Skeletal muscle differentiation is a tightly regulated process, and the importance of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling family for regulation of genes involved in skeletal myogenesis is well-established. Our prior work showed that bromodomains of mSWI/SNF ATPases BRG1 and BRM contribute to myogenesis by facilitating the binding of mSWI/SNF enzymes to regulatory regions of myogenic and other target genes. Here, we report that pathway analyses of differentially expressed genes from that study identified an additional role for mSWI/SNF enzymes via the regulation of the Wnt signaling pathway. The Wnt pathway has been previously shown to be important for skeletal muscle development. To investigate the importance of mSWI/SNF enzymes for the regulation of the Wnt pathway, individual and dual knockdowns were performed for BRG1 and BRM followed by RNA-sequencing. The results show that BRG1, but not BRM, is a regulator of Wnt pathway components and downstream genes. Reactivation of Wnt pathway by stabilization of β-catenin could rescue the defect in myogenic gene expression and differentiation due to BRG1 knockdown or bromodomain inhibition using a specific small molecule inhibitor, PFI-3. These results demonstrate that BRG1 is required upstream of β-catenin function. Chromatin immunoprecipitation of BRG1, BRM and β-catenin at promoters of Wnt pathway component genes showed binding of BRG1 and β-catenin, which provides further mechanistic insight to the transcriptional regulation of these genes. The skeletal muscle system is the largest organ of the muscular system, comprising about 30%–40% of the body mass. Skeletal muscles are responsible for voluntary body movements in coordination with the skeletal and nervous systems. These muscles originate from stem cells in the mesodermal layer during embryonic development . In adulThe SWI/SNF enzymes are the largest family of ATP-dependent chromatin remodelers in eukaryotes, and their roles in transcriptional regulation have been well-documented . MutatiomSWI/SNF complexes are also known to regulate signaling pathways in a tissue-specific manner by coordinating gene activation with help from signature transcription factors. Simone et al. had shown that, in skeletal muscle, p38/MAP kinase activity is required for phosphorylation of the BAF60c subunit, which promotes recruitment of mSWI/SNF . In the 2+/Calmodulin-dependent pathways . Dulbecco’s modified Eagle’s medium (DMEM) was purchased from ThermoFisher Scientific (#11965118). Vectastain elite ABC (PK-6200) and HRP DAB substrate (SK-4100) kits were purchased from Vector Laboratories, United States.Primary antibodies used for WB against BRG1 , LaminB1  and β-catenin  were purchased from Santa Cruz Biotech, United States. Myosin Heavy Chain (MHC)  was purchased from the Developmental Studies Hybridoma Bank, University of Iowa, United States. Antibodies used for chromatin IPs against β-catenin , BRG1  and BRM antisera (3–4 μL per μg of chromatin) have been previously described . PFI-3  and BRM  and non-targeting control  were purchased from Dharmacon Horizon Discovery Ltd., United States. C2C12 cells were transfected as previously described  using inC2C12 cells were stained and analyzed for immunofluorescence as previously described . Immunosg for 10 min at 4 °C and supernatants were collected. Protein concentrations were determined using a Pierce™ BCA protein assay kit  according to the manufacturer’s protocol. 20–50 μg of total protein with 5X loading dye  was boiled at 95 °C for 10 min and subjected to 8% SDS-polyacrylamide gel electrophoresis followed by electroblotting onto Immobilon-P PVDF membranes . The membranes were then blocked using 5% non-fat milk in PBS for 30 min followed by overnight incubation at 4 °C with primary antibody against protein of interest at the desired dilution in 2% non-fat milk prepared in PBS or TBS. This was followed by 3 washes with TBS containing 0.1% Tween-20 for 5 min each at room temperature. The membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies  diluted in 2% non-fat milk prepared in PBS for 1 h at RT followed by 3 washes with TBS containing 0.1% Tween-20 for 5 min each at room temperature. Immuno-reactive bands were visualized by chemiluminescence with ECL Plus  using an Amersham Imager 600 . ImageJ software (NIH)  supplemented with 1X protease inhibitor cocktail . The lysates were incubated on a rocker for 15 min at 4 °C to allow lysis of cell membranes, followed by sonication in an ultrasonic bath sonicator for 3 cycles of 30 s on/off. Samples were centrifuged at 14,000 re (NIH)  was usedCells grown on 10 cm dish were treated according to the relevant treatment group. On the day of harvest, culture media was aspirated, and cells were washed with ice-cold PBS twice to wash off residual media. Cells were scraped, resuspended in 1 mL ice-cold PBS, and collected in 1.5 mL Eppendorf tubes. Cells were pelleted at 2500 rpm for 5 min and the cell pellet was resuspended in 300ul of hypotonic buffer ). After 10 min of incubation on ice, with gentle vortexing 2-3 times in between, the cell suspension was centrifuged at 10,000 rpm for 1 min at 4 °C. The supernatant comprises the cytosolic lysate and was collected in a fresh Eppendorf tube. The nuclei in the pellet were washed gently with 1 mL hypotonic buffer and then resuspended using 300ul of RIPA buffer  fresh). The suspension was incubated on ice for 10–15 min followed by lysis using 27G/28G syringe needles. The samples were centrifuged at 10000rpm for 5 min at 4 °C and supernatant (nuclear lysate) was used for co-immunoprecipitation experiments.To set up co-IP, protein concentrations of the lysates were determined using Pierce™ BCA protein assay kit  according to the manufacturer’s protocol. Equal amounts of protein was aliquoted to fresh pre-cooled Eppendorf tubes. Relevant antibodies for desired proteins were added and reactions were incubated overnight with rotation at 4 °C. Next morning, 40ul of equilibrated protein A beads  were added to each IP mix and incubated with rotation for additional 1–2 h. The beads were washed with 500ul cold RIPA lysis buffer thrice to remove unbound proteins. The pulled down protein-Ab complex was eluted in 50ul of RIPA buffer by incubating the beads at RT for 2 h. The eluant was collected and mixed with 5X loading dye  and electrophoresed using 8% SDS-polyacrylamide gels.Eef1α1) from that of the target gene. ΔΔCT of each target gene was then calculated by subtracting the average of the ΔCT obtained in the control samples from ΔCT for each test sample. Relative gene expression was calculated using the 2−ΔΔCT method (RNA was extracted using Trizol (ThermoFisher) as per the manufacturer’s protocol. cDNA was prepared using 2 μg of total RNA quantified using a Nanodrop1000 spectrophotometer (ThermoFisher Scientific) using Superscript III First Strand Synthesis Kit (Invitrogen) according to manufacturer’s protocol. At least three independent biological replicates for qRT-PCR were performed with technical duplicates for each sample using 1 μL each of forward and reverse primers (10 μM stocks), 1 μL cDNA, 5 μL of Fast SYBR Green 2X Master Mix (Applied Biosystems), and final reaction volume was adjusted to 10 μL per reaction. All qPCRs were run in QuantStudio 3 RT-PCR machine (Applied Biosystems). ΔCT was calculated by subtracting the CT value for housekeeping gene ) and shown as relative to a control region from a gene desert on mouse chromosome 6 as performed previously . Significance is displayed with *in vivo (We recently reported that the bromodomains of mSWI/SNF ATPases BRG1 and BRM are crucial for regulation of myoblast differentiation in culture and skeletal muscle regeneration following injury in vivo . Inhibitin vivo . Mechaniin vivo  to identin vivo . All sigin vivo . Both ofin vivo .Brg1 or Brm transcripts (Volcano plots of all identified DEGs from 24 h to 48 h differentiated C2C12 myoblasts were generated, and some of the identified myogenic, cell cycle-related and Wnt-associated genes were labeled . Wnt patnscripts , as shownscripts . A heatmnscripts . The Wntnscripts .Western blots indicate an increase in the β-catenin levels in differentiating cells compared to proliferating myoblasts and demonstrate that bromodomain inhibition decreases β-catenin protein expression levels . InteresPFI-3 is a selective inhibitor of the bromodomains of BRG1 and BRM, both of which have been known to be crucial for skeletal muscle differentiation . To specA heatmap of Wnt pathway-associated genes supports the idea that BRG1, but not BRM, regulates the expression of Wnt pathway and related genes. Expression of these genes were significantly altered in cells where BRG1 and BRM or BRG1 only were knocked down, whereas knockdown of BRM alone had minimal effects on gene expression . VolcanoAlthough mSWI/SF enzymes remodel chromatin structure, they often physically interact with sequence-specific DNA-binding transcription factors  to targeIf inhibition of the BRG1 bromodomain or a deficiency in cellular BRG1 levels contributes to myoblast differentiation via regulation of Wnt signaling pathway components, agents that stabilize β-catenin may permit differentiation even in the presence of PFI-3 or BRG1 knockdown. Lithium chloride has been historically used as a Wnt pathway agonist for this purpose to understand the role of Wnt signaling pathway, as it inhibits GSK3β activity and therefore stabilizes β-catenin . A selecAs shown previously , PFI-3 tSimilar experiments were performed using cells subjected to knockdown of BRG1. Two different siRNAs that target BRG1 were used . KnockdoAxin2 gene. Binding was significantly impaired in the presence of the PFI-3 bromodomain inhibitor, thereby identifying the bromodomain as a contributor to the ability of the ATPase to interact with the chromatin at these promoter sequences. There was no indication of interaction of BRM with any of these sequences except at the Fzd4 promoter, where it bound in a differentiation-specific and PFI-3 sensitive manner. This agrees with Fzd4 transcript expression levels as seen in Fzd4 might be co-regulated by BRG1 and BRM. We conclude that BRG1 interacts with the regulatory sequences of most Wnt pathway genes.We asked whether BRG1 or BRM binds to Wnt pathway gene promoters to determine if they likely act in a direct manner on the