as toughness, heat resistance, chemical resistance, and adhesion. Graphene–epoxy or rGO–epoxy composites have been used in new and interesting material applications for advanced technology ,20. The The poor alignment/dispersion of graphene/rGO in a polymer matrix can have a negative impact on the expected properties of graphene/rGO–epoxy composites. Various dispersion methods are applied in the preparation of polymer resins with graphene/rGO. The pi–pi stacking of graphene/rGO is a driving force for aggregation. The dispersion and stability of the coating paste are the crucial issues for polymer composite applications. A better alignment for the graphene/rGO results in a higher Young’s modulus. Greater interaction between the polymer matrix and graphene/rGO results in better tensile and elongation properties. Greater interfacial interaction produces better enhancement of the mechanical, electrical, and thermal properties . A simil4 is widely used for the production of alloys, cement, ceramics, and glass as a luminophoric material or pigment in various coatings [4 powder works as the carrier in the thermal control coating for space vehicles [BaSOcoatings ,24. Furtvehicles . It has vehicles . For comvehicles .2·6H2O, 9.75 wt% Na2SO4, 2.765 wt% CaCl2, 1.645 wt% KCl, 0.477 wt% NaHCO3, 0.238 wt% KBr, 0.071 wt% H3BO3, 0.095 wt% SrCl2·6H2O, and 0.007 wt% NaF) to simulate a seawater environment. TAFEL analyses were conducted to estimate the anti-corrosion properties of the rGO–B–epoxy composites. In this study, we report the results of studies on rGO–B–epoxy composites used to coat 7205 aluminum alloy to improve its corrosion resistance and thermal properties. The use of corrosion-proof coatings is a significant method to increase the lifetimes of metal applications. The corrosion resistances of different rGO–B–epoxy composites were investigated using a voltammeter on samples immersed in 3.5 wt% synthetic seawater  was mixed into a bisphenol-A (BPA) epoxy resin , which then formed a cross-linked structure through the ring-opening polymerization (ROP) of the epoxy groups. This was then used to apply a pure epoxy coating on a 1 mm-thick aluminum alloy  specimen with a 1 hp air compressor connected to a nozzle with a 100 mL container. The chemical composition of the 7205 alloy is listed in 4, 99%), sulfuric acid (H2SO4), nitric acid (HNO3), hydrogen peroxide (H2O2), and hydrochloric acid (HCl) purchased from Sigma-Aldrich.The graphite used in this study was purchased from Sigma-Aldrich Co. . Hammer’s method was used to synthesize the graphene using potassium permanganate (KMnO4 (10 g), concentrated H2SO4 (72 mL), and concentrated HNO3 (65%) for an oxidation reaction and stirred for 40 min in an ice bath. Then, the residual slurry was washed with 12 mL of H2O2 solution (65%) after washing with 400 mL of deionized (DI) water multiple times (more than three times), until the solution was close to neutral (pH ~7). Finally, graphite oxide (GO) was peeled off through ultrasonic vibration. The GO nanosheets (80 mg in 10 mL of DI water) and ammonia water (70 mL) were mixed in an appropriate ratio, and the solution was placed in a hydrothermal tank and heated to 200 °C for 12 h. Then, the solution was repeatedly washed with 0.1 M HCl, DI water, and alcohol, and centrifuged several times, until the solution was close to neutral. Finally, the resultant product was dried in a vacuum oven at 60 °C for 24 h. Dried reduced graphene oxide (rGO) was produced [Hammer’s method was modified for the graphene synthesis in this study as follows. First, 2.0 g of graphite was added to a mixture of KMnOproduced .4 into 100 mL of the epoxy resin separately, the B–epoxy composites were produced by gradually adding the dry rGO with 5 wt% and 2 wt% into the 12.5 wt% and 25.0 wt% B–epoxy mixtures in the fast stirring, respectively. The hardening agent with a volume ratio of approximately twice as much epoxy resin was gradually and simultaneously added to the above-mentioned rGO–B–epoxy resins. Then, different amounts of the rGO–B–epoxy pastes were dispensed using an ultrasonic dispenser for 10–20 min. The rGO–B–epoxy composite coatings were applied using a one-horse power air-compressor paint sprayer. The thickness of the composite coatings was approximately 40 µm with two layers of 3M scotch tape used to control the thickness of the rGO–B–epoxy on the surface of the 7205 alloy. They were cured in an 80 °C oven for 4 h, then at 120 °C for 6 h. After adding 45 wt%, 25 wt%, and 12.5 wt% BaSOTransmitting electron microscopy  was used to analyze all the specimens. Dual-column ultra-high resolution field emission scanning electron microscopy  was used for the preparation of the TEM specimens, which had the dimensions of 4 mm × 2 mm and a thickness of 30–50 nm. The rGO–B–epoxy composites were examined using an X-ray diffractometer with a Cu Kα  with a scan rate of 0.2°/s in 2θ range in 20°–60°. D/IG band ratio and the identification of the functional groups. The rGO surface binding energy for the contained elements was determined using a Shimadzu ESCA 750 spectrometer  with an X-ray source of Mg Kα (8 kV and 30 mA). The thickness of the rGO was characterized using atomic force microscopy (VEECO Nanoscope 3100 AFM with tapping mode).The prepared rGO was characterized using vibrational spectroscopy, Raman spectroscopy , and Fourier-transform infrared spectroscopy  for the I2 corrosion surface area in the electrolyte and a plate corrosion cell from BSA Inc. . A 1 cm2 platinum foil was used as the counter electrode, and the reference electrode was Ag/AgCl. The test parameter was controlled by a computerized Autolab PSTAT 30 potentiostat  with a scan rate of 10 mV/s and scan ranges of −1.0 to +1.0 V (for the B–epoxy composite) and −0.1 to 0.0 V (for the graphene–epoxy coatings). The corrosion potential (Ecorr) and corrosion current density (jcorr) were determined by the TAFEL equation based on the LSV results.An Altolab potentostat 30 was employed for linear cyclic voltammetry (LSV) examinations conducted to characterize the graphene–epoxy’s corrosion resistance. The 2 cm × 2 cm rGO–B–epoxy composite on alloy 7205 coupons were set into the three-electrode corrosion cell. The electrochemical analysis was conducted by LSV with 3.5 wt% aqueous synthetic seawater at ambient temperature. The working electrode had a 10 mmA Netsch LFA 467 HyperFlashTM  laser flash thermal analyzer was used. Two sample holders were employed: the 10 mm standard and in-plane holders for the vertical (z-axis) and the xy-plane thermal diffusivities, respectively. The measurements were made at room temperature (25 °C) and ambient atmosphere. The in-plane holder could measure the surface thermal diffusivity, as shown in The thermogravimetric analyzer  was used to examine the epoxy composite coatings to reveal the additive amounts. A symmetrical hang-down balance was used to continually record the weight changes in the coatings with a ±0.1 μg resolution during the measurements. The temperatures of the samples were controlled and measured with a measurement accuracy of ±0.1 °C. Argon (Ar) gas was purged into the heating chamber at a flow rate of 20 sccm to protect the heating device.D/IG band ratio (the D band is at ~1296 cm−1 and G band was at~1604 cm−1) was approximately 1.04, and the D+G band was approximately 2886 cm−1, as shown in the Raman results of −1. There is C=C sp2 stretching for un-oxidized graphite at ~1662 cm−1. The alcohol C~OH stretching peak is approximately 1225 cm−1. Furthermore, the peak at 1056 cm−1 is defined as the C-O-C stretching mode. In comparison to the GO vibrational spectral peaks, the ID/IG ratio is approximately 1.21 and the carboxyl ~OH stretching at 3000~3500 cm−1 is more obvious in the FTIR spectrum of rGO. The characterization results with rGO are shown in In The SEM examination results for rGO are shown in The rGO–B–epoxy coatings were not very smooth like the epoxy-only coatings. The SEM images of the rGO–B–epoxy composites were examined and showed no significant differences. We conducted a TEM analysis after FIB sample preparation in a cross-sectional investigation. The three samples with 0, 2, and 5 wt% rGO added to the B–epoxy matrix were examined.4. The volume-to-mass ratio of rGO was much larger than those for the epoxy and BaSO4. To make better coatings, the amounts of rGO and BaSO4 added were approximately 2 wt% rGO with 25 wt% BaSO4 and 5 wt% rGO with 12.5 wt% BaSO4. The B–epoxy in 4. The TEM images for graphene with larger magnification show the typical morphologies. 