APC and CTNNB1. Collectively, our data identifies a previously unappreciated role for the HBV pre-core protein p22 in elevating Wnt signaling. Understanding the molecular mechanisms of p22 activity will provide insight into how Wnt signaling is fine-tuned in cancer.An emerging theme for Wnt-addicted cancers is that the pathway is regulated at multiple steps via various mechanisms. Infection with hepatitis B virus (HBV) is a major risk factor for liver cancer, as is deregulated Wnt signaling, however, the interaction between these two causes is poorly understood. To investigate this interaction, we screened the effect of the various HBV proteins for their effect on Wnt/β-catenin signaling and identified the pre-core protein p22 as a novel and potent activator of TCF/β-catenin transcription. The effect of p22 on TCF/β-catenin transcription was dose dependent and inhibited by dominant-negative TCF4. HBV p22 activated synthetic and native Wnt target gene promoter reporters, and TCF/β-catenin target gene expression in vivo. Importantly, HBV p22 activated Wnt signaling on its own and in addition to Wnt or β-catenin induced Wnt signaling. Furthermore, HBV p22 elevated TCF/β-catenin transcription above constitutive activation in colon cancer cells due to mutations in downstream genes of the Wnt pathway, namely  CTNNB1) gene, the gene that codes for β-catenin, occurs in up to 40% of cases making it the most frequent mutation in HCC  , mouse anti-αTubulin , mouse anti-active β-catenin  and mouse anti-β-actin . Secondary antibody was rabbit anti-mouse polyclonal antibody HRP . Cells were seeded into two-well Nunc Lab-Tek (Thermofisher) chamber slides to reach 60–70% confluence overnight. Cells were transfected with 200 ng plasmid as described above. After 48 h, cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton-X100 and blocked with 1% FBS and stained with control antibody or anti-HBcAg [C1]  primary antibody and detected with goat anti-mouse alexa fluor 488 (1:1000 Invitrogen A11029). DAPI (1:2000) was used for nuclear staining and the cells analyzed using Zeiss LSM700 as previously described .n is at least three independent experiments with cell lines or tissue from at least three mice per cohort, unless stated otherwise. The Student t-test was used for comparisons and significance was defined as p < 0.5.The data represent mean ± SEM, where APC and CTNNB1 are the most frequent mutations in colon and liver cancer, respectively, and are thought to initiate cancer. Here we demonstrate that the HBV precore protein p22 can activate Wnt signaling in these cancer contexts. The ability of p22 to additionally activate Wnt signaling in the context of these mutations indicates oncogenic interplay between HBV infection and Wnt signaling in liver cancer. Furthermore, it is now clear that Wnt-addicted cancers harbor aberrations to Wnt signaling via both intracellular and cell-surface mechanisms [Mutations to chanisms , thus ouchanisms ,25. Unde


S‐EX consisted of a once‐off 40‐min moderate‐intensity bout without rest interval. To evaluate IC, the color‐word Stroop task was administered before exercise, immediately after exercise, and every 10 min during the 30‐min postexercise recovery period. The reverse‐Stroop interference score, which is a parameter of IC, significantly decreased immediately after both R‐EX and S‐EX compared with that before each exercise (both Ps < 0.05). The degree of changes in IC following exercise did not differ between the two protocols. By contrast, the results of the present study showed that R‐EX may have more beneficial effects on cardiac and perceptual responses than S‐EX. Therefore, the present study determined that R‐EX changes postexercise IC similar to S‐EX. We suggest that R‐EX can be used as safe and effective exercise protocol to improve cognitive function in various populations.We previously demonstrated that duration of aerobic exercise plays an important role in improving cognitive inhibitory control (IC). Repeated bouts of aerobic exercise (R‐EX), which are performed with a rest interval, is a useful strategy in improving physical health parameters in similar manners to a single bout of aerobic exercise (S‐EX). However, whether R‐EX would be effective in improving IC remains unknown. This study compared the effect of R‐EX versus S‐EX of moderate‐intensity exercise on postexercise IC. Twenty healthy, young males performed both R‐EX and S‐EX in a crossover design. R‐EX consisted of two 20‐min moderate‐intensity bouts (60% of peak oxygen consumption) for 20 min Aerobic exercise improves postexercise cognitive function. Effective aerobic exercise protocols for improving cognitive function are poorly understood. A repeated bout protocol of aerobic exercise can be used as safe and effective exercise protocol to improve cognitive functioin. Prior to this study, we calculated the required sample size utilizing an effect size (0.30), an α‐level of 0.05, and a β‐level of 0.2 (80% power), based on the data of our previous study  for 20 min, which were separated by a 20‐min sitting rest. S‐EX consisted of a once‐off 40‐min bout of moderate‐intensity exercise. Mean value of exercise volume for both protocols was 355 ± 9 kJ.Experimental procedures of R‐EX and S‐EX protocols are presented in Figure 2.32 peak, which was used to calculate the workload of moderate‐intensity exercise for both R‐EX and S‐EX.All subjects completed a familiarization visit where they practiced the three types of the color‐word Stroop task (CWST) for each a minimum of 10 times until they achieved consistent scores. During this visit, the subjects also performed a ramp‐incremental test on a cycle ergometer to determine their VOOn the day of the experiment, the subjects practiced the three CWST types for each a minimum of five times before experimental session to minimize learning effect. Then, the subjects rested for 5 min before undergoing measurements of cardiovascular parameters and blood metabolites. Once this was completed, the subjects performed the CWST before exercise session . In order to ensure consistency in time intervals in both protocols between CWSTs before and after exercise session, S‐EX included a 20‐min rest period in the sitting position following the preexercise CWST before exercise session.Subjects completed either R‐EX or S‐EX. The CWST was performed again immediately after completion of the exercise session and then was repeated three times at 10‐min intervals during the 30‐min postexercise recovery period to evaluate the sustainable effect of postexercise IC improvements induced by R‐EX and S‐EX, as in our previous studies  and perceived exertion  parameters were measured eight times during both R‐EX and S‐EX. During the R‐EX, cardiovascular and perceived exertion parameters were measured every 5 min  over each 20‐min exercise bout. During the S‐EX, cardiovascular and perceived exertion parameters were measured every 5 min throughout a once‐off 40‐min exercise bout . Additionally, cardiovascular and blood metabolites parameters were measured immediately before all five CWSTs to assess the effects of cardiovascular and metabolic condition on IC. Felt arousal scale (FAS) and visual analog scale (VAS) were measured immediately after the five CWSTs to assess the effect of psychological conditions on IC.2.42 peak on a cycle ergometer, as previously described . Systolic blood pressure and diastolic blood pressure were measured using a mercury manometer . MAP was calculated as ([systolic blood pressure−diastolic blood pressure]/3 + diastolic blood pressure).2.6Blood glucose and lactate levels were measured using a glucose  and lactate analyzer , respectively.2.7The Borg's RPE scale was measured to assess perceived exertion expended during exercise, and this scale was ranged from 6 (no exertion) to 20  Borg, . The Bor2.8FAS was measured to assess arousal during the CWST, and this scale is a 6‐point, single‐item scale ranging from 1  to 6   and perceptual  responses during R‐EX and S‐EX were compared using a paired Student's t test. Changes in measured variables throughout experimental session between the two conditions were analyzed using two‐way (condition × time) repeated‐measures analysis of variance. If the sphericity assumption was not met, Greenhouse–Geisser corrections were used. Specific differences were identified with a paired Student's t test or a Bonferroni post‐hoc test. The statistical significance level was defined at p < .05. All statistical analyses were conducted using IBM SPSS software .The data are expressed as the mean ± d effect size using the pooled standard deviation was calculated to determine the magnitude of a difference in the reverse‐Stroop interference score between before  and after  R‐EX and S‐EX. The Cohen's d effect size were interpreted as small (0.20–0.49), moderate (0.50–0.79), and large (>0.80)  values were determined as a measure of the effect size for main effects of condition and time or interaction effect.Cohen's ) Cohen, . Partial33.1p = .041, d = 0.53).Mean values of HR (149.7 ± 2.1 and 153.2 ± 2.8 bpm) and MAP (95.0 ± 1.4 and 97.2 ± 1.7 mmHg) throughout exercise did not differ significantly between R‐EX and S‐EX. Similarly, mean values of RPE (13.6 ± 0.3 and 13.9 ± 0.3) and lower limb discomfort (4.9 ± 0.3 and 5.0 ± 0.3) throughout exercise did not differ significantly between R‐EX and S‐EX. However, mean RPE during the second bout of R‐EX was significantly lower than that during the latter 20‐min of S‐EX  = 1105.21, p < .001, ηp2 = 0.98) and a significant interaction effect  = 3.80, p = .024, ηp2 = 0.18). A trend against significance was also obtained in main effect for condition  = 3.40, p = .081, ηp2 = 0.15). HR significantly increased immediately after R‐EX and S‐EX compared with that before each exercise , and the increased HR remained significant until the 30‐min postexercise recovery period for both protocols . Although HR immediately after exercise did not differ significantly between R‐EX and S‐EX , HR at the 10‐min postexercise recovery period was significantly lower for R‐EX than for S‐EX . MAP analysis revealed a significant main effect for time  = 53.89, p < .001, ηp2 = 0.74). MAP significantly increased immediately after R‐EX and S‐EX compared with that before each exercise .Changes in physiological variables throughout R‐EX and S‐EX sessions are shown in Table F = 17.92, p < .001, ηp2 = 0.49). Blood glucose significantly decreased immediately after R‐EX compared with that before exercise , the decreased blood glucose remained significant until the 30‐min postexercise recovery period for this protocol . Blood glucose immediately after S‐EX was a lower trend than that before exercise , but did not reach statistical significance. Blood glucose levels at 20 and 30 min after S‐EX were significantly lower than that before exercise . Blood lactate analysis revealed a significant main effect for time  = 51.85, p < .001, ηp2 = 0.73) and a significant interaction effect  = 4.02, p = .033, ηp2 = 0.17). Blood lactate significantly increased immediately after R‐EX and S‐EX compared with that before each exercise , and the increased blood lactate remained significant until the 10‐min postexercise recovery period for both protocols . Blood lactate immediately after exercise was a lower trend for R‐EX than for S‐EX , but did not reach statistical significance.With regard to blood metabolite responses, blood glucose analysis revealed a significant main effect for time  = 2.85, p = .030, pη2 = 0.13 for congruent task; F = 11.80, p < .001, ηp2 = 0.38 for neutral task; F = 28.04, p < .001, ηp2 = 0.60 for incongruent task). Congruent reaction time significantly shortened immediately after only an R‐EX compared with that before exercise . Neutral and incongruent reaction times significantly shortened immediately after R‐EX and S‐EX compared with those before each exercise , the decreased neutral and incongruent reaction times remained until the 10‐min postexercise recovery period . Response accuracy analyses for all three tasks revealed no significant main effects for time and condition or no significant interaction effects.Changes in reaction times and response accuracies on three types of the CWST throughout R‐EX and S‐EX sessions are summarized in Table F = 5.10, p = .001, ηp2 = 0.21). The reverse‐Stroop interference score significantly decreased immediately after R‐EX and S‐EX compared with that before each exercise .Changes in the reverse‐Stroop interference score  throughout R‐EX and S‐EX sessions are presented in Figure 3.4F = 6.78, p = .017, ηp2 = 0.26) and time  = 17.51, p < .001, ηp2 = 0.48) and a significant interaction effect  = 3.44, p = .040, ηp2 = 0.15). Arousal significantly increased immediately after R‐EX and S‐EX compared with that before each exercise , and the increased arousal remained until the 10‐min postexercise recovery period in only an S‐EX . Arousal levels immediately and 10 min after exercise were significantly lower for R‐EX than for S‐EX . All three VAS‐measured psychological conditions analyses revealed significant effects for time  = 27.47, p < .001, ηp2 = 0.59 for mental fatigue; F = 7.11, p < .001, ηp2 = 0.27 for concentration; F = 3.75, p = .008, ηp2 = 0.17 for motivation). Mental fatigue significantly increased immediately after R‐EX and S‐EX compared with that before exercise , and the increased mental fatigue remained until the 30‐min postexercise recovery period for both protocols . There were no significant differences for concentration and motivation between conditions or time points throughout experimental session.Changes in psychological conditions throughout R‐EX and S‐EX sessions are shown in Table 4In this study, we expected that R‐EX would be more effective in changing postexercise IC than S‐EX because of an additive effect induced by the two bouts of exercise. Unexpectedly, the primary finding of the present study was that although the IC  significantly changed following R‐EX and S‐EX, the degree of changes in IC throughout the experimental session did not differ between the two protocols. In a potential explanation of this finding, R‐EX was separated by a 20‐min rest interval, and thus, the rest interval caused a wash‐out of the IC changes induced by the first bout of exercise. Our previous study determined that although postexercise IC improvements induced by moderate‐intensity exercise were slightly more sustained following a 40‐min bout than a 20‐min bout of exercise, the degree of the exercise‐induced IC improvements throughout the 30‐min postexercise recovery period did not differ between the two duration protocols . The faster HR recovery after R‐EX than that of S‐EX may be beneficial in reducing the likelihood of a cardiovascular event following exercise in older individuals and patients with chronic diseases  has no effect on changes in IC (Tsukamoto, Suga, et al., In a perspective, Goto et al.  found th5This study is the first to determine the potential impact of R‐EX on cognitive function. The present study determined that the degree of changes in IC following exercise was similar between R‐EX and S‐EX. In addition, the findings of the present study and those of previous studies suggest that R‐EX may have more beneficial effects on certain factors, such as cardiovascular and perceptual responses, than S‐EX. Therefore, we conclude that R‐EX can be used as a safe and effective exercise protocol to improve cognitive function, in addition to physical function in various populations.The authors declare no conflict of interest.Take S. and Tada S. conceived and designed the experiment, analyzed the data, and wrote the manuscript. Take S., Tada S., H.T., K.T., and K.D. performed experiments. Take.S., Tada.S., H.T., K.T., K.D., T.H., and T.I. interpreted results of experiments. T.H. and T.I. edited and revised the manuscript. All authors have read and approved the manuscript.


PRMT5 gene was transcriptionally activated by YY1. Collectively, our results unravelled the important role of PRMT5 in laryngeal cancer tumorigenesis and metastasis. The dysregulation YY1/PRMT5/LATS2/YAP axis may contribute to laryngeal cancer progression; thus, PRMT5 may be a potential therapeutic strategy for patients with laryngeal cancer.Metastases lead to high mortality in laryngeal cancer, but the regulation of its underlying mechanisms remains elusive. We identified Protein arginine methyltransferase 5 (PRMT5) was significantly up‐regulated in laryngeal cancer tissues, which predicts poor patient prognosis. Functional assays demonstrated that PRMT5 overexpression promoted the invasive capacity and lymph node metastasis in vitro and in vivo. Mechanistic experiments suggested that LATS2 was a downstream target of PRMT5. PRMT5 inhibition increased the expression of LATS2 and YAP phosphorylation in laryngeal cancer cells, thereby promoting laryngeal cancer metastasis. Furthermore, informatics and experimental data confirmed that  Accumulating evidence shows that PRMT5 is overexpressed in several human cancers and correlates with poor prognosis.Transcription factors (TFs) play a leading role in cancer initiation.The Hippo pathway is highly conserved and has multiple biological functions in regulating organ size, tissue homeostasis and the regeneration of tissues.In the present study, we investigated the role of PRMT5 in regulating laryngeal cancer progression and metastasis. We found that PRMT5 was transcriptionally activated by YY1 and triggered proliferation and invasion via inhibiting LATS2 and phos‐YAP of Hippo signalling pathway in laryngeal cancer cells. Our results demonstrate that inhibition of PRMT5 could represent an promising therapeutic stragety for the treatment of laryngeal cancer.22.1https://portal.gdc.cancer.gov/projects/). This study was conducted according to the Declaration of Helsinki.Fresh tissues from 19 laryngeal cancer specimens were collected for determination of mRNA and protein levels of PRMT5 from the Sun Yat‐sen University Cancer Center of Sun Yat‐sen University. Informed consent was obtained from the patients before the study began, none of whom had received chemotherapy or radiotherapy prior to surgery. Another archival formalin‐fixed paraffin‐embedded tissues with primary and lymph node metastatic tumour and normal tissues from 80 patients, who underwent laryngectomy and/or metastasectomy, were collected from January 2013 to August 2016 in Medical College of Jiaying University. None of the patients received radiotherapy or/and chemotherapy before surgery. The clinicopathological information was retrieved and summarized. Laryngeal cancer patients gene expression was obtained from the TCGA Data Portal website . CAL‐27, HOK and SCC‐15 cells were obtained from Beina Biological Technology . All cell lines were validated by STR (short tandem repeat) assays and cultured in RPMI 1640  supplemented with 10% FBS  and 1% penicillin‐streptomycin solution  in a humidified atmosphere of 5% CO2 at 37°C.2.2PRMT5 short hairpin RNA (shRNA) and overexpression lentivirus were cloned into the pCDH‐CMV‐MCS‐EF1‐Puro or pEZ‐Lv217.1‐Luc‐Puro vectors, respectively. PRMT5‐targeting shRNAs oligonucleotide sequences are listed in Table 2.3The SYBR green qPCR Master Kit (Thermo Fisher Scientific) was used for PCR amplification. Total RNA was isolated and reverse‐transcribed according to the protocol as previously described.2.4Western blot analysis was performed as previously described.2.5Immunohistochemistry (IHC) was performed to detect PRMT5, E‐cadherin, Vimentin, MMP9, YY1 and Ki‐67 expression. Paraffin‐embedded tumour tissues were deparaffinization and antigen‐retrieval, sectioned and stained with PRMT5 , E‐cadherin , Vimentin , MMP9 , YY1  antibodies at 4 ℃ overnight, respectively. Then, after washed three times with PBS, the sections were incubated with secondary antibody at room temperature for 2 hours. The immunostaining was visualized by diaminobenzidine staining solution . The expression of PRMT5 was evaluated and quantified by two independent pathologists according to a staining scoring system. Five microscopic visual fields (X200) were randomly selected from each slice to ensure representation of the sample. Photographs were taken with Olympus BX53 Microscope system. The median IHC scores >4 were classified as high expression, and scores of 0‐4 were classified as low expression.2.6p value that was more than 0.05. Pathway analysis was used to elucidate the significant pathway according to the KEGG database.Total RNAs were extracted from laryngeal cancer cells using TRIzol reagent (Invitrogen). cDNA library construction and high‐throughput sequencing results analysis were performed by Shanghai Sinotech Genomics. The differentially expressed genes were screened by the threshold of 1.5‐fold change and 2.73/well cells were seeded in 96‐well plates at 37℃ for 24 hours. The proliferation absorbance at 450 nm was measured using a Spark® multimode microplate reader . Experiments were repeated three times. For cell cycle assay were performed with propidium iodide‐stained laryngeal cancer cells by flow cytometry  and analysed using the Modfit software.The CCK8 assay was performed to detect cell proliferation. For transient transfection experiments, 1 × 102.84 cells were suspended in serum‐free medium and seeded into the upper chambers  with or without precoated matrigel (BD Biosciences). 500 μL RPMI‐1640 medium containing 20% foetal bovine serum (FBS) was added to the lower chamber of each well. After 36 hours incubation, cells in the upper chamber were removed using a cotton swab and cells located on the underside of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution (Beyotime Biotechnology). Five randomly selected fields at ×200 magnification were imaged and counted. Three independent experiments were carried out for this.For in vitro invasion and migration assays, 5 × 102.9PRMT5 promoter activity. Briefly, cells on a 24 well plate were co‐transfected with the firefly luciferase reporter (50 ng) along with the Renilla luciferase reporter (Promega) (20 ng) for 24 hours using Lipofectamine 3000 according to the protocol provided by manufacturers. The luciferase activity was measured in cellular extracts using a Dual‐luciferase Reporter Assay Kit (Promega). The luciferase activity of each well was normalized to Renilla luciferase activity. The experiment was repeated in triplicate.A dual‐luciferase assay was performed to confirm the impact of YY1 on 2.106 cells Tu686 cells with or without PRMT5 suppression were inoculated into the right lower footpads of BALB/c‐nu mice (4‐5 weeks of age). The tumour growth and metastasis were monitored and imaged by injecting D‐luciferin substrate (Promega) and Bruker In‐Vivo FX PRO System  every one week after injection. Six weeks later, the mice were killed and the tumours were embedded in paraffin for later detection. To evaluate the effect of PRMT5 on tumorigenesis, BALB/c‐nu mice were subcutaneously injected 1 × 107 cells into the right flank. The tumour size was measured every 3 days, and tumour volume was calculated as ( width2 × length)/2. On day 30, the mice were killed and tumour tissues were collected, fixed in 4% paraformaldehyde and embedded in paraffin. The animal experiments were approved by the Institute Animal Care and Use Committee of Cancer center of Sun Yat‐sen University Cancer Center according to the protocols.For lymphatic metastasis, 2 × 102.11Cells were grown to 90% confluence and then treated with 1% formaldehyde to cross‐link proteins to DNA. The crosslinking, immunoprecipitation, washing, elution, reverse crosslinking and proteinases K treatment were performed according to the manufacturer's directions described in the Simple ChIP Enzymatic Chromatin IP Kit #9003 . Purified immunoprecipitated DNA was used for real‐time qPCR. Primers for ChIP PCR were as follows: sense CGAGACCAGCCTGACCAACA, antisense CTTCCAGCAACCAATCAGAT.2.12P < .05, **P < .01 and ***P < .001.The data are presented as the mean ± SD from three independent experiments. The GraphPad Prism 6 software  was used for statistical analysis. Comparisons between two independent groups were analysed with two‐tailed Student's t test. Statistically significance was assigned at *33.1high and PRMT5 low) according to the median IHC score of PRMT5 expression (4.0). The correlations between the clinicopathologic features of laryngeal cancer patients and PRMT5 were summarized in supplementary Table To understand the expression profile of PRMT5 in laryngeal cancer, we first assessed the expression of PRMT5 in fresh laryngeal cancer tissues and paired non‐cancerous tissues. The mRNA level of PRMT5 was frequently up‐regulated in 13 of the 19 paired tissues Figure . The up‐3.2PRMT5, which could efficiently reduce the endogenous expression of PRMT5 in Tu686 and CAL‐27 of laryngeal cancer cells  achieved the same reduction in Figure To ascertain the function of PRMT5 in laryngeal cancer, we performed Western blot analysis in a panel of cell lines, which included CAL‐27, Tu212, Tu686, SCC‐15 and human oral keratinocytes (HOK). As shown in Figure s Figure . As illuTo study the effect of PRMT5 knockdown in vivo, we designed a popliteal lymph node metastasis model in nude mice. A stable PRMT5 down‐regulation Tu686 cells with stably expressing firefly luciferase were inoculated into the footpads of nude mice. After six weeks, shPRMT5‐Luc cells effectively inhibited the metastasis to the popliteal lymph node through bioluminescence image scanning Figure . In addi3.3Gain‐of‐function study was performed by stably transfecting PRMT5 expression vectors into Tu212 and SCC‐15 cells. Ectopic expression of PRMT5 in these cells, as shown in Figure 3.4PRMT5  assay was used to verify whether YY1 binds directly to the PRMT5 promoter (located at −1671~‐1660 bp) in Tu212 and SCC‐15 cells, as shown in Figure We next investigate the mechanism underlying transcriptional activation of PRMT5 in laryngeal cancer cells. As predicted by the JASPAR database,5 Figure . In addi5 Figure . TherefoIn addition, IHC was performed to assess PRMT5 and YY1 expression in the context of laryngeal cancer tissues. The results indicated that YY1 was markedly increased in PRMT5‐positive laryngeal cancer tissues Figure . H‐scori3.5Given that EMT is one of the hallmarks during cancer metastasis. Previous studies revealed that YY1 can act as a metastasis inducer and promote EMT in hepatocellular cancer.PRMT5 targets the Hippo signalling pathway to regulate laryngeal cancer metastasis.PRMT5 expression was negatively correlated with LATS2 expression pathway enrichment analysis showed that the Hippo signalling pathway was enriched upon PRMT5 overexpressed Figure . MeanwhionFigure . To furtonFigure . MoreoveonFigure . These f4Most cancer‐related deaths are caused by tumour metastasis.PRMT5 is a member of the PRMTs family, which methylates arginines in both histones and non‐histone proteins.PRMT5 and promoted the expression of PRMT5 to trigger EMT. In this YY1/PRMT5 axis, how do they bind to each other still needs further verification.Transcription factors (TFs) are considered to play a key role in regulating gene expression. Abnormally expressing TFs are found in many types of cancers and gained increasing attention. YY1 itself is functionally diverse because it can act as an activator or inhibitor, which is based on the tumour type and its interacting partners.et al indicated that metastasis of head and neck cancer related to the hippo pathway.Poma In summary, this study showed PRMT5 promoted tumour metastasis and correlated with a poor prognosis in laryngeal cancer. The underlying mechanism reveals YY1/PRMT5 axis enhances laryngeal cancer cell growth and invasive capacity via LATS2 and YAP signalling pathway Figure . TherefoThe authors have declared that no competing interest exists.Nan Wang: Data curation (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Di Wu: Formal analysis ; Writing‐original draft . Qian Long: Formal analysis (supporting); Methodology ; Software ; Writing‐original draft . Yue Yan: Formal analysis (supporting); Investigation (supporting); Software (supporting). Xiaoqi Chen: Methodology ; Software (supporting); Validation . Zheng Zhao: Methodology ; Resources ; Validation (supporting). Honghong Yan: Validation . Xinrui Zhang: Investigation (supporting); Software ; Validation (supporting). Meilan Xu: Formal analysis ; Investigation ; Validation (supporting). Wuguo Deng: Supervision ; Writing‐review & editing . Xuekui Liu: Funding acquisition (lead); Project administration ; Supervision ; Writing‐original draft (supporting); Writing‐review & editing .Figure S1Click here for additional data file.Figure S2Click here for additional data file.Table S1Click here for additional data file.Figure LegendsClick here for additional data file.


Arginine methylation is an essential post-translational modification (PTM) deposited by protein arginine methyltransferases (PRMTs) and recognized by Tudor domain-containing proteins. Of the nine mammalian PRMTs, PRMT5 is the primary enzyme responsible for the deposition of symmetric arginine methylation marks in cells. The staphylococcal nuclease and Tudor domain-containing 1 (SND1) effector protein is a key reader of the marks deposited by PRMT5. Both PRMT5 and SND1 are broadly expressed and their deregulation is reported to be associated with a range of disease phenotypes, including cancer. Hepatocellular carcinoma (HCC) is an example of a cancer type that often displays elevated PRMT5 and SND1 levels, and there is evidence that hyperactivation of this axis is oncogenic. Importantly, this pathway can be tempered with small-molecule inhibitors that target PRMT5, offering a therapeutic node for cancer, such as HCC, that display high PRMT5–SND1 axis activity. Here we summarize the known activities of this writer–reader pair, with a focus on their biological roles in HCC. This will help establish a foundation for treating HCC with PRMT5 inhibitors and also identify potential biomarkers that could predict sensitivity to this type of therapy. Signal transduction is the process by which information is relayed through a cell. Extracellular signals, such as growth factors or contact points with other cells, stimulate receptors on the cell surface to initiate this process by converting one stimulus (ligand binding) into another (phosphorylation). This signal initiation event is then propelled through the cytoplasm and into the nucleus using a series of sequential PTM events that rely on “reader” proteins, or effector molecules, to dock onto a specific PTM and then promote the deposition of a new PTM downstream, which in turn is read and relayed by another effector. There is a vast array of different PTMs including, but not limited to, phosphorylation, acetylation, ubiquitination and methylation, which can all be read by specialized protein domains in effector molecules . These gArginine residues are frequently methylated post-translationally, and these modifications come in one of three flavors: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), or symmetric dimethylarginine (SDMA) . These mDrosophila melanogaster Tudor protein which contains repeating domains that are present in a number of other proteins in many different species . Si. Si14]. The melanoma B16F10 cell line has been used to investigate the role of SND1 in facilitating immune evasion of tumor cells . B16F10-Like PRMT5 , SND1 isLiver cancer comes in a variety of types and frequencies, from the common HCC and cholangiocarcinoma to the rare liver angiosarcoma and pediatric hepatoblastoma. HCC is cited as constituting approximately 75% of all liver cancers according to the Cancer Treatment Centers of America. Among all cancers, HCC is the fourth leading cause of cancer mortality worldwide . TraditiAtypical lipid accumulation is characteristic of hepatic damage and malignancies and HCC is no exception ,126. HalPRMT5 has, in recent years, become an increasingly prominent character in HCC research. Multiple studies have reported a worse prognosis of HCC patients with increased PRMT5 expression ,76,131. EMT and invasion are key hallmarks of metastasis and PRMT5 has been identified as important in both. In vitro knockdown of PRMT5 in HCC and colon cancer cells decreases matrix metalloproteinase-2 expression . This imA cellular defense against EMT in hepatocytes is the transcription of hepatocyte nuclear factor 4α (HNF4α). HNF4α has been shown to re-differentiate HCC cells towards hepatocytes and repress EMT, thereby blocking hepatocarcinogenesis ,132,133.Another tumor suppressor, BTG2, is known to suppress proliferation, and is typically inhibited in cancers. While tumor suppressors are commonly inactivated by genetic mutations, deleterious mutations in BTG2 have not to date been identified, suggesting its downregulation may be a result of epigenetic reprogramming. PRMT5 has recently been linked to repressing BTG2 expression  through ERK signaling, though the mechanism of repression remains unknown .As previously mentioned, lipid accumulation is concurrent with hepatic damage . Lipid aLong non-coding RNAs (lncRNAs) have emerged as key regulators of normal physiology as well as pathogenesis. Long intergenic non-coding RNA 1q21.2 (LINC01138) has been shown to correlate with PRMT5 expression as well as HCC tumor size, AFP levels, and hepatitis B surface antigen levels. PRMT5 and LINC01138 were shown to interact in HCC, which allowed PRMT5 to evade proteasomal degradation . IncreasRecent findings reveal that PRMT5, in combination with SND1, promotes the dynamic regulation of E2F1 target genes. PRMT5 has been shown to promote cell growth, which contrasts to PRMT1 asymmetric dimethylation of E2F1 which promotes apoptosis ,81. PRMTWhile the role that PRMT5 plays in the development of HCC is largely circumstantial—elevated PRMT5 levels clearly correlate with the promotion of HCC and poor prognosis of these cancer patients—its effector molecule, SND1, is more solidly implicated as a driver of HCC. Indeed, a landmark study of SND1 in HCC emerged from a mouse model with SND1 overexpression . TransgeSND1 contributes to alterations in the signaling cascades within HCC that control both transcriptional and post-transcriptional regulation. Angiotensin 2 receptor 1 (AT1R) mRNA stability is augmented through overexpression of SND1 . ImportaNF-κB is a transcription factor that regulates innate immunity, and activation of this pathway promotes an inflammatory response and cellular growth. Chronic inflammation has been linked to many cancers, including HCC ,144,145.In order for cancer cells to persist and multiply, they evolve aggressive survival responses to stress signals to evade cell death pathways, which represents one of the major hallmarks of cancer. Stress granules are membraneless organelles that collect in the cytoplasm of cells, and serve as a means of stalling cellular machinery while the cell responds to the strain . This suA third survival pathway for cancer cells is DNA damage response. Using a laser microirradiation approach, SND1 is clearly localized to the laser-induced DNA damaged stripes . MechaniCHPT1, LPGAT1, PTDSS1 and LPIN1) are regulated by SND1, in response to proinflammatory TNFα signaling [Like PRMT5, SND1 has been noted to have a variety of functions in lipogenesis. SND1 facilitates lipid droplet formation in mammary cells and hepatocytes. This association is lost in milk globules suggesting that SND1 association is specific to formation, but not maintenance of fat droplets ,155. SNDignaling . SND1 isignaling .As highlighted above, PRMT5 is overexpressed in a large number of different tumor types, and the inhibition of PRMT5 has been linked to tumor regression in mouse models ,16,54. TIn addition to the panel of 13 different PRMT5 inhibitors that have been developed, it may be possible to target elevated PRMT5 levels using inhibitors for PRMT1 and perhaps even CARM1 (PRMT4). This is because there is clear evidence of redundancy between different PRMT family members. Indeed, we have shown that PRMT1 and PRMT5 share many substrates . FurtherHCC is a major health concern worldwide. It has a 20% five-year survival rate and 1% of the global population are expected to develop HCC in their life time. This disease poses a significant health burden which urges additional study. The majority of therapeutic options are hepatic resection and transplant, though transplant needs far outweigh available organs . Accordi


Fiber-reinforced epoxy composites are used in various branches of industry because of their favorable strength and thermal properties, resistance to chemical and atmospheric conditions, as well as low specific gravity. This review discusses the mechanical and thermomechanical properties of hybrid epoxy composites that were reinforced with glass, carbon, and basalt fabric modified with powder filler. The modification of the epoxy matrix mainly leads to an improvement in its adhesion to the layers of reinforcing fibers in the form of laminate fabrics. Some commonly used epoxy matrix modifiers in powder form include carbon nanotubes, graphene, nanoclay, silica, and natural fillers. Fiber fabric reinforcement can be unidirectional, multidirectional, biaxial, or have plain, twill, and satin weave, etc. Commonly used methods of laminating epoxy composites are hand lay-up process, resin transfer molding, vacuum-assisted resin transfer molding, and hot or cold pressing. The following review is a valuable source of information on multiscale epoxy composites due to the multitude of technological and material solutions. Polymer hybrid materials contain fillers or modifiers that have different functionalities, thanks to which they are characterized by unique usable, technological, and processing properties. Designing a composite material requires taking the processing technique, the susceptibility of all components to heat, mechanical treatment, durability, and the ability to create compounds into account ,2,3,4,5.IC fracture toughness decreased when compared to the reference sample. The authors attributed this effect to the MWCNTs agglomerates and the GPOSS aggregates present in the epoxy resin. Qi et al. also described a synergistic effect on the enhancement of mechanical features of composites after introducing graphene oxide and carbon nanotubes into liquid resin [Numerous studies focus on the assessment of the impact on the characteristics of the epoxy materials of powder additives, such as: carbon nanotubes (CNTs), graphene; graphite, graphene, graphene nanosheets, fullerenes , metal cid resin . In addition to different types of powder modifiers, the epoxy matrix is often reinforced using many types of fibers, such as glass fiber (GF), carbon fiber (CF) , basalt The impact properties under low impact velocity of glass/basalt woven fabric reinforced epoxy hybrids were reported in work . Basalt The main purpose of this article is to describe epoxy hybrid composites that contain both powder and fiber filler in their structure. The modification of the epoxy matrix by particles and simultaneous reinforcement with fibers allows for obtaining the unique properties of such composite materials.Glass fiber-reinforced plastics, in particular epoxy glass-reinforced composites, are used in the construction, aviation, and maritime industries due to their advantageous mechanical properties, lightness, resistance to atmospheric conditions, and low price. That is why they remain the subject of numerous studies into fiber arrangement and modification of the epoxy matrix through nano- and micro-additives, despite other types of reinforcing fibers appearing on the market ,50.2 and multi-walled carbon nanotubes at a low concentration (0.3 wt.%) [Böger et al. studied the fatigue properties of the epoxy laminates containing two different types of E-glass fibre non-crimp-fabrics and modified with fumed silica SiO.3 wt.%) . For the.3 wt.%) . For theThe thermal properties of epoxy laminates that were reinforced with E-glass fabric and modified by graphene nanoplatelets (GNPs) at 2 wt.% depending on the post-curing time and temperature were evaluated by Seretis et al. . DiffereAdditionally, the application of epoxy with the addition of GNPs, CNTs, hexagonal boron nitride nanosheets (BNNS), and boron nitride nanotubes (BNNT) nanoparticles as the matrix for the composites reinforced with glass fiber fabric was described in another study . The autZeng et al. studied the influence of graphene oxide (GO) dimensions on interlaminar shear property of glass fabric/epoxy (GF/epoxy) structural materials . The resThe effect of montmorillonite (MMT) clay particles (1% and 3% nanoclay doping ratios) on the incubation period in solid particle erosion of glass fibers fabric/epoxy nanocomposites was presented in work . The unmCellulose microcrystals (CMCs) at concentration of 1–3 wt.% were successfully used to improve the fiber-matrix interface, mechanical, dynamic mechanical, and thermal behavior of epoxy laminates reinforced with glass woven fabric . Figure Naidu et al. evaluated the mechanical properties of epoxy hybrid composites that were reinforced with woven glass fiber and modified with the graphitic carbon nitride (g-C3N4) at concentrations of 1%, 1.5%, 2%, 2.5%, and 3% . The besShah et al. applied different bamboo powder (BP) concentrations to modify the glass fiber-reinforced epoxy hybrid composites with a sandwich-structure from the top layer, and the bottom layer from woven E-glass fiber . The comBased on the above examples, it can be stated that the main use of hand lay-up and compression hot press techniques allowed for producing stable fiber glass reinforced laminate structures. In order to obtain a hybrid structure, the epoxy matrix was modified by introducing powder fillers into it and then using it in the manufacturing process. When introducing a nano or micro powder filler into the epoxy system, its concentration should be selected in order to enable the modified resin to be produced in a specific production process. By introducing carbon materials in nanometric sizes, the highest efficiency for hybrid composites was obtained while using modifier concentrations in the range of 0.1–0.5 wt.%. When used as MWCNT modifier, both the mechanical high-speed mixer and sonication methods allowed for obtaining a homogeneous suspension for the lamination process. Favorable mechanical properties were obtained by introducing MWCNT in a concentration of up to 0.3 wt.%. For epoxy laminates reinforced with glass fabric and modified graphene nanoplates, improved mechanical properties  were obtained by introducing this modifier into the resin at 0.1 wt.%. Additionally, the interlaminar shear strength composites increased when graphene oxide at low concentration (≤0.1 wt.%) is introduced into the epoxy matrix. As the modifier’s particle size increases, its higher concentrations (1–5 wt.%) should be used to improve the desired properties. Explicit enhancement in the tensile strength and flexural strength was obtained after the introduction of MMT into the epoxy matrix. Cellulose microcrystals have also proved to be an effective modifier in relation to the epoxy polymer, enabling the improvement of the mechanical properties of laminates that were made using the vacuum bag method. Techniques for producing fiber glass-reinforced epoxy composites are well developed. However, the methods of improving the adhesion between the fiber and matrix and the mechanical properties of the composite through the introduction of powder fillers each time require the development of a special technology for mixing components, methods for applying them to fibers, and a curing scheme for epoxy materials.Carbon fibers are infusible high strength and stiffness materials made of graphitic and non-crystalline regions. They are chemically inert, electrically conductive, and have a relatively low coefficient of thermal expansion . That isS showed higher shear strength, and the epoxy structural materials with GO showed higher humidity resistance. CNT coating improved the surface roughness of the carbon fiber and increased the contact area between the fiber and the matrix. On the other hand, the GO coating interacted with resin and created interfacial mechanical interlocking and chemical bonding. The synergistic effect of modifying the carbon fabric-reinforced epoxy composites with layered graphene nanoplatelets and multi walled carbon nanotubes was described in work [IC) was the highest for 0.5 wt.% GNPs + 1 wt.% MWCNTs addition (45% higher than the reference sample). The same hybrid composites indicated the highest value of (GIIC) Mode II fracture toughness (25% higher than the reference sample). Likewise, Sánchez et al. presented the interesting results of their study into carbon fiber composites with modified epoxy resin matrices via amino-functionalized and non-functionalized carbon nanotubes [Jiang et al. proposed an interesting method of epoxy matrix modification through introducing graphene oxide sheets onto carbon fiber surface. The team managed to significantly improve the interfacial shear strength of the CF-GO/epoxy composite in comparison to composites without GO . Yao et  in work . In all anotubes . These hanotubes . The car3O4/MWCNTs showed enhanced mechanical properties, such as tensile strength, the impact strength of epoxy matrix at 77 K, as well as micro-cracks resistance, and a lower coefficient of thermal expansion (decreased by 37.6%) of the epoxy matrix.Composites that were based on epoxy resin reinforced with carbon fiber, polyhedral oligomeric silsesquioxane (POSS), and CNT were also presented in work . GraftinDorigato et al.  applied 2 particles and short carbon fibers. Moreover, nano-SiO2 particles were grafted with copolymer of styrene and maleic anhydride. The synergetic effect of both fibrous and powder filler in hybrid composites was observed, together with improved surface hardness and tribological performance. On the other hand, Landowski et al. [2 content. Moreover, a decrease in permanent deformation by ~15% and absorbed energy by ~8%, as well as better fiber/matrix interfacial strength were observed. In other studies, prepregs of carbon fibers with and without surface-modified silica nanoparticle (SiO2) were used to prepare a laminate via compression molding [Guo et al.  preparedi et al.  used ind molding . The lam molding . The aut molding . The add2O3, fillers (10 vol.%) improved the mechanical properties of these materials [2O3 filler.In turn, the hybridization of the carbon fiber-reinforced epoxy composites by nano- and micro-sized Alaterials . UnidireBunea et al. studied the effect of epoxy resin features, amount of carbon and aramid sheets, and fiber direction on low velocity impact properties of the structural hybrid composites . The epo2 particles into the epoxy system in an amount of up to 8 wt.%, allows for improving the interfacial strength, impact toughness, and flexural strength of layered materials.Carbon fiber reinforced laminates exhibit high specific stiffness and strength in various structural applications. Therefore, the method of modification of these materials cannot lead to a deterioration of their original properties. Basalt fiber is obtained through molting basalt rock and fiber spinning, which is a more eco-friendly process than producing other fibers, such as glass and carbon. Basalt is made of plagioclase, pyroxene, and olivine. Its fibers display high strength and modulus in comparison to glass fiber and, moreover, they are chemically stable, non-toxic, and non-combustible . TherefoChen et al. manufactured and investigated cross-ply composites reinforced with basalt fibers and functionalized MWCNTs at concentration of 0.5 vol.% or 1.5 vol.% from unidirectional epoxy prepregs . The disIC) and critical strain energy release rate (GIC). Composites with 20 wt.% of NGF showed the best mechanical properties and thermal stability. Thermal conductivity increased with the NGF concentration in the composites.Study  assessedJamali et al.  studied 2, 3 wt.% clay or 1,5 wt.% TiO2, and 1,5 wt.% clay) [Moreover, halloysite nanotube (HNT) at concentrations of 1.0, 2.0, and 3.0 wt.% was applied to modify the epoxy resin matrix for basalt fiber reinforced composites . These h.% clay) . The fil.% clay) , basalt Subagia et al. used tourmaline (TM) micro/nano particles from a group of silicate minerals (0.5–2 wt.%) with and without surfactant to modify the epoxy laminate with basalt fiber . The lamSurana et al. examined the effect of the addition of sawdust particles  on the tensile and vibration properties of BF/epoxy composites . Six layA natural origin basalt fiber is more environmentally friendly than other reinforcing fibers. Additionally used in composites, it allows for obtaining similar results to materials that were reinforced with carbon fiber. Therefore, the largest number of studies referred to in this review concerned the use of basalt fibers in hybrid composites. In most cases, vacuum assisted and hot pressing techniques were used to produce this group of materials. The characteristics of basalt fiber reinforced hybrid composites are presented in 2 into epoxy resin (up to 8 wt.%) contributed to improving the properties of these laminates. The epoxy matrix should be modified in such a way as to ensure good adhesion between all components in the laminate in order to provide increased mechanical and thermomechanical properties of hybrid composites. It should be emphasized that for all these types of analyzed hybrid composites reinforced with glass, carbon and basalt fibers, respectively, carbon-based nanomodifiers proved to be the most effective powder filler. Their small addition significantly improved the adhesion of the fiber to the matrix, which translated into the observed effect of strengthening and improving their thermal and mechanical properties.This review presents various studies into the mechanical and thermo mechanical properties of hybrid epoxy composites with both powder and fiber fillers. In particular, composites that were reinforced with glass, carbon, and basalt fabrics obtained by various lamination techniques are characterized. Laminates are composed mainly of: fibers that are responsible for transferring the load to the composite, a matrix that provides the volume of the composite and transfers the load between the fibers, and a fiber-matrix interface that should provide adequate interphase strength of the composite. Therefore, selecting the accurate method of epoxy matrix modification requires the consideration of many factors that are related to the structure of individual components and the way they are processed. Most literature reports concern the use of nano- or micro-powder fillers, such as carbon nanotubes, graphene derivatives, nanoclays, and silica, to modify the epoxy matrix. Using the simultaneous modification of the epoxy matrix with powder filler and reinforcing fiber, beneficial improvement of such properties as the tensile and flexural strength, interlaminar shear strength, impact strength, fracture toughness, ballistic impact behavior, viscoelastic, and tribological were obtained. In the case of nanofillers, the typically applied percentages of additives did not exceed 1 wt.%, while for other microfillers they were significantly higher. In most cases, the use of carbon nanotubes introduced into the resin (up to 1 wt.%) improved the mechanical properties of composites and the adhesion between the fiber and the matrix. Favorable results were also noted for hybrid materials that were modified with graphene nanoplatelets (up to 2 wt.%). On the other hand, materials modified with montmorillonite in an amount of up to 5 wt.% showed superior properties. In addition, for carbon fiber composites, the introduction of nano-SiO


Therapeutic normal IgG intravenous immunoglobulin (IVIG) is a well-established first-line immunotherapy for many autoimmune and inflammatory diseases. Though several mechanisms have been proposed for the anti-inflammatory actions of IVIG, associated signaling pathways are not well studied. As β-catenin, the central component of the canonical Wnt pathway, plays an important role in imparting tolerogenic properties to dendritic cells (DCs) and in reducing inflammation, we explored whether IVIG induces the β-catenin pathway to exert anti-inflammatory effects. We show that IVIG in an IgG-sialylation independent manner activates β-catenin in human DCs along with upregulation of Wnt5a secretion. Mechanistically, β-catenin activation by IVIG requires intact IgG and LRP5/6 co-receptors, but FcγRIIA and Syk are not implicated. Despite induction of β-catenin, this pathway is dispensable for anti-inflammatory actions of IVIG in vitro and for mediating the protection against experimental autoimmune encephalomyelitis in vivo in mice, and reciprocal regulation of effector Th17/Th1 and regulatory T cells. Karnam et al. show that the β-catenin pathway is dispensable for the anti-inflammatory actions of intravenous immunoglobulin (IVIG) in human dendritic cells and in protecting against experimental autoimmune encephalomyelitis. This study implicates an unknown signaling pathway as a mediator of the anti-inflammatory effects of therapeutic IVIG. In addition to its application in the replacement therapy of primary and secondary immune deficiencies, high-dose (1–2 g/kg) IVIG is used for the immunotherapy of large number of systemic autoimmune and inflammatory diseases like immune thrombocytopenic purpura, Kawasaki disease, Guillain-Barré syndrome, transplantation, inflammatory myopathies, and many others. In addition, IVIG therapy has been explored over 100 different pathologies as an off-label manner6. The signaling pathways implicated in anti-inflammatory actions of IVIG are however not completely known and are the subjects of intense research.Based on the studies performed both in human and experimental models, several mechanisms have been proposed for IVIG. The current evidence suggests that IVIG suppresses the activation and function of various innate immune cells (like dendritic cells (DCs), neutrophils, monocytes, macrophages) and effector T and B cells. On the other hand, IVIG promotes the anti-inflammatory processes such as enhancing the tolerogenic properties of antigen presenting cells, increasing the secretion of anti-inflammatory molecules like IL-1RA and IL-10, and expansion of regulatory T cells (Tregs)13. In addition to nonimmune cells, β-catenin is expressed in all immune cells including DCs, macrophages and T cells, and regulate their functions16. In fact, conditioning of DCs with Wnt ligands promotes the expansion of Tregs and limit the expansion of Th1/Th17 cells17. In experimental autoimmune encephalomyelitis (EAE) model, lack of β-catenin in DCs leads to increased severity of the disease18. Conversely, treatment with β-catenin agonists protects the mice from EAE with reduced frequency of IFN-γ and IL-17-expressing cells12.β-catenin, the central component of the canonical Wnt signaling pathway plays a major role in balancing the immune aggression verses tolerance33. In view of reports on the central role of β-catenin/Wnt pathway in mediating these anti-inflammatory effects, we hypothesized that IVIG induces the activation of β-catenin/Wnt pathway to exert anti-inflammatory effects.It is interesting to note that IVIG has been reported to suppress the inflammatory cytokines in DCs and reciprocally regulate Tregs and effector Th1/Th17 responses in vitro, in vivo in EAE model, and in treated autoimmune patientsIn the present report, we show that IVIG indeed activates the β-catenin pathway along with Wnt5a secretion in human DCs in a sialylation independent manner. Mechanistically, β-catenin activation by IVIG requires low-density lipoprotein receptor related proteins (LRP) 5 and 6 co-receptors but FcγRII and Syk pathway are not implicated. However, despite induction of β-catenin/Wnt signaling, we found that this pathway is dispensable for the anti-inflammatory action of IVIG both in vitro, and in vivo in mediating the protection against EAE in mice and reciprocal regulation of effector Th17/Th1 and Tregs.34. Non-phosphorylated forms of β-catenin are functionally active and translocate to nucleus and regulate the expression of target genes. In view of critical role of DCs in mediating both immune response and tolerance, we first investigated whether IVIG activates β-catenin signaling in these innate immune cells. Monocyte-derived DCs were treated with 25 mg/ml of IVIG  for 24 h and activation of β-catenin signaling was analyzed by immunoblotting for active form of β-catenin (non-phospho β-catenin).Glycogen synthase kinase-3 beta (GSK-3β) acts as a negative regulator of β-catenin pathway by phosphorylating β-catenin at Ser33, Ser37 and Thr41, and directing it to proteasomal degradationWe found that IVIG significantly increased the levels of active β-catenin, which is not phosphorylated at Ser33, Ser37, and Thr41 residues Fig. . On the We have also assessed the levels of triple phosphorylated β-catenin . Consistent with the data on active β-catenin Fig. , IVIG siCTNNB1 gene (that encodes β-catenin) expression analyses by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and found that IVIG significantly enhanced the CTNNB1 mRNA in DCs  sialic acid linkages of Fc region in mediating the anti-inflammatory actions of IVIG41. To investigate if IVIG induces β-catenin activation via C-type lectin receptors, we treated the DCs with Ca2+ chelating agent ethylenediaminetetraacetic acid (EDTA) followed by IVIG treatment. However, IVIG-induced β-catenin activation remained intact upon treatment of DCs with EDTA  and equimolar concentrations of F(ab′)2 or Fc fragments. In line with the data on the lack of implications of sialylation of IgG, Syk and FcγRIIA in mediating β-catenin activation by IVIG, Fc fragments of IVIG failed to induce β-catenin activation ion Fig. .Fig. 5In2 fragments. However, we did not observe β-catenin activation even with F(ab′)2 fragments of IVIG VIG Fig. . Togethe47.We then explored if Wnt-receptor interaction is required for the activation of β-catenin by IVIG. Signaling by Wnt ligands upon binding to transmembrane Frizzled (Fz) receptor and its co-receptors, LRP5/6 leads to the transduction of signals and accumulation of β-catenin in the cytoplasm by preventing GSK-3β-mediated phosphorylation and proteosomal degradation of β-catenin. Eventually, β-catenin translocate into the nucleus to activate Wnt target genes WNT3A, WNT1, and WNT10A were significantly upregulated upon 12 h of IVIG treatment of DCs  Fig. . These r50. Therefore, to unequivocally prove that IVIG triggered β-catenin activation in DCs through LRP5-mediated signaling of Wnt5a, we resorted to silence LRP5 gene by siRNA method. Although LRP6 might not be binding to Wnt5a, to prevent the binding of other canonical Wnt ligands, we decided to knock-down both LRP5 and 6 co-receptor genes to ensure complete inhibition of β-catenin activation signals. In line with our hypothesis, the ability of IVIG to activate β-catenin was compromised when DCs are treated with siRNA against LRP5/6  in DCs. As shown in Fig. 52.The critical question was whether β-catenin signaling is important for the anti-inflammatory actions of IVIG. We addressed this by using FH535, a small molecule inhibitor of Wnt/β-catenin signaling54. Therefore, by using TLR4-activated DCs , we investigated if β-catenin signaling is implicated in the anti-inflammatory actions of IVIG. As depicted in Fig. 19. However, inhibition of Wnt/β-catenin signaling by FH535 had no repercussion on the inhibitory actions of IVIG on the production of these inflammatory cytokines. These results suggest that β-catenin though induced by IVIG, this pathway is dispensable for the anti-inflammatory action of IVIG.Previous reports have shown that β-catenin pathway regulates the inflammatory cytokine response in toll-like receptor 4 (TLR4)-activated DCs26.To validate the role of β-catenin signaling in the anti-inflammatory actions of IVIG in vivo, we resorted to EAE model. Previous reports have shown that IVIG protects the mice from the disease and is associated with the peripheral expansion of Tregs and suppression of pathogenic Th1 and Th17 cellsWe first wanted to confirm that IVIG induces activation of β-catenin in mouse immune cells. Culturing of splenocytes from C57/BL6 mice with IVIG for 24 h induced active-β-catenin while HSA had no such effect Fig. . Thus, iFurthermore, inhibition of β-catenin signaling in vivo in EAE model by daily injection of β-catenin antagonist (FH535) along with IVIG did not compromise the protective effects of IVIG. The clinical scores were similar to IVIG injected in combination with dimethyl sulfoxide  Fig. . The con+ T cells in the spleen of mice on the day of onset of disease. Confirming the previous reports, IVIG suppressed both Th1 and Th17 cells and reciprocally enhanced Tregs , neutralization of pathogenic autoantibodies and inflammatory cytokines, and complement scavenging are functioning during early phase of IVIG therapy; regulation of immune cells like innate cells  and adaptive immune cells (T and B cells) occur during subsequent phase to ensure immune homeostasis27 The ability of IVIG to suppress DC activation also had impact on the DC-mediated T cell responses and the pathogenesis of autoimmune diseases62. The signaling pathway(s) that are implicated in the regulation of DC functions by IVIG is not well understood. Previous reports have shown that preconditioning of human monocyte-derived DCs with IVIG impairs TLR4-mediated activation of extracellular signal–regulated kinases 1/2 (ERK1/2)63. Although ERK1/2 was not inhibited by IVIG in murine bone marrow-derived DCs, when these cells were stimulated with LPS along with IVIG, p38 mitogen activated protein kinase (p38MAPK) activation was suppressed23. Possibly, differences in the concentrations of IVIG and/or experimental conditions might have contributed to the differences observed between human and mouse DCs. Type II C-type lectin receptors appear to play an important role in transducing the signaling events by IVIG in DCs. However, the nature of the lectin receptor varies depending on the subset of DCs65. By signaling via DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), IVIG and its F(ab’)2 fragments induced prostaglandin E2 in human DCs by activating cyclooxygenase-2 pathway and mediated Treg expansion26. The IL-3-produced from these activated Tregs might license basophils to undergo activation by IVIG leading to the secretion of IL-4 and further suppression of effector Th17 and Th1 cells66. On the other hand, in the humanized DC-SIGN mice model, Fc fragments containing terminal α- sialic acids interacted with DCs and induced IL-3335 though in human, DC-SIGN-positive DCs failed to produce IL-33 upon exposure to IVIG67. Ligation of DCIR (Dendritic cell immunoreceptor) by IVIG induced phosphorylation of immunoreceptor tyrosine-based inhibition motif (ITIM)-linked phosphatases Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase-1 (SHIP-1) and SH2-containing tyrosine phosphatase-2 (SHP-2) in pulmonary CD11c+ DCs of mice68. Thus, IVIG activates various signaling pathways in DCs to mediate anti-inflammatory actions.DCs play a crucial role as professional antigen presenting cells in balancing the immune response and tolerance. Investigations on the mode of action of IVIG have revealed that it inhibits the activation of both human and murine DCs and the production of inflammatory cytokines8, several recent studies have emphasized the role of Wnt/β-catenin pathway in the regulation of systemic and mucosal inflammatory immune responses by mediating tolerogeneic properties in DCs and ensuing T cell responses71. β-catenin signaling in DCs suppresses inflammatory cytokines like IL-6, induces anti-inflammatory cytokines, and reciprocally regulates Treg and Th1/Th17 responses in vivo in experimental models of autoimmune and inflammatory diseases like EAE and inflammatory bowel disease69. In line with these data on the role of β-catenin in promoting tolerance in DCs, treatment of these cells with Wnt3a proteins that trigger β-catenin activation also suppress TLR-induced pro-inflammatory cytokines in DCs17. As both IVIG and Wnt/β-catenin pathway mediate similar anti-inflammatory actions on DCs and T cell responses, inspired us to explore if normal IgG (IVIG) targets Wnt/β-catenin pathway to exert therapeutic benefits.Though Wnt/β-catenin pathway is generally studied for their role in embryonic development and tumorigenesis72.In line with our proposition, IVIG induced activation of β-catenin in DCs. However, inhibition of β-catenin pathway did not compromise the ability of IVIG to suppress IL-6 and IL-8 production in DCs, suggesting that β-catenin pathway is redundant for IVIG. Our data thus affirm that IVIG is not bound by a single mechanism to exert anti-inflammatory effects. The mutually nonexclusive pathways induced by IVIG ensure that a deficiency in a particular pathway could be compensated by others. β-catenin pathway however is not only the pathway dispensable for anti-inflammatory actions of IVIG. In fact, heme oxygenase-1 (HO-1) pathway that regulates inflammatory responses by catabolizing the free heme and releasing carbon monoxide and biliverdin, is also found be not essential for the anti-inflammatory effects of IVIG50. In the presence of Fzd4 and LRP coreceptors, Wnt5a could indeed activate β-catenin-mediated canonical pathway50. Secretion of Wnt5a in IVIG-treated DCs and abrogation of IVIG-induced β-catenin activation upon silencing of LRP 5/6 indicate Wnt5a-induced β-catenin activation by IVIG.How does IVIG induce β-catenin activation? Mechanistically, our data imply that IVIG induces Wnt5a in DCs that signals via frizzled and LRP5 receptors to activate β-catenin pathway. Though Wnt5a is a noncanonical Wnt ligand, various reports have indicated its involvement to induce β-catenin pathway73. In our report, pre-treatment of DCs with EDTA that chelates Ca2+ and inhibits C-type lectin receptor-mediated binding of ligands including Dectin-1, DC-SIGN and DCIR, did not prevent the ability of IVIG to induce β-catenin activation. The retention of ability to induce β-catenin activation by desialylated IVIG suggest that type II Fc receptors are also not implicated in the process74. The involvement of TLR2 was also precluded in view of the fact that IVIG-could induce β-catenin activation in DCs independent of TLR signaling.Several reports have demonstrated that β-catenin could be activated by Dectin-1, TLR2, or FcγR-mediated signaling2 fragments could recapitulate these functions. As human monocyte-derived DCs mainly express low affinity FcγRII, explains the inability of Fc fragments of IVIG to induce β-catenin activation. However, despite having the ability to bind DCs19, F(ab′)2 fragments did not promote β-catenin activation. Therefore, it is likely that cooperation between Fab and Fc regions lead to β-catenin activation by IVIG. As reported earlier, IVIG contains antibodies to various self-motifs including those antigens expressed on DCs6. Thus, binding of Fab region of IVIG to DCs might license Fc region to bind FcγRII of adjacent DCs and signal β-catenin activation in those cells. Human DCs display a balanced expression of activating FcγRIIA and inhibitory FcγRIIB43. Data from the FcγRIIA blocking experiments and Syk inhibition assays provide a pointer towards lack of participation of activating FcγRs including FcγRIIA in IVIG-induced β-catenin activation. These data thus suggest a possible role for FcγRIIB in inducing β-catenin activation although, we do not exclude the implication of yet another non-identified receptor. In fact, reports have demonstrated that interaction of IgG with certain receptors like FCRL5 (Fc receptor-like protein 5) on B cells requires intact IgG75.We found that induction of β-catenin activation by IVIG requires intact IgG while neither Fc nor F(ab′)26. Activation of the β-catenin pathway is also reported to suppress the severity of the EAE and to mediate reciprocal regulation of effector CD4+ T cells and Tregs12. EAE has been used by several groups to explore the anti-inflammatory mechanisms of IVIG. Although a recent report suggested possible neutralization of Mycobacterial antigens by F(ab′)2 fragments as a mechanism of IVIG-mediated protection in EAE76, other lines of evidences suggest that IVIG mechanisms in EAE go beyond mere neutralization of Mycobacterial antigens. For inducing EAE, Mycobacterial antigens are emulsified in Freund’s adjuvant and then injected to the mice. Mycobacterial antigens in emulsion are not freely accessible to antibodies to get neutralized. It is contrary to the report of Quast et al.76 who used free Mycobacterial antigens in ELISA to test binding of IVIG. Furthermore, other reports have shown the importance of Tregs, IL-11 receptor, IL-33 receptor in IVIG-mediated protection against EAE. Depletion of Tregs or deficiency of IL-11R, IL-33R, SIGN-R1 (Specific ICAM-3 grabbing nonintegrin-related 1), all led to abrogation of protection by IVIG78.To explore the importance of β-catenin signaling in IVIG-mediated anti-inflammatory effects in vivo, we used EAE model. Our previous data show that IVIG has the ability to delay onset of the disease and to reduce the severity of EAE. The protection was associated with the inhibition of Th1/Th17 responses and peripheral expansion of Tregs2 or Fc fragments79, others like β-catenin activation as shown here requires intact IgG that were either not damaged or subjected to structural modifications. Moreover, the structural integrity of IgG is critical for the half-life of IVIG in the treated patients. Though both IVIG and β-catenin mediate similar actions, from the current perspective it is evident that IVIG does not link β-catenin to exert therapeutic benefits, as IVIG is able to reduce the severity of EAE and reciprocal regulation of Th1/Th17 and Treg responses independent of β-catenin. These data thus highlight that a single mechanism might not be entirely responsible for the sustained beneficial effects of IVIG in autoimmune and inflammatory diseases.Our data also advocate IgG structure-dependent diversity in the mechanisms of IVIG. While some mechanisms are dependent on F(ab′)CD14 MicroBeads, basophil isolation kit II, rhGM-CSF, rhIL-4 were from Miltenyi Biotec . Rabbit MAbs to non-phospho (active) β-catenin (Ser33/37/Thr41) , GSK-3β , GAPDH (HRP-conjugated) , β-Actin (HRP-conjugated) ; polyclonal protein A and peptide affinity chromatography purified rabbit antibodies to phospho-β-catenin (Ser33/37/Thr41) , β-catenin , mouse PCNA MAb  and HRP-conjugated affinity purified horse anti-mouse IgG secondary antibody  were from Cell Signaling Technology . HRP-conjugated human adsorbed goat anti-rabbit IgG secondary antibody  was purchased from Southern Biotech .FITC-conjugated anti-mouse MAbs to IFN-γ  and CD25 , anti-human CD32 , anti-human CD86 ; PE-conjugated anti-mouse MAb to IL-17A ; Per-CP/Cy5.5-conjugated anti-mouse MAb to CD4 ; APC/Cy7-conjugated anti-human MAb to CD69  were from BD Biosciences . APC-conjugated MAb to FOXP3 , APC-conjugated anti-human HLA-DR  and Fixable Viability Dye eFlour506 (1:500 dilution) were from eBioscience . Alexa Flour 488-conjugated goat anti-rabbit IgG (H + L) was from Invitrogen . Blocking MAb to FcγRIIA (clone IV.3) was purchased from Stem Cell Technologies .E. coli 055:B5) was purchased from Sigma-Aldrich .Syk inhibitor R406 and β-Catenin/TCF Inhibitor, FH535 were obtained from InvivoGen  and Merck Chimie  respectively. LPS  and Dr. V Aimanianda . IL-3 was purchased from ImmunoTools .39. Lectin-blot analyses was performed to validate the desialylation of IVIG  were used. IVIG was dialyzed against large volumes of RPMI 1640 at 4 °C for 4 h. For desialylation of IgG, IVIG was treated with recombinant neuraminidase  as previously described2 and Fc fragments of IVIG were prepared by pepsin and papain digestion respectively56. The F(ab′)2 and Fc fragments were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses for the verification of purity.F(ab′)+ cells were positively selected using CD14 MicroBeads (Miltenyi Biotec). Monocytes were cultured for 5 days in the presence of GM-CSF (1000 IU/106 cells) and IL-4 (500 IU/106 cells) to obtain DCs80. The gating strategy for DCs is provided in the Supplementary Fig. 2a.Peripheral blood mononuclear cells (PBMCs) were obtained by subjecting the buffy coats of healthy donors to Ficoll density gradient centrifugation . CD1426. Briefly, mice were immunized on day 0 by subcutaneously injecting 200 µg of MOG35–55 peptide  emulsified in complete Freund’s adjuvant, containing 880 µg of killed Mycobacterium tuberculosis strain H37RA. Further, all mice received 300 ng pertussis toxin on day 0 and day 2. Mice were observed every day for the development of clinical signs of EAE. Clinical signs in control mice typically appeared between 10–12 days post immunization. Mice were scored based on the following scoring system where, 0, no signs; 1, tail paresis; 2, hind limb paresis; 3, hind limb paralysis; 4, tetraplegia; and 5, moribund. Any animal reached the score 4 was euthanized for ethical reasons.C57BL/6J female mice were obtained from Envigo laboratories, France. All the experiments on mice were done after approval from the ethical committee for animal experimentation and French Ministry of Higher Education and Research (APAFIS#10539-2017070715163055 V4). EAE was induced in 8-week-old mice as described in our earlier studiesMice were divided into three groups. Control group, IVIG + DMSO group and IVIG + inhibitor group. Mice received by intraperitoneal route, 0.8 g/kg of IVIG in combination with either 50 μl of DMSO (Sigma-Aldrich) or 15 mg/kg of β-catenin inhibitor (FH535) every day until peak of the disease.+ T cells is provided in the Supplementary Fig. On the day of onset of the disease, mice were sacrificed to collect spleen. Splenocytes were isolated and red blood cells were lysed using ACK (Ammonium-Chloride-Potassium) lysis buffer. Cells (0.5 million/ml) were stimulated with phorbol myristate acetate (PMA) (50 ng/ml/0.5 million cells) and ionomycin (500 ng/ml/0.5 million cells), along with GolgiStop for 4 h. Th1, Th17, and Treg populations were analyzed by combination of surface and intracellular staining for various markers. Surface staining was performed with fluorescence-conjugated MAbs to CD4 and CD25. After fixation and permeabilization by intracellular staining kit (eBioscience), intracellular staining with fluorescence-conjugated MAbs to IFN-γ, IL-17A, and FOXP3 were carried out. Samples were acquired using LSR-II flow cytometer (BD Biosciences) and data were analyzed by FACS-DIVA (BD Biosciences). The gating strategy for CD42 fragments, Fc fragments or HSA (irrelevant protein control) for 24 h. Activation of the β-catenin signaling was analyzed by immunoblotting.DCs (0.5 million cells/ml) were cultured in RPMI-1640 containing 10% fetal calf serum (FCS) alone or with 25 mg/ml or 15 mg/ml of IVIG or desialylated IVIG (25 mg/ml) or equimolar concentration of F(ab′)For the experiments involving treatment of pharmacological inhibitors, concentrations were chosen after titration. β-Catenin/TCF inhibitor, FH535 was used to inhibit β-Catenin at the concentration of 15 μM, 2 h prior to the treatment of IVIG.In other experiments, DCs were pretreated with pre-determined concentrations of EDTA (0.5 mM) or with Syk inhibitor R406 (10 μM) or with blocking MAb to FcγRIIA (10 μg/0.5 million cells) for one hour before culturing with IVIG. We confirmed that aforementioned concentrations of EDTA, Syk inhibitor and FcγRIIA MAb were functional , and expression was measured with TaqMan Gene Expression Assays  and Hs02786624_g1 .For the analyses of gene expression, cells were treated with IVIG (25 mg/ml) or HSA for 12 h. Untreated cells were used as control. Total RNA from the different conditions was isolated using the RNeasy Mini Kit . cDNA was synthesized using High-Capacity cDNA Reverse Transcription kit . Quantitative RT-PCR for Wnt genes, SYBRTM Green PCR Master Mix (ThermoFisher Scientific) was used. Gene expression was calculated relative to the housekeeping gene GAPDH. The primers used in the study are as follows:For the analyses of expression of WNT1- F: CAGCGACAACATTGACTTCG, R: GCCTCGTTGTTGTGAAGGTT;WNT3A- F: CCTGCACTCCATCCAGCTACA, R: GACCTCTCTTCCTACCTTTCCCTTA;WNT5A- F: CTCACTGAAATGCGTGTTGG, R: AATGCCCTCTCCACAAAGTG;WNT7A- F: CCCACCTTCCTGAAGATCAA, R: GTCCTCCTCGCAGTAGTTGG;WNT7B- F: GGCACAAGGACCTACCAGAG, R: CCTGATGTGTTCTCCCAGGT;WNT10A- F: CCACTCCGACCTGGTCTACTTTG, R: TGCTGCTCTTATTGCACAGGC;GAPDH- F: CGACCACTTTGTCAAGCTCA, R: GGTGGTCCAGGGGTCTTACT.6 cells/0.5 ml of Accell Delivery media. One micrometer of nontargeting control siRNA or LRP5 and LRP6 siRNA were introduced into cells for 72 h. After 72 h, cells were cultured in RPMI with 10% FCS and IVIG for 24 h followed by FACS analysis. Surface staining of DC was performed with fluorescence-conjugated MAb to HLA-DR. For active-β-catenin detection, cells were stained with rabbit MAb to non-phospho (active) β-catenin (Ser33/37/Thr41) and followed by Alexa Flour 488-conjugated goat anti-rabbit IgG (H + L) by using Cell Signaling Buffer Set A (Miltenyi Biotec).LRP5 and LRP6 predesigned Accell SMART Pool siRNA was purchased from Dharmacon (Thermo Fisher Scientific) and introduced into cells according to manufacturer’s protocol. Briefly, DCs were cultured in 12 well plate at 0.5 × 10TM WestDura Extended Duration substrate (Thermo Fisher Scientific). β-actin or GAPDH were used as a loading control. The western blot images were analyzed by myImageAnalysis software v 2.0 (Thermo Fisher Scientific). The full western blot images are provided in the Supplementary Fig. Total cell lysates were obtained by lysing cells in RIPA buffer  containing protease and phosphatase inhibitors (Sigma-Aldrich). Proteins were quantified using Bio-Rad protein assay reagent  and an equal amount of proteins were resolved on SDS-PAGE and transferred on to nitrocellulose, iBlot™ Transfer Stack (ThermoFisher Scientific). The membrane was blocked using 5% bovine serum albumin (BSA) in TBST  and Polysorbate 20 or Tween 20) for 60 min. The blots were incubated overnight at 4 °C with various primary antibodies diluted in TBST-5% BSA followed by incubation with secondary antibody for 2 h. After washing blots in TBST thrice with 10 min interval, blots were developed with SuperSignal2, 1 mM EDTA, and 240 mM Sucrose). After incubating for 10 min on ice, cells were centrifuged at 13,000 rpm for 10 min at 4 °C to pellet down the nuclei. Supernatant was collected and used as a cytosolic extract. Pellets were resuspended in ice cold Buffer C . Pellets were incubated on ice for 30 min followed by centrifugation at 13,000 rpm for 20 min at 4 °C to collect the nuclear extract. Immunoblotting of cytosolic and nuclear extracts was performed as detailed above.DCs were cultured alone or in presence of IVIG or equimolar concentration of HSA for 24 h. Cells were harvested and pellets were washed twice with ice cold PBS and resuspended in hypotonic lysis buffer  for 24 h. The cells were hen treated with β-catenin antagonist FH535 for 2 h followed by culture with IVIG (25 mg/ml) for additional 24 h. Cell-free supernatants were analyzed for the secretion of IL-8 and IL-6 by ELISA .Secretion of Wnt3a and Wnt5a was analyzed in cell-free supernatants from IVIG-treated DCs with the help of Human Protein Wnt-3a and Wnt-5a ELISA Kit .t-test for in vivo experiments. P < 0.05 was considered significant.As highlighted in the figure legends, the experiments were repeated several times by using independent donors or mice to ensure reproducibility. Human data were from three to seven donors except for nuclear translocation of β-catenin that was representative of two donors. Mice data were from two independent experiments. Statistical analyses were performed by using one-way ANOVA followed by Tukey’s multiple comparisons test or by two-way ANOVA with Bonferroni post-Further information on research design is available in the Description of Additional Supplementary FilesSupplementary Data 1Supplementary InformationPeer Review FileReporting Summary