regulatory sequences of these genes. We examined two ligand gene promoters, two receptor gene promoters, two antagonist gene promoters and two other gene promoters . ProlifeWe subsequently examined β-catenin binding to Wnt pathway gene promoters. β-catenin binding occurred in a differentiation-specific manner in PFI-3 treated and control cells at all of the tested loci . AlthougSkeletal muscle differentiation is regulated by a wide range of signaling pathways. Using the P19 trans-differentiation model, Petropoulos et al.  showed tA link between BRG1 and Wnt signaling was reported over 20 years ago, when it was determined that BRG1 and β-catenin interact to activate target genes . Mouse mPharmacological stabilization of cellular β-catenin protein levels activated downstream Wnt signaling which, in turn, rescued the muscle differentiation phenotype and myogenic gene expression even upon BRG1 inhibition or knockdown. This indicates BRG1 control of Wnt signaling is upstream of β-catenin-dependent signaling. Chromatin immunoprecipitation assays showed binding of BRG1 on the regulatory regions of Wnt pathway genes in a differentiation-induced manner, and co-immunoprecipitation experiments showed that BRG1 and another mSWI/SNF subunit, BAF250A, interacted with the β-catenin effector of Wnt gene expression in nuclear extracts. These findings support a model where BRG1-based mSWI/SNF enzymes promote the appropriate regulation of Wnt pathway component genes and cooperate with β-catenin in the activation of Wnt signaling via direct stimulation of Wnt pathway genes.Fzd6 promoter during tumor inducing cell self-renewal (Inhibition of BRG1 bromodomain function using PFI-3 was previously shown to inhibit myogenesis and to affect the recruitment of BRG1 to myogenic gene promoters and enhancers and the recruitment of BRM to the promoters of cell cycle regulators important in muscle differentiation . BRG1 re-renewal , therebyβ-catenin is a key effector component of the canonical Wnt pathway. Interestingly, we saw no change in expression of β-catenin at the transcript level due to BRG1 knockdown or PFI-3 induced inhibition ; 2E. HowAxin2 expression (Axin2 expression by BRG1-and BRM-containing mSWI/SNF complexes that is tilted one way or the other in the absence of one of the ATPases. Inhibition of bromodomain function by PFI-3 resulted in a modest reduction of Axin2 mRNA expression, suggesting that inhibiting BRG1 bromodomain function had a greater impact than simultaneous inhibition of BRM bromodomain function. The reason for Axin2 sensitivity to BRM while most of the other Wnt signaling pathway components did not show regulation by BRM is not known. The difference in transcript output could possibly be a result of variable mode of BRG1/BRM functional manipulation. We note that neither BRG1 nor BRM bound to Axin2 sequences even though β-catenin binding was detected (Axin2 locus. An alternative hypothesis is that BRG1 and/or BRM bind elsewhere on the Axin2 locus.Axin2 is a component of the β-catenin-degradation complex and is also a downstream Wnt target . We obsepression . There wdetected , suggest2+ levels (It is worth noting that other signaling pathways, such as integrin and Rho GTPase signaling, are also altered due to BRG1 knockdown or inhibition of bromodomain function . The rol+ levels . The exp+ levels  also depIt has long been appreciated that mSWI/SNF enzymes contribute to myogenesis, but prior work has largely focused on the functions of these enzymes in the regulation of myogenic genes required for differentiation and maintenance of the muscle phenotype and of genes involved in cell cycle control. Our findings indicate a previously unappreciated role for BRG1 and the mSWI/SNF enzymes in the regulation of the Wnt signaling pathway during skeletal muscle differentiation. This function is largely independent of the homologous BRM ATPase. mSWI/SNF enzymes containing BRG1 transcriptionally regulate Wnt pathway genes via binding to sequences upstream of these genes and through a post-transcriptional mechanism that impacts the protein levels of the β-catenin regulatory protein that is central to Wnt signaling. The integration of mSWI/SNF function in promoting myogenic differentiation through multiple mechanisms reflects the complexity of differentiation signaling and the important roles for these chromatin remodeling enzymes.


Objective. Atherosclerosis is a leading cause of mortality and morbidity. Optical endoscopy, ultrasound, and X-ray offer minimally invasive imaging assessments but have limited sensitivity for characterizing disease and therapeutic response. Magnetic resonance imaging (MRI) endoscopy is a newer idea employing tiny catheter-mounted detectors connected to the MRI scanner. It can see through vessel walls and provide soft-tissue sensitivity, but its slow imaging speed limits practical applications. Our goal is high-resolution MRI endoscopy with real-time imaging speeds comparable to existing modalities. Methods. Intravascular (3 mm) transmit-receive MRI endoscopes were fabricated for highly undersampled radial-projection MRI in a clinical 3-tesla MRI scanner. Iterative nonlinear reconstruction was accelerated using graphics processor units connected via a single ethernet cable to achieve true real-time endoscopy visualization at the scanner. MRI endoscopy was performed at 6-10 frames/sec and 200-300 μm resolution in human arterial specimens and porcine vessels ex vivo and in vivo and compared with fully sampled 0.3 frames/sec and three-dimensional reference scans using mutual information (MI) and structural similarity (3-SSIM) indices. Results. High-speed MRI endoscopy at 6-10 frames/sec was consistent with fully sampled MRI endoscopy and histology, with feasibility demonstrated in vivo in a large animal model. A 20-30-fold speed-up vs. 0.3 frames/sec reference scans came at a cost of ~7% in MI and ~45% in 3-SSIM, with reduced motion sensitivity. Conclusion. High-resolution MRI endoscopy can now be performed at frame rates comparable to those of X-ray and optical endoscopy and could provide an alternative to existing modalities, with MRI’s advantages of soft-tissue sensitivity and lack of ionizing radiation. Atherosclerosis is a prevalent factor in cardiovascular disease and a leading cause of mortality and morbidity -3. Whileμm resolution for real-time visualization in blood vessels [IV magnetic resonance imaging (MRI) is a newer approach that employs miniature MRI detector coils mounted on guidewires or catheters for use in higher-field MRI scanners. It can provide high-resolution and soft-tissue contrast for characterizing different stages of vessel disease without using X-rays -18. An “ vessels . The spe vessels . Thus, tμm, and an imaging field-of-view (FOV) of 2-3 cm without inducing significant local heating. After calibration and safety testing, a uniform MRI excitation flip angle (FA) is excited by the tiny coil at the end of the endoscope using adiabatic “BIR-4” RF pulses [ex vivo and from animal vessels in vivo are demonstrated, and the structural and temporal information in the highly accelerated image streams is compared with fully sampled MRI endoscopy scans using image similarity metrics and with tissue dissections and histology.A highly accelerated real-time MRI system was recently developed for conventional multichannel MRI with transmitters and receivers that are fixed to the scanner frame of reference and its localizing gradient system -23. The F pulses , and theμm nominal (in-plane) resolution. Frames in rows (a1-a4) are recorded using highly undersampled radial acquisitions at five different positions at 6 fps and visualized in real time. Frames (b1-b4) are recorded in real time from approximately the same locations but at 10 fps. These are, respectively, 20 and 33 times faster than 0.3 fps 200 μm resolution images acquired using a “conventional” fully sampled endoscopy sequence depicted in frames (c1-c4). Videos of the image streams are presented in Supplementary Materials. Figures Figure μm endoscopy sequences, respectively, at 6 fps and 10 fps. As the endoscope is withdrawn through the artery, the branching of the internal iliac is seen as a lateral opening in the vessel wall  are reference scans from fully sampled 0.3 fps endoscopy stream, and those in row (d) derive from high-resolution  static 3D endoscopic images acquired in 112 s. Poststudy dissections of the vessel wall in (e1) and (e5) show atherosclerosis with fibrocalcific plaques and intimal thickening corresponding to images (d1) and (d5), respectively.Figure In vivo MRI endoscopy frames from a pig inferior vena cava (IVC) are shown in Figure μm, and each column of images is from approximately the same location for comparison.μm nominal resolution) highly undersampled images and the slow “conventional” fully sampled scans. MI is widely used for registering images from the same source that have different contrasts such as medical images acquired with different modalities [μm nominal resolution; 50-370 s scan time) and fully sampled endoscopy at 0.3 fps . Two-way ANOVA showed significant differences in MI between the high-resolution static 3D reference scans acquired in 50-370 s which used Cartesian spatial encoding and the 0.3-10 fps dynamic scans acquired with radial encoding  and the three-component weighted structural similarity index measure (3-SSIM) were used to compare the similarity between the high-speed 6-10 fps . Compared to the fully sampled experiments, there was no significant difference in MI between 6 and 10 fps. The small difference in the 3-SSIM metric for the 10 fps and the 6 fps data streams, as compared to the 0.3 fps scans  pulses, which is not well gauged by the volume-averaged RF-specific absorption rates used for general MRI , 34. Witl injury , 17. Whethe coil , 17 and the coil , 36.In conclusion, high-speed MRI endoscopy, unlocked from the scanner frame of reference, can be combined with undersampling and real-time image reconstruction technologies to match the speed of existing clinical catheterization and endoscopy procedures and could provide a useful complement to established minimally invasive imaging modalities including X-ray fluoroscopy, IVUS, OCT, and optical endoscopy . It has Prisma, Siemens Healthineers AG, Munich, Germany). The endoscopes had 3-5 turn transmit/receive coils that were tuned to the 123.2 MHz MRI frequency with microchip capacitors and mounted on 1/4-wavelength (42 cm), 50 Ω, flexible, 1.25 mm diameter, silver-plated microcable and enclosed in a 2.4 mm polymer sheath  GPUs and a “bypass” computer  described previously [The MRI acceleration unit was comprised of 8 eviously -23. It weviously  which joeviously . The fileviously  programmeviously .ex and in vivo studies, to provide effective FOVs of ≥20 mm and ≥30 mm, respectively  were obtained from commercial research suppliers . Deidentified human vessels harvested postmortem from elderly voluntary donors were obtained from this institution’s autopsy service. These commonly show evidence of atherosclerotic lesions and calcifications detectable by MRI endoscopy. Vessels were mounted on a rubber platform at the center of a (0.35%) saline solution placed in the MRI scanner. The MRI endoscope was placed at the distal end of the vessel, the endoscopy sequence commenced, and the coil retracted by an operator from the end of the scanner  and in diseased human blood vessels (7 samples) μm; μm; μm; phase encoding steps, Real-time BIR-4 FLASH MRI endoscopy of porcine carotid arteries was performed at 6 and 10 fps   of each MI, 3-SSIM, and