4 connecting or touching each other, shown in 4. The 0.246 nm and 0.303 nm may be he lattice distances for rGO. Others may be for BaSO4.The TEM images with 2 wt% and 5 wt% rGO-B-epoxy composites. For 10,000 magnification, the rGO layer-by-layer structures could be found in 4 and graphene–epoxy composites are shown in Furthermore, select area electron diffraction (SEAD) images of the epoxy–BaSO4 was multi-crystalline, and we could find marked facets corresponding to the degree, shown in 4 crystalline facets (041) and (200) were found with both analysis techniques. However, the XRD results do not easily identify the peak for reduced graphene oxide in Based on the XRD results, the BaSOEcorr and jcorr values for the 2 wt% rGO–B–epoxy coating on the 7205 alloy surface were −0.3118 V and −9.4694 μA/cm2, respectively. The 5 wt% rGO–B–epoxy coating had values of −0.1906 V and −9.3724 μA/cm2 for Ecorr and jcorr, respectively. For an anti-corrosion protective coating layer, a more positive Ecorr indicates better protection. The jcorr values of the rGO–B–epoxy composites with 2 wt% and 5 wt% rGO added were almost the same. Thus, the 5 wt% rGO–B–epoxy coating was more corrosion-proof than the 2 wt% rGO–B–epoxy coating because of the higher Ecorr. Nevertheless, the B–epoxy coating did not show a redox curve in the TAFEL analysis, which may imply that this coating is the best corrosion-inert layer for certain applications. The 45 wt% BaSO4 could extend the epoxy composite lifetime for corrosion applications.An experiment to test the corrosion-proofing abilities was conducted in 3.5 wt% synthetic seawater (ASTM D 1141-98) using the electrochemical cell with LSV. The 7205 alloy LSV result shows a negative corrosion potential of approximately −1.0 V (versus the Ag/AgCl reference electrode). For the B–epoxy composite coating, the redox curve could not be found using the LSV result with a TAFEL analysis, as shown in 2/s and 0.121 W/m K, respectively. When we touched the rGO–B–epoxy coatings in this study with our hands, the surfaces of the coatings felt cold. The thermal diffusivities of the rGO–B–epoxy composites should have been much higher than that of the B–epoxy coating. The in-plane thermal properties  found using the laser flash method for the 2 wt% rGO–B–epoxy coating were 51.46 mm2/s and 133.0 W/m K, respectively. The best thermal properties were found for the 5 wt% rGO–B–epoxy composite coating, which had a thermal diffusivity and thermal conductivity of 71.38 mm2/s and 165.0 W/m K, respectively. In contrast to the thermal properties found using the standard method (heat flow from bottom to top), the 2 wt% rGO–B–epoxy composite coating had thermal diffusivity and conductivity values of 9.78 mm2/s and 25.28 W/m K, respectively. Furthermore, the thermal diffusivity and conductivity of the 5 wt% rGO–B–epoxy were 15.83 mm2/s and 36.60 W/m K, respectively. It was impressive that adding 2 wt% and 5 wt% graphene to the B–epoxy could dramatically improve its thermal properties (diffusivity and conductivity).The thermal diffusivities and conductivities at 25 °C for the B–epoxy and rGO–B–epoxy composites are listed in 4 and rGO added to the epoxy resin were calculated. The real amounts added to the coatings were investigated using a thermogravimetric analyzer (TGA). The TGA examination showed that there was approximately 39.7 wt% residual BaSO4 in the B–epoxy coating. It was different from the 5.3 wt% used for the preparation of the B–epoxy composite resin. The TGA examinations of the 2 wt% and 5 wt% rGO–B–epoxy coatings showed 11.6 wt% and 26.3 wt% residual BaSO4 values, respectively. These represented differences of 0.9 wt% and 1.3 wt% from the values using in the preparation of the B–epoxy resins, as shown in 4–epoxy composite from the TGA results, as shown in The amounts of BaSOi is the initial temperature (25 °C), and Tf is the final temperature (800 °C), as listed in 4 contents had the following order: B–epoxy coating (~45 wt% BaSO4) > 2 wt% rGO–B–epoxy coating (~26.3 wt% BaSO4) > 5 wt% rGO–B–epoxy coating (~11.6 wt% BaSO4). A larger amount of BaSO4 provided better thermal stability with the ceramic additive as a thermal barrier. The IDPT results were the opposite of the thermal diffusivity and conductivity results, where the 5 wt% rGO–B–epoxy had the best thermal diffusivity and conductivity with the least amount of BaSO4 added. In 4 particles. Because graphene has great surface electron conductivity, the thermal conductivity was also great. With the good dispersive preparation of the composite epoxy resin, the rGO–B–epoxy was well connected and had good thermal flow tunnels or channels. The 5 wt% rGO–B–epoxy showed the best thermal properties out of the three samples. This was because of the good dispersion of the BaSO4 and graphene in the epoxy resin. The amount of BaSO4 decreased with an increase in the graphene added. This was crucial for the ultrasonic dispersive method because the energy transferred through the resin had to be equal and homogeneous throughout the 40 μm coating thickness.The TGA results could be used with the first-order derivative to show the decomposition temperature. A thermal stability assessment used the integral procedure decomposition temperature (IPDT) by Dolye : IPDT = 4 powder and added rGO to enhance the corrosion-proof and thermal properties. The epoxy–BaSO4 composite coating was the best corrosion protection coating, beyond the 2 wt% and 5 wt% rGO–B–epoxy composite coatings on the 7205 alloy. Based on the LSV and TAFEL analyses, the epoxy–BaSO4 composite was very stable in the corrosion test solution, and no redox curve could be found. The rGO–B–epoxy composites showed good corrosion protection ability, with higher Ecorr values  than the 7205 alloy (~−1.0 V). The thermal properties in the vertical direction of the coatings were not very different for the different samples. However, the in-plane thermal properties were very impressive, with values of 160.0 W/m K for the thermal conductivity and 71.38 mm2/s for the thermal diffusivity of the 5 wt% rGO–B–epoxy composite coating. The 2 wt% rGO also showed values of 51.46 mm2/s and 133.0 W/m K for the in-plane thermal properties. A further study of the thermal conductivity and diffusivity should be conducted on the ultimate compositions of epoxy resin-based composites. We reported our recent epoxy-based composites with BaSO


In this study, we aimed to explore the potential molecular mechanisms underlying the protective effect of STR on AS both in vitro and in vivo. Male DBA/1 mice were employed to establish an AS animal model. Hematoxylin-eosin staining showed that STR reversed pathological damages in bone tissues and the total antioxidant capacity of AS mice and increased the antioxidant capacity by upregulating superoxide dismutase and malondialdehyde expression in DBA/1 mice. The MTT, RT-qPCR, and Western blotting results further indicated that STR improved the survival rate of cells by downregulating the expression of target genes in the Wnt/β-catenin pathway and by inhibiting cell inflammation and apoptosis. In conclusion, our findings indicated that STR treatment might be an effective therapeutical strategy for AS.Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by invasion of the joints of the central axis that involves soft tissues and joints surrounding the spine. Stretching training rehabilitation (STR) has been widely applied for the treatment of AS. The Wnt/ Ankylosing spondylitis (AS) is a prototypical disease of spinal arthritis (SpA) . It is aβ-catenin signalling pathway is closely related to AS  × 100% ÷ 50% × V1 ÷ V2 × n. Here, V1 is the total volume of the reaction solution (mL), V2 is the measured sample volume (mL), n is the sample diluent multiple, ODA is the autooxidation rate of pyrogallol, and ODB is the change rate of the sample optical density value [Serum samples were prepared using a standard venous blood sampling protocol. Blood in mice was collected through the tail vein (0.1 mL/mouse) after anaesthesia. After centrifugation at 3,000 × g for 10 min at 4°C, the serum was obtained then transferred to a clean tube, followed by storage at −80°C until use. In an alkaline environment, pyrogallol produces its own oxidation color reaction. The intensity of the color reaction caused by self-oxidation is high or low, depending on the concentration of superoxide anion radicals released during the reaction. Therefore, a high concentration of superoxide anion radicals is released during the reaction, and vice versa, a low concentration of superoxide anion radicals. Briefly, 4.5 mL of Tris-HCl-EDTA buffer, pH 8.2, was placed in a 10 mL colorimetric tube, incubated at 25°C for 10 min, and then added to 10 mL of a 45 mmol/L pyrogallol solution at 25°C. The optical density value was measured at a wavelength of 325 nm once every 30 s for a total of 4 min to obtain the autooxidation rate of pyrogallol in ODty value .4 solution was added to catalyse the oxidation reaction, mixed, and placed in a constant temperature water bath shaker at 37°C in the dark for 1 h, followed by the addition of 1 mL of trichloroacetic acid solution. Next, 1 mL of TBA was added, shaken well, boiled in a water bath for 15 min, cooled quickly, and centrifuged at 5000 r/min for 10 min, and the supernatant was collected, and the absorbance Ax was measured at 535 nm. The blank tube contained distilled water instead of the sample to be tested, the operation method was the same as that of quality control, and the absorbance A0 of the blank tube was measured. Three parallel measurements were performed [Serum samples were prepared using a standard venous blood sampling protocol. Blood in mice was collected through the tail vein (0.1 mL/mouse) after anaesthesia. After centrifugation at 3,000 × g for 10 min at 4°C, the serum was obtained then transferred to a clean tube, followed by storage at −80°C until use. Malondialdehyde (MDA) was heated with thiobarbituric acid (TBA) under acidic conditions  to produce pink substances, and the maximum absorption peak at 535 nm was recorded to calculate the amount of lipid peroxidation. Briefly, 0.4 mL of the test solution was added to 3.6 mL of lecithin solution; then, 0.4 mL of FeSOerformed .2 cell incubator for 2 to 4 h. The sample was filtered using a 100 μm pore size filter and a large and small cell strainer and then centrifuged to pellet the cells. DMEM  containing 10% FBS (Invitrogen) was used to resuspend the cells and transferred to a cell culture flask for culture; the medium was changed every 2 to 4 days. The cell morphology and growth of the primary and postpassage cells were observed. When the cells reached passages 3-4, they were digested to prepare a single cell suspension with a density of 1 × 105 cells/mL. Additionally, LiCl  or IWR  was added to cells of the model or model+stretching exercise groups [Synovial tissues were minced from mice in four different groups  with ophthalmic scissors, digested with type I collagenase (1 mg/mL), and digested in a 37°C, 5% COe groups .μL of medium was added to each well and incubated overnight. The MTT reagent  was added to each well and incubated for 4 h in the dark; the culture solution was discarded, DMSO solution was added and shaken for 10 min, and finally, the absorbance was measured at 570 nm using a microplate reader  [Three replicate wells in each group were prepared; 100 es, USA) . All expμL of forward and reverse primers , 2 μL of cDNAs, and 20 μL of nuclease-free water were included in the amplification reaction mixture (50 μL), and the PCR conditions were as follows: 95°C ; 35 cycles of 95°C (30 s), 58°C (30 s), and 72°C (1 min) (21) [β-actin gene was used as an internal control. The following primer sequences were used: β-catenin, F: 5′-CCACTCCAGGAATGAAGG-3′, R: 5′-AGCAGTCTCATTCCAAGC-3′; TNF-α, F: 5′-ATAAGAGCAAGGCAGTGGAG-3′, R: 5′-TCCAGCAGACTCAATACACA-3′; IL-17, F: 5′-AGCCAGAGTCCTTCAGAGAG-3′, R: 5′-TCCTTAGCCACTCCTTCTGT-3′; Bcl-2, F: 5′-CTGGTGGACAACATCGC-3′, R: 5′-GGAG-AAATCAAACAGAGGC-3′; and β-actin, F: 5′-TCACCATCTTCCAGGAGCGAG-3′, R: 5′-TGTCGCTGTTGAAGTCAGAG-3′. All experiments were performed in biological triplicates (n = 6), and data are representative of three independent experiments. The relative transcript abundances (2ΔΔCT−) were calculated based on the equation 2−∆CT (∆CT = CTtarget gene–CTβ‐actin). The differences were determined using unpaired Student's t-test for comparisons between different groups. A P value of <0.05 was regarded as statistically significant.Total RNA was isolated from bone tissues and synovial cells of mice in each group using the TRIzol reagent (Invitrogen) and converted cDNAs using the OneScript Reverse Transcriptase OneScript cDNA Synthesis Kit . Twenty-five microliters of Dream Taq PCR Master Mix (Abcam), 1.5 in) (21) ; and a fμL was added to each well for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)  followed by wet transfer to polyvinylidene fluoride (PVDF) membranes  at 120 mA (2-3 h). The membrane was blocked with a solution containing 5% skim milk powder at room temperature for 1 h and rinsed with TBST 3 times. The membrane was incubated with antibodies against β-catenin , TNF-α , IL-17 , and β-actin  at 4°C overnight, rinsed with TBST 3 times, incubated with the secondary antibody  in the dark at room temperature for 2 h, and rinsed with TBST 3 times in the dark. Bands were visualized with electrochemiluminescence (ECL) , and analysed by ImageJ  [Western blotting was used to detect the relative protein expression. After extracting the total protein from the bone tissue, 10-25  Health) .P < 0.05 indicated a significant difference.Statistical analyses were performed using GraphPad Prism software (version 7.0). Data are presented as the means ± SD (standard deviations). Differences and comparisons between multiple groups were analysed using one-way ANOVA followed by a post hoc test (Tukey's test). The Achilles tendon showed no infiltration of inflammatory cells and fibroblasts and a normal morphological structure in the normal group. The model group presented varying degrees of cartilage and bone formation, inflammatory cells, and fibroblast-like infiltration of attachment points. The mice in the stretching exercise group had small Achilles tendon tissues that more frequently contained scattered infiltrating lymphocytes, and cartilage and bone formation were rare Figures . TherefoWe examined the antioxidant effect of stretching exercise on mice with AS and found a significantly lower serum level of the antioxidant TAS in model mice than in normal mice, while mice in the stretching exercise group had significantly increased TAS levels . Based oα and IL-17 in the model group were increased significantly and were then significantly reduced after stretching exercise. Meanwhile, the expression level of the β-catenin mRNA in the model group was significantly higher than that in the control group, while β-catenin expression in the stretching exercise group was significantly decreased. The relative protein levels were detected using Western blot assays  and added LiCl  and IWR  to the cells of the model group and the model+stretching exercise group to study the mechanism underlying the therapeutic effect of stretching exercise on AS. MTT results showed that cell in the model group has lower viability than that in the normal group. LiCl decreased cell viability while IWR or stretching exercise increased cell viability. Stretching simulation reversed the effect of LiCl and aggravated the effect of IWR on cell viability .α, IL-17, and β-catenin mRNA expression. Stretching exercise decreased TNF-α, IL-17, and β-catenin mRNA levels in both normal synovial cells and model cells. LiCl increased TNF-α, IL-17, and β-catenin mRNA expression while IWR decreased TNF-α, IL-17, and β-catenin mRNA expression in model cells. Stretching exercise rescued the effects of LiCl and aggravated the effects of IWR on TNF-α, IL-17, and β-catenin mRNA expression in model cells  and added LiCl and IWR to the cells of the model group and the model+stretching exercise group. The RT-qPCR results revealed that cells in the model group showed increased TNF- Figures .  