N-glycan more than the high mannose type. In conclusion, PRMT5 overexpression is related to the development of BLV infection with a high proviral load and lymphoma stage and PRMT5 inhibition enhances BLV gene expression. This is the first study to investigate the role of PRMT5 in BLV infection in vivo and in vitro and to reveal a novel function for a small-molecule compound in BLV-gp51 glycosylation processing.Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle and is closely related to human T-cell leukemia viruses. We investigated the role of a new host protein, PRMT5, in BLV infection. We found that PRMT5 is overexpressed only in BLV-infected cattle with a high proviral load, but not in those with a low proviral load. Furthermore, this upregulation continued to the lymphoma stage. PRMT5 expression was upregulated in response to experimental BLV infection; moreover, PRMT5 upregulation began in an early stage of BLV infection rather than after a long period of proviral latency. Second, siRNA-mediated PRMT5 knockdown enhanced BLV gene expression at the transcript and protein levels. Additionally, a selective small-molecule inhibitor of PRMT5 (CMP5) enhanced BLV gene expression. Interestingly, CMP5 treatment, but not siRNA knockdown, altered the gp51 glycosylation pattern and increased the molecular weight of gp51, thereby decreasing BLV-induced syncytium formation. This was supported by the observation that CMP5 treatment enhanced the formation of the complex type of  Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, a B-cell leukemia and lymphoma ,2, whichDeltaretrovirus genus of the Retroviridae family and is closely related to human T-cell leukemia viruses types 1 and 2 (HTLV-1 and -2) [5 cells and 18,000 copies/105 cells in blood samples secreted BLV into the nasal mucus [5 cells [5 peripheral blood cells is a relatively high number and indicates BLV spreading within the whole body. Thus, a PVL of 10,000 copies/105 cells was used in our study as a threshold to classify BLV-infected cattle into high-PVL (HPVL) or low-PVL (LPVL). In contrast, a low PVL (LPVL) is known to prevent natural BLV infection in cattle [BoLA). Several studies have identified BoLA-DRB3 alleles and single-nucleotide polymorphisms in the BoLA region that are associated with BLV PVL and disease progression [BoLA-DRB3 polymorphism, remain to be identified.BLV belongs to the  and -2) . Similar and -2) ,8, but a and -2) ,9,10. Fo and -2) ,9. Addit and -2) . A previal mucus . Further[5 cells . These fn cattle . One of gression ,18,19,20gag, pro, pol, and env, the BLV genome contains the regulatory genes tax and rex and genes R3 and G4 [gag of BLV is translated as the precursor Pr70 Gag and is processed into three mature proteins: p15 is the matrix protein, p24 is the most abundant capsid protein, and p12 is the nucleocapsid protein [env of BLV encodes a Pr72 envelope (Env) precursor that is glycosylated in the rough endoplasmic reticulum and Golgi apparatus [N-glycosylation sites [N-glycosylation appeared to regulate viral replication, antigenic conformation, and syncytium forming capacity in vitro [In addition to the structural genes 3 and G4 ,2. gag o protein ,22. env pparatus ,23. Thispparatus ,24. BLV pparatus . This gpin vitro ,27 and iin vitro . In genein vitro ,29,30. Iin vitro .N-methyltransferase (PRMT) family. PRMTs are divided into four types of enzymes. Type I, which is the most common, induces asymmetric dimethylation; Type II catalyzes symmetric dimethylation; Type III produces monomethyl arginine as its final product; and Type IV is found only in fungi [Arginine methylation is an important posttranslational modification with crucial roles in chromatin regulation, transcription control, RNA processing, nuclear/cytoplasmic shuttling, DNA repair, and other biological processes ,33. Argiin fungi . In the in fungi , hepatitin fungi ,37,38, hin fungi ,41,42,43in fungi , Epsteinin fungi ,46, and in fungi .PRMT5 is a type II arginine methyltransferase that was recently shown to be crucial in EBV-driven B-cell transformation  and HTLVIn this study, we investigated the role of PRMT5 in various aspects of BLV infection in vivo and in vitro. First, we focused on the correlation between the PRMT5 expression level and BLV PVL, an index of virus infectivity, in peripheral blood from infected animals in various stages, such as the lymphoma and asymptomatic stages. Second, we revealed the impact of PRMT5 inhibition on BLV gene expression, gp51 glycosylation, and syncytium formation. This is the first study to investigate the role of PRMT5 in BLV infection.FLK-BLV cells, which are permanently infected with BLV , were cultured in Dulbecco’s modified Eagle’s Medium  containing 10% heat-inactivated fetal bovine serum . PK15-BLV cells, which were produced by stably transfecting the pig kidney-15 (PK15) cells  with CMV∆U3-pBLV-IF2, were cultured in Minimum Essential Medium Eagle (Thermo Fisher Scientific) containing 10% fetal bovine serum and 1% non-essential amino acids . CMV∆U3-pBLV-IF2 is the modified version of the BLV-infectious molecular clone pBLV-IF2, which was used previously ,49. CMV∆For transfection of small interfering RNA (siRNA), we used Lipofectamine RNAiMax Reagent (Thermo Fisher Scientific) according to the manufacturers’ instructions and RNA was extracted from these cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol.N = 20), (ii) BLV-infected cattle with LPVL , and (iii) BLV-infected cattle with HPVL . Blood samples were also collected from BLV-infected Holstein cows with lymphoma (N = 20). Lymphoma was diagnosed by both gross and histological observation and by detecting atypical mononuclear cells in the slaughterhouse. All animal experiments were conducted in accordance with the guidelines for Laboratory Animal Welfare and Animal Experiment Control established by the RIKEN Animal Experiments Committee . For experimental infection, five BLV-negative one-year-old Japanese black calves carrying susceptible alleles BoLA-DRB3*1601/*1601 were experimentally challenged intravenously with blood containing a PVL of 4 × 107 copies/105 cells. This study was approved by the Animal Ethical Committee and Animal Care and Use Committee of the Kyoto Biken Institute.Blood samples were collected from 62 Holstein cattle including BLV-negative cattle and BLV-infected cattle, which were maintained in Japan. These cows were classified into three groups according to their PVL as follows: (i) BLV-negative cattle  according to the manufacturer’s instructions. Genomic DNA for PCR amplification was isolated from EDTA-treated whole blood samples using the Wizard Genomic DNA Purification Kit .Blood samples were collected and diluted in a 1:1 ratio with AmbionBoLA-DRA  was amplified using the primer pair DRA-F and DRA-R. FAM-DRA was used as a probe. The PVL was calculated using the following equation: (number of BLV-LTR copies/number of BoLA-DRA copies) × 105 cells.The BLV PVL was measured by BLV-CoCoMo-qPCR-2  using genomic DNA as described previously . Brieflygag, and tax were designed using the primer-designing tool provided by the National Center for Biotechnology Information. The primer list is shown in ® FAST qPCR Kit  using an Applied Biosystems 7500 Fast Real-Time PCR system . The following thermal cycling program was used: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Samples were evaluated in duplicate and data analysis was performed using the comparative CT method (∆∆CT) with normalization to GAPDH mRNA expression.RNA was reverse-transcribed for cDNA synthesis using a High Capacity RNA-to-cDNA kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Primers for PRMT5, GAPDH, ® select Product # s20376 was used. For PK15-BLV knockdown, Silencer® select Product # s20377 was used. siRNA negative control (si-NC) was constructed by Ambion . On the day of transfection, 3 × 105 cells of FLK-BLV or PK15-BLV were seeded into a 6-well plate and transfected with si-PRMT5 or si-NC using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 48 or 72 h of incubation, cell lysates were prepared and knockdown efficiency was evaluated by RT-PCR or Western blotting analysis.siRNA designed to target PRMT5 (si-PRMT5) of FLK-BLV or PK15-BLV was constructed by Silencer Select siRNAs . To knock down PRMT5 in FLK-BLV cells, SilencerCMP5, a selective PRMT5 inhibitor , was purTM software (Alpha Innotech Corporation).Cells were lysed for 30 min on ice in 20 mM Tris-HCl (pH 7.4), 300 mM NaCl, 2 mM EDTA, and 2% NP40 supplemented with a protease inhibitor cocktail . Lysates were mixed with SDS buffer and boiled for 5 min. Protein concentrations were determined with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were electrophoresed via SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were then transferred onto a polyvinylidene difluoride membrane  using a Trans-Blot Turbo apparatus  and incubated with anti-BLV gp51 monoclonal antibody (Mab) (BLV 2) , anti-BLV Gag Mab (BLV 3) , polyclonal anti-PRMT5 , or anti-α-tubulin clone B-5-1-2 Mab . After washing, the membranes were incubated with horseradish peroxidase-conjugated AffiniPure goat anti-mouse IgG  or horseradish peroxidase-conjugated goat anti-rabbit IgG . Signals were visualized after treating the membrane with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). Images were acquired using a MultiImage TM light Cabinet . Band intensity was quantitated using the analysis tool provided with the AlphaEaseFCFLK-BLV or PK15-BLV were cultured in the absence or presence of 20 µM CMP5 for 48 h. Thereafter, the cell lysates were treated in the absence or presence of PNGase F  for 3 h