There are new evidences that protein arginine methyltransferase 5 (PRMT5) is widely involved in the progression of various diseases, but its effect is unclear on Primary Sjogren's syndrome (pSS). The main purpose of this study is to explore the regulatory effect of PRMT5 on pSS and its potential mechanisms.CD40L treated CD19 + B cells to construct a cell model of pSS. CCK‐8 assay and Annexin V‐FITC/PI kits were used to measure cell proliferation and apoptosis. ELISA assay was used to determine the contents of IL‐6 and TNF‐α in CD19 + B cells. And commercial kits were used to detect the levels of immunoglobins  in CD40L‐treated CD19 + B cells. And successfully constructed a pSS mouse model.The results revealed an increase in the expression of PRMT5 in CD19 + B cells from patients with pSS. After CD40L treatment, the knockdown of PRMT5 prominently decreased cell viability, the production level of immunoglobulins , and the content of IL‐10, increased the content of IL‐6 and IL‐8, and promoted the apoptosis of pSS CD19 + B cells. Mechanistically, PRMT5 negatively regulated the RSAD2 and nuclear factor kappa‐B (NF‐κB) signaling pathway. Furthermore, overexpression of RSAD2 and p65 significantly rescued the effect of PRMT5 knockdown on proliferation, immunoglobin production and secreting cytokines in CD40L‐treated CD19 + B cells. More importantly, inhibition of PRMT5 significantly inhibited the symptoms of pSS mice.Low‐expression of PRMT5 through inactivation of RSAD2/NF‐κB signalling pathway alleviates the hyperactivity of B cells, which may provide theoretical basis and potential therapeutic targets for clinical treatment of pSS. Low‐expression of protein arginine methyltransferase 5 (PRMT5) through inactivation of RSAD2/NF‐κB signaling pathway alleviates the hyperactivity of primary Sjogren's syndrome (pSS) B cells, which may provide theoretical basis and potential therapeutic targets for clinical treatment of pSS. It can be divided into pSS and secondary Sjogren's syndrome.Protein arginine methyltransferase 5 (PRMT5), a member of the methyltransferase family, is a type II arginie methyltransferase that catalyzes symmetric dimethylation labeling on histones and non‐histones.According to reports, when PRMT5 is inhibited, S‐adenosylmethionine domain 2  is significantly inhibited. Therefore, PRMT5 may regulate the expression of RSAD2.The main purpose of this study is to explore the role and mechanism of PRMT5 in the occurrence and development of pSS. The results of this study indicate that PRMT5 via RSAD2 mediate nuclear factor kappa‐B (NF‐κB) signaling pathway inhibits the activity of B cells, improve the symptoms of pSS in mice, and provide preliminary theoretical basis and therapeutic targets for clinical treatment of pSS.22.1Forty‐seven patients with pSS (pSS group) were selected from the General Hospital of Ningxia Medical University from June 2017 to 2021. All patients were newly diagnosed or did not receive glucocorticoid or immunosuppressive therapy. According to the pSS disease assessment index (ESSDAI), patients with pSS were evaluated.2.2C57BL/6 mice aged 8 weeks were purchased from the Experimental Animal Center of Yangzhou University. Mice were raised in a specific pathogen free animal facility, and all experiments were approved by the Living Animal Research and Teaching Committee of General Hospital of Ningxia Medical University. The ESS mouse model was induced according to previously described.2.35/mL in RPMI (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) upon stimulation with 5 µg/mL soluble recombinant human CD40L (BD Biosciences) in a 24‐well culture plate. Then the small interfering RNAs against PRMT5 (si‐PRMT5‐1 and si‐PRMT5‐2) and the overexpression plasmids of p65 (pcDNA3.1‐p65) and RSAD2 (pcDNA3.1‐ RSAD2), synthesized by GenePharma Co., Ltd., were cotransfected into cells with Lipofectamine 2000 reagent (Invitrogen).Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by Ficoll‐Paque density centrifugation, and CD19 + B cell isolation kit (Miltenyi Biotec) was used to purify CD19 + B cells from PBMCs according to the manufacturer's instructions. The purity was over 90% by flow cytometry. The CD19 + B cells were cultured at 2.5 × 102.44 cells per well) and cultured for 24 h, and 10 µL CCK‐8 solution was supplemented for each well. Then, the cells were incubated at 37°C for 2 h, and the absorbance at 450 nm was measured by a SpectraMax M5 fluorimeter.CD19 + B cells were stimulated with CD40L for 72 h, and then were seeded into 96‐well plates (1 × 102.55 cells per well) were washed twice by cold PBS and resuspended in 200 μL buffer. Then, adding 10 μL Annexin V‐FITC and PI into cell samples, and the samples were incubated at room temperature in the dark for 1 h. At last, apoptotic cells were measured by a flow cytometer (BD Bioscience).After CD40L stimulation, the apoptotic rate was quantitatively analyzed by Annexin V‐FITC/PI apoptosis detection kit (BioVision). Treated CDl9 + B cells (5 × 102.65/mL in RPMI (Miltenyi Biotec) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) upon stimulation with 5 µg/mL soluble recombinant human CD40L (BD Biosciences) in a 24‐well culture plate. Centrifuge the cells and collect the cell supernatant. According to the instruction for manufacturers, the concentrations of IgG, IgA, and IgM in the supernatant were measured by using the commercial ELISA kits. The optical density of the samples was read at 450 nm.The CD19 + B cells were cultured at 2.5 × 102.7TM qPCR RT kit (DBI). RT‐qPCR analyses were performed with BestarTM qPCR MasterMix (DBI) on an Agilent Stratagene Mx3000P fluorescence quantitative PCR instrument. Gene expression was analyzed by the 2‐ΔΔCt method. The primer sequences used were: PRST5, 5′‐AAAGTCCTAGGAAACGCCCG‐3′ and 5′‐GTGACGATGCACACGAAACC‐3′; RSAD2, 5′‐GCTCCACATCAGTCCTTCC‐3′ and 5′‐GGTCGTCTTCGTCCTCATC‐3′; GAPDH, 5′‐AATCCCATCACCATCTTC‐3′ and 5′‐AGGCTGTTGTCATACTTC‐3′. The fold‐change for target genes normalized by internal control was determined by the formula 2‐ΔΔCt.All RNA in cells was isolated using TRIzol regent (Miltenyi Biotec). Isolated RNA was synthesized to cDNA using a Bestar2.8The protein concentration was determined using BCA assay kit (Miltenyi Biotec) after cell lysate extraction using RIPA buffer (Biyuntian). Twenty micrograms of proteins was separated by SDS‐PAGE, transformed into PVDF membranes, and incubated with primary and secondary antibodies. Primary antibodies were obtained from the following companies: anti‐GAPDH , anti‐PRMT5 , anti‐caspase‐3 , anti‐Bcl‐2 , anti‐Bax , anti‐p50 , anti‐RSAD2 , anti‐p65 , and anti‐p‐p65 . Band intensities were measured using ImageJ (NIH).2.9Saliva flow rates were measured as previously described.2.10p < .05 was used as a cutoff for statistical difference.All experiments had three biological replicates. The data were shown as mean ± SD. All statistics were performed using GraphPad Prism 8.4.2 (GraphPad Software). Tukey's post‐multiple tests using ANOVA was selected to comprise difference of groups. 33.1In the present study, the EULAR ESSDAI score was used to assess the activity of the disease. In the pSS group, the ESSDAI score was higher than that in the healthy group. Anti‐SSA and anti‐SSB autoantibody status was assessed in both persons with pSS and controls, and controls were all negative for these autoantibodies. In addition, in the pSS group, 25 persons (53.2%) developed lymphadenopathy, 40 persons 85.1%) suffered from arthralgia, 5 persons (10.6%) with splenomegaly, and 38 persons (80.1%) from cardiopulmonary symptoms. Among people who did not suffer from pSS, 3 (6.4%) demonstrated arthralgia and 2 (4.3%) demonstrated cardiopulmonary arthralgia. The demographic, immunological, and clinical data on the 94 people are presented in Table % suffere3.2The expression of PRMT5 in peripheral blood CD19 + B cells of patients with pSS was detected by qRT‐PCR and western blot analysis. The results revealed that the mRNA and protein expression of PRMT5 increased in CD19 + B cells from patients with pSS compared to healthy individuals Figures . We used3.3The next research investigated the effect of PRMT5 on CD40L‐induced apoptosis in CD19 + B cells. As presented in 2 A, si‐PRMT5 significantly decreased the protein expression of PRMT5 Figure . Meanwhi3.4Next, we investigated the effect of PRMT5 on production of cytokines of CD40L‐treated CD19 + B cells. We found that PRMT5 siRNA significantly reduced the levels of immunoglobulins IgG, IgM, and IgA in CD40L‐induced CD19 + B cells Figure . At the 3.5In the following study, we will explore the potential mechanism of PRMT5 in the progression of pSS. It has been reported that PRMT5 regulates the expression of RSAD2.3.6Further research found that overexpression of RSAD2 or p65 significantly reversed the inhibition of RSAD2 on CD19 + B cells activity Figure . Meanwhi3.7n = 10) was immunized with adjuvant alone; the second group was pSS model group: mice were immunized with SG protein; the third group  or pcDNA3.1‐PRMT5 (ov‐PRMT5) for 24 h. (A) The cell proliferation of CD19 + B cells in different group.Click here for additional data file.