Figure Figures . These rAS is a systemic disease that is mainly caused by chronic inflammation of the joints , 25. Itsβ-catenin signalling pathway. AS is a type of rheumatism characterized by chronic inflammation of the axial joint and may involve the internal organs and other tissues [α, IL-6, IL-8, and IL-17 have been identified to be overexpressed in AS patients [α, IL-17, and β-catenin expression. The Wnt pathway has been confirmed to be closely associated with AS development [β-catenin pathway [β-catenin inhibitor or agonist. Compared with the model group, the expression of the TNF-α, IL-17, and β-catenin was increased by LiCl and decreased by IWR. Stretching exercise rescued the effects of LiCl and aggravated the effects of IWR on TNF-α, IL-17, and β-catenin expression. Expression of antiapoptotic factor Bcl-2 showed the opposite trend under the same treatment. These results indicated that the therapeutical effect of stretching exercise on AS is mediated by the Wnt/β-catenin axis. Stretching exercise inhibits cell inflammation and apoptosis through blocking the Wnt/β-catenin signalling pathway.In this paper, DBA/1 mice were used as a focus for follow-up research to verify the specific mechanism by which stretching training inhibits inflammation and apoptosis through the Wnt/ tissues . TNF-α, patients . Furtherpatients . Naringipatients . We founelopment . For exaelopment . miR-22- pathway . We subsin vivo. This provides an interesting point worthy of discussion and further investigation. Many diseases, such as cardiovascular diseases, neurodegenerative disease, cancer, diabetes, atherosclerosis, inflammatory diseases, and premature aging, are all related to oxidative stress [Based on this study, stretching exercise could significantly improve the antioxidant capacity of AS mice by increasing the serum level of TAS, SOD, and MDA e stress . Howeverβ-catenin signalling and its inhibition of inflammation and the occurrence of apoptosis. Stretching exercise improves the antioxidant capacity of AS mice by regulating Wnt/β-catenin signalling (In summary, we experimentally verified the regulatory effect of stretching exercise on Wnt/gnalling . Our res


Richter’s Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases. Richter’s Transformation is a treatment-resistant and fatal progression from Chronic Lymphocytic Leukemia (CLL) to an aggressive lymphoma. Here, the authors show that PRMT5 is upregulated months prior to and after transformation, PRMT5 overexpression in a CLL mouse model leads to increased risk of transformation, and that targeted PRMT5 inhibition prolongs survival and delays disease development. Definitive molecular mechanisms driving the establishment and progression of chronic lymphocytic leukemia (CLL) have yet to be fully understood. Contributing to this lack of understanding, select patients experience a more aggressive disease course in the absence of known high-risk genetic alterations, suggesting the existence of some undiscovered factors influencing CLL pathogenesis and outcome.2. Unlike de novo DLBCL, generally chemotherapy-responsive and potentially curable, RT is chemotherapy-resistant and associated with a dreadful prognosis3. While initially described in 1928, the molecular, genetic, and epigenetic events driving RT remain poorly characterized. At transformation, between 25 and 60% of RT tumors display inactivation of TP53 and ~50% acquire epigenetic changes affecting c-MYC expression, suggesting dysregulation of these pathways contribute to the pathogenesis of RT7. Complex karyotype, CDKN2A/B gene locus deletion, and aberrant NOTCH1 activation are also associated with RT11. While these discoveries contributed to the understanding of RT biology, comprehensive studies underlying CLL-to-RT progression are needed to identify patients at risk of transformation and to discover targeted therapeutic approaches.Richter’s transformation (RT) is the morphologic evolution from CLL/small lymphocytic lymphoma to a high-grade diffuse large B-cell (DLBCL) or immunoblastic lymphoma, occurring in up to 10% of CLL patients. RT is the most common disease progression in CLL patients within the first 18 months of receiving targeted therapies , with overall incidence expected to rise as more patients receive these treatments in the front line and relapse/refractory setting18. The major type II protein arginine methyltransferase 5 (PRMT5) mediates symmetric dimethylation by catalyzing the transfer of a methyl group from S-adenosylmethionine (SAM) to arginine residues on histones  and non-histone proteins  thereby influencing gene transcription, splicing19, and protein activity. PRMT5 is essential for the eIF4E-mediated 5′-cap-dependent translation of c-MYC and CYCLIN D120 and is required for the establishment and maintenance of c-Myc and NOTCH1-driven lymphomas22. Moreover, co-deletion of MTAP and CDKN2A as a result of del(9)(p21.3), seen in up to 30% of RT tumors24, has been shown to confer vulnerability to PRMT5-targeted therapies, suggesting dependence on PRMT5 for the proliferation of these tumors27. While literature strongly supports a requisite role for PRMT5 in the context of large cell lymphomas, emerging evidence now suggests aberrant PRMT5 expression additionally disrupts critical regulatory mechanisms and contributes to tumor progression in CLL28. Accordingly, we hypothesized mechanisms stemming from dysregulated PRMT5 activity to play a major role in facilitating CLL-to-RT evolution.Arginine methylation regulates critical cellular processes, including proliferation and differentiationWe herein demonstrate that RT tumors overexpress PRMT5, and PRMT5 upregulation may be observed in some cases up to one year prior to RT diagnosis. We then developed and characterized a murine model of B-cell-specific expression of human PRMT5 (Eµ-PRMT5), producing an overt B-lymphoid expansion eventually developing a CLL-like phenotype. When crossed with the Eµ-TCL1 model, double transgenic mice uniformly developed a highly aggressive disease with select focal lymphoid tumors transforming to histologically resemble RT, altogether proposing a direct cause-effect relationship for PRMT5 in the pathogenesis of RT and identifying an attractive therapeutic target for RT and CLL at risk of RT. Lastly, we report the development of a small molecule inhibitor (PRT382) with enhanced PRMT5 selectivity, improved tolerability, and optimal in vitro and in vivo anti-tumor activity compared to currently available PRMT5 inhibitors.CD79A+) sequenced contained identical VDJ sequences, confirming the RT-phase tumor in this patient derived as a clonal progression from the existing CLL  from CLL patients and lymph node (LN) biopsy with PBMCs was collected for those with progression to RT. In a CLL patient with three previous lines of therapy, we obtained PBMC and LN samples at RT diagnosis in addition to CLL-phase PBMCs 2 months prior to RT and conducted single-cell RNA-sequencing . The small lymphocytes within typical CLL cases stained negative for PRMT5 expression, however, small PC areas showed weak PRMT5 expression  or did not (CLL) transform to RT within one year from the time the sample was collected. PRMT5 expression was variable in CLL patients who did not undergo transformation, ranging from not expressed to weakly expressed, whereas PRMT5 was consistently overexpressed in pre-RT patients Fig. . We thenion Fig. . In starion Fig. . Furtherion Fig. .MYC locus as identified by FISH while “RT Pt 1” did not present any identifiable rearrangement. Transcriptional heterogeneity was observed in B cells between samples; noting that PRMT5 was not abundantly expressed in all B cells, but was instead restricted to distinct subpopulations also displaying strong MYC and MKI67 expression , indicative of a highly proliferative population within the overall tumor burden. Clustering of cells with strong PRMT5 and MYC expression was observed in both patients regardless of identifiable MYC rearrangement.To further elucidate a role for PRMT5 in RT, we evaluated scRNA-seq in LN biopsies from two patients collected at the time of RT diagnosis Fig. . Notably31 and plotted the aggregate gene