Kochia scoparia) and its principle saponin, Momordin Ic, have been reported to protect against inflammation. Herein, we demonstrated that Momordin Ic could inhibit HaCaT cell proliferation and enhance cell apoptosis. In the meantime, Momordin Ic alters Wnt/β-catenin pathway activation by affecting β-catenin nuclear distribution. The Wnt/β-catenin signaling activator LiCl partially reversed the effects of Momordin Ic on HaCaT phenotypes and the Wnt/β-catenin pathway factors. Altogether, we demonstrate the inhibitory effects of Momordin Ic, one of the major saponin constituents of Fructus Kochiae, on HaCaT cell proliferation and Momordin Ic-induced alteration within the Wnt/β-catenin pathway. Momordin Ic might act on HaCaT cells by modulating the Wnt/β-catenin pathway.Psoriasis is a chronic, recurrent, immunoinflammatory disease. For a long period, Traditional Chinese Medicine (TCM) is considered a reliable alternative therapy for patients with psoriasis. Fructus Kochiae (or  Psoriasis is a chronic, recurrent, immunoinflammatory disease. The most common syndromes in patients consist of pain, itching, and bleeding. The burden of disease continues to be enhanced by a number of comorbidities, including metabolic syndromes and cardiovascular disorders caused by the syndromes , 2. MoreKochia scoparia (L.) Schrad., a broom cypress fruit, which has been used as an edible and topical drug in the treatment of skin, urinary, and eye diseases for more than 2000 years in China [Kochia scoparia) has been reported to protect against skin inflammation related to evil heat, humidity, and wind, including atopic dermatitis [Traditional Chinese Medicine (TCM) is characterized by multiple pathways, target points, and elements, and its complex composition can further enhance the acting surface and effect intensity of the drug target. For a long time, TCM has been considered a reliable alternative therapy for patients with psoriasis. A variety of prescriptions for psoriatic treatment were documented in ancient Chinese medical books, such as The Peaceful Holy Benevolence Formulae and Taiping Holy Prescriptions for Universal Relief . Fructusin China , 9. Frucrmatitis –14. Repormatitis , inhibitrmatitis , antirhermatitis , and redrmatitis . Howeverβ-catenin pathway exerts an important effect on activating and maintaining the proliferation of keratinocytes during hair follicle cycling [β-catenin transcriptional activity are essential [β-catenin pathway was involved in the proliferation of HaCaT cells under IL-22 stimulation [β-catenin pathway, has been reported to be a critical event that should be considered for psoriasis treatment.Notably, Momordin Ic has been reported to be associated with the apoptosis (proliferation) of multiple cell types, including hepatocellular carcinoma HepG2 cells –19 and p cycling . In promssential , 28. As mulation  or withomulation . Thus, tβ-catenin pathway. To validate the speculation, we first treated the human immortalized epidermal cell line, HaCaT, with increasing concentrations of Momordin Ic and chose the minimum efficient concentration. The effects of Momordin Ic on HaCaT cell viability, apoptosis, cell cycle distribution and the key factors of the Wnt/β-catenin pathway were examined. We also cotreated HaCaT cells with Momordin Ic and Wnt/β-catenin pathway activator (LiCl) for verifying that the Wnt/β-catenin pathway was involved. Taken together, we examined the specific effects of Momordin Ic on HaCaT cell proliferation and the Wnt/β-catenin pathway, attempting to provide a rationale for further studies on Momordin Ic cellular functions in HaCaT cells.Considering the above-mentioned findings, we speculate that Momordin Ic might also inhibit keratinocyte proliferation, possibly via the Wnt/2.Human immortalized epidermal cell line, HaCaT (3142C0001000001712), was obtained from China Center for Type Culture Collection  and cultured in Minimum Essential Medium  added with 10% FBS  at 37°C in 5% COμmol/L) or 30 mM LiCl for 24 or 48 h. The same amount of PBS was used as control. Then, cells were harvested for further experiments.Momordin Ic was purchased from Yuanye Biotech . LiCl was purchased from Sigma-Aldrich . HaCaT cells were treated with different concentrations of Momordin Ic  to incubate the cells for 4 h. At the end of the incubation, 100 μl DMSO solution was added to incubate the cells for 10 min. Then, detect the OD value at 490 nm with a microplate reader. The lowest effective concentration of Momordin Ic was selected.Cells were transfected and/or treated with Momordin Ic, washed with normal culture medium, and added with 10 Cells were treated with Momordin Ic for 24 h, 48 h, and 72 h, centrifuged with PBS, and added with RNaseA. Resuspend the cells in Annexin V-FITC binding solution, add Annexin V-FITC fluorescein solution and propidium iodide (PI) staining solution for cell apoptosis detection, or add PI staining solution for cell cycle detection. The experiment was repeated three times.