Burkholderia mallei antibody titers. The diagnosis was established through a multi-pronged approach encompassing microbiological culture, mass spectrometry, and genome sequencing. The outbreak occurred in 2019 in Tatuí, São Paulo, Brazil, and the infected mare, despite displaying no clinical symptoms, had multiple miliary lesions in the liver, as well as intense catarrhal discharge in the trachea. Samples were collected from various organs and subjected to bacterial isolation, molecular detection, and identification. The strain was identified as B. mallei using PCR and confirmed by MALDI-TOF mass spectrometry. Whole-genome sequencing revealed a genome size of 5.51 Mb with a GC content of 65.8%, 5871 genes (including 4 rRNA and 53 tRNA genes), and 5583 coding DNA sequences (CDSs). Additionally, 227 predicted pseudogenes were detected. In silico analysis of different genomic loci that allow for differentiation with Burkholderia pseudomallei confirmed the identity of the isolate as B. mallei, in addition to the characteristic genome size. The BAC 86/19 strain was identified as lineage 3, sublineage 2, which includes other strains from Brazil, India, and Iran. The genome sequencing of this strain provides valuable information that can be used to better understand the pathogen and its epidemiology, as well as to develop diagnostic tools for glanders.This manuscript elucidates the occurrence of glanders in an asymptomatic mare from Brazil presenting positive  Burkholderia mallei is a Gram-negative bacterium that causes glanders, a contagious and potentially fatal disease in equids such as horses, mules, and donkeys . This may be partly attributed to the use of more sensitive and specific diagnostic methods for glanders diagnosis compared to the complement fixation test [In Brazil, from 1999 to 2022, 3385 cases of glanders were reported. Of these, 715 cases occurred in the last 3 years, according to reports from the Brazilian Ministry of Agriculture and Livestock [ion test .In Brazil, equids that work in sugar cane fields in the forest zone of the Northeast are more frequently reported to exhibit symptomatic cases of glanders ,8,9. ConDiagnostic tests for glanders worldwide heavily rely on serology. In Brazil, the previous law  mandated euthanasia for seropositive equids. However, relying solely on serology as the diagnostic criterion, especially in asymptomatic horses, has caused distrust within the equine sector. Consequently, legal disputes arose when horse owners opposed mandatory euthanasia.B. mallei in a sample from an equine or detecting specific antigens, genetic material, or antibodies related to B. mallei in a sample from an equine displaying clinical or pathological signs compatible with glanders.Recently, the legislation in Brazil has changed  and suspected cases of glanders are identified as equine that are susceptible and show clinical or pathological signs compatible with glanders or when there is an epidemiological link to a confirmed outbreak or case. Confirmation of cases involves isolating and identifying B. mallei to better comprehend its transmission and virulence aspects [B. mallei in a seropositive mare that displayed no clinical symptoms, employing a comprehensive approach that integrates microbiological culture, mass spectrometry, and genome sequencing.There is an increasing need to isolate and characterize Brazilian strains of  aspects ,11,12. TIn 2019, an outbreak of glanders in horses was identified in the city of Tatuí, São Paulo, Brazil, by state agricultural defense services. During the outbreak, one young mare was found to have a positive result in both the complement fixation (CF) screening test (cold procedure) and Western blot (WB), which were performed in an official laboratory according to the Brazilian Equine Health Program. In Brazil, the CF test employs antigens from the USDA, USA, but undergoes an in-house standardization process, resulting in variable test accuracy. Conversely, the WB test  boasts a diagnostic sensitivity and specificity of 100%. Despite having a healthy body condition score and displaying no clinical symptoms, the mare was found to have multiple miliary lesions in the liver and intense catarrhal discharge in the trachea see  during nw/v, and 10 µL of the suspension was plated on 5% sheep blood agar supplemented with 1% glycerin and 2500 IU of potassium benzylpenicillin. The plates were then incubated at 37 °C for 48–72 h [B. mallei, colonies were resuspended in 0.85% saline, inactivated at 100 °C for 10 min, and stored at −20 °C for molecular analysis. PCR was performed, targeting fliP-IS407 using primers described by Abreu et al. (2020) [fliP-IS407 F: 5′ TCAGGTTTGTATGTCGCTCGG 3′ and fliP-IS407 R: 5′ GCCCGACGAGCACCTGATT 3′. A reference strain of B. mallei INCQS 00,115 (ATCC 15310) from the Collection of Reference Microorganisms in Sanitary Surveillance, FIOCRUZ-INCQS, Rio de Janeiro, Brazil, was used as a positive control, and sterile deionized water was used as a negative control.All tissue samples were suspended in 0.85% sterile saline at a concentration of 1:5  48–72 h ,14. The  48–72 h  and Winng at room temperature. The cell pellet was suspended in 300 µL of sterile water and inactivated by the addition of 900 µL absolute ethanol. After centrifugation at 10,000× g at room temperature for 2 min, bacterial proteins were extracted with 70% formic acid and pure acetonitrile. The supernatant containing proteins was collected after centrifugation at 10,000× g at room temperature for 2 min, and 1 µL was spotted in a clean stainless steel MALDI target  in triplicate. Upon air drying, bacterial proteins were overlaid with 1 µL of a-cyano-4-hydroxy-cinnamic acid (5 mg/mL) matrix solution and allowed to dry in the air. Mass spectra were acquired with an Autoflex III Smartbeam  in a mass to charge (m/z) range of 2000–20,000 Daltons after calibration with an E. coli standard , as described [B. mallei and B. pseudomallei MSPs, we have included reference mass spectra for those species using B. mallei ATCC 23344 and B. pseudomallei mass spectra from Robert Koch Institut (RKI), Berlin, Germany, available at Zenodo [B. mallei ATCC 15310 and seven other B. mallei and B. pseudomallei clinical isolates previously characterized by our group [B. mallei MSP for the BAC 86/19 clinical isolate described in this work was generated with the MALDI Biotyper™ standard method after processing 30 mass spectra using 70 m/z signals with a frequency higher than 50%.Bacterial samples were inactivated with ethanol, and protein profiles were acquired and analyzed by MALDI Biotyper™ , as described ,16. Brieescribed . For bact Zenodo , and MSPur group . The B. The Whole Genome Sequence (WGS) analysis for BAC 86/19 was conducted at the NGS multiuser platform of Oswaldo Cruz Foundation (FIOCRUZ), located in Rio de Janeiro, Brazil. First, DNA quantification was performed using the Qubit™ dsDNA HS Assay Kit  and the Agilent High Sensitivity DNA Kit . The WGS procedure was carried out on a HiSeq instrument  using HiSeq Rapid SBS Kit v2 (200 cycles) chemistry and the Nextera DNA Flex Library preparation kit  following the manufacturer’s guidelines.The resulting reads were deposited in SRA (Sequence Read Archive), NCBI  under accession number SRR19621772.B. mallei ATCC 23344 (Accession numbers: CP000010.1 and CP000011.2). The assembled genome was deposited in NCBI and annotated with PGAP (Prokaryotic Genome Annotation Pipeline) (Accession number: JANCTE01).Reads were evaluated using FastQC  before aB. mallei and B. pseudomallei were used to confirm the bacterial species of the isolate. Briefly, primer and probe sequences used to detect orf11 and orf13 genes  [mprA-associated regions  [serB genes of the isolate sequenced herein were retrieved and subjected to a global alignment against the 23S rRNA and serB genes of the reference genome B. pseudomallei Mahidol-1106a .Primers and probes used in real-time PCR assays to differentiate between rf13pro) , as wellrf13pro)  and mprA5, 14R5) , were seB. mallei BAC 86/19 was identified following a scheme previously reported by Girault et al. (2018) [B. mallei BAC 86/19 were mapped to the reference genome B. mallei ATCC 23344 using BWA-MEM [The lineage of . (2018) . The sch BWA-MEM  with def BWA-MEM ,30, sele BWA-MEM . All 15  BWA-MEM  were invB. mallei growth on a selective blood-enriched medium, as previously described by Abreu et al. (2020) [During necropsy, a total of eleven samples were collected. Among these, one sample from the tracheal secretion showed suggestive colonies of . (2020) . The colB. mallei and B. pseudomallei MSPs of reference from Zenodo.org [B. mallei BAC 86/19 MSP, Biotyper™ score values were above 2.3, consistent with reliable identification at the species level, which was observed in all 86/19 strain samples tested  and an estimated genome size of 5.51 Mb was formed, a size consistent with expectations.The genome was initially analyzed using Kaiju software v.1.9.0 , showingi. Krona  analysesB. mallei BAC 86/19 was identified as lineage 3, sublineage 2  WB, among other recommended methods [In this study, an asymptomatic mare was diagnosed with glanders, initially relying on serology using CF as a screening test and WB as a confirmatory test. The World Organization for Animal Health (OIE) guidelines recommend that a diagnosis of glanders should be supported by positive serological test results. This typically involves an initial screening with CF, followed by confirmation through a second test with equal or higher sensitivity and higher specificity, such as the  methods .B. mallei in microbiological cultures, or the identification of specific antigens or genetic material in biological samples. However, there is accumulating evidence supporting the high specificity of the serological tests currently utilized in Brazil, such as ELISA and WB, as documented by [Recently, Brazilian legislation has introduced additional criteria for confirming glanders in seropositive animals. These criteria include the presence of clinical or pathological signs consistent with glanders, direct detection of ented by .Bacterial colonies were chosen with characteristic morphology: round, grayish, non-hemolytic, and isolated. In addition, to characterize the bacteria of interest, biochemical tests were used. Previous studies in Brazil with a strain from the Brazilian Northeast reported small differences in the fermentation of some carbohydrates, but without compromising bacterial identification .B. mallei from B. pseudomallei has been historically difficult due to their high genomic similarity [IS407A element in the fliP gene, B. mallei lost its motility capacity. Accordingly, the fliP gene of the strain sequenced herein has been predicted by PGAP as a pseudogene (locus_tag = “LV178_14020”). Genetic events leading to pseudogenization are classified by PGAP as frameshift, internal stop codon or incomplete. The fliP pseudogene is described as incomplete, which agrees with a potential disruption caused by an insertion sequence. The genome size can also be used to distinguish B. mallei from B. pseudomallei; B. mallei has a smaller genome size than B. pseudomallei (~7.2 Mb) [B. mallei BAC 86/19 showed an estimated genome size of 5.51 Mb, which is in accordance with other B. mallei strains and smaller than that of B. pseudomallei [The differentiation of milarity . A major~7.2 Mb) . Accordidomallei .B. mallei and B. pseudomallei [mprA-associated regions were not detected, which also confirms the isolated species as B. mallei. Finally, the global sequence alignment between the 23S rRNA and the serB genes of the sequenced strain with that of the reference genome of B. pseudomallei Mahidol-1106a also showed the presence of SNPs previously identified as specific to B. mallei [Many PCR assays have been developed to differentiate between domallei . Exact m. mallei ,37.B. mallei, including 9902 RSC, BM_campo 1, BM_campo 3, and UFAL2, which were obtained from the Northeast region of Brazil. Laroucau et al. (2018) also confirmed that the 16-2438_BM#8 strain, collected from the same region, is part of this lineage. Additionally, this lineage encompasses isolates from India  and Iran (A200) [According to Falcão et al. (2022) , the L3Bn (A200) .The L3B2 lineage in Brazil includes isolates that have caused either clinical infections (such as UFAL 2 and 16-2438_BM#8) or asymptomatic infections  .B. mallei, such as the need for tissue collection and preservation, slow growth on media without glycerol supplementation, and the requirement of a biosafety laboratory, bacteriological analyses are not always feasible or successful [B. mallei, MALDI-TOF is a safe and fast alternative for accurate identification, due to its inactivation with ethanol, to extract proteins at a biosafety level [Due to the challenges involved in culturing ccessful . To reduty level .The analysis of the 86/19 strain using MALDI-TOF involved its characterization across various culture media, resulting in species-level identification. This step effectively mitigated a previously acknowledged bias . The proB. mallei in different regions of Brazil [Expanding the isolation of f Brazil  will ena