expression profile defined therein against CLL PBMCs, RT PBMCs, and RT LN tumor scRNA-seq expression profiles from the two patients described in our study , CDK1, and TCL1A, among others, likely contributing to CLL-to-RT progression by stimulating proliferation and anti-apoptotic signaling with enhanced activity along the BCR-signaling axis . Gene set enrichment analysis revealed that RT cells were in a proliferative state with enhanced MYC and E2F activity while the CLL cells maintained upregulated interferon-gamma response signaling genes, likely in support of their growth and survival32  under the control of a VH promoter-IgH-Eμ enhancer  was significantly shorter compared to wild-type (WT) littermates  was collected monthly and monitored for disease development by flow cytometry. Abnormal expansion over time of hematopoietic cell populations was commonly observed in Eµ-PRMT5 mice, including a Cd5+Cd19+B220dim B-cell population with similarity to the CLL-like disease in Eµ-TCL1 mice33  was observed in the dominant clones across all samples, consistent with classification as unmutated IgHV  between Eµ-PRMT5 and Eµ-TCL1 splenic B cells, suggesting a similar transcriptome profile within CLL-like cells in these two models  and Cd69 positivity, indicating these as activated B1a lineage lymphocytes and supporting description as a CLL-like phenotype. Cd93 expression was exclusively observed in a subset of Eµ-TCL1 B cells. By K-means clustering, n = 10 distinct cell clusters were observed in varying ratios within Eµ-PRMT5 and Eµ-TCL1 spleen populations  is implicated in the pathogenesis of several large cell lymphomas37, suggesting a role for hyperactive MAPK activity in CLL progression. Clusters 3.4 and 3.6 also featured upregulation of genes implicated in large cell lymphomas, and notably overlapping with several genes enriched in RT tumors compared to matched CLL precursors, including Birc5, Cdk1, Mki67, and Npm1 , a rare event in Eµ-TCL1 mice . All Eµ-PRMT5/TCL1 mice between ages 6–15 months became visibly ill and developed a neoplasia with a similar CLL-like immunophenotype to Eµ-TCL1 mice; although Eµ-PRMT5/TCL1 mice frequently presented with large cells circulating in the blood , identifying convergence on pathways highlighted by MYC and E2F targets, cell cycle checkpoints, and cell cycle control  —a marker for Myc-driven cancer stem cells in human and mouse lymphoma models38, Myc, and Il-10 as well as increased expression of markers indicating activated cell populations, including Cd274 (Pd-L1), Mki67, and Ccr7 . Of these, cluster LN.2 was predominantly present in Eμ-PRMT5/TCL1 mice while cluster LN.4 was almost exclusively present in Eμ-TCL1 mice, although these two clusters have very similar gene expression profiles . This included leukemogenic genes including Myc, Cd69, Cd83, AP-1 components including Fos, Jun, and Junb, tubulin-encoding genes Tuba1b and Tubb5, genes encouraging lymphoid homing including Ccr7 and Cxcr5, and mediators of immune suppression including Il-10, Ctla4, and Cd274 (Pd-L1), cumulatively providing supporting evidence for a role for PRMT5 in the advanced progression of CLL.Overall, a high degree of overlap in the most enriched genes from Eµ-PRMT5/TCL1-dominant lymph node cluster LN.7, Eµ-PRMT5/TCL1-dominant spleen cluster 5.6, and Eµ-PRMT5-dominant spleen clusters 3.4 and 3.6, was observed with cells from human RT lymph node tumors with abundant 39) and competitive  inhibition of SAM-mediated PRMT5 enzymatic activity. However, these compounds have been reported with limited specificity for PRMT5 among other methyltransferases, restricting the potential for clinical translatability as off-target effects disrupting processes essential in normal hematopoiesis are of significant concern.The emerging role for PRMT5 activity in both solid and hematologic tumors has spurred the development of several drug candidates with uncompetitive , and other members of the PRMT family , and high AUC levels (1175 h*kg*ng/mL/mg) in mice. Delivery of PRT382 at 10, 30 and 100 mg/kg was well-tolerated in WT CD-1 mice, leading to peak plasma levels of 12, 32, and 75 µM, respectively, without observable toxicity after 5 daily oral doses Fig. .39, which coincided with only a modest decrease in global symmetric dimethyl arginine residues at higher concentrations  with and without TLR9 stimulation using synthetic CpG oligodeoxynucleotide, 72 h PRT382 treatment resulted in a dose-dependent reduction in proliferative potential in vitro  to evaluate PRT382 efficacy in vivo and to further compare efficacy against the non-competitive PRMT5 inhibitor EPZ015666. PRT382 treatment reduced the rate of peripheral disease early in disease progression (weeks 2-7 post engraftment) compared to mice treated with EPZ015666 or vehicle  have been identified, however, these mutations do not entirely explain the molecular pathogenesis of CLL or transformation to RT. Animal models mimicking large B-cell lymphomas that arise from CLL are completely missing, posing a major hurdle in RT research. Importantly, given the poor survival seen in patients with RT, a model for testing therapeutics in the setting of both CLL and aggressive lymphoma is of high interest.A number of recurrent clonal and subclonal genetic mutations of potential leukemogenic relevance , and Il-10 suggest aberrant activation of PRMT5 in B lymphocytes predisposed to CLL may further manipulate the surrounding environment to facilitate tumorigenic outgrowth in secondary lymphoid niches. Importantly, DEG analysis in Eμ-PRMT5/TCL1 mice at 6 months of age revealed dysregulation of genes including Myc, Il-10, Sox4, Ltk, Bcl11b, and Runx3 compared to Eμ-TCL1 mice at this age and both Eμ-PRMT5/TCL1 and Eμ-TCL1 mice at 3 months of age. Interrogating the differential transcriptome at this early time point, while the CLL-like disease in Eμ-PRMT5/TCL1 mice is evolving through the pre-RT phase to morphologically resemble a large B-cell lymphoma, provides insight to mechanisms driving this progression. Understanding the dysregulated gene signature in the pre-RT phase, like that identified herein, can assist in the early detection of evidence indicating CLL-to-RT is likely in patients monitored in the clinic, and further characterization of this pre-RT phase gene signature should be prioritized to better define this phenomenon.A CLL-to-RT phenotype is rarely observed in the Eμ-TCL1 mouse model, even in the presence of conditional B-cell-specific Trp53 deficiency or with simultaneous co-expression of 31 against the scRNA-seq from RT patients data in this study, we observed a high degree of overlap between the gene signature in PBMC and LN samples from RT patients described here with those described previously, notably varying from samples from the CLL phase in our study and in previous studies >2] were CCNA2, MYC, and NR4A1; each identified as genes also enriched in B-cell clusters predominantly in the spleen and lymph nodes of Eμ-PRMT5 and Eμ-PRMT5/TCL1 mice evaluated in this study  may be drivers of this phenotype.These limitations were also evident in recent publications evaluating gene expression from paired circulating CLL vs nodal RT tumorssue Fig. , indicatdy Figs. . OverlapNR4A1 gene encodes a member of the nuclear receptor subfamily 4 (Nur77) that behaves as a ligand-activated transcription factor and nuclear co-factor. NR4A1 is reported to be largely absent in leukemia and lymphoma yet highly expressed in a myriad of solid tumors, specifically in solid tumors with enhanced metabolic rate, with elevated NR4A1 expression acting as a negative prognostic marker in affected patients50. ChIP analysis has demonstrated NR4A1 interactions bound to the GC-rich promoter sequence of BIRC5 (survivin), and Nr4a1 knockout mice developed expansions of myeloid progenitors and hemopoietic stem cells with decreased expression of c-Jun and Jun-B52. In our studies, Birc5 and Junb were repeatedly observed among the most enriched genes in B-cell clusters predominantly in the spleen and lymph nodes of Eμ-PRMT5 and Eμ-PRMT5/TCL1 mice, possibly implicating a PRMT5-NR4A1 axis as a major mechanism supporting the CLL-to-RT evolution. Further work to describe this interaction will be necessary to