β-catenin, c-myc, and VEGF were detected, as shown in Logarithmic phase HaCaT cells were collected to extract total RNA using a TRIzol™ Plus RNA Purification Kit ; then, the reverse transcription into cDNA with a High-Capacity cDNA Reverse Transcription Kit was performed , and the mRNA expressions of β-catenin , anti-c-Myc , and anti-VEGF . Then, protein samples were incubated with the secondary antibody. Band signals were visualized by the enhanced chemiluminescence  method according to the manufacturer's instructions. Relative protein expression was normalized to actin.The total protein of HaCaT cells was extracted with a Total Protein Extraction Kit . The protein concentration was determined using a bicinchoninic acid (BCA) method with a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples were separated on a 10% SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% (w/v) skimmed milk at room temperature for 1 h, protein samples were incubated with the corresponding primary antibody at 4°C overnight. The following antibodies were used: anti-β-catenin  at 4°C overnight. Then, the cells were stained with a proper FITC-conjugated secondary antibody (1 : 1000) in a dark room at 37°C for 1 h. Then, DAPI was used to stain the nucleus. The cells were observed under the inverted fluorescence microscope.The HaCaT cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and permeabilized with 0.1% Triton X-100 for 10 min. Then, the cells were blocked in PBS containing 10% BSA at room temperature for 1 h. At the end of the blocking, cells were incubated with anti-P < 0.05 indicated statistically significant difference.The data were analyzed with GraphPad software. The measurement data were expressed as mean ± standard deviation (SD). Among-group and intragroup data comparisons were performed with the ANOVA followed by Tukey's post hoc test. μmol/L Momordin Ic for 0, 24, or 48 h and examined the cell viability. Compared with the nontreated group (control), after 48 h of treatment, 25, 50, and 100 μmol/L significantly reduced the HaCaT cell viability. The IC50 of Momordin Ic is 168.70 and 76.40 μmol/L for 24 and 48 h, respectively. Thus, 25 μmol/L Momordin Ic treatment for 48 h was selected for further experiments.Before investigating the specific effects of Momordin Ic  on HaCaTμmol/L Momordin Ic for 48 h and examined for the morphological and phenotype changes. Compared with the blank control and DMSO treatment groups, HaCaT cells in the Momordin Ic group were shrunk, the volume was reduced, and the number of dead cells increased  could reverse Momordin Ic effects on HaCaT cells. Compared to the PBS control group, LiCl treatment increased the cell viability, while Momordin Ic reduced the cell viability and increased cell apoptosis  proteins, thereby stimulating the expression of Wnt target genes, including cyclinD1 and c-myc [β-catenin, increased β-catenin, c-myc, and VEGF proteins, promoted HaCaT cell proliferation, and inhibited cell apoptosis. The cellular and molecular functions of Momordin Ic were all partially reversed by LiCl, suggesting the involvement of the Wnt/β-catenin pathway in Momordin Ic functions on HaCaT cells.To further validate that the Wnt/-catenin . Once innd c-myc –35. Hereβ-catenin pathway. Momordin Ic might act on HaCaT cells by modulating the Wnt/β-catenin pathway.Altogether, we demonstrate the inhibitory effects of Momordin Ic, one of the major saponin constituents of Fructus Kochiae, on HaCaT cell proliferation and Momordin Ic-induced alteration within the Wnt/