or Endo H (Promega) overnight according to the manufacturer’s protocol , respectively.4 cells were seeded into a 96-well plate at a final volume of 100 µL in the absence or presence of different concentrations of CMP5. The assay was conducted according to the manufacturer’s instructions.A Premix WST-1 Cell Proliferation Assay Kit  was used to assess the effect of CMP5 on cell viability. For each cell line, 3 × 10FLK-BLV cells were grown on coverslips with or without CMP5 treatment. After 48 h, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature; next, they were permeabilized with 0.5% Triton X for 5 min and then blocked with 5% skim milk for 30 min. The gp51 staining in the cell membrane was performed without the permeabilization step. The cells were incubated with anti-BLV gp51 Mab (BLV 2)  or anti-BLV Gag Mab (BLV 3)  for 1 h at room temperature followed by 30 min incubation with Alexa Fluor 488 rabbit anti-mouse for Gag or Alexa Fluor 594 rabbit anti-mouse for Env . Nuclei were stained with Hoechst 33342  for 5 min in the dark. After each staining step, the samples were washed three times with PBS. The coverslips were mounted on glass slides and fluorescence images were obtained using an FV1000 confocal laser-scanning microscope .5 cells were stained with anti-BLV gp51 Mab (BLV 2)  for 1 h on ice. The cells were then stained with APC rat anti-mouse  for 30 min on ice. Propidium Iodide (PI) was used for live/dead staining. Stained cells were analyzed using a BD AccuriTM C6 Plus with a sampler flow cytometer . The data were analyzed using FlowJo v10 .FLK-BLV cells were collected using 2 mM ethylenediaminetetraacetic acid (EDTA)/PBS for 5 min at 37 °C and washed twice with PBS and 5 × 105 cells) were co-cultured with 5 × 104 FLK-BLV cells in a 12-well plate in LuSIA medium, after which the indicated concentrations of CMP5 were added. After 48 h incubation, the cells were washed with PBS and fixed with 4% formaldehyde/PBS containing 10 µg/mL Hoechst 33342 (Thermo Fisher Scientific). The fluorescent syncytia were visualized by EVOS2 fluorescence microscopy (Thermo Fisher Scientific) and 9 fields of view in each well were automatically scanned with a 4× objective. Syncytia were detected by enhanced green fluorescent protein (EGFP) expression and counted using HCS Studio Cell Analysis software (Thermo Fisher Scientific).The luminescence syncytium induction assay (LuSIA) using CC81-GREMG cells was performed as described previously . CC81 GRt-test. Differences were estimated to be significant at p ≤ 0.05 (*) and strongly significant at p ≤ 0.01 (**) and p ≤ 0.001(***). The p value in the animal experiments was calculated by the Kruskal-Wallis test and Dunn’s multiple comparisons test.All data were expressed as the mean ± standard deviation based on at least 3 independent experiments. Statistical significance was evaluated using Student’s 5 was used as a threshold to distinguish between the HPVL and LPVL groups. Next, we evaluated PRMT5 expression at the RNA levels by qRT-PCR. The mean fold-change in PRMT5 was 1.12 ± 0.62 in the control group, 1.18 ± 0.64 in the LPVL group, and 1.64 ± 0.62 in the HPVL group (p = 0.0014) and LPVL group (p = 0.012) (r = 0.52)  . Our res = 0.52) B.BoLA-DRB3*1601/1601, which are related to a high PVL in BLV infection [5 cells before infection (0 week), 34 copies/105 cells after 3 days, 1158 copies/105 cells after 1 week, 26,760 copies/105 cells after 2 weeks, 90,150 copies/105 cells after 3 weeks, and 62,440 copies/105 cells after 4 weeks (p = 0.0008) and then the expression slightly decreased after 4 weeks  (r = 0.79) (As shown in nfection  and we c = 0.79) B. Thus, r = 0.524).Finally, we investigated whether the change in PRMT5 expression in BLV-infected cattle occurred in the lymphoma stage of disease. We compared PRMT5 expression at the RNA level among three groups of cattle: 20 BLV-negative cattle, 42 BLV-infected but clinically normal cattle, and 20 BLV-infected cattle with lymphoma. The fold-change in PRMT5 expression was 1.12 ± 0.62 in BLV-negative cattle, 1.48 ± 0.66 in the asymptomatic group, and 2.45 ± 1.1 in the lymphoma group . This stgag, which is produced by the un-spliced mRNA, and tax, which is produced by the double splicing event  and of tax was 1.33 (p = 0.04). In the PK15-BLV cell line, the fold-change of gag was 1.72 (p = 0.01) and of tax was 1.6 (p = 0.0014).The data shown in ng event . We founp = 0.02)  (To confirm the finding shown in  = 0.02) A. Moreovctively) B. The baNext, we examined the effect of a pharmacological PRMT5 inhibitor on BLV protein expression. We used a small-molecule PRMT5 inhibitor (CMP5), which was described previously by Alinari et al. . We examUnexpectedly, we found that CMP5 treatment altered gp51 electrophoretic mobility in SDS-PAGE and formed gp51 with a higher apparent molecular weight than gp51 in untreated cells A,B. The N-Glycan structures are classified into three types: high mannose, hybrid, and complex types. All types share a common tri-mannosyl core (Man3GlcNAc2). However, they differ in the structure of the remaining branches [N-glycan typically runs slower in SDS-PAGE than the high mannose type. To further illustrate the effect of CMP5 treatment on gp51 electrophoretic mobility in SDS-PAGE, we examined whether the slow migration of gp51 in the presence of CMP5 resulted from an increase in the complex type of N-glycan. We cultured FLK-BLV or PK15-BLV in the absence (CMP5–) or presence of 20 µM of CMP5 (CMP5+) for 48 h. Cell lysates of each cell line were collected and divided into three parts: one part was not treated (PNGase F−/Endo H−), the second part was treated with PNGase F (PNGase F+), and the third part was treated with Endo H (Endo H+). As illustrated in N-linked glycosylation from the glycoprotein: high mannose, hybrid, and complex. Additionally, PNGase F severs the bond between N-acetylglucosamine (GlcNAc) and asparagine (Asn), liberating the entire sugar chain and converting asparagine (Asn) to aspartic acid (Asp). In contrast, Endo H cleaves only the high mannose and hybrid types of N-glycans and the complex type remains resistant to Endo H deglycosylation. Endo H cleaves the bond between the two GlcNAc residues in the core region, leaving one GlcNAc bound to the protein [branches ,55. The  protein .N-glycan of gp51 may increase after CMP5 treatment  or presence of 20 µM of CMP5 (CMP5+) for 48 h; Gag was stained with a green fluorescent marker, Env was stained with a red fluorescent marker, and DAPI was used to stain the nucleus. Gag and Env localization were evaluated by fluorescence confocal microscopy. The cellular membrane staining of gp51was performed without the permeabilization step and the intracellular staining was conducted after the permeabilization with 0.5% Triton X-100 for 5 min. We observed a large accumulation of Gag after CMP5 treatment A. AdditiN-linked glycan structure of the HIV and HTLV-1 envelope proteins affects syncytia formation [Previous studies showed that perturbation of the ormation ,58. Therormation . Becausep = 0.017) and 20 µM (p = 0.0002)  B. In con 0.0002) C. In add 0.0002) D. These BLV-mediated pathogenicity depends on a delicate balance between viral gene expression and certain host-related genetic and epigenetic events . The mut5), but not in those with LPVL   . This fiies/105) . We alsonfection A and itsnfection B. Finallnfection A. This fOur present study clearly demonstrates that PRMT5 negatively regulates BLV expression as PRMT5 inhibition including siRNA-mediated PRMT5 knockdown and treatment with CMP5 enhanced BLV gene expression at the transcriptional and protein levels in BLV-expressing cell lines, indicating that PRMT5 had an additive inhibitory effect on BLV gene expression . This reN-glycan over the high-mannose type. Therefore, following CMP5 treatment, gp51 traveled more slowly than untreated gp51 in SDS-PAGE. The effects of CMP5 on gp51 glycosylation processing involve a sophisticated process requiring a large number of enzymes and organelles, named as the glycosylation machinery. The glycosylation machinery is a group of enzymes, chaperones, transporters, sugar donors, and accessory molecules necessary to form a specific glycan structure [Alinari and co-workers  investigtructure . Therefotructure . Thus, CN-glycan over the high mannose type. In FLK-BLV, however, the results did not reveal that the irregular type of N-glycan resulted from CMP5 treatment. Similarly, a previous report clearly showed distinct differences in the glycosylation profiles of HIV-1 ENV gp120 expressed in CHO, which originated from hamsters, and 293T cells, which originated from humans [N-linked glycans attached to a glycoprotein is determined not only by the peptide backbone of a protein, but also by the cell in which it is expressed.Notably, the effect of CMP5 treatment on the gp51 glycosylation pattern differs between the two cell lines (FLK-BLV and PK15-BLV), which belong to different host species. In the PK15-BLV cell line, CMP5 treatment enhanced the formation of the complex type of m humans . TherefoAlthough CMP5 affects the gp51 apparent molecular weight, siRNA knockdown of PRMT5 did not appear to induce the same effect . The reaN-glycosylation appeared to regulate the syncytium-forming capacity in vitro [Our study showed that CMP5 treatment impeded BLV-induced syncytium formation, which was measured by LuSIA ,51. Howein vitro ,27. TakeIn conclusion, we determined various roles for PRMT5 in BLV infection in vivo and in vitro; CMP5 and other PRMT5 inhibitors should be further investigated and evaluated in clinical studies as a novel antiretroviral therapy.