Various weight fractions of the reinforcement materials, ranging from 0 to 0.5 wt.%, were incorporated into the epoxy. The aim is to enhance the characteristics and durability of the polymers for potential utilization in different mechanical applications. The addition of hybrid additives consisting of Al2O3 nanoparticles and graphite to the epoxy resin had a noticeable effect on the performance of the epoxy nanocomposites. The incorporation of these additives resulted in increased elasticity, strength, toughness, ductility, and hardness as the concentration of reinforcement increased. The enhancement in the stiffness, mechanical strength, toughness and ductility reached 33.9%, 25.97%, 25.3% and 16.7%, respectively. Furthermore, the frictional tests demonstrated a notable decrease in both the coefficient of friction and wear with the rise of the additives’ weight fraction. This improvement in the structural integrity of the epoxy nanocomposites led to enhanced mechanical properties and wear resistance. The SEM was utilized to assess the surfaces of tested samples and provide insights into the wear mechanism.The main target of this study is to evaluate the impact of hybrid reinforcement using Al Lately, numerous industries have been increasingly utilizing polymers as viable alternatives to metals and metal alloys due to their advantages that can be used in various applications. Polymers have many attractive characteristics, such as lightweightness, affordability, non-toxicity, corrosion resistance, and ease of manufacturing or production, which allow outstanding design flexibility . Among tRecently, polymer nanocomposites have gained significant attention from both researchers and industrial sectors as they present promising materials with outstanding behavior. This performance arises from the interconnection between the polymer matrix and incorporated nanoparticles ,7. ThrouTo improve polymer properties and mechanical, thermal, electrical, and tribological characteristics, a lot of researchers have focused on incorporating nano-additives (nano-fillers) into epoxy resins. In an investigation into the thermomechanical performance of epoxy/block-copolymer/core-shell rubber nanocomposites conducted by Bajpai et al. , the aut2O3) are ceramic fillers that are popular for their remarkable behavior such as superior resistance to wear, high stiffness, corrosion resistance, and scratch resistance. Additionally, they have outstanding mechanical performance, high toughness, and creep resistance [2O3 nanoparticles (high loading) was examined to evaluate its mechanical properties [2O3. They evaluated the tribological and mechanical performance of the epoxy for up to 0.4 wt.% weight fraction of Al2O3. The outcomes illustrated a considerable enhancement in the mechanical performance and the wear resistance.The inclusion of ceramic fillers is usually employed to preserve the mechanical performance of the composites such as elasticity modulus and strength . The utisistance . Epoxy coperties . The invoperties  tried toCarbon materials such as graphite, graphene nanoplatelets, and carbon nanotubes, are famous for their ability in the production of multifunctional engineering components. Such carbon materials possess the ability to minimize friction and improve wear behavior . In addiThe utilization of hybrid nanofillers in reinforcing polymer nanocomposites proved that the combination of different filler types is able to improve the physical, tribological and mechanical performance of the nanocomposite relative to the utilization of a single filler type . It has 2O3 and graphite with different loading fractions on the mechanical and tribological performance of the epoxy nanocomposite. The epoxy nanocomposite’s mechanical properties  were determined as a function in the nanoparticle loading fraction. Additionally, the coefficient of friction and the wear of the epoxy nanocomposites were assessed under the variation of the normal loads and sliding distances. In order to gain insights into the wear mechanism of the produced composites, the morphological properties of the nanocomposite surfaces were examined through scanning electron microscopy (SEM). Additionally, X-ray diffraction (XRD) analysis was employed to assess the phase composition of the hybrid epoxy nanocomposite.Based on the literature survey, the inclusion of hybrid nanoparticles in the epoxy can result in a significant impact on the produced polymer’s nanocomposite properties. Each type of nanoparticle can contribute to the enhancement of a specific property. Furthermore, the nanoparticle loading fraction has a crucial function in controlling the performance of the produced nanocomposite. The primary purpose of the current investigation is to study the influence of incorporating hybrid nanoparticles of Al2O3 nanoparticles were obtained from US Research Nanomaterials, Inc., Houston, TX, USA. The Al2O3 has the following specifications: purity of 99% or greater, 100% alpha phase, particle size 80 nm, and density of 3.97 g/cm3. The previous specifications were obtained from the company datasheet. On the other hand, the graphite (Gr) was obtained from the same company, with the following specifications: fixed carbon 99.9+%; moisture: <0.3%; ash: <0.5%; particle size 400 nm–1.2 µm diameter, and with a flaky morphology with a thickness of 80 nm and 8–6 m2/g surface area.For the current investigation, the epoxy was obtained from Graffiti Resin Company, Saudi Arabia. The package consisted of two components: a non-pigmented liquid epoxy which is the resin and a hardener which is used for curing the epoxy resin. These two compounds are typically mixed in specific ratios (1:1) to initiate the curing process and achieve the desired properties of the epoxy. The Al2O3 nanoparticles and graphite were subjoined into the epoxy. The epoxy/Al2O3/Gr mixture was mechanically stirred at room temperature for a duration time of 30 min, with a rotational speed of 150 rpm. The previous process aimed to distribute the Al2O3 nanoparticles and Gr uniformly inside the epoxy matrix. Secondly, the hardener was carefully blended, in a 1:1 ratio, with the epoxy/Al2O3/Gr mixture for 3 min. The previous process is crucial to achieve inclusive homogenization of the mixture components without creating air bubbles inside the composite and to ensure uniform nanoparticle distribution throughout the epoxy matrix. The weight fractions of both the Al2O3 nanoparticles and the graphite are provided in 2O3 nanoparticles, graphite, and the hardener, were poured into molds that were prepared beforehand. The molds were designed specifically for the production of samples used in compression and tribological tests. The mixtures were left for 24 h at room temperature to harden and be ready for tests. The curing time was identified based on the recommendations provided in the epoxy datasheet. The waiting time is allowed to give the mixture a proper crosslinking and hardening, and such a process leads to the formation of solid specimens and composite specimens ready for subsequent testing and characterization. Furthermore, a reference sample without any additives was also fabricated. This reference sample serves as a baseline for comparison purposes, and allows a direct evaluation of the impact of the Al2O3 and Gr nanoparticles on the mechanical and tribological performance of the epoxy composite. The sample production process was conducted based on standard precautions to ensure the accurate mixing and homogeneity of epoxy formulation. Firstly, Al2O3/Gr samples underwent compression, utilizing a computer-controlled servo-hydraulic universal testing machine. The machine had a maximum load capacity of 30 tons and the compression rate was adjusted to 1.5 mm/min. From the stress–strain curves achieved, various mechanical characteristics of the epoxy nanocomposites were derived. In accordance with ASTM D2240 [The compression specimens were designed following the standard of ISO 604 Plastics . The speTM D2240 , the har2O3 nanoparticles and graphite. The examination revealed that the Al2O3 nanoparticles displayed an approximately spherical shape, as shown in 2O3 nanoparticles and graphite aligned with the data provided on the data sheet obtained from the manufacturing company. These analyses confirmed the consistency between the observed properties of the nanoparticles and the specifications provided by the supplier.SEM and TEM analyses were utilized to investigate the morphology of the Al2O3 nanoparticles and graphite. Additionally, XRD was utilized to examine the chemical composition of the epoxy/Al2O3/Gr nanocomposites. 2O3 nanoparticles utilized in the current study. The analysis reveals that the alumina powder exclusively consisted of the α-Al2O3 phase. It is noteworthy that these Al2O3 peaks align with those documented in a prior investigation carried out by Mulpur et al. [2O3 nanoparticles and graphite in the epoxy resin is reflected in the XRD pattern of the epoxy composite. However, the analysis confirms that no chemical reaction occurred between the Al2O3, the graphite, and the epoxy matrix. The presence of the nano additives did not modify the crystal structure of the epoxy, and the epoxy retained its distinctive characteristics and identity within the resulting composite.XRD (X-ray diffraction) analysis was employed to explore the chemical compositions of the materials used in the current study: epoxy, as well as the Alr et al. . In contr et al. . Moreove2O3 and graphite, the hardness consistently and significantly rises. The D-index scale was used to measure the hardness of the nanocomposite samples, and the average hardness of the pure epoxy was recorded as 73.4. As the concentration of additives increased in the nanocomposite, the epoxy nanocomposite exhibited an increase in hardness. The highest level of hardness was observed at a weight fraction of 0.5 wt%, measuring 85.1 on the D-index scale. This represented a significant enhancement of 16%, compared to the hardness of the pure epoxy. The observed increase in composite hardness can be attributed to the favorable interface between the Al2O3 and graphite particles and epoxy resin. This interface facilitates the efficient load transfer, resulting in enhanced hardness. Moreover, the hardness of the composite relies on the intermolecular bonds formed among the matrix and the reinforcement material. The noticeable increase in hardness indicates that the additive particles are uniformly distributed within the epoxy resin, further supporting their role in reinforcing the composite structure.According to 2O3 and graphite particles within the epoxy resin. During the compression tests, the stress and strain values were carefully recorded for the epoxy nanocomposites containing various weight fractions of Al2O3 and graphite particles. During the investigation of the impact of adding Al2O3 and graphite particles onto the epoxy composites, several compressive properties were examined, including Young’s modulus, yield strength, ductility and toughness. By incorporating Al2O3 and graphite particles into the epoxy, the nanoparticles were effectively dispersed into the epoxy chains. This arrangement resulted in a reduction in the epoxy chain flexibility, leading to an improvement in the compressive properties of the epoxy nanocomposite.In order to examine the load-carrying capacity of the epoxy nanocomposites, a compression test was performed. The aim was to explore the compressive properties resulting from the addition of the Al2O3 and graphite weight fraction increased from 0.1 to 0.5 wt%. The pure epoxy modulus of elasticity was recorded as 2.77 GPa. However, at a weight fraction of 0.5 wt% for both additives, the Young’s modulus experienced a significant enhancement of 33.9%, reaching 3.71 GPa. Furthermore, the findings indicate that as the content of Al2O3 and graphite particles in the epoxy increased, there was a corresponding increase in the nanocomposite strength. Specifically, when the weight fraction of Al2O3 and graphite reached 0.5 wt%, the strength exhibited a significant improvement of 25.97% when compared to the pure epoxy.2O3. Aluminum oxide possesses a high Young’s modulus of approximately 1400 GPa, which contributes to the overall stiffness of the composite. During the test, the applied compressive force is distributed between the epoxy and the aluminum oxide. In the presence of nanoparticles, when a crack is initiated within the composite, the nanoparticles play a crucial role in healing the crack and preventing its propagation. This crack-healing mechanism effectively increases the overall strength of the composite [The enhanced strength of the epoxy nanocomposites can be attributed to the superior characteristics of Alomposite .2O3 and graphite particles, the plastic deformation area of the composite expanded. Consequently, the toughness of each epoxy nanocomposite was assessed by computing the area for each nanocomposite under the stress–strain diagram. To quantify the toughness enhancement, the relative rise in toughness for each nanocomposite was estimated in comparison to the neat epoxy, as presented in 2O3 and graphite. This interface promotes interfacial adhesive bonding, which facilitates the transfer of load from the epoxy to the Al2O3 and graphite particles. As the reinforcement content was increased in the composite, there was a steady increment in the toughness of the material. Specifically, when 0.5 wt.% for both Al2O3 and graphite were added to the epoxy, the toughness of the nanocomposites was improved by 25.3%. The recorded results are in line with the research conducted by Emiroglu et al. [2O3 and graphite content, reaching a relative ductility of 16.7% compared to the pure epoxy.With the increase in the concentration of the Alu et al. , where tu et al. . The rel2O3 and graphite particles on the epoxy/Al2O3/Gr nanocomposite coefficient of friction, the nanocomposite specimens were subjected to rubbing against a stainless-steel counterpart. The rubbing was performed in the existence of different loads, specifically between 10 and 40 N, at 0.4 m/s sliding speed. The coefficient of friction for each condition was recorded. In order to investigate the impact of loading fractions of Al2O3/Gr nanocomposites, friction tests were carried out for various durations: 10, 20, 30, and 40 min, at a constant load of 40 N. In order to investigate the effect of sliding distance/time on the tribological characteristics of epoxy/Al2O3 nanoparticles in the epoxy/Al2O3/Gr nanocomposites played a significant role in achieving a more uniform and consistent transferred film on the steel counterpart. This uniformity of the transferred film further facilitated a dropping in the coefficient of friction as the sliding duration time increased [The observed decrease in the coefficient of friction accompanied by increasing friction duration time can be attributed to several factors. Firstly, the long friction duration time and rubbing against the counterpart contribute to the smoothing of the composite surface, leading to a reduction in friction. Additionally, prolonged sliding duration generates sufficient heat to induce localized melting of the nanocomposites . This mencreased .2O3/Gr nanocomposites  were measured under different normal loads. The results, in 2O3 and graphite particles led to a decrease in weight loss. This decrease in weight loss occurred due to the improved mechanical characteristics of the nanocomposites. Specifically, the increased concentration of additive particles enhanced the strength of the epoxy nanocomposites. As a result, the strength of bonding among the epoxy resin and the additive particles prohibited the surface degradation during the friction test and improved the wear strength of the nanocomposites. However, with the rise in applied load, a significant amplification in the weight loss of the nanocomposite samples was noticed. This increase in wear may have occurred due to the generation of heat resulting from the kinetic energy which resulted from the relative motion between the rubbing surfaces. The increase in temperature affects the contact region, leading to surface softening. Consequently, high shear resistance is created, resulting in a notable rise in nanocomposite weight loss and the formation of some transferred layers. Additionally, the elevated temperature in the contact area during the sliding test can degrade the shear strength of the epoxy sample. This resulted in escaping reinforcement particles from the epoxy sample throughout the friction test, which may act as abrasive particles on the surface of the epoxy nanocomposite, consequently contributing to increased weight loss [2O3 may work as rolling balls, effectively functioning as a solid lubricant. This behavior helps to reduce the friction coefficient of the nanocomposites in comparison with the pure epoxy [2O3/Gr nanocomposites. It can be observed that increasing the sliding distance/time had a minimal impression on the wear rate of the nanocomposite samples. This limited effect can be attributed to the increased shear resistance among the nanocomposite sample surfaces and the counterpart, which occurs as layers of nanocomposite are transferred to the counterpart.To assess the wear performance of the fabricated nanocomposites, the weight loss of both the pure epoxy (Epoxy0) and epoxy/Alght loss . Neverthre epoxy . Figure 2O3/Gr nanocomposites, the surface of samples after the tribology tests were examined utilizing SEM (scanning electron microscopy), as illustrated in 2O3 and graphite particles exhibit a relatively smooth surface. This indicates that the presence of these additive particles improves the nanocomposite surface strength; consequently, the wear and the coefficient of friction for the nanocomposites decreased, even at low-loading content. Moreover, the graphite particles work as a solid lubricant, reducing the shear resistance throughout the tribology test. In 2O3 and graphite is a fatigue delamination mechanism.In order to gain further insights into the improved wear properties of the epoxy/Al2O3 and graphite particles up to 0.2 wt.% resulted in a relatively smooth sample surface with a decrease in microcracks. This change in the wear mode is attributed to micro-plowing, as shown in 2O3 and graphite particles to 0.3, 0.4, and 0.5 wt.% led to an improvement in the bonding strength and hardness of the proposed epoxy nanocomposites. This in turn enhanced the interfacial adhesion among the matrix and the additive particles. The improved interfacial adhesion facilitated the efficient stress–energy transfer and consequently enhanced the wearing out of the sample surface. As a result, Increasing the concentration of Al2O3 nanoparticles and graphite additives on the various properties of epoxy nanocomposites was investigated. The study outcomes can be concluded as follows:An enhancement of 16% in nanocomposite hardness was achieved, due to uniform dispersion of added particles.2O3 and graphite particle concentration from 0.1 to 0.5 wt.% resulted in 33.9% increase in stiffness, relative to pure epoxy.Increased Al2O3 led to 25.97% increase in mechanical strength compared to neat epoxy.Adding 0.5 wt.% of AlThe toughness is enhanced by 25.3% and the ductility by 16.7% in the 0.5 wt.% composite.An enhancement in the tribological performance was achieved where the coefficient of friction was reduced by 20.75%.A noticeable enhancement in the wear resistance under various frictional conditions is achieved.2O3 and graphite particle concentration reduced surface shear resistance, leading to decreased wear in the nanocomposites.The worn surface examination revealed that increased AlIn the presented experimental study, the impact of reinforcing epoxy with a com-bination of Al


The banana quality evaluation system is not sufficiently mature in China and cannot meet the demand of producing high-quality processed banana products. In order to screen banana varieties suitable for low-temperature vacuum frying and extend the banana deep processing industry chain, banana slices from 15 varieties planted in China were prepared by low-temperature vacuum-frying (VF) technology in the present study. After factor analysis on 20 indicators of sensory, flavor, nutritional and processing quality from different varieties of banana slices, comprehensive quality evaluation models were constructed for banana slices. It was concluded that Meishijiao No. 1 had the highest overall score among the 15 banana varieties; hence, it was deemed suitable for processing. Meanwhile, in order to investigate the difference between flavor substances in banana slices before and after processing, a flavor histology study was conducted with solid-phase microextraction (SPME) and comprehensive two-dimensional gas chromatography coupled to time-of flight mass spectrometry (GC×GC-TOFMS). It was found that the content differences of 2,3-pentanedione, hexanal and pentanal may cause the weakened fruitiness and the increased oil flavor of banana taste. Banana is one of the main crops in the tropical and subtropical regions . CurrentThe quality of fruit slices is directly influenced by the variety of raw materials, maturity and processing technology ,9. ThereThe flavor of a banana slice is an indicator for evaluating the quality of banana slices. Generally, during the frying process of food, the formation mechanism and types of volatile metabolites are very complex and are related to the decomposition and interaction of substances inside the food, as well as the frying medium . BananasTherefore, the objective of this study was to comprehensively screen suitable banana varieties for slice processing through building a quality evaluation model, as well as to identify flavor components of banana slices after low-temperature vacuum frying using the SPME and GC×GC-TOFMS method. The research findings can offer useful references in the development of the banana processing industry.w/v), drained and ripened at 20 °C to the ripening stage 5 (the skin color of the bananas was yellow with a trace of green) [The sample information regarding the names and origins of the banana varieties are listed in Chromatographic 2-octanol was obtained from Shanghai Yuanye Biotechnology Co., Ltd. , and 4,7-diphenyl-1,10-phenanthroline was obtained from Shanghai Yuanye Biotechnology Co., Ltd. . Folin phenol and a starch content detection kit were obtained from Beijing Solebar