appropriately define the oncogenic potential of this relationship. Overall, comparative evaluation of Eμ-PRMT5 and Eμ-PRMT5/TCL1 mouse tumors and human RT lymph nodes in our limited cohort, in addition to similar recently described human RT cohorts, validates the use of the Eμ-PRMT5 mouse as a promising laboratory model of Richter’s Transformation and warrants further evaluation with additional current and ongoing studies in RT.Interestingly, the PRMT5 in CLL cells decreased cell proliferation, supporting the inhibition of PRMT5 as a promising therapeutic target in CLL. Recently, a suite of SAM-competitive PRMT5 inhibitors have been developed42, however, the diverse array of targets affected by these compounds may limit their progression in the clinical setting due to unwanted cytotoxicity in healthy tissue. To address this issue, we herein report here the development of a small molecule PRMT5 inhibitor with a predicted SAM-competitive mechanism of action, PRT382, which is highly selective for PRMT5 against a wide range of other methyltransferases. PRT382 demonstrated in vitro and in vivo efficacy against CLL cell lines, isolated CLL patient cells, and the CLL-like cells from the Eμ-PRMT5/TCL1 mouse model of CLL/RT, overall providing strong evidence supporting the use of targeted PRMT5 inhibition in CLL with elevated PRMT5 expression at high risk of RT. As expected, due to restricted selectivity, PRT382 demonstrated reduced cytotoxic potency against CLL cells compared to compounds with a similar mechanism of action. However, when compared with the SAM non-competitive PRMT5 inhibitor EPZ015666, PRT382 demonstrated superior in vitro anti-tumor activity and improved survival in the Eμ-PRMT5/TCL1 mouse model of CLL/RT in vivo.Critically, we show that genomic ablation of 53. Studies to understand mechanisms by which PRMT5 overexpression may cooperate with recurrent genomic lesions to contribute to CLL pathogenesis and progression are also necessary as the critical targets for response to PRMT5 inhibition are likely context-dependent for each cancer type54.The ability of PRMT5 inhibitors to influence multiple cellular pathways and restore the function of tumor suppressors via demethylation may be attractive as a single agent in CLL to induce durable responses and prevent or delay acquired resistance, however, care must be taken evaluating this target in the clinical setting as PRMT5 is essential in normal hematopoiesisPRMT5 along with other key genes 55 in CLL and DLBCL, suggesting BRD4 activity may control PRMT5 gene transcription. To explore the PRMT5 gene interactome, we employed a targeted 4C-seq approach in a CLL cell line (HG3) using bait primers specific for the PRMT5 gene promoter region. In addition to multiple trans interactions with this site  to down-modulate 5, prospective screening for diagnostic markers such as PRMT5 may be pivotal for improving the bleak outcomes associated with this disease.In summary, our data indicate that PRMT5 promotes CLL development, facilitates CLL progression to aggressive lymphoma through altered epigenetic programming, and provides a preclinical model and a therapeutic candidate to study CLL-to-RT progression. Upregulated PRMT5 thus presents as a biomarker for CLL patients at risk of transformation several months before a definitive RT diagnosis can be made. As patient mortality rates post-RT diagnosis are reported among the most rapid of all cancer types56. Sex and age of the patients who provided samples was not available. Human peripheral blood mononuclear cells (PBMC) were isolated from CLL patients using Ficoll density gradient centrifugation57. . Enriched B-cell fractions were prepared using the RosetteSep™ B-cell kit  according to the manufacturer’s instructions. Isolated cells were incubated in RPMI 1640 media  supplemented with 10% heat-inactivated fetal bovine serum , 2 mM l-glutamine (Invitrogen), and 100 U/mL penicillin/100 μg/mL streptomycin (Sigma-Aldrich) at 37 °C in an atmosphere of 5% CO2. The purity of enriched populations of CLL was routinely checked using CD19-PE  staining by flow cytometry.Our research complies with all relevant ethical regulations. De-identified human CLL cells were isolated as previously described after obtaining informed consent on protocols approved by The Ohio State University Cancer Institutional Review Board in accordance with the Declaration of Helsinki. All patients examined had CLL as defined by the 2008 IWCLL criteriaMEC-1 and HG3 lines were obtained from DSMZ . MEC-1 cell identity was reconfirmed via fluorescence in situ hybridization (FISH) using CLL probe panel, i.e., CEP12/13q14/13q34, ATM/p53, BCL6, MYC, IGH/CCND1 , and 6q21  . Lymphoma cell lines JVM-2, U-2932, RL, KARPAS-422, Jeko, Jurkat, Pfeiffer, SUDHL were obtained from commercial sources . CCMCL-1 was provided by Dr. Selina Chen-Kiang from the Department of Pathology and Laboratory Medicine, Weill Cornell Medicine. All cell lines were authenticated by short tandem repeat DNA profiling. All cell lines were confirmed to be mycoplasma negative using the MycoAlert™ Mycoplasma Detection Kit from Lonza  according to manufacturer instructions and used within 3–4 weeks from thawing. For experiments, cells were resuscitated, cultured in RPMI  supplemented with 10% fetal bovine serum (FBS), and used within 3-4 weeks from thawing.55. CpG685 oligonucleotide was obtained from the OSU Cytogenetics Core Lab. Antibodies used for immunoblots included anti-human PRMT5 (Abcam #ab109451), GAPDH (Millipore Sigma #MAB374), SDMA , c-Myc , and β-Actin . Cas9-mediated doxycycline-inducible knockout of PRMT5 utilized a targeted sgRNA .Proteins extracted from whole-cell lysates were resolved by SDS-PAGE and transferred on nitrocellulose membrane as previously described59. CLL populations in mouse peripheral blood were determined as Cd45+/Cd5+/Cd19+ and Cd45+/Cd19+/B220dim populations. Immunophenotyping of tumor cells in peripheral blood and spleen of Eµ-PRMT5, Eµ-PRMT5/TCL1, and Eµ-TCL1 mice by flow cytometry was performed as follows: APC rat anti-mouse Cd45 , FITC rat anti-mouse Cd45R/B220 , BV421 rat anti-mouse Cd19 , PE rat anti-mouse Cd5 , BV510 hamster anti-mouse Cd3e , BV650 rat anti-mouse Cd11b , BB515 rat anti-mouse Cd19 , BUV737 rat anti-mouse Cd5 . Immunophenotyping was conducted using a Beckman-Coulter Gallios 3-laser-10-color (B5-R3-V2) cell analyzer and BD LSRFortessa Cell Analyzer (Cat #649225). Peripheral blood from Eµ-PRMT5, Eµ-PRMT5/TCL1 and Eµ-TCL1 transgenic mice was collected monthly via check punch. Cells from the spleens of Eµ-PRMT5, Eµ-PRMT5/TCL1 and Eµ-TCL1, having met predefined euthanasia criteria, were processed and isolated as single-cell suspensions. Mouse B cells were isolated from whole spleen suspensions using EasySepTM mouse pan B-cell isolation kit . All flow cytometry data were analyzed with Kaluza v2.0 software (Becton Dickinson).All gates were set using fluorescent antibody controls prepared for each individual experiment using strategies following published data and technical resource publications60. Cell apoptosis was evaluated by Annexin V-FITC or PE and propidium iodide (PI) staining using FC500 cytometer  following the manufacturer’s procedure 55. For cell cycle analysis, cells were fixed with cold ethanol overnight and then stained before analysis with 30 μg/mL propidium iodide (Invitrogen) and 200 μg/mL RNase (Sigma-Aldrich) in 0.1 % v/v Triton X-100 (Sigma-Aldrich). Cell cycle data analysis was performed using ModFit LT software .Cell proliferation was assessed via MTS  assay (Sigma), using the tetrazolium dye 3’-2,5-diphenyl-tetrazolium bromideEµ-PRMT5 mice: A cDNA fragment possessing the entire human PRMT5 coding region was generated by PCR and cloned into pBluescript II KS (pEμ) plasmid containing a mouse VH promoter (V186.2) and the IgH-μ enhancer along with the 3′ untranslated region and the poly(A) site of the human β-globin gene. The pBluescript II KS (pEμ) plasmid containing full length of human PRMT5 cDNA were injected into mouse embryos at the single-cell stage and implanted into pseudopregnant B6 females. Mice were screened for the presence of the transgene by PCR on tail DNAs. Three founders were obtained  and bred. Eµ-PRMT5 mice