Glaucoma is one of the leading causes of worldwide irreversible blindness. Lowering elevated intraocular pressure (IOP) is currently the only effective approach for controlling the progress of glaucoma. Angiopoietin-like 7 (ANGPTL7) takes a key part in elevated outflow resistance of aqueous humor in dysfunctional trabecular meshwork (TM), along with the formation of cross-linked actin networks (CLANs), leading to high IOP. In this study, we explored the role of the ANGPTL7 signaling pathway in CLAN formation. We detected the expression of ANGPTL7 in cultured primary TM cells treated with dexamethasone (DEX) and ethanol as a control using qRT-PCR and western blotting. Actin filaments were revealed by phalloidin staining. ANGPTL7 short hairpin RNA (shRNA) was applied to TM cells to examine the effect of ANGPTL7 on DEX-induced CLAN formation. Western blotting was used to assess the effect of ANGPTL7 on the RhoA/Rho-associated kinase (Rho-kinase/ROCK) signaling pathway. Bioinformatics, dual-luciferase reporter assays, and chromatin immunoprecipitation were employed to identify the transcription factors of ANGPTL7. Transcription factor specificity protein 1 (SP1) overexpression and silencing were performed to determine their roles in the modulation of ANGPTL7 expression. We found DEX-induced ANGPTL7 expression and stress fiber rearrangement in TM cells. ANGPTL7 knockdown effectively inhibited the formation of CLANs. Moreover, it was involved in the regulation of the RhoA/ROCK signaling pathway, further affecting DEX-induced CLAN formation. SP1 was identified as a transcription factor of ANGPTL7 which regulated ANGPTL7 level to mediate CLAN formation through the RhoA/ROCK signaling pathway. This study contributes to revealing the molecular mechanisms of ANGPTL7 in CLAN formation, which is involved in TM dysfunction and glaucoma pathogenesis. Glaucoma is a leading cause of worldwide irreversible vision loss, characterized by progressive optic neuropathy . The mosHigh IOP is caused by increased outflow resistance of aqueous humor (AH) . AccumulPrevious studies have found that the concentration of angiopoietin-like 7 (ANGPTL7) is increased in glaucomatous AH and that overexpression of ANGPTL7 in the TM alters the components of the extracellular matrix (ECM) , 10. A rIt is imperative to elucidate the molecular mechanisms of ANGPTL7 that lead to glaucoma pathology and find new potential therapeutic targets. A previous study reported that transcription factor specificity protein 1 (SP1) is involved in promoter activity and further regulates the F-actin architecture of the Schlemm’s canal endothelium, suggesting that SP1 may function in the development and progression of glaucoma . In thisTo assess the expression of steroid-induced glaucoma and POAG-related genes, the expression data from the GEO database (GSE65240) were analyzed first. The heatmaps and volcano plots identifying the differentially expressed genes showed that ANGPTL7 expression was distinctly upregulated in the TM cells with DEX treatment Fig. . Next, tTo study the effect of ANGPTL7 on CLAN formation, we first identified the transfection effect of ANGPTL7 shRNA by measuring the mRNA and protein expressions of ANGPTL7 in TM cells which were transfected with scr shRNA and ANGPTL7 shRNAs  before DEX treatment. The results showed that both ANGPTL7 shRNA1 and ANGPTL7 shRNA2 substantially reduced the expression of ANGPTL7 compared with scr shRNA Fig. . IF staiThe KEGG pathway enrichment analysis of GSE65240 showed that differentially expressed genes took part in some pathways, such as calcium signaling pathway, neuroactive ligand-receptor interaction, tyrosine metabolism, and regulation of actin cytoskeleton Fig. . Actin cWe confirmed the effect of ANGPTL7 on the RhoA/ROCK signal pathway through a series of experiments. First, the protein levels of RhoA and p-MLC were elevated in DEX-treated TM cells. The knockdown of ANGPTL7 evidently reduced the protein expression of RhoA as well as p-MLC Fig. . MoreoveTo further explore the upstream mechanism of ANGPTL7 in CLAN formation, potential transcription factors of ANGPTL7 were predicted using the UCSC Genome Browser Fig.  and siteNext, we explored the regulatory effect of SP1 on ANGPTL7. The results showed that SP1 overexpression upregulated the mRNA and protein expressions of SP1 and ANGPTL7 compared with the empty vector group, while SP1 knockdown downregulated the expression of SP1 and ANGPTL7 compared with the scr shRNA group Fig. . This suIn this study, we detected high levels of ANGPTL7 in DEX-induced TM cells, which promoted the formation of CLANs. Furthermore, ANGPTL7 facilitated CLAN formation by activating the RhoA/ROCK pathway. SP1 was found to mediate the expression of ANGPTL7 as a transcription factor and further modulated CLAN formation  are members of generalized angiopoietin family and are secreted proteins known as angiogenic factors . Some ofBased on previous reports , 24 and To study the exact molecular mechanisms of ANGPTL7 in CLAN formation, we applied the UCSC Genome Browser to predict SP1 as a transcription factor of ANGPTL7 due to its more binding locations and a higher prediction score than those of other transcription factors and a previous report that SP1 activates the molecular pathway of actin cytoskeleton as a transcription factor . We veriThis is our first attempt to reveal the role of ANGPTL7 and the relevant pathway in CLAN formation. Many questions remain unanswered, and the study is not without limitations. First, the dynamics and functions of CLANs in TM cells are not fully known. It could provide valuable information if live-cell imaging of the actin cytoskeleton were carried out to study the actin dynamics in TM cells. Second, we observed the ultrastructure of CLAN formation by IF staining and qualitatively verified the effect of molecules involved in the signaling pathway. However, a quantitative analysis taking into account the proportion of CLAN-positive cells, the CLAN size, and the dimensions of hubs and spokes would have produced more detailed results. Third, it is uncertain whether IOP elevation is the result or the cause of CLAN formation. Our findings suggest that the former is probably the case. However, the effect of IOP fluctuations on CLAN formation requires further investigation. Moreover, it would be important to confirm the molecular mechanisms using an animal model.This study shows that ANGPTL7 transcriptionally regulated by SP1 can modulate CLAN formation via the RhoA/ROCK pathway and provides novel insights into how the TM ultrastructure and functions may be altered in some types of glaucoma. Future studies will explore the possibility of regulating the SP1/ANGPTL7/ROCK axis as targeted therapies for steroid-induced glaucoma and POAG.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65240). The datasets consisted of the effects of DEX for primary TM cell gene expression from three lots of TM cells treated with DEX as cases and three lots without DEX as controls. The pheatmap and limma packages of R software were used to draw heat maps and volcano plots, respectively. The R package was applied to calculate the false discovery rate and the log2 fold change (FC). An absolute value of log2 FC > 1 was selected, and an adjusted value of P < 0.05 was considered statistically significant.The microarray data of GSE65240 were obtained from the publicly available Gene Expression Omnibus (GEO) database (P < 0.05 was used as the cutoff criterion.For Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the clusterProfiler KEGG package of R software was used. A value of Primary TM cells were purchased from ScienCell Research Laboratories . The cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 2 mM L-glutamine (Sigma-Aldrich), 0.05% gentamicin , 1% amphotericin B (Mediatech), and 1 ng/mL FGF-2 . The TM cells were grown to confluence. To study the effects of DEX on ANGPTL7 expression, the cells were treated with 100 nM DEX or solvent (ethanol) as a control for 14 days.The primary antibodies were anti-ANGPTL7 , anti-RohA , anti-p-MLC (CST), anti-MLC (CST), anti-SP1 (Abcam), phalloidin-488 , and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam). Y27632, a ROCK inhibitor, was purchased from Selleck Chemicals . Dexamethasone (Sigma D4902) was purchased from Sigma-Aldrich.For ANGPTL7 and SP1 knockdown, short hairpin RNA (shRNA) and scrambled (scr) shRNA were designed and supplied by Genomeditech . The lentiviral vectors were transfected into the TM cells according to the manufacturer’s instructions. All the sequences of shRNAs are presented in Table For ANGPTL7 and SP1 overexpression, lentiviral overexpression vectors and Lv-empty vectors as controls (PGMLV-CMV-MCS-EF1-mScarlet-T2A-Blasticidin) were also constructed and supplied by Genomeditech. The lentiviral vectors were transfected into the TM cells according to the manufacturer’s instructions. Then 100 nM DEX was added into the medium and the cells were cultured for 14 days.All cells used to perform immunofluorescent (IF) staining were cultured on coverslips in 12-well plates. Actin filaments (F-actin) were revealed by phalloidin staining. Cells were rinsed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min. They were then rinsed with PBS and permeabilized using 0.5% Triton X-100 (Sigma-Aldrich) for half an hour. The cells were rinsed again with PBS and blocked with 1% bovine serum albumin for 1 h. And then the cells were incubated with Alexa Fluor 488-phalloidin (1:500) for 1 h at room temperature. 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) was used for nuclei staining. Imaging was carried out using a confocal microscope .−ΔΔCt method was used for calculation. All primer pairs are displayed in Table Total RNA was extracted from the TM cells with a Total RNA Extraction Kit  and reverse transcribed into cDNA with a Synthesis Kit . QRT-PCR was carried out by using a qPCR Detection Kit . Gene expression was normalized against GAPDH expression. The 2http://imagej.nih.gov/ij/; made available in the public domain by the National Institutes of Health, Bethesda, MD, USA).Proteins were extracted using a radioimmunoprecipitation assay lysis buffer  according to standard protocol. Protein concentrations were detected by using a bicinchoninic acid (BCA) protein assay (ThermoFisher). Then, they were isolated using 12% SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Sigma-Aldrich). Blots were blocked by incubation with 5% nonfat milk at room temperature for 1 h and incubated at 4 °C overnight with the following primary antibodies: anti-ANGPTL7 (1:1000), anti-RohA (1:2000), anti-p-MLC (1:2000), anti-MLC (1:2000), anti-SP1 (1:2000), and anti-GAPDH (1:2000). The membranes were rinsed three times and then incubated with the corresponding peroxidase-conjugated secondary antibodies at room temperature for 2 h. An enhanced chemiluminescence system  was employed to visualize the proteins. Densitometric signal intensity of the western blotting was performed using ImageJ software (http://genome.ucsc.edu/). SP1 was chosen since it had more DNA-binding locations and a higher prediction score than other transcription factors. The JASPAR database (http://jaspar.genereg.net/) was used to predict sites where SP1 might bind to the promoter of ANGPTL7.The underlying transcription factors of ANGPTL7 were identified by employing the UCSC Genome Browser (ANGPTL7 promoter reporter vectors (ANGPTL7-WT) and the corresponding control reporter vectors (ANGPTL7-MUT) were separately obtained from the process that the fragments of ANGPTL7 containing the DNA-binding sites between ANGPTL7 and SP1 or mutation sites were cloned into luciferase reporter vectors . These reporter vectors together with SP1 overexpression plasmids (SP1-OE) or corresponding control plasmids (empty vector) were co-transfected into TM cells. The co-transfected cells were lysed and the luciferase activities were assessed using the Dual-Luciferase Reporter Assay System (Promega).The Magnetic ChIP Kit (ThermoFisher) was used to perform chromatin immunoprecipitation according to the manufacturer’s protocol. Anti-SP1 antibodies and control rabbit immunoglobulin G (IgG) were used. After immunoprecipitation, the DNA fragments were purified and the DNA-binding domain of ANGPTL7 with SP1 was detected by using qRT-PCR.t-test was applied to analyze data between two groups, and one-way ANOVA was applied to analyze results between multiple groups. P < 0.05 was considered statistically significant.SPSS 17.0  was used to perform statistical analyses. The data were expressed as means ± standard deviations from three independently repeated experiments. The paired cddiscovery-author-contribution-form


Serendipita indica and bamboo fungus (Dictyophorae echinovolvata) culture substrates on the growth and wilt disease resistance of banana. S. indica colonization significantly enhanced the growth of the two banana cultivars, while banana seedlings grown in nutrient soil containing bamboo fungus culture substrates inoculated with D. echinovolvata showed suppressed growth. Moreover, both S. indica colonization and D. echinovolvata culture substrates addition can alleviate the disease symptoms caused by Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4), and their combined application showed the best disease resistance enhancement effect. The results obtained in this study can provide a basis for the application of S. indica and bamboo fungus in the prevention and control of banana Fusarium wilt disease in the future.By using ‘Zhongjiao No.3’ and ‘Zhongjiao No.4’ banana seedlings as plant materials, we investigated the influences of Serendipita indica and bamboo fungus (Dictyophorae echinovolvata) culture substrates on the growth and Fusarium wilt disease resistance of banana were investigated. Results showed that the plant height, leaf length, leaf width, root length and root thickness, aboveground part fresh weight, root fresh weight, and relative chlorophyll content and nitrogen content in leaves of banana seedlings colonized with S. indica were all greater than those of non-colonized controls, while these parameters of banana seedlings grown in nutrient soil containing D. echinovolvata culture substrates were significantly suppressed. Both S. indica non-colonized and colonized seedlings cultivated in nutrient containing 1/4 D. echinovolvata culture substrates showed much milder symptoms compared with those cultivated in normal nutrient soil, indicating that the addition of bamboo fungus substrates to the soil can enhance the Fusarium wilt resistance of banana. The results obtained in this study can provide a basis for the application of S. indica and bamboo fungus in the prevention and control of banana Fusarium wilt disease.Recently, many control methods have been tried and applied in the Fusarium wilt disease control of banana and have achieved definite progresses. In this study, by using ‘Zhongjiao No.3’ and ‘Zhongjiao No.4’ banana seedlings as materials, the effects of  Serendipita indica (named formerly as Piriformospora indica) is an arbuscular mycorrhizal (AM)-like fungus isolated from the shrub roots in a desert of India by Verma et al. [S. indica associations with host plants promote growth and development as well as nutrient absorption and enhanced stress resistance of host plants [S. indica was first applied to bananas by Madaan et al. [Musa indica cv. Grand Naine) [S. indica significantly promoted the rooting and photosynthetic pigment synthesis and greatly accelerated the growth of tissue cultured ‘Tianbaojiao’ banana plantlets [S. indica can also enhance the resistance of banana to both abiotic and biotic stresses. For example, Li et al. reported that colonization of S. indica improved the banana cold resistance by stimulating antioxidant capacity, improving SS accumulation, and by regulating the expression of cold-responsive genes in banana leaves [S. indica improved the thermal resistance of banana [Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4) by regulating the antioxidant enzyme activities of banana root [S. indica has great potential to be applied as a production and resistance improvement agent in the banana industry.a et al. ,2. The fa et al. . S. indit plants . It has n et al. , and itsd Naine) . Later, lantlets . In addia leaves ; Bodjrenf banana ; Cheng eana root . All thiDictyophora genus with high nutritive values, immunostimulatory activity [Dictyophorae echinovolvata pilei, volva, and stipes all showed certain inhibitory effects on the growth of FocTR4 on the PDA medium, and the pilei crude extract showed the best effect [Bamboo fungus (named as ‘Zhusun’ (ZS) in Chinese), known as ‘the queen of fungi’ and ‘the king of delicacies’, is a kind of edible mushroom in the activity ,10, and activity . In the t effect , suggestS. indica and the anti-microbial activity of bamboo fungus, in this study we investigated the effects of S. indica colonization and D. echinovolvata culture substrates addition into the nutrient soil on the growth and Fusarium wilt resistance of two banana cultivars, ‘Zhongjiao No.3’ (ZJ3) and ‘Zhongjiao No.4’ (ZJ4). Growth-related parameters, including plant height, leaf length, leaf width, root length and root thickness, aboveground part fresh weight, root fresh weight, relative chlorophyll content and nitrogen content in leaves of banana seedlings colonized and non-colonized with S. indica (CK and S+ groups), and banana seedlings grown in nutrient soil containing different volume ratios of bamboo fungus culture substrates were measured and compared. Moreover, CK and S+ seedlings cultured in nutrient soil containing no and 1/4 bamboo fungus culture substrates were subjected to FocTR4 inoculation experiment to show their influences on the appearance and development of the Fusarium wilt disease symptoms. The results obtained in this study can provide a basis for the application of S. indica and bamboo fungus in the prevention and control of banana Fusarium wilt disease in the future.Banana is an important fruit and food crop in the world . As a chS. indica and FocTR4 isolates used in this experiment were kept in our lab. The bamboo fungus culture substrates  that have been inoculated with Dictyophorae echinovolvata for one month were purchased from a mushroom market in Nanping city, Fujian province of China.The tissue-cultured ‘Zhongjiao No.3’ (ZJ3) and ‘Zhongjiao No.4’ (ZJ4) banana seedlings used in this study were kindly provided by Fruit Research Institute, Guangdong Academy of Agricultural Sciences. ZJ4 was reported to be a banana Fusarium wilt resistant banana cultivar, while ZJ3 was somewhat sensitive to banana wilt disease. The S. indica agar discs with a diameter of 5 mm were added into a triangular flask containing 100 mL PDB medium and cultured in the dark at 200 rpm at 28 °C for three days. Next, the obtained S. indica fermentation solution was diluted with distilled water for about 3 times and used as inoculation solution (containing approximately 60 g mycelia per liter and 1 × 105 spores per milliliter) [FocTR4 fermentation solution was prepared. Then, FocTR4 inoculation solution were prepared according to the method described by Wang et al. [FocTR4 fermentation solution was filtered using two layers of lens papers to remove mycelia, then spores were collected by centrifugation at 4000 rpm for 5 min, re-suspended in Hoagland nutrition solution, and adjusted to a final concentration of 1 × 107 spores/mL.Three round g et al. . BrieflyS. indica inoculation solution for 6 h, and transplanted into plastic breeding bags containing nutrient soils . To better ensure its colonization, the S. indica inoculation solution was watered into the soil nearby the banana roots (S+ group) for three times [S. indica colonization, at two weeks post-inoculation, roots of S+ plants were collected and cut into 1 cm segments, soaked in 10% KOH solution for 24 h, washed with sterile water for 5 times, treated with 1% hydrochloric acid solution for 4 min, then stained using 0.05% Trypan blue staining solution for 1 min [Roots of tissue-cultured banana seedlings were slightly wounded with surgical blades, soaked in ee times . Seedlinor 1 min  and obseS. indica on the growth and development of banana, seedlings with and without S. indica colonization were transplanted into nutrient soil. To study the effects of bamboo fungus culture substrates on banana growth and development, half of the S. indica colonized and non-colonized seedlings were transplanted into nutrient soil containing no (CK group), 1/4 (1/4 group), 1/2 (1/2 group), and complete (ZS group) bamboo fungus culture substrates that have been inoculated with D. echinovolvata for one month, respectively. Then, all the seedlings were cultured in a greenhouse at 28 °C and transplanted into flowerpots at 2 months post-transplanting (mpt). At 1, 2, and 3 mpt, growth parameters such as plant height, leaf length, and leaf width were measured. At 3 mpt, root length, root number, root thickness, and fresh weight of overground and roots were also measured. Moreover, the chlorophyll relative content and nitrogen content in banana leaves were measured using a chlorophyll meter  at 3 mpt.To investigate the effects of S. indica colonization were divided into two groups. One group was transplanted into nutrient soil (CK and S+ groups), and the other group was transplanted into nutrient soil containing 1/4 of bamboo fungus culture substrates (1/4Z and 1/4SZ groups). All the seedlings were cultured in a greenhouse at 28 °C for one month. Then, roots of CK and 1/4Z groups were directly immersed in FocTR4 inoculation solution for 24 h (F and ZF groups) [S. indica can delay the appearance of Fusarium wilt symptoms [S. indica colonized banana seedling, the roots of S+ and 1/4SZ seedlings were firstly wounded using surgical blades, then washed and soaked in FocTR4 inoculation solution for 24 h (SF and SZF groups). After FocTR4 inoculation, seedlings were replanted back, and the FocTR4 inoculation solution was watered into the soil nearby the root system. Then, they were cultured in a greenhouse at 28 °C with a photoperiod of 12 h light/12 h dark. Five weeks later, disease symptoms of each banana seedling group were observed.Banana seedlings without and with  groups) . Since S® SPSS® Statistics 25 . Difference significance analysis among different groups was performed by using Duncan’s multiple-range test at 0.05 level. GraphPad Prism v6.01  was used for figure drawing.For each group and each treatment, at least 10 seedlings of both the two banana cultivars were used, and all the parameters were measured for at least three times. Results for the detected parameters are presented as the mean ± standard deviation (SD). All the data analyses were performed using Microsoft Excel 2019 and IBMS. indica colonization in banana roots was observed  (p < 0.05) (p < 0.05), which was 1.47-fold and 1.27-fold of their corresponding CK, respectively.After transplanting, the plant height, leaf length, and leaf width of banana seedlings were measured monthly and for a total of three times. The plant height, leaf length, and leaf width of  < 0.05) . At 1 mp < 0.05) A, and th < 0.05) B. At 3 m < 0.05) . The roop < 0.05)  . MoreoveThe plant height, leaf length, and leaf width of banana seedlings grown in nutrient