Technology Co., Ltd. . Carbazole was obtained from Shanghai Maclean Biochemical Technology Co., Ltd. . Potassium hydrogen phthalate, trichloroacetic acid, phosphoric acid, ethanol, ferric chloride, ascorbic acid, sodium carbonate, gallic acid, phenol, concentrated sulfuric acid, sucrose, potassium sulfate, copper sulfate pentahydrate, boric acid, hydrochloric acid and galacturonic acid were obtained from Chengdu Kelong Chemical Co., Ltd. . All reagents were analytical reagents.Bananas at ripening stage 5 were washed, peeled and then cut into small pieces. Fresh banana pieces were put into liquid nitrogen for quick freezing, crushed into fresh banana powder at high speed, and then lyophilized  for 48 h to obtain dried fresh banana powder. The powder was stored at −20 °C.Bananas at the ripening stage 5 were washed, peeled, sliced (the thickness was 4 mm) and subjected to color protection with hardening for 5 min. Banana slices were obtained by low-temperature vacuum frying with a vacuum fryer . The vacuum degree was 0 MPa, and the frying temperature was 85 °C. The frying time and the centrifuge time were 50 min and 7 min, respectively. The banana slices were crushed in a high-speed mill , sieved by a 60-mesh sieve and vacuum-packed and frozen at −20 °C. All banana samples were processed in the same way. However, the samples for the determination of sensory quality index were banana slices, and they were not crushed.The color was measured with a spectrophotometer , and the results showed L*, a* and b* values, with L* representing lightness , a* representing red (+) or green (−), and b* representing yellow (+) or blue (−). The L*, a* and b* parameters were can be read directly from the device . HardnesTotal soluble solid (TSS) was determined with a refractometer  . SolubleProtein content was determined using the Kjeldahl method, and a digestion furnace  and Kjeldahl azotometer  were used, with the results presented as % . Starch The degree of expansion was the ratio of banana slice thickness after frying to that before frying, with 10 replicates for each banana sample. The output ratio was the ratio of the quality of banana slices after frying to that before frying (after slicing and before pretreatment). The calculation method of rehydration was the ratio of the weight of banana slices after (they were drained for 5 min after soaking for 30 min) to before soaking. Moisture content was determined with a halogen moisture meter .The SPME—GC×GC-TOFMS method was used to determine flavor substances in the banana ,33,34. Am/z, and ionization energy was 70 eV. The voltage for the detector was 1680 V. The acquisition rate was 50 spectra/s. Mass spectra were probability matched to the NIST  2014 Mass Spectral Library .GC×GC-TOFMS was performed using a comprehensive two-dimensional gas chromatography  coupled to a time-of-flight mass spectrometer  with a TG-WAXMS column  and a DB-17MS column  used as the second column. Helium (purity > 99.9999%) was used as the carrier gas at a flow rate of 1.0 mL/min, and the injector split ratio of the split/splitless injector was set to 1:5. The injection temperature was 250 °C. The initial oven temperature was 40 °C, and this temperature was held for 5 min, then raised to 70 °C at 2 °C/min, raised to 250 °C at 8 °C/min and then held for 7.5 min. The second oven was always 15 °C hotter than the first oven. The modulation period was 4.0 s, and a modulator temperature offset of 15 °C above the second oven was applied. The injector port was heated to 270 °C, and the ion source was maintained at 230 °C. The mass range was 20–450 The factor analysis method was used to analyze the quality indexes of banana slices. All the experiment were repeated at least three times, and the results are expressed as means with standard deviations. SPSS  software and R  software were used for data analysis.The evaluation of the sensory quality of the banana slices is shown in The horizontal analysis of the flavor quality of the banana slices is shown in The horizontal analysis of the nutritional quality of the banana slices is shown in As shown in The complexity of the indicators of banana slices makes analysis difficult. Factor analysis was applied to obtain a few common factors, to explain the variables from the multiple indicators of banana slices in this paper and finally to achieve the idea of dimensionality reduction. Factor analysis was carried out on 14 selected banana slices indicators using SPSS software. The results were shown in The percent of variance for common factor 2 was 18.748%. It gathered information on pectin and b* and a* values indicating color, output ratio, and polyphenols. Polyphenols were significantly correlated with the output ratio (R = −0.547 *), overlapping with common factor 1. The a* value was significantly correlated with b* values (R = 0.524 *), and the b* value had the largest factor loadings. The b* value has practical significance because it can indicate the degree of blue–yellow in banana slices. Therefore, b* values and pectin were chosen as the representative indicators for common factor 2.In common factor 3, the percent of variance was 14.015% and mainly included information on sugar–acid ratio and starch content. The contribution of the sugar–acid ratio was much greater than that of starch, which overlapped with the information on factor 1. Therefore, the sugar–acid ratio was chosen as a representative indicator for the common factor 3.The percent of variance for common factor 4 was 13.232% and was mainly related to Vc and crude fiber contents. Despite the component information Vc factor loadings reaching as high as 0.929, the Vc to output ratio was highly significantly correlated (R = −0.691 **), and the output ratio indicator in the common factor 1 was duplicated. Therefore, crude fiber content was the representative indicator for common factor 4.The percent of variance for common factor 5 was 11.500% and consisted mainly of moisture information with factor loadings up to 0.922. Therefore, moisture was a representative indicator of common factor 5. In summary, b* value, brittleness, sugar–acid ratio, crude fiber, pectin, output ratio and moisture were finally selected as the core indicators for the comprehensive quality evaluation of banana slices, which basically cover the sensory, flavor, nutrition and processing quality of banana slices.comprehensive score = b* value × 0.1742 + brittleness × 0.2201 + sugar-acid ratio × 0.2724 + crude fiber × 0.2093 − pectin × 0.1668 + output ratio×0.1220 + moisture × 0.1689.nQ is the weight value of each index calculated by factor analysis, jnZ is the factor loadings of the j-th common factor of the nth index, jλ is the eigenvalue of the j-th common factor, jE is the ratio of the percent of variance to the cumulative percent of variance for the jth common factor, and m is the number of common factors with eigenvalues greater than 1, with a fixed value 5.With the sensory, flavor, nutritional and processing quality index matrix of banana slices taken as variables, the eigenvalues of common factor, percent of variance and factor loadings of each variable in the corresponding common factor were obtained using factor analysis. Based on the research methods of Jing et al. , the weixyF is the original measured value of the y-th index for the x-th banana slices.To avoid the impact of the different measurement units and data scales for the core indexes of banana slices, the above indexes were converted for data normalization. Among them, the brittleness and moisture of banana slices were negative indicators, with a lower value indicating the better quality of banana slices. The other indexes were positive indicators because a greater proportion of them had better quality. The data of each index were converted according to Formulas (2) and (3). The standardized data of the core indicators of banana slices are shown in The standardized data on the core indexes were substituted into the evaluation model of comprehensive quality to obtain comprehensive scores of all banana slices. Qualitative analysis was then carried out by means of systematic cluster analysis. The suitability of banana slices for processing was divided into five categories, as detailed in A variety of domestically grown bananas were selected, dimensionality reduction analysis was performed on 14 quality indicators through factor analysis, 7 core indicators were obtained, and these covered the quality of banana slices such as color and taste. These indicators can comprehensively reflect the comprehensive quality of slices and can predict the processing suitability of unknown banana varieties. Thus far, little research has been conducted on the suitability of banana processing; rather, experts from China and other countries have conducted suitability studies on other crops, such as pear , peach , citrus The formation of banana aroma substances is closely related to its variety, cultivation environment, maturity and storage method ,42,43. TOrthogonal projections to latent structures discriminant analysis (OPLS-DA) was carried out based on the obtained data to reveal the distribution of the samples, the score of which plots are shown in 2X was 0.929, the R2Y was 0.999, and the Q2 was 0.932. The predictability of the model and the interpretability of independent variable and dependent variable were higher than 0.900. This indicated that the model was stable and the data were reliable. OPLS-DA could reduce the complexity and improve the interpretation ability of the model, and it maximized the differences between data groups. However, it could lead to the overfitting problem of the OPLS-DA model. Therefore, the data needed to be replaced to evaluate the current model fitting phenomenon, with the specific replacement inspection diagram being seen in 2 intercept value is  and Q2 intercept value is . All the blue points on the left are lower than the original blue points on the far right of Q2, indicating that OPLS-DA model had no overfitting and was an effective model, which could be used to screen different flavor substances in this study.In OPLS-DA, the Rp < 0.01), the change range of VIP was 1.03~5.31, which indicated that the substances changed significantly before and after VF of banana, and had an important impact on the flavor change of bananas before and after processing.Generally, VIP could be used to measure the influence of the expression pattern of each metabolite on the classification and discrimination of each group of samples and the explanatory power. According to the VIP size, the variable (characteristic peak) could explain the importance of the X dataset and the associated Y dataset. The variable with VIP > 1 was used as one of the screening conditions for potential biomarkers. As shown in The heatmap and cluster analysis of key different flavor substances in bananas before and after VF are shown in By comparing the 79 obtained compounds described in Many studies have been conducted on the changes in volatile components during banana ripening and on the volatile components in banana powder ,46, banaIn this study, the comprehensive quality of banana slices was evaluated using factor analysis, and the evaluation model for the comprehensive quality of banana slices was established. The most suitable banana variety for processing was Meishijiao No. 1, followed by Guijiao No. 6 and Guijiao No. 2. Through the GC×GC-TOFMS method, the change of banana flavor composition before and after VF was explored, and it was found that the key substances affecting the sensory flavor of VF bananas might be 2,3-pentanedione, hexanal and pentanal. This study offers a theoretical foundation for the adjustment of banana sensory flavor and the optimization of VF process conditions. Further studies on the mechanism of flavor change of banana before and after frying should be pursued.


PRMT5 is overexpressed and activated in various human cancers, including breast cancer. This study aims to dissect the mechanism underlying how PRMT5 is dysregulated in cancers. Our results demonstrate that TRAF6-mediated ubiquitination plays an important role in the regulation of PRMT5 activity and cell proliferation. Thus, inhibition of TRAF6 is a possible strategy for improving PRMT5 targeted therapy.Protein arginine methyltransferase 5 (PRMT5) is the primary enzyme generating symmetric dimethylarginine (sDMA) on numerous substrates, through which it regulates many cellular processes, such as transcription and DNA repair. Aberrant expression and activation of PRMT5 is frequently observed in various human cancers and associated with poor prognosis and survival. However, the regulatory mechanisms of PRMT5 remain poorly understood. Here, we report that TRAF6 serves as an upstream E3 ubiquitin ligase to promote PRMT5 ubiquitination and activation. We find that TRAF6 catalyzes K63-linked ubiquitination of PRMT5 and interacts with PRMT5 in a TRAF6-binding-motif-dependent manner. Moreover, we identify six lysine residues located at the N-terminus as the primarily ubiquitinated sites. Disruption of TRAF6-mediated ubiquitination decreases PRMT5 methyltransferase activity towards H4R3 in part by impairing PRMT5 interaction with its co-factor MEP50. As a result, mutating the TRAF6-binding motifs or the six lysine residues significantly suppresses cell proliferation and tumor growth. Lastly, we show that TRAF6 inhibitor enhances cellular sensitivity to PRMT5 inhibitor. Therefore, our study reveals a critical regulatory mechanism of PRMT5 in cancers. Arginine methylation is acknowledged as one of the most common post-translational modifications (PTMs). It occurs on approximately 7% of all arginine residues in arginine-methylated proteins, which is comparable to phosphorylation at 9% of serine residues and ubiquitination at 7% of lysine residues ,2,3,4. EProtein arginine methyltransferases (PRMTs) are the writers responsible for transferring the methyl group to the guanidino nitrogen atoms of arginine, forming three types of methylated arginine residue: monomethylarginine (MMA), asymmetric dimethylarginine (aDMA), and symmetric dimethylarginine (sDMA) ,11. In mPRMT5 is the main type II enzyme that deposits the sDMA on histones and non-histone substrates . PRMT5 wUbiquitination is another of the most common PTMs which covalently conjugates the small protein ubiquitin (Ub) to the lysine residue through a sequential enzymatic cascade consisting of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and E3 ubiquitin ligases (E3s) ,20. UbiqHere, we investigated the interplay between PRMT5 and ubiquitination. Our findings demonstrate that TRAF6 catalyzes non-proteolytic K63-linked ubiquitination of PRMT5 and consequently promotes PRMT5 activity and its role in cell proliferation and tumorigenesis.HEK293T, MCF-7, and MDA-MB-231 cells were obtained from American Type Culture Collection. All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. GSK3326595 (HY-101563) and C25-140 (HY-120934) were purchased from MedChemExpress .For protein expression in cells, transfection was performed using Lipofectamine 3000 and Plus reagents following manufacturer’s instructions. For lentiviral virus production, 293T cells were transfected with target and packaging plasmids (pMD2G and psPAX2) with Polyethylenimine (PEI). Twenty-four hours post-transfection, fresh medium was replaced. Virus-containing supernatants were harvested at forty-eight hours post-transfection and filtered with 0.45 μm PES filter. Targeted cells were infected with viruses and selected with hygromycin (200 μg/mL), puromycin (2 μg/mL), or blasticidin (10 μg/mL) for 4 days to eliminate the non-infected cells.https://www.synthego.com (accessed on 13 March 2023) and were cloned into the lentiCRISPR v2 vector . PRMT5 sgRNA was described previously ) and sonicated. The lysates were incubated with nickel–nitrilotriacetic acid (Ni-NTA) matrices  for three hours at room temperature. The His pull-down products were washed twice with buffer A, twice with buffer A/TI (1 volume buffer A and 3 volumes buffer TI), and one time with buffer TI (25 mM Tris-HCl and 20 mM imidazole [pH 6.8]). The pull-down proteins were resolved by SDS-PAGE for immunoblotting.In vivo ubiquitination assay was performed as previously described . Cells w2, 1 mM β-mercaptoethanol, and 100 mM sucrose) with 1 μL S-adenosylmethionine  at 30 °C for 1 h, stopped by adding 3× SDS loading buffer, resolved by SDS-PAGE, and immunoblotted with H4R3me2s antibody.In vitro methylation assays were performed as previously described . BrieflyCells were seeded in 96-well plates (1000–2000 cells/well) for 24 h and treated with TRAF6 inhibitor and/or PRMT5 inhibitor for 96 h. Assays were performed using the Cell Titer-Glo luminescent cell viability assay kit according to the manufacturer’s instructions .Cells were seeded in 6-well plates (200–300 cells/well) and cultured for 10 to 15 days until formation of visible colonies. Colonies were washed with PBS and fixed with 10% acetic acid/10% methanol for 20 min, then stained with 0.4% crystal violet for 20 min. After staining, the plates were washed with distilled water and air-dried. The colonies were counted manually.L × W2 × 0.5, where L is the longest diameter and W is the shortest diameter. Mice were euthanized at day 23 and the solid tumors were dissected. All mice are housed in 22 °C, 50–60% humidity, and a 12 h light/12 h dark cycle. All mouse experiments were conducted under Protocol IACUC-2018-00604-1 approved by the MUSC Institutional Animal Care and Use Committee.MDA-MB-231 cells depleted of endogenous PRMT5 and then stably expressing PRMT5-WT or PRMT5-3A mutants were injected into the flank of 5-week-old female nude mice . Tumor size was measured every other day with an electronic caliper. The tumor volume was calculated by t test or two-way analysis of variance (ANOVA). p < 0.05 was considered significant.All quantitative data are presented as the means ± SD of three biologically independent experiments. Statistical analyses were performed using GraphPad Prism 9 . Significance was determined by two-tailed Student’s Ubiquitination is one of the most common PTMs and serves as a key mechanism controlling protein functions . HoweverTo further validate TRAF6 is the E3 ubiquitin ligase of PRMT5, we examined their interactions by immunoprecipitation assays. In agreement with TRAF6 promoting PRMT5 ubiquitination, TRAF6, but not other members of the TRAF family, interacted with PRMT5 A. We furIntegrated proteomic analysis by mass spectrometry identified at least 13 lysine (K) residues of PRMT5 being ubiquitinated . DeletioGiven that ubiquitination is a key mechanism regulating enzymatic activity ,22, we wWe then aimed to understand the molecular mechanism by which TRAF6-mediated ubiquitination triggers PRMT5 activation. Generally, K63-linked ubiquitination regulates protein interaction or cellular localization, controlling protein activity and substrate specificity in turn . StructuOur previous study demonstrated that depletion of PRMT5 significantly suppressed breast cancer cell proliferation and tumor growth . To invePRMT5 gene [A growing amount of evidence reveals that PRMT5 is overexpressed or activated in a variety of cancers . Whole-gMT5 gene , suggestMT5 gene ,37. NotaMT5 gene . In contMT5 gene . MoreoveMT5 gene . AdditioMT5 gene ,42. AsidMT5 gene . In the current study, we identified that TRAF6 acts as an E3 ubiquitin ligase to catalyze non-proteolytic K63-linked ubiquitination of PRMT5 and subsequently promote PRMT5 activation. Our findings elucidate a novel ubiquitination-dependent regulatory mechanism of PRMT5. We are aware that additional evidence, such as in vitro ubiquitination data, is required to further support TRAF6 as a direct E3 ubiquitin ligase of PRMT5. Moreover, as a versatile protein, PRMT5 methylates numbers of substrates to regulate many fundamental cellular processes, including DNA repair, transcription, and cell signaling transduction. Further studies are warranted to investigate the impacts of TRAF6-mediated ubiquitination in regulating these cellular processes downstream of PRMT5. For example, it will be critical to explore how PRMT5 ubiquitination affects H4R3me2s-mediated transcription. Furthermore, growing evidence indicates that a protein can be modified by various PTMs simultaneously to orchestrate the protein function . It woulTRAF6 belongs to the tumor necrosis factor receptor-associated factor (TRAF) family and functions as either an adaptor protein or E3 ligase to regulate cell signaling transduction, such as the NF-κB pathway , AKT sigBoth PRMT5 and TRAF6 are frequently overexpressed in various types of human cancers, including breast cancer, colorectal cancer, gastric cancer . Their hTRAF6 interacts with and catalyzes K63-linked ubiquitination of PRMT5, which in turn promotes PRMT5-MEP50 complex formation, activation, cell proliferation, and tumor growth L. These