were genotyped using the following primer sequences: . Transgenic heterozygote mice issued from these founders were studied and compared with nontransgenic siblings raised in identical conditions. Generation of the Eµ-PRMT5/TCL1 double transgenic leukemia model was achieved by crossing Eµ-PRMT5 mice with homozygous Eµ-TCL1 mice on the C57BL/6 J background61. Disease progression was monitored by Cd45+/Cd5+/Cd19+ flow cytometry of peripheral blood and manual assessment of peripheral blood smears, spleen size, and weight loss. Whole blood was collected via cheek punch at time of euthanasia, as well as at monthly intervals for each transgenic colony and at weekly intervals for each arm of the transplant model. Manual assessment of peripheral blood smears were confirmed by two independent pathology experts. Organs were collected at time of euthanasia and preserved in 10% formalin. Formalin-fixed tissues from mice were paraffin-embedded, sectioned onto glass slides at 4 µM, then deparaffinated, hydrated with distilled water, and stained with hematoxylin and eosin (HE), or Ki67. Mixed female and male populations were used across all experiments unless otherwise stated.Experiments were carried out under protocols approved by OSU Institutional Animal Care and Use Committee (IACUC). +Cd5+ cells (5 × 106) from the spleen of an Eµ-PRMT5/TCL1 transgenic mouse with active CLL-like leukemia  and palpable splenomegaly were engrafted by tail vein into immunocompetent C57/BL6J female mice. Engrafted mice were randomly assigned to treatment conditions: PRT382 10 mg/kg, EPZ015666 50 mg/kg, or vehicle  at 1-week post engraftment. All treatments were administered by oral gavage 4 contiguous days per week (Mon-Thurs). Predefined euthanasia criteria for mice in all transgenic colonies and murine transplant models were followed and included lethargy, impaired motility, splenomegaly, enlarged lymph nodes and/or superficial lymphoid tumors greater than 1.6 cm in diameter (or cumulative diameter of 1.6 cm if multiple tumors), decrease in body weight (>20%), ruffled fur, hunched back, failure to nest, and loss of appetite.For adoptive transfer, viable Cd19TM mouse pan B-cell isolation kit (STEMCELL Technologies). Total RNA was isolated from TRIzol suspensions using a chloroform/ethanol extraction method and quantified via Qubit RNA HS Assay kit (Invitrogen). Total RNA was quantified using the Nanodrop 2000 Spectrophotometer (ThermoFisher Scientific). Sequencing libraries were prepared using Illumina TruSeq stranded mRNA library preparation kit and sequenced on HiSeq 4000 instrument targeting ~60–80 × 106 read pairs per sample. The 150 bp paired-end reads were trimmed for adapters and low-quality bases with Cutadapt and mapped to the mouse reference genome GRCm38 with STAR aligner v2.6.062. The featureCounts utility63 from the subread package64 (v1.4.6) was used to generate gene counts based on Ensembl gene annotation release 9565. Pairwise comparisons were performed to identify DEGs using the DESeq2 package (v1.24.0)66.Mouse B cells were collected using the EasySepn = 4), Eµ-TCL1 (n = 4), and Eµ-PRMT5xTCL1 (n = 3) mice meeting euthanasia criteria. In total, 1 × 106 isolated B cells were washed with PBS, and RNA was extracted using the Monarch Total RNA Miniprep Kit (New England Biolabs) according to manufacturer instructions. Total RNA was quantified using the Nanodrop 2000 Spectrophotometer (ThermoFisher Scientific). The Clontech SMARTer v4 kit  was used for global preamplification. Illumina sequencing libraries were derived from the resultant cDNA using the Illumina Nextera XT DNA Library Prep Kit following the manufacturer’s instructions. Libraries were sequenced using an Illumina HiSeq 4000 sequencer paired-end 150 bp protocol to approximately 12 million passed filter clusters per sample. Data processing was performed according to the CLEAR workflow, which identifies reliably quantifiable transcripts in low-input RNA-seq for differentially expressed gene (DEG) transcripts using gene coverage profiles. BCR reads were binned by specific heavy (H) and light-chain V genes. First, we quantified the percentage of reads with heavy, kappa, and lambda chains compared to the number of total sequencing reads. MiXCR67 (v3.0.5) was used with default parameters to identify preprocessed reads containing CDR3 regions from B-cell heavy, kappa, and lambda chains, generating a list of CDR3 sequences associated with their relative abundances and specific V(D)J gene usage. Global DEG analysis was performed using DESeq2 (v1.20.0)66. The count’s table containing CLEAR transcripts from all replicates was used for comparisons between groups. The resultant data were then used for gene-specific analysis for a targeted list of oncogenes.B cells were isolated via EasySep Mouse Pan-B Cell Isolation Kit (Stemcell Technologies) from spleen suspensions of diseased Eµ-PRMT5  and centrifuged for 10 min at 4 °C. On centrifugation, the pellet was washed briefly in 50 μl MgCl2 buffer (10 mM Tris pH 8.0 and 5 mM MgCl2) before incubating in the transposase reaction mix  and 10.5 μl nuclease-free water) for 30 min at 37 °C. After DNA purification with the MinElute kit, 1 μl of the eluted DNA was used in a qPCR reaction to estimate the optimum number of amplification cycles. Library amplification was followed by SPRI size selection to exclude fragments larger than 1200 bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). Library amplification was performed using Nextera primers. The libraries were sequenced by the Genomics Services Laboratory at Nationwide Children’s Hospital using an Illumina HiSeq 4000 platform. The 150 bp paired-end reads were trimmed for adaptors and low-quality bases with Cutadapt and mapped to the mouse reference genome GRCm38 using the Bowtie2 algorithm70 with a maximum fragment length of 1000 and no-mixed, no-discordant and very-sensitive options. After the removal of duplicate reads, deepTools271 were used to generate normalized genome coverage files. ATAC-seq peak locations were determined using the MACS2 algorithm (v2.1.1.)72 with a cutoff P value of 1E-5 with “–broad–nomodel–shift -100–extsize 200” options. Consensus peak regions from all samples were generated as the union of the overlapping peaks using R GenomicRanges package73, then annotated using R ChIPpeakAnno package74. Using annotatePeakInBatch function from ChIPpeakAnno package, each ATAC peak was assigned to closets/overlapping gene within 5000 bp.Accessible chromatin mapping was performed using the ATAC-seq method75. The library was subjected to next-generation sequencing on the Illumina platform. The sequencing reads were analyzed with the R.4Cker pipeline and visualized with the RCircos package76 mapped to the UCSC.HG19.Human.CytoBandIdeogram .HG3 cells were fixed with paraformaldehyde, and chromosome segments in physical proximity were cross-linked. Chromatin was digested by DpnII and ends of interacting chromatin regions were ligated with T4 ligase. Cross-linking was removed and secondary digestion was done with Csp6I primers  to specifically amplify interacting partners with the bait for library construction77. For Ki67 staining, following hydration, sections were treated with citrate buffer pH 6.0 at 125 °C for 15 min. In all, 3% hydrogen peroxide with methanol was used to block endogenous peroxidase. A serum-free protein block was applied, incubated for 10 min and rinsed. Staining was performed by sequential application and incubation with anti-Ki67 primary antibody  using 1:100 dilution at room temperature, biotinylated goat anti-rabbit secondary antibody  for 30 min at room temperature, ABC biotinylated antibody linker (Vector Labs) for 30 min at room temperature, and DAB substrate  for 5 min. Hematoxylin was used as a counter-stain. All photographs were taken using an Olympus SC30 camera with an Olympus BX53 microscope.Formalin-fixed spleens, lungs, hearts, and lymph nodes from study mice were paraffin-embedded, sectioned onto glass slides at 4 µM, then deparaffinated, hydrated with distilled water, and stained with hematoxylin and eosin (HE), or Ki67 as previously describedA 10X Genomics Chromium machine was used for 3000–5000 single-cell capture. Cells were washed and