In this case-controlled study, a total of 53 patients undergoing chronic HD were identified having PEW based on established diagnostic criteria and were age- and sex-matched with non-PEW (n = 53) HD patients. Fasting predialysis plasma samples were analyzed. Partial least square discriminant analysis demonstrated a significant separation between groups for specific metabolic pattern alterations. Further quantitative analysis showed that the level of 3-hydroxybutyrate, acetate, arabinose, maltose, ribose, sucrose and tartrate were significantly increased whilst creatinine was significantly decreased  in PEW subjects. Pathway analysis indicated that PEW-related metabolites reflected perturbations in fatty acid mechanism and induction of glyoxylate and dicarboxylate pathway attributed to gluconeogenesis. These results provide preliminary data in understanding metabolic alteration of PEW and corresponding abnormal metabolites that could potentially serve as biomarkers of PEW.End-stage renal disease patients undergoing maintenance hemodialysis (HD) are vulnerable to the protein energy wasting (PEW) syndrome. Identification and diagnosis of PEW relies on clinical processes of judgment dependent on fulfilling multiple criteria drawn from serum biochemistry, weight status, predictive muscle mass, dietary energy and protein intakes. Therefore, we sought to explore the biomarkers’ signature with plasma metabolites of PEW by using  Protein energy wasting (PEW) syndrome is characterized by a persistent depletion of protein stores and energy fuels in the body, caused by multiple nutritional and metabolic alterations  arising The term PEW as a uniform nomenclature was proposed by the International Society of Renal Nutrition and Metabolism (ISRNM 2008), after increasing research reported on pathophysiological mechanisms leading to the syndromes of muscle wasting, malnutrition and inflammation in CKD patients ,7. AccorIdentification of PEW thus far has relied on clinical judgment processes based on an extensive patient assessment. However, the application of the ISRNM diagnostic criteria to diverse hemodialysis (HD) populations is challenging , due to 1H-nuclear magnetic resonance (1H-NMR)-based metabolomics approach to facilitate identification of plasma metabolites related to the catabolism of muscle and fat tissues associated with facets of PEW development in HD patients. The metabolomics approach would facilitate the identification of a consistent, precise and reliable marker to improve the diagnosis of PEW. This unbiased quantitative method would detect abnormal metabolites related to PEW potential biomarkers, as well as clarify the underlying pathophysiological mechanism. The fact is, the metabolome represents the ultimate downstream products of cellular metabolism [In line with this need, we propose to use the cutting edge tabolism ,19 and ttabolism  to elucitabolism .1H-NMR based metabolomics approach to understand the metabolic alteration associated with PEW in maintenance HD patients.Metabolomics via the NMR platform offers a simple sample preparation for a convenient but comprehensive analysis for the identification of metabolite biomarkers for the diagnosis of PEW. Importantly, no study pertaining to identification of PEW metabolites using the metabolomics approach has been published to date. Therefore, this study sought to identify biomarker signatures of PEW using the p > 0.05). On the other hand, the PEW group exhibited a significantly higher trend towards a longer time on dialysis (p = 0.041), but lower trends of creatinine (p < 0.001) and blood urea (p = 0.036) levels compared to the NPEW group. The PEW group also demonstrated heightened inflammatory responses with higher IL-6 values (p = 0.006), although high sensitivity C-reactive protein (hsCRP) levels between groups was not significant (p > 0.05). Insulinemic status as per plasma insulin levels and homeostasis model assessment index of insulin resistance (HOMA-IR) scores were both significantly lower (p < 0.001) for the PEW compared to the NPEW group.The demographic characteristics, clinical and biochemistry data of the study population are presented in p < 0.05) except for total cholesterol (TC) and dietary protein intake (DPI)  .1H-NMR spectra were obtained for all 106 samples and spectra profiles of plasma samples from hemodialysis patients with PEW and non-PEW (NPEW) are presented in 2X(cum) = 0.342, R2Y(cum) = 0.426 and Q2(cum) = 0.222. This model was acceptable as the difference between R2Y(cum) and Q2(cum) was <0.3, indicating each sample was equally and uniformly contributing to the observed group separation [2Y-intercept value that was less than 0.3–0.4 and the Q2Y-intercept value of <0.05 supported the PLS-DA model validity, as values larger than 0.05 indicated overfitting [p value of <0.05 denoting a statistically significant model of PLS-DA [Further analysis using supervised partial least square-discriminant analysis (PLS-DA) was performed to maximize the group separation and facilitate the detection of discriminating metabolites in PEW and NPEW patient samples. paration . The apprfitting  in the of PLS-DA . The PLSp < 0.05) in PEW patients as compared to NPEW patients including 3-hydroxybutyrate (p < 0.001), acetate (p = 0.027), arabinose (p = 0.029), maltose (p = 0.021), ribose (p = 0.041), sucrose (p = 0.008) and tartrate (p = 0.018). In contrast, creatinine (p < 0.001) was the only metabolite that showed a significantly lower plasma concentration in PEW patients compared to NPEW patients  database were referenced to identify metabolic network(s) and biological relevance of the significant metabolite changes in PEW patients. The significant pathways identified by KEGG were fatty acid β-oxidation and gluconeogenesis. However, other metabolic pathways such as glyoxylate and dicarboxylate metabolism and carbon metabolism were also implicated . It may p < 0.05) between the groups, even after adjustment for these covariates (p > 0.05) between the group and gender, implying that the effect of group differentiation was not driven by this factor.Additionally, a one-way analysis of covariance (ANCOVA) was also performed to identify significant discriminating metabolites between the two groups after adjustment for covariates of age and dialysis vintage. Results indicated that specific metabolites 3-hydroxybutyrate, acetate and creatinine were significantly different , mid-arm muscle area (MAMA) and dietary energy intake (DEI). Some other parameters including serum creatinine and urea are also lower when compared to their counterpart suggesting more progressive muscle wasting , and in In this study, metabolomic analysis showed a significant separation of the metabolites profile in PLS-DA with a stronger clustering for the PEW group on the negative side of PC2 but on the positive side for the NPEW group. This suggests that the PEW syndrome induced that a specific metabolite pattern alteration resulted in different profiles between the groups. As metabolomics identifies and quantifies various groups of endogenous metabolites , metabolMetabolic pathway identification revealed that the PEW condition indicated systemic perturbations of fatty acid metabolism as well as inducing the glyoxylate and dicarboxylate metabolism attributed to gluconeogenesis. This is based on the following postulation: chronic suboptimal intake of the dietary protein and energy as evidenced in PEW patients will promote gluconeogenesis, which mobilizes fatty acids and amino acids from adipose tissue and muscle respectively ,34. On tIt is interesting however to observe that the PEW group in our study had lower insulinemic status (plasma insulin levels and HOMA-IR scores) when compared to the NPEW group. We postulated that this could be due to suboptimal food intake leading to overall reduction in insulin secretion . In ordep < 0.001) in PEW patients. About 95% of the creatinine is found in the muscle [In the current study, the level of serum creatinine (SCr) was significantly lower  and acetate (p < 0.05), which are implicated in the fatty acid β-oxidation pathway in patients with PEW. Gluconeogenesis is also fuelled by rapid lipolysis and release of fatty acids from adipose tissue [We also found significant elevation of the metabolites β-hydroxybutyrate  in PEW patients, although after adjustment with covariates this significance disappeared. Tartrate accumulation in PEW subjects may be ascribed to the endogenous production from either bacterial degradation in the gut or via tissue metabolism or from exogenous substrates such as medicine and food [p < 0.05) in the arabinose, maltose, ribose and sucrose concentrations in patients with PEW compared to NPEW patients, although after an adjustment with covariates this significance disappeared. The implication of this finding is unknown although it has been shown that longer dialysis vintage is related with unfavorable changes in the nutritional status of HD patients [An important observation from our study is that the level of tartrate was significantly increased  in the levels of 3-hydroxybutyrate, acetate, tartrate and creatinine to discriminate PEW from NPEW patients, clearly indicating there were aberrations in fatty acid metabolism and evidence for the presence of the gluconeogenesis pathway. Whereas NPEW patients who did not experience a persistent loss of protein and energy stores in their body demonstrated significantly opposite trends  for these metabolites indicating that utilization of fatty acids and amino acids as the primary energy-yielding fuel was non-essential. As this is a hypothesis generating study with no baseline data on the PEW syndrome, these discriminating metabolites between PEW and NPEW patients could potentially serve as biomarkers of PEW in HD patients.In response to suboptimal intakes of dietary protein and energy associated with the PEW condition, the body will promote gluconeogenesis and fatty acid oxidation to supply energy. This would affect the level of specific metabolites involved in the mechanisms. We found significant differences . All patients provided written informed consent.n = 53) and non-PEW (n = 53) HD patients.This was a case-control study based on a subset of data collected for the multicenter Hemodialysis Nutrition Study (HDiNS). The inclusion criteria of the HDiNS study were HD patients above 18 years old, dialyzed for at least 3 months and willing to provide fasting blood samples. Patients with poor adherence to the HD regime, cognitive impairment or terminal illness were excluded. The ISRNM criteria  were useTypically, pre-dialysis blood samples were collected from patients after 8–10 h fasting through their dialysis access site during a mid-week dialysis session. Then plasma was isolated by centrifugation and divided into aliquots. For this case-control study, a total volume of 0.5 mL of plasma aliquot collected in heparin tubes was reserved for metabolomics analysis, as other anti-coagulants such as EDTA and citrate or other added stabilizers give additional signals in the NMR spectra . SamplesSerum urea and creatinine data was obtained from patients’ medical records. Additionally, the serum high-sensitivity C-reactive protein (hsCRP) was measured by an immunoturbidimetric assay and serum insulin by the electrochemiluminescence assay method by an independent laboratory using automated clinical chemistry . IL-6 concentrations were measured in-house by the sandwich enzyme-linked immunosorbent assay (ELISA) method using a commercial kit, IL-6 High Sensitivity Human Elisa Kit . HOMA-IR was calculated using both serum glucose and serum insulin levels using the following equation :HOMA-IR 2PO4) prepared in deuterium oxide (D2O) containing 0.2% trimethylsilyl propanoic acid  and 0.1% imidazole  in 1:2 ratio (200 μL filtrate + 400 μL D2O). A total of 600 μL of prepared samples were then transferred to 5 mm NMR tubes.Upon thawing of frozen samples, a total of 500 μL plasma were vortexed for a minute followed by a centrifugation at 10,000 rpm for 2 min in order to remove solid debris. Plasma supernatant (400 μL) was then transferred into a 0.5 mL 3 kDa centrifugal filter  to remove macromolecules such as proteins, lipids, lipoproteins and the protein-bound form of molecules  and spun1H-NMR spectra were acquired at 499.887 MHz on a 500 MHz Varian INOVA NMR  at a temperature of 26 °C. Water signals and broad protein resonances were suppressed by the combination of PRESAT and the Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence. NMR spectra of 10 ppm spectral width were obtained from a total of 128 scans and an acquisition time of 536 s. NMR spectra were then processed with Chenomx NMR suite version 8.3 software  by applying a consistent setting for all sample spectra; 0.50 Hz of line broadening, autophasing, baseline-corrected (Whittaker spline), referenced to TSP as an internal standard and referenced to imidazole as a pH indicator. Intelligent binning was used to divide the spectral region from 0.50 to 10.00 ppm into equal bins (0.04 ppm). The peak ppm values were calibrated with reference to the TSP signal at 0 ppm. Prior to the analysis, the region of spectra associated with residual water (4.77–4.86 ppm) and imidazole (7.29–7.33 ppm and 8.24–8.32 ppm) were removed. Subsequently, the respective spectra data were converted into a table of common integrals containing a non-negative value for multivariate data analysis.NMR analysis method was adapted and optimized from Maulidiani et al. . BrieflyMultivariate data analysis of the processed spectra was performed using SIMCA-P software version 14.1 . Firstly, data were mean-centered, and Pareto scaled by dividing each variable by the square root of its standard deviation. Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were used to analyze the spectra data. PCA is an unsupervised statistical analysis used to display the dominating trends of clustering inherent in the data set as well as the outliers . A regreChenomx NMR suite version 8.3 software  was used to identify and quantify metabolites. Based on multivariate data analysis, parts of the spectra (ppm) responsible for the difference between groups were identified using Chenomx software by matching the corresponding peak based on the position, intensity and line-width with the 500 MHz metabolites library available from the software. The area under the peak corresponded to the relative concentration of the metabolites. Once the changes in metabolite concentrations had been determined, the metabolic pathway identification was then performed based on the Kyoto Encyclopedia of Genes and Genomes database .p value of <0.05 considered to be significant. Data for categorical variables were reported as number and percentage values, while continuous data were reported as mean ± SD. An independent t-test was performed to analyze continuous data whilst Chi-square (χ2) statistics was used to evaluate differences between categorical variables. A one-way ANCOVA with the Bonferroni post-hoc test was performed to determine any significance between the groups for the identified metabolites when adjusted for covariates.Normality of the data was checked prior to data analysis. Data that was not normally distributed was log transformed. The SPSS statistical software  was used for statistical analysis with a In summary, the metabolomics analysis revealed that the plasma metabolic profiles of PEW subjects were altered in response to the syndrome that distinguished PEW from the NPEW group. Significant differences in the levels of 3-hydroxybutyrate, acetate, tartrate and creatinine were clearly ascribed to the presence of PEW, indicating there were aberrations in fatty acid metabolism and the presence of the gluconeogenesis pathway. Overall, these metabolites can be considered as the potential biomarker signature of PEW in HD patients. Although the results need further validation, this study demonstrated the metabolomics approach has the potential to develop a diagnostic tool for PEW syndrome.


Wnt signaling is well-known to play major roles in the hematopoietic system, from embryogenesis to aging and disease. In addition to the main β-catenin-dependent pathway, it is now clear that Wnt5a and the structurally related Wnt5b are essential for hematopoiesis, bone marrow colonization and the final steps of hematopoietic stem cell (HSC) maturation via β-catenin-independent signaling. Wnt5a and Wnt5b ligands prevent hematopoietic exhaustion , induce the proliferation of progenitors, and guide myeloid development, in addition to being involved in the development of aging-related alterations. The aim of this review is to summarize the current knowledge on these roles of Wnt5a and Wn5b signaling in the hematopoietic field. Hematopoiesis is the process in which all blood cells are produced, starting from a unique and scarce population of hematopoietic stem cells (HSCs). This population is characterized by multipotency (ability to form mature cells of various lineages of same tissue), capacity for self-renewal, and long-term hematopoietic maintenance after transplantation ,2. As reAccording to the classical model of hematopoietic differentiation, the multipotent progenitors separate into two branches from which myeloid or lymphoid lineages will arise ,10. ThesAlthough the classical model is well accepted for adults and illustrates the steps involved in hematopoiesis, it is noteworthy that numerous other models were proposed and are often being revised to include new findings, such as the uneven time point in progenitors branching from HSC ,15,16,17It is known that cytokines and growth factors are key players in all steps of hematopoietic regulation and development, but new factors in this context are not commonly described. Recent findings point to an important role for Wnt signaling in HSC maintenance and differentiation, showing that the Wnt pathway is crucial to subsequent events in myeloid and lymphoid differentiation. Whereas, originally, Wnt/β-catenin-dependent signaling was a main area of focus, it is now clear that signaling through β-catenin independent signaling with the involvement of Wnt5a and Wnt5b plays major roles in hematopoietic development, differentiation and ageing. The aim of this review is to summarize the current knowledge on the role of Wnt5a and -b signaling in the hematopoietic field. Here, when HSC are addressed, this refers to ST-HSC, except when specified to besomething else.Wnt ligands are secreted lipid-modified glycoproteins, which rely on their post-translational modifications for the secretion and activation of their receptors  ,22. The The β-catenin pathway has been the most described and studied, being triggered when a Wnt ligand binds to a Fzd receptor in the presence of low-density, lipoprotein-receptor-related protein (LRP)5/6 co-stimulatory receptors ,27. β-caIn the absence of an extracellular Wnt ligand, the cytoplasmic abundance of β-catenin is kept low due to phosphorylation, ubiquitination, and proteasome degradation . This isWhen an extracellular Wnt ligand couples to its Fzd receptor, it triggers intracellular alterations that induce the stabilization of β-catenin. For Wnt/β-catenin signaling, the Fzd receptor has to be juxtaposed with an LRP family molecule. LRP is recruited in the presence of Wnt, forming a heterotrimeric complex responsible for intracellular signaling ,31. LRP β-catenin activity in the nucleus is normally inhibited by gene repressors (the most well-known is Groucho), but its accumulation competitively drives these repressors away from transcriptional target sites, allowing TCF and LEF-1 DNA-binding and gene transcription, stimulated by β-catenin . It is i2+ [In addition to Wnt/β-catenin signaling, Wnt ligands can trigger several β-catenin-independent pathways . As observed for Wnt/β-catenin signaling, these pathways are activated by extracellular Wnt ligand binding to a plasma membrane receptor, triggering intracellular changes. In vertebrates, β-catenin-independent Wnt signaling is primarily categorized as two main pathways: PCP and Wnt/Ca2+ , althoug2+ .The planar cell polarity (PCP) pathway, first identified in Drosophila, is the best characterized β-catenin-independent pathway, and is essential in cellular polarity. As for the Wnt/β-catenin pathway, the PCP pathway is widely conserved evolutionarily and uses Fzd receptors, but without the need for LRP molecules .The PCP signaling pathway is involved in the directional instruction of cells in a tissue to support its functional properties. In the first studies on Wnt/PCP signaling ,40, Fzd Drosophila, in vertebrates, they are known as Celsr and Vangl, respectively .Interestingly, the asymmetrical distribution of these complexes forms an axis that orientates cellular function. The Wnt/PCP pathway is also known as the Wnt/JNK pathway , because2+ pathway is widely active in the hematopoietic system and has been a topic of increasing interest in recent years. The proposed pathway involves the activation of a G-protein after Wnt/Fzd binding [2+-release, CaMKII  and PKC (protein kinase C) phosphorylation [2+ signaling events driving subsequent gene transcription [Unlike the PCP pathway, the Wnt/Ca binding  and resurylation ,49. The cription .2+ release from the endoplasmic reticulum, leading to increased cytoplasmic Ca2+ concentrations [For this pathway, the above-mentioned co-receptor ROR2 plays a key role. ROR2 functions as a co-receptor for Fzd, and induces membrane PLCβ activation and second messenger production, such as inositol 1,4,5-trisphosphate (IP3) and 1,2 diacylglycerol (DAG), which results in Catrations . When Wntrations , which m2+ signaling, involving two possible mechanisms: (1) activation of G-proteins (similar to ROR2), and (2) activation of transient receptor potential (TRP) receptors, causing extracellular Ca2+ influx [Ryk has also been associated with Wnt/Ca+ influx .Studies on Wnt signaling in the hematopoietic field started a few years after the initial discovery of Wnt and were mainly focused on the β-catenin-dependent branch. Wnt signaling appears to play key roles in the ontogenesis of the hematopoietic tissue, from the embryonic period to adult stages.The primitive endoderm and primitive streak are particularly important in the ontogeny of the hematopoietic system, since they are precursors of the yolk sac, in which the first wave of hematopoietic production takes place (primitive hematopoiesis) . HoweverYolk sac and blood island formation is crucial in hematopoietic initiation and, at this point, Wnt5a gene expression peaks and its signaling appears to play an important role. Interestingly, this initial hematopoietic commitment can be accelerated by DMSO treatment and repressed by retinoic acid . AccordiStudies using embryonic stem cell lines describe stage-specific waves of Wnt (the in vivo relevance of this is not established). In these cells, right before the formation of embryonic bodies (a stage that resembles the embryonic three germ layers), there is a peak in Wnt5a expression that seems to be related to hematopoietic initiation, reflecting the observations in vivo. Embryonic bodies can differentiate to blast-like colonies, immunophenotypically expressing Sca-1 and c-Kit (CD117), recognized markers of primitive hematopoietic development . At thisAs discussed previously, it may be too simplistic to extrapolate pathway activation only on the basis of ligand gene expression . LRP5 waDefinitive hematopoiesis originates in the fetal liver  with the colonization of progenitor cells, and subsequently, after maturation, migration to the spleen and bone marrow . Bone maStudies in zebrafish showed that there is a peak in Wnt16 expression, a β-catenin-independent pathway activator, as soon as the cells arrive in the bone marrow ,66. It sAfter bone marrow colonization, a flow of hematopoietic cell production is established, which is maintained for most of life, even though the fetal origin of adult HSCs is more accepted for lymphopoiesis than myelopoiesis . Primiti2+ concentration is increased in peripheral bone areas and cells expressing membrane Ca2+ sensors are attracted to these areas, characterized by reduced irrigation and hypoxia [In addition to the HSCs and progenitors themselves, the microenvironment and extracellular molecules are important to hematopoiesis as well . Ca2+ co hypoxia ,68. In t hypoxia .2+, but a higher presence of blood vessels and, as a consequence, are normoxic. The more active HSCs and multipotent progenitors reside in these vascular niches  , and is 2+ pathway. It can be hypothesized that Wnt/Ca2+ signaling would be important for the LT-HSC population, considering the increased levels of this ion in the endosteal niche, and the little available evidence for nuclear β-catenin accumulation in these cells.Flamingo is commonly associated with the Wnt/PCP pathway, whereas Fzd4, Fzd8, Ror2 and Ryk seem more related to the Wnt/CaThe intracellular mechanisms involved in Wnt5a-driven quiescence maintenance are not completely elucidated, but Ryk participation and a consequent decrease in reactive oxygen species formation seem important , in addiFor the ST-HSC, however, cell cycle activity as well as nuclear β-catenin accumulation is observed ,89. FurtFor this population, Wnt5a now acts as a growth factor and, instead of inhibiting, it promotes cell cycle progression . IncreasAdditionally, Wnt5a also seems to have a role in reducing proliferation by inducing apoptosis in LT-HSCs , which mWith hematopoietic branching in myeloid and lymphoid lineages, Wnt receptor and co-receptor expression patterns seem to change drastically and, as a consequence, responsiveness to one or several ligands may change as well. Knockout models for Wnt signaling were of great help to clarify the involvement of these pathways in hematopoiesis. For the lymphoid branch, Wnt5a knockout mice present decreased thymic cellularity and higher apoptosis of double positive thymocytes , coincidLymphoid Enhancer Binding Factor 1 (LEF-1), an important transcription factor for lymphopoiesis , is tranFor myelopoiesis, on the other hand, some roles for Wnt5a have been described , includiMyelopoiesis is responsible for the formation of granulocytes, monocytes, platelets and erythrocytes, originating from a common myeloid progenitor, as schematized in 2+ signaling was proposed [Erythroid progenitors are highly responsive to Wnt5a, entering the cell cycle and proliferating (by PI3K activation)  or diffeproposed .Little is known about Wnt5a (or b) in monocyte differentiation. Stimulation with Wnt5a had similar effects as Wnt3a treatment regarding IL-3 presence, causing an inhibition of monocytic differentiation . InteresInterestingly, granulopoiesis seems less affected in Wnt5a and -11 knockdown models . It is dThere is increasing evidence that well-known hematopoietic alterations during aging  are related to changes in Wnt signaling. The scenario for adult subjects seems to pass through gradual changes, in which the expression levels of environmental Wnt5a decrease, whereas the opposite takes place in HSCs, which acquire more Wnt5a with age . These gThe increase in Wnt5a signaling activation in aged HSCs is proposed to inhibit HSC cell cycle entering and hamper hematopoietic differentiation, and it seems to affect both, LT and ST-HSC . In addiFor the cells in the committed progenitor phase, this change in Wnt status may act to skew the lineage commitment in favor of myeloid cells, as lymphoid cells are more susceptible to Wnt5a mediated inhibition than myeloid cells. In fact, for the myeloid lineage, the increase in Wnt5a does not hinder differentiation; instead, it stimulates it, since granulocyte–monocyte progenitors as well as granulocytes are increased in aged subjects .+/−) provides more evidence that Wnt5a is indeed one of the main molecules related to aging-related myeloid skewing, since these animals do not present increased Wnt5a expression with aging and their blood cell counts are similar to young control animals [Interestingly, analyzing aged mice haploinsufficient for Wnt5a , chronic lymphocytic leukemia (CLL), and lymphoma are of lymphoid origin, whereas chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and myelodysplastic syndrome (MDS) are of myeloid origin. The development of hematological malignancies requires malignant clones to lose their regulatory mechanisms, resulting in the production of a large number of dysfunctional cells and the disruption of normal hematopoiesis.Most of the investigation about Wnt signaling and leukemogenesis focuses on β-catenin dependent signaling. There is evidence that β-catenin overexpression and its increased or constitutive activation drives leukemia initiation in some types of myeloid malignancies ,100,101,In CML, whilst in vivo leukemic stem cells’ (LSC) survival was not affected by the inhibition of β-catenin signaling, it was in vitro , suggestAlthough most Wnt signaling research is focused on the Wnt/β-catenin pathway, interest in the independent branch is growing. In lymphoid leukemia, there is evidence that Wnt5a induces ROR1/ROR2 hetero-oligomerization and Rac1 activation , and thaThere is evidence pointing at Wnt5a as a therapeutic molecule against leukemias; by inhibiting β-catenin-dependent signaling, it would promote differentiation and reduce aberrant proliferation ,101,119.Other studies in B-ALL and CLL models revealed a positive correlation between ROR1 expression and STAT3 activation ,122. Rec+) presented increased chemoresistance, which correlated with Wnt5b signaling activation. The study also demonstrated that the generation of anti-EpCAM antibodies promotes the effective elimination of AML cells through the immune system [Interestingly, a study of the gene expression pattern in AML revealed that cells exhibiting high levels of the epithelial cell adhesion molecule  for CLL, after promising results were obtained in preclinical studies . Recent Chimeric antigen receptor T (CAR-T) cell therapy is another type of ROR1-targeted therapeutic approach that has been explored, with the advantage of low off-target cytotoxicity due to the aforementioned unique expression of ROR1 in cancer cells, such as CLL and MCL cells, but not in normal B cells . This stThe effects of ROR1 inhibition have also been studied in other preclinical models, such as MCL, which expresses high levels of ROR1 as well. Studies using drug combinations revealed that inhibitors of several kinases, such as BTK , FAK , and PKC  were more effective in killing sensitive MCL cells after shRNA-mediated ROR1 inhibition .In contrast to ROR1, therapies able to target RYK have not yet been successfully demonstrated/developed. Recently, however, a monoclonal antibody with high affinity for the RYK WIF domain was developed by phage library, which could block Wnt5a signaling, and as such could hold promise for future therapies .Despite the significant progress made over in recent decades in understanding the mechanisms by which Wnt signaling affects hematological malignancies, its full role in the pathogenesis of these diseases has not yet been completely established, mainly when considering the β-catenin independent branch and Wnt5 ligands. A deeper insight into the molecular mechanisms of Wnt signaling in hematological malignancies will pave the way for the development of innovative (combination) therapies in the near future.Molecules involved in the modulation of hematopoiesis have been known for decades, but the introduction of Wnt signaling in this context changed how we understand this system, from early development to aging and disorders . Wnt sigThe ability to frequently rely on switches between β-catenin-dependent and -independent pathways suggests a highly interconnected system. In addition, multiple mechanisms of action of ligands, such as Wnt5a, further reinforce the idea of an interdependent system.2+, PI3K/AKT, STATs), extracellular molecules involved in Wnt signaling modulation (proteoglycans and extracellular matrix molecules) and interaction between growth factors and Wnt ligands, transcriptional targets of β-catenin-independent signaling, not to mention the mechanisms involved in Wnt-driven hematopoietic senescence, which is a high interest field. Aside from this, it may be of interest to focus on intermediate molecules (not only ligands and receptors), as it is undoubtable that the individual components involved in Wnt signaling are critically important in hematopoiesis, and that further understanding of the complex mechanisms involved in Wnt signaling and its relation to hematopoiesis, both in health and disease, will provide novel insights leading to future therapeutic strategies.By now, the understanding of Wnt ligands in the hematopoietic system is under construction and, mainly when considering the β-catenin-independent pathway and the roles of Wnt5a and b in lineage specification and aging. Several points of attention still need to be addressed and elucidated, such as Wnt signaling crosstalk with other pathways involved in hematopoietic proliferation and differentiation (Ca


Three thermoset resin systems—bio-epoxy, epoxy, and polyester-with 30 v% flax fiber reinforcement have been studied to identify the optimal fiber–resin combination in a typical composite structure. Tensile, interface and interlaminar shear strength together with flexural and impact damage tolerance were compared in this study. The results revealed that mechanical and interfacial properties were not significantly affected by the different resin systems. Microscopy studies reveal that epoxy laminates predominantly fail by fibre linear breakage, polyester laminates by fiber pull-out, and bio-epoxy laminates by a combination of the two. The higher failure strains and pull-out mechanism may explain the better impact damage tolerance of polyester composites. Flow experiments were also conducted, revealing faster impregnation and lower void content with polyester resin, followed by bio-epoxy, due to their lower viscosities. Overall, bio-epoxy resin demonstrates comparable performance to epoxy and polyester resins for use in (semi-)structural bio-composites. The composites industry has drawn significant attention to utilizing the benefits of lightweight, low-cost natural fibers such as flax, jute, kenaf, hemp and sisal to replace glass fiber in specific stiffness-limited design applications . In addiBesides the resin system, it is also necessary to select the best fiber architecture to tailor the performance of natural fiber composites. To utilize maximum mechanical properties of fiber in composites, all the fibers have to be placed parallel to the loading direction . Natural2) was sourced from Tilsatec Ltd, Flanshaw Lane, UK. All the flax fibers used in this process have a unidirectional arrangement without any twist. A small percentage of poly-lactic acid polymer binder (3 w%) was used to hold the flax in tape format. The fiber assembly was passed through a heating and pressing unit during the manufacturing of the tapes , EL2 Epoxy Laminating Resin (resin: hardener-100:30), and low viscosity pre-accelerated isophthalic IP2 polyester resin with 2% MEKP catalyst. The physical, mechanical and flow properties of the different resins are given in Twelve layers of flax tape preform were cut to size (170 mm × 350 mm) and then cross-laminated in 0/90 fashion. The sequenced laminates were placed on the mold surface, sandwiched between layers of peel ply. A net mesh infusion medium was placed on top to ensure uniform flow distribution. A nylon bag was used to seal the reinforcement under full vacuum, with the inlet pipe clamped. The inlet pipe was then placed into the de-gassed resin bath to resume the vacuum reinfusion process. According to the manufacturer’s instruction, the infused panel was left with a sealed bag for 24 h at room temperature in order to fully cure the composites panel. x) and filling time (t) were recorded, according to the recommendations of Subbiah et al ..32]. Genp > 0.05). The only statistically significant difference (p < 0.05) is in the peak force for bio-epoxy composites, which is found to be much lower than that of epoxy and polyester composites.The falling weight impact (IFW) test is one of the test methods used to assess the impact properties (fast fracture or rapid crack propagation) of composites. There are a limited number of studies in the literature assessing the damage tolerance properties of natural fiber composites. However, this test is one of the essential ways of assessing the suitability of composites materials for (semi-)structural application. We conduct tests at two impact energies (5 J and 10 J) to investigate the damage process in flax tape thermoset composites. Load-deflection curves obtained from the impact test can provide some meaningful information to evaluate the damage tolerance of composite structures . Figure At 10 J impact, energy epoxy and polyester composites once again outperform the bio-epoxy composites in terms of peak loads b. In add2 at 5 J and 10 J, respectively). For the bio-epoxy composites, the damaged area under both energy levels is much higher (15 and 25.5 cm2). No inter-ply delamination is visible for any of the tested samples which may occur due to non-woven architecture that generates fiber flaws between 00 and 900 directions. With regards to energy absorption, polyester composites offer the best performance  void content, with polyester composites, owing to the low viscosity of polyester, exhibiting the lowest void content (1%) and fastest filling times. The interfacial and mechanical study revealed that bio-epoxy resin is highly comparable with epoxy and polyester resin systems. A noticeable difference was observed in the failure modes of composite laminates with three resin systems. Epoxy composites, for instance, formed a relatively stronger bond with the flax fibers, leading to more fiber breakage in tension. In contrast, polyester composites exhibit more fiber pull-out and debonding. Bio-epoxy composites show mixed modes of failure, if not more similar to epoxy composites. Similarly, polyester composite exhibit more ductile behavior, with relatively larger failure strains in tension and flexure, but also substantially greater dent depth in falling weight impact tests. Overall bio-epoxy composites proved to be a suitable alternative to conventional resins in terms of mechanical behavior.


Cholangiocarcinoma (CCA) is a fatal malignant tumor of biliary epithelial cells involving intra- or extra-hepatic bile ducts. The prognosis of CCA is generally poor due to its diagnosis at the late stages. The currently employed chemotherapeutic agents do not increase the survival rate in patients with unresectable CCA. Accordingly, there is a need to identify new therapeutic agents for the effective management of intra- and extra-hepatic CCA. Clinical as well as preclinical studies have suggested the key role of the activation of Wnt/β-catenin signaling pathway in the induction and progression of CCA. There is an up-regulation of different Wnt ligands including Wnt2, Wnt3, Wnt5, Wnt7 and Wnt10 along with redistribution of β-catenin (more expression in the nucleus and lesser on the cell surface due to nuclear translocation of β-catenin) in different types of malignant biliary tumors. Apart from the role of this pathway in the induction and progression of CCA, this pathway is also involved in inducing multidrug resistance by inducing the expression of P-glycoprotein efflux pump on the cancer cells. These deleterious effects of Wnt/β-catenin signaling are mediated in association with other signaling pathways involving microRNAs (miRNAs), PI3K/AKT/PTEN/GSK-3β, retinoic acid receptors (RARs), dickkopf-1 (DKK1), protein kinase A regulatory subunit 1 α (PRKAR1A/PKAI), (SLAP), liver kinase B1 (LKB1) and CXCR4. The selective inhibitors of Wnt/β-catenin signaling may be potentially employed to overcome multidrug-resistant, fatal CCA. The present review discusses the role of Wnt/β-catenin along with its relation with other signaling pathways in the induction and progression of CCA. Cholangiocarcinoma (CCA) is a fatal malignant tumor of biliary epithelial cells involving intra- or extra-hepatic bile ducts. The prognosis of CCA is generally poor due to its typical late diagnosis at the advanced stage, the ability to metastasize and relapse ,2. The iCTNNB1 gene-encoded multifunctional β-catenin proteins [Wnt/β-catenin signaling comprises Wnt ligands, which are 350–400 amino acids long lipid-modified glycoproteins and proteins . Furtherproteins . The preSurgery is the preferred treatment option for all types of CCA. Since most of the patients are diagnosed at the advanced stage, therefore, a minority of patients have the option of cure with the surgical removal of this aggressive . Liver tn=29), which was again correlated to the high grade of tumor and metastasis [n=31). Accordingly, it is suggested that reduced immunoexpression of β-catenin on the plasma membrane and its overexpression in the nucleus may be used as prognostic markers in cholangiocarcinogenesis [There have been a number of studies showing a decrease in the expression of β-catenin on the plasma membrane with a corresponding increase in the expression in the nucleus ,26. It htastasis  also eluogenesis .The data from the study of 37 biliary tract specimens obtained from biliary tract patients have shown mutations in the β-catenin gene suggesting the role of this gene in the poor prognosis in these cancer patients . AnotherIt has been proposed that intramembranous proteolysis of epithelial cell adhesion molecule (EpCAM) may release an epithelial cell intracellular domain (EpICD) into the cytoplasm, which subsequently translocates in the nucleus to form a complex with β-catenin. The data obtained from the surgical specimens of extra-hepatic CCA patients and CCA cells have shown a correlation between the nuclear expression of EpICD and β-catenin. An increase in the nuclear expression of EpICD and β-catenin was also significantly correlated with histologic grade of the tumor, increased tumor growth and proliferation. On the other hand, small interfering RNA (siRNA)-mediated inhibition of EpCAM decreased cellular proliferation, migration and invasion suggesting the importance of localization of EpICD and its interaction with β-catenin in the progression and invasion of ECC  Table 1Table 1.It has been proposed that β-catenin is involved in inducing malignancy in normal cholangiocytes. The study by Dutta et al.  describeIn CCA, the features of epithelial–mesenchymal transition (EMT) have been identified and EMT phenotypes are associated with poor survival rate in these patients . It is sThe role of Wnt/β-catenin signaling in the development of multidrug resistance in CCA has also been described. In human multidrug-resistant CCA cells (QBC939/5-FU), the expression of P-glycoprotein and β-catenin was up-regulated. Inhibition of the Wnt/β-catenin signaling pathway by β-catenin siRNA overcame the multidrug resistance by decreasing the expression of the P-glycoprotein efflux pump . In anotOpisthorchis viverrini -induced cholangiocarcinogenesis in a hamsters, an up-regulation of Wnt/β-catenin signaling with an increase in the expression of Wnt3, Wnt3a, Wnt5a, Wnt7b and nuclear β-catenin in CCA tissues has been described [The role of different Wnt ligands in the development and progression of CCA has been described . Based oescribed  (Table 3escribed .The role of Transcribed-ultraconserved regions (T-UCR), non-coding RNAs, downstream of the Wnt/β-catenin pathway has also described in biliary cancers. It was shown that there is overexpression of T-UCR uc.158- in CCA cells with nuclear localization of β-catenin. However, the expression of uc 158 was reduced following treatment with Wnt/β-catenin inhibitors. Furthermore, co-transfection of uc.158- inhibitor prevented cellular proliferation . BoulterDavaadorj et al.  describen=44) using immunohistochemistry. The authors reported that immunoreactive Wls were detected in normal cholangiocytes, but not in normal hepatocytes. Moreover, the intensity for immunoreactive Wls was directly correlated with the tumor stage, tumor-node-metastasis stage and lymphatic invasion. Accordingly, it may be suggested that the expression of Wls is positively correlated to tumor stage and lymphatic invasion and it may be employed as a potential marker to diagnose the tumor stage and metastasis.Wntless (Wls) are transmembrane proteins that function to transfer the palmitoylated Wnt proteins from the endoplasmic reticulum to the plasma membrane. Since these proteins function to increase cell proliferation, decrease apoptosis and promote cell survival, therefore, the increased expression of these proteins is associated with the development and progression of different types of cancers ,53. The Scientists have explored the interrelationship between Wnt/β-catenin with other signaling pathways leading to the development of CCA .MicroRNAs (miRNAs) are approximately 20–25 nucleotide-long non-coding RNAs and these function to control the gene expression by binding to 3′-UTR of target mRNA . There hin vitro and in vivo studies, it was reported that overexpression of miRNA Let-7c inhibits the tumorigenic properties of CCA by modulating β-catenin signaling pathway [In contrast with the above-described roles of miRNA in biliary cancers, the expression of MIR22HG was found to be significantly down-regulated in CCA tissues and cell lines. Moreover, MIR22HG negatively regulated the mRNA and protein expression of β-catenin. The overexpression of MIR22HG inhibited cell proliferation, migration and invasion in CCA along with a decrease in the expression of β-catenin . The dec pathway   participates in the progression of CCA via EP3 receptors, which trigger PI3K/AKT/GSK-3β pathway to up-regulate the expression of β-catenin proteins [2 leads to the activation of β-catenin in human CCA cells. Moreover in this study, administration of docosahexaenoic acid (DHA) was shown to dephosphorylate GSK-3β (increase in enzymatic activity), reduce the expression of β-catenin protein and attenuate the growth of tumor cells again. It suggests that the dephosphorylation of GSK-3β and increase in its enzymatic activity may induce protective effects by reducing the expression of β-catenin in CCA [The interrelationship between GSK-3β/β-catenin has been described in the development of multidrug resistance in CCA and it is shown that Wnt3a activates GSK-3β/β-catenin signaling to up-regulate the expression of P-glycoprotein efflux pump on the tumor cells . Anotherproteins . The resn in CCA . In anotn in CCA .Retinoic acid, a bioactive metabolite of vitamin A, is involved in regulating a large number of biological processes including cellular proliferation, differentiation and apoptosis. RARs , and RXRs are the retinoid receptors ,72. StudIn vitro studies showed that the genetic depletion of DKK1 in the HCCA cells significantly reduces the expression of β-catenin along with inhibition of cellular proliferation and colony formation.The family of Dickkopf (DKK) comprises DKK1, DKK2, DKK3 and DKK4. Among the four members, the role of DKK1 is widely studied, including the progression of different cancers. In different types of cancers, the up-regulation of DKK1 has been positively correlated with the progression of cancers ,79. Moreviz. PKA regulatory subunit 1 α (PRKAR1A/PKAI) and PKA regulatory subunit 2 β [The cAMP‐dependent protein kinase A (PKA) constitutes an important signaling molecule in a variety of cells and its enzymatic activity is regulated by two different regulatory domains, unit 2 β . The impunit 2 β . Moreoveunit 2 β .Studies have also suggested the interrelationship of Wnt/β-catenin with other signaling pathways in the induction and development of CCA. It has been shown that there is decrease in the expression of SRC-like adaptor protein (SLAP)  and liveThere is up-regulation of different Wnt ligands including Wnt2, Wnt3, Wnt5, Wnt7 and Wnt10 along with differential changes in the expression of β-catenin (more expression in the nucelus and less expression on the membrane) in different types of malignant biliary tumors. Accordingly, activation of Wnt/β-catenin pathway not only participates in the induction and progression of CCA, rather it may also induce multidrug resistance in these cancer cells. These deleterious effects of Wnt/β-catenin signaling are mediated in association other signaling pathway involving miRNAs, PI3K/AKT/PTEN/GSK-3β, RARs, DKK1, PRKAR1A/PKAI, SLAP, LKB1 and CXCR4. The employment of selective inhibitors of Wnt/β-catenin signaling may be potentially employed to overcome multidrug resistant, fatal CCA.


WNT family member 6 (WNT6) is a member of the highly conserved WNT protein family. It plays an essential role in the normal development process, not only in embryonic morphogenesis, but also in post-natal homeostasis. WNT6 functions in mice and humans. This review summarizes the current findings on the biological functions of WNT6, describing its involvement in regulating embryogenesis, decidualization, and organ development. Aberrant WNT6 signaling is related to various pathologies, such as promoting cancer development, lung tuberculosis, and kidney fibrosis and improving the symptoms of Rett syndrome (RTT). Thus, due to its various functions, WNT6 has great potential for in-depth research. This work not only describes the signaling mechanism and function of WNT6 under physiological and pathological conditions, but also provides a theoretical basis for targeted therapy. WNT family member 6 (WNT6), a member of the Wingless/integrase 1 (WNT) family comprising at least 19 members in mammals, is a secreted glycoprotein. The WNT signaling family is highly conserved and in several species play essential roles in various physiological processes during which it is either stimulated or inhibited. WNT signals mediate various cellular functions by binding to different receptors on the cell membrane including 10 members of human Frizzled (FZD) receptors, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) receptor, and many non-class FZD receptors . The WNTThe WNT/β-catenin pathway is stimulated by an extracellular WNT ligand that binds to a FZD receptor and a LRP5/6 co-receptor (FZD and LRP5/6) on the cell membrane and recruits disheveled (DVL) proteins to promote the dissociation of the β-catenin destruction complex, leading to an intracellular signaling cascade. The β-catenin destruction complex consists of the scaffold protein Axin, Adenomatous Polyposis Coli (APC), Casein Kinase-1 (CK-1), and Glycogen Synthase Kinase-3β (GSK-3β). In the absence of WNT signaling, the β-catenin destruction complex phosphorylates β-catenin, leading to subsequent proteasomal degradation of the protein. However, in the presence of WNT signaling, WNTs inactivate the β-catenin destruction complex and prevent β-catenin degradation, thereby allowing β-catenin accumulation in the cytoplasm and translocation into the nucleus. Then β-catenin binds to the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors, mediating the activation of downstream targets  such as 2+ pathways. The WNT/PCP pathway is associated with the downstream activation of c- Jun N-terminal kinase (JNK) and ras homolog family member A (RhoA) (2+ pathway is associated with the activation of phospholipase C (PLC) and leads to the formation of inositol 1,4,5-triphosphate (IP3) and 1,2 diacylglycerol (DAG), thereby increasing Ca2+ level and activating downstream targets. The WNT/Ca2+ pathway is associated with muscle contraction, gene transcription, and enzyme activation  . The WNTA (RhoA) . Converstivation  and actitivation .WNT6, a WNT family member that is highly conserved in various species, mainly considered to be a member of the β-catenin-dependent WNT signaling pathway . Howeverin vivo mouse models, in vitro mouse and human cell systems, and clinical data in humans.Because WNT6 is closely associated with many diseases, understanding its biology is important. Our review discusses the functions of WNT6 and its various signaling pathways, identified through studies of various wnt6 gene is located in the 2q35 region of the human chromosome, and encodes a 365 amino-acid polypeptide with an N-terminal signal peptide, a WNT core domain and a RGD motif. Wnt6 is fused with wnt10a in a head-to-tail manner, with an interval less than 7.0 kb , Pax8, and E-cadherin is markedly increased in the mesenchyme , an extracellular matrix protein in the connective tissue of dentin that is primarily released by fibroblasts and osteoblasts , the precursors of odontoblasts in human dental papilla tissue, have the potential to differentiate into odontoblasts and dental pulp cells , indicatin vitro . The Brdin vitro . It alsoeoblasts . WNT6 preoblasts . These rwnt6, wnt10a, and wnt10b was markedly reduced in adipocytes both in vitro and in mice. The ectopic mRNA expression of wnt6, wnt10a, and wnt10b suppresses the expression of two adipogenic transcription factors, peroxisome proliferator-activated receptor-γ (PPARγ), and CCAAT/enhancer binding protein-α (C/EBPα) in 3T3-L1 pre-adipocytes and completely inhibits neutral lipid accumulation  differentiate into various cell types including adipocytes and osteoblasts. The dysregulation of these two cell types strongly correlates with obesity, type 2 mellitus diabetes, and osteoporosis. Therefore, maintenance of the normal functioning of MSCs is important. WNT/β-catenin signaling is an important regulator of mesenchymal stem cells (MSCs). mulation , indicatmulation . Thus, dogenesis . This inogenesis . This inwnts mRNA not only upregulate the expression of Twist1, a transcription factor involved in the regulation of osteoblastogenesis, but also increases the expression of alkaline phosphatase, an osteoblast marker. Moreover, they also increase the calcium content in cells, thereby promoting the differentiation of stem cells (ST cells) into osteoblasts. However, β-catenin loss prevents this stimulation. Similarly, a block by short hairpin RNA of three WNT expression suppresses osteoblastogenesis. In summary, WNT6, WNT10a, and WNT10b inhibit adipogenesis and induce osteoblastogenesis by activating the WNT/β-catenin pathway , and the degree of inflammation induced by M. tuberculosis. A mutual relationship was identified between WNT6 expression and macrophages in TB infection. The level of wnt6 mRNA in the macrophages of infected lung tissues depends on toll-like receptor (TLR), myeloid differentiation primary response88 (Myd88), and nuclear factor kappa (NFκB). Furthermore, wnt6 mRNA increases the level of Arginase-1 (Arg-1) and mannose receptor 1 (MRC-1). Arg-1 is the mouse marker for alternatively activated macrophages (M2), and MRC-1 is a marker for alternative macrophage activation. These indicate that WNT6 promotes inflammation in the infected macrophages, and in uninfected macrophages by increasing anti-apoptotic factors such as B-cell lymphoma 2 (BCL-2). WNT6 changes macrophages into the M2-like phenotype by increasing Arg-1 and decreasing TNF-α , caused by clerosis . Infecting TNF-α . Additioferation . c-Myc rferation ; howeverrophages .wnt6 mRNA expression is significantly reduced in MECP2 mutant mice models, controlled by MeCP2 SUMOylation  to IGF-1 and BDNF to increase the expression of two genes. WNT6 may increase MECP2 SUMOylation by increasing the expression of IGF-1 and BDNF. Thus, the overexpression of WNT6 increases the expression of both IGF-1, BDNF and MECP2 SUMOylation to partly improve abnormal behaviors. Additionally, in mutant mice, environmental enrichment (EE) also upregulates WNT6 protein expression by mediating the NMDA receptor and increases the expression of IGF-1, BDNF, and MECP2 SUMOylation. Thus, a positive regulatory loop exists between MECP2 SUMOylation and WNT6 expression, and this loop is composed of multiple molecules that improve RTT. However, the exact molecular mechanism requires further investigation. One signaling pathway might be a β-catenin-dependent WNT pathway, since the overexpression of WNT6 restores the level of β-catenin and GSK-3β phosphorylation in mutant mice. However, lentivirus-WNT6 transduction, which shows limited expressed in the amygdala, partially but not completely, rescues motor function deficits and social behavior deficits in MECP2 T158A mutant mice. The activated neuron in the amygdala is insufficient to recovery the full behavior. Therefore, it is possible that maybe other molecules, in addition to WNT6, have the potential to improve RTT (Rett syndrome (RTT) is a neurological and developmental disorder caused by a mutation in the methyl-CpG-binding protein 2 (MECP2) gene. Patients usually exhibit normal development at the infant stage; however, abnormal behaviors, such as locomotion impairment, cognitive function deficits, and other intellectual disability-related symptoms appear later in life . MECP2, Oylation . MeCP2 iOylation . AdditioOylation . WNT6 ovrove RTT . Neverthrove RTT . Collectwnt6 mRNA is not overexpressed. Analysis of TCGA and other dataset of glioma tissues has shown that certain GBM patients show high expression of wnt6 mRNA and protein in tumor cells. Analysis of various GBM subtype patients in independent queues has revealed that wnt6 mRNA is overexpressed in several GBM molecular subtypes   in both intracranial mouse models and patients. Mice with WNT6-overexpressing tumors show GBM-related neurological symptoms early on. The body weight loss in WNT6 over-expressing tumors is faster than that in low expressing tumors  is the most lethal tumor of the central nervous system. Grade IV glioma has the strong ability to invade and spread . Analysioneural) . These rg tumors . These rg tumors . WNT6 sig tumors . In addig tumors .In WNT6-silenced human GBM cells, the activation of Src family kinases (SFK), heat shock protein (HSP) family (HSP27), and the total levels of β-catenin are inhibited or decreased, indicating that WNT6 acts through the β-catenin-dependent WNT pathway, the SFK/STAT pathway, and the PI3K/AKT/mTOR pathway. However, the WNT6 target in each pathway is different in different cell lines, but the effect is the same. In the PI3K/AKT/mTOR pathway, WNT6-silenced U373 cells directly inhibit the activation of AKT and mTOR, while WNT6-silenced SNB19 cells inhibit the downstream targets such as p70 S6K and eNOS. In the SFK/STAT pathway, WNT6-silenced U373 cells increase serine 727(S727) phosphorylation to decrease signal transducer and activator of transcription 3 (STAT3) activity. WNT6-silenced SNB19 cells decrease the phosphorylation of tyrosine 705 (Y705) to decrease STAT3 activity . In GBM,Wnt6 mRNA expression is increased in HCT116 and SW480 human CRC cells compared to other three colon cancer cell lines   is one of the most common malignant tumors worldwide. nd HT29) . The hignd HT29) . Therefond HT29) . PLAGL2,nd HT29) , is a prnd HT29) . The accnd HT29) . Howeverwnt6 mRNA expression, while MKN45/Cav1 cells overexpressing Cav1 had a high level of wnt6 mRNA expression. Other GC cell lines (AGS and HEK293) also had the same expression profile, indicating that Cav1 can upregulate the expression of WNT6. MKN45/RNAiCav1 contributes to chemoresistance. MKN45/RNAi cells are more sensitive to anthracycline (epirubicin) than Cav1-expressed cells might by activating apoptosis. MKN45/RNAi cells with WNT6 knockdown have a similar effect as anthracycline. When Cav1 is not expressed, in the absence or presence of wnt6 mRNA expression, cells are not significantly different regarding their sensitivity to anthracycline (epirubicin). By contrast, the overexpression of both WNT6 and Cav1 decreases cell death, indicating that Cav1 activates WNT6 to prevent tumor cell apoptosis, and induces chemoresistance through the β-catenin-dependent WNT pathway. These data suggest that WNT6 and Cav1 are potential therapeutic targets to improve chemoresistance. A study found that cells expressing WNT6 at low levels, when treated with anthracycline, expressed increasing levels of wnt6 mRNA and protein in a time-dependent manner, by activating the WNT/β-catenin pathway. These results indicate that Cav1 and WNT6 increase chemoresistance by activating the WNT/β-catenin pathway. Moreover, anthracycline promotes the expression of WNT6 to increase chemoresistance. This mechanism differs from that of the classical multidrug resistance transporters (Gastric cancer (GC) is one of the largest causes of cancer-related deaths worldwide. WNT6 expression is low in some primary human GC cell lines compared to cell lines derives from distant metastases such as MKN45, MKN7, and NCIN87. Not all GC cells express high levels of WNT6. In GC, caveolin-1 (Cav1) can regulate the expression of WNT6. Caveolin, a plasma membrane micro-domain enriched in cholesterol and sphingolipids, is an essential structural protein . Cav1 issporters . Additiosporters , due to WNT6 belongs to the family of WNT ligands and is a highly conserved molecule that plays various roles in mice and humans. Its expression and function begin from the early stage of embryonic formation, and play a role in several stages of life. Importantly, WNT6 has the ability to become a therapeutic target, because it plays an important role in human diseases. However, further studies are needed to elucidate the role of WNT6 in diseases. Current research has mainly used animal disease models and clinical studies to provide comprehensive evidence of the role of WNT6. Meanwhile, the research on the mechanisms underlying WNT6 in disease is are still limited; thus, additional investigations are needed to provide a better understanding of the biology and function of WNT6 and its potential as a therapeutic target in these diseases.MW, QW, XD, and HZ wrote, performed the revisions, and reviewed the manuscript. CZ and YT conducted the literature research. All authors gave the final approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of largely unknown pathophysiology, characterized by the progressive loss of motoneurons (MNs). We review data showing that in presymptomatic ALS mice, MNs display reduced intrinsic excitability and impaired level of excitatory inputs. The loss of repetitive firing specifically affects the large MNs innervating fast contracting muscle fibers, which are the most vulnerable MNs in ALS. Interventions that aimed at restoring either the intrinsic excitability or the synaptic excitation result in a decrease of disease markers in MNs and delayed neuromuscular junction denervation. We then focus on trans‐spinal direct current stimulation (tsDCS), a noninvasive tool, since it modulates the activity of spinal neurons and networks. Effects of tsDCS depend on the polarity of applied current. Recent work shows that anodal tsDCS induces long‐lasting enhancement of MN excitability and synaptic excitation of spinal MNs. Moreover, we show preliminary results indicating that anodal tsDCS enhances the excitatory synaptic inputs to MNs in ALS mice. In conclusion, we suggest that chronic application of anodal tsDCS might be useful as a complementary method in the management of ALS patients. Motoneuron activity is disrupted during Amyotrophic lateral sclerosis (ALS), depriving these cells of activity‐dependent neuroprotection. Anodal tsDCS can modify motoneuron excitability promising a new approach for ALS management. Most interestingly, interventions with pharmacological or chemogenetic tools that aim at correcting the firing of the most vulnerable MNs prove to have some beneficial impact on the disease. After reviewing these data, we focus on trans‐spinal direct current stimulation (tsDCS) as a potential alternative therapeutic method in ALS. Indeed, electrical polarization by direct current is well‐known to modify spinal networks. We review recent work suggesting that tsDCS could be used to compensate for the changes in intrinsic excitability of MNs, or synaptic excitation, and hopefully to deliver some neuroprotection in ALS.1.1In ALS, some motor pools are more vulnerable than others  prolongs survival in patients with ALS, albeit modestly  display a normal excitability. A similar conclusion was reached by Venugopal et al. . In these conditions, Delestrée et al. ‐derived MNs from human patients have consistently reported the same time‐dependent excitability changes as in ALS mice. After an initial hyperexcitability , or to glycine‐receptor transmembrane domain for neuronal hyperpolarization (to decrease the excitability)  (either from Ia spindle afferents or from descending systems) are functionally depressed in spinal MN  activated the cAMP/PKA signaling pathway eliciting membrane insertion of GluR4 subunits and restoration of excitatory synapses. This elicits an improvement of disease markers  through the enhancement of neuronal firing  massively and specifically target MN, (b) control the cellular and immune responses, and (c) avoid side effects such as neoplastic tumors. There is evidently a real interest to develop noninvasive methods that could restore intrinsic excitability or synaptic excitation of MNs. In this framework, we will present new data showing that tsDCS may actually induce long‐lasting restoration of MN excitability and synaptic inputs, with the hope that tsDCS has the potential to deliver some neuroprotection in ALS.1.2cranial direct current stimulation (tDCS) . Altogether these results indicate that in SOD1G93A mice tsDCS evokes polarity‐dependent MN plasticity. Although the functional and survival analysis of tsDCS effects on ALS mice are ongoing, these preliminary findings already provide a proof of concept for further tsDCS application in ALS management.The main advantage of DC polarization is that it is not invasive and can be easily used in humans. However preclinical animal studies have to be performed before starting clinical trials. Preliminary results indicate that anodal tsDCS can enhance the excitatory synaptic inputs to MNs in the SOD1S Figure . InteresS Figure . Moreove2G93A mice. Pharmacological and chemogenetics interventions that aimed at restoring either the intrinsic excitability or the synaptic strength were shown to ameliorate the disease phenotype. In parallel, other experiments showed that anodal tsDCS enhances the intrinsic excitability of spinal MNs and the effect outlasts the stimulation period. Moreover, preliminary results show that anodal tsDCS also elicits long‐lasting enhancement of EPSPs in SOD1G93A mice, compensating the EPSP impairment observed in these mice. We suggest that chronic anodal tsDCS may be useful in the management of ALS patients.Recent experiments suggest that vulnerable MNs become intrinsically hypoexcitable (as seen in a loss of their ability to discharge repetitively) and that their excitatory synapses are impaired in the SOD1