partitioned into nanoliter-scale Gel Bead-In-EMulsions (GEMs), where all generated cDNA share a common 10x Genomics Barcode but uses a pool of ~750,000 barcodes to separately index each cell’s transcriptome. The silane magnetic beads and Solid Phase Reversible Immobilization beads were used to clean up the GEM reaction mixture and the barcoded cDNA was then amplified in a PCR step. For mouse scRNA-seq, libraries were prepared using the Chromium Single Cell 3′ Reagent Kits (v2): Single Cell 3′ Library & Gel Bead Kit v2 (PN-120237), Single Cell 3′ Chip Kit v2 (PN-120236) and i7 Multiplex Kit (PN-120262) (10x Genomics). Murine libraries were sequenced on an Illumina Nextseq 500 to achieve 75 bp reads. Optimal cDNA amplicon size was achieved using Covaris machine prior to library construction. The P7 and R2 primers were added during the GEM incubation and the P5, and R1 during library construction via end repair, A-tailing, adapter ligation and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification. Following the Single Cell 3′ Reagent Kits (v2) user guide , data was processed by the Cell Ranger pipeline . Count matrices were analyzed using the Seurat package (v2.2.1) in R. Read counts were normalized to library size, scaled by 10,000, log-transformed, and filtered.blaseRtools (https://github.com/blaserlab/blaseRtools). The Doubletfinder package78 was used to remove high-likelihood doublets. UMAP dimensionality reduction, clustering, and top marker analysis was achieved using functions from Monocle380. Leiden clustering81 and k-Means10 clustering was utilized for human and murine analyses, respectively. BCR V(D)J single-cell transcriptome analysis was analyzed using blaseRtools R package. Gene expression profiles for stated clusters  PN-1000263): Library Construction kit (PN-1000190), Chromium Next GEM Chip K Single Cell Kit (PN-1000286), Chromium Single Cell Human BCR Amplification Kit (PN-1000253), and Dual Index Kit TT Set A (PN-1000215) (10x Genomics). Human libraries were sequenced on a NovaSeq 6000 with 300 cycles and paired 150 bp reads and resulted in a minimum of >10,000 reads per cell. The data were processed by the Cell Ranger pipeline . Quality control removed low-quality cells with a high percentage of reads mapped to mitochondrial genes (mito counts >2 median absolute deviations above the median) and/or low numbers of genes detected (feature counts >2 median absolute deviations below the median) using functions from 000263: L). Chemical identity for compound PRT382 is as follows: HNMR: 1H NMR  δ 8.13 , 7.68–7.53 , 7.38 , 6.67 , 6.04 , 4.79 , 4.49 , 4.08 , 3.97 , 3.82 . 1H NMR was carried our under ambient temperature. LC-MS (m/z): [M]+ calcd. for C18H18Cl2N4O5, 441.3; found, 443.3 -2-[(R)--hydroxy-methyl]-5-pyrimidin-7-yl]tetrahydrofuran-3,4-diol hydrochloride, was synthesized for this study by Prelude Therapeutics , LLY-283 (cat #HY-107777), and PLX-51107 (cat #HY-111422) were obtained from MedChemExpress . PF-06855800 (cat #PZ0401) was obtained from Sigma-Aldrich Inc. .For in vitro assays, the compound was stored in a 10 mM solution in DMSO. For in vivo assays, PRT382 was dissolved in a 0.5% methylcellulose, 0.1% tween solution, and administered as oral gavage. For Pharmacokinetic studies, PRT382 was formulated in 0.5% carboxymethylcellulose sodium salt + 0.5%Tween80 as a 1 mg/mL suspension for 10 mg/kg and was administered in a 10 mL/kg dose volume by oral gavage to CD-1 mice. Blood was collected at various time points after dosing into EDTA-K2 tubes, placed on ice, and stored at −80 °C prior to analysis by LC-MS/MS. For biochemical potency and selectivity, PRT382 was solubilized in 100% DMSO and diluted serially threefold. For assessment of selectivity, enzyme activity against a panel of 37 methyltransferases was evaluated using 3H-SAM as a radioligand probe with filtration binding. PRT382 was tested in duplicate at a final concentration of 10 µM. The percent of enzyme activity relative to DMSO control condition was determined for each enzyme.50 curve at a concentration tenfold greater than the desired assay concentration. Standard reactions were performed in a total volume of 30 μl in assay buffer, with 300 nM histone H4-based AcH4-23 (Anaspec: AS-65002) as substrate. To this was added the PRMT5/MEP50 complex diluted to provide a final assay concentration of 2.5 nM, and the compounds were allowed to preincubate for 20 min at 37 °C. The reaction was initiated by adding S-[3 H-methyl]-adenosyl-l-methionine (PerkinElmer: NET155001MC) to a final concentration of 1 μM. Following a 30 min of incubation at 37 °C, the reaction was stopped by adding 25 μL of 8 M Guanidine HCl. To each reaction, 150 µL of SPA beads suspension (Perkinelmer: RPNQ0012) was added and incubated while shaking at room temperature for 30 min. The plate was centrifuged at 100 xg for 30 s before reading in a scintillation counter. IC50 values were determined by fitting the data to with Hill Slope using GraphPad Prism software (v9.2.0).For biochemical potency and selectivity, PRT382 was solubilized in 100% DMSO and diluted serially threefold. The diluted compound was further diluted in assay buffer  for 10-point IC50 values were calculated using Graphpad Prism (v9.2.0) .For western blot analysis of Cellular sDMA in Granta-519, one day before the experiment, Granta-519 cells were passaged to a density of 0.5 × 106 cells/ml. The next day, Granta-519 cells were spun down at 1500 rpm for 4 min, resuspended in fresh medium at 0.5E6 cells/ml and 3 mL of culture (1.5E6 cells) were seeded into a six-well plate. Eight-point, threefold serial dilutions of compound working stocks were added to cells  and incubated for 3 days. Cells incubated with DMSO was used as vehicle control. Cells were harvested 3 days later, resuspended in 15 µL PBS, lysed in 4% SDS, and homogenized by passing through homogenizer column . Total protein concentrations were determined by BCA assay . Lysates were mixed with 5× Laemmli buffer and boiled for 5 min. Forty micrograms of total protein was separated on SDS-PAGE gels , transferred to PVDF membrane, and blocked with 5% dry milk  in TBS with 0.1% v/v Tween20 (TBST) for 1 h at room temperature (RT), and incubated with primary antibodies  in 5% dry milk in TBST at 4 °C overnight. The next day, membranes were washed with TBST, 5 × 5 min, and incubated with HRP conjugated seconded antibody  for 2 hours at RT, followed by 5 × 5 min washes with TBST, and incubation with ECL substrates . The chemiluminescent signal was captured with Fluochem HD2 imager (Proteinsimple). SmD3me2s bands were quantified by ImageJ. Signals were normalized to β-Actin and DMSO control. ICFor cell growth curves in HG3 and Mec-1 cells, 3E5 cells were plated in 2 mL culture media with indicated dose PRT382 or comparator compound. Live cells/mL were counted on days 3, 6, 10, and 12. At indicated time points, cells were pelleted and 3E5 live cells were removed and resuspended in 2 mL fresh culture media with fresh dose of PRT382 or comparator compound. The split factor was calculated at indicated time points by dividing the counted cells/mL by 3E5. Cell growth was extrapolated over time by multiplying the subsequent cell count by the previous split factor. Cell growth is plotted as fold change relative to DMSO-treated cells.57 using SAS/STAT software . For survival experiments, survival curve estimates were calculated using the Kaplan–Meier method, and differences in curves were assessed using the log-rank test. Spleen volume was compared between groups using ANOVA, and mixed effects models were used to assess changes in %CD19+/CD5+ cells over time. Differences in PBL at different time points were assessed using a mixed effects model, and a two-sample t test assuming unequal variances was used to compare spleen weight. Where applicable, data were log-transformed to reduce skewness. P values were adjusted for multiple comparisons using Holm’s stepdown procedure or Dunnett’s test, where appropriate. Chemical structures were generated using ChemDraw JS for the web (PerkinElmer Informatics).All analyses were performed by the OSU Center for Biostatistics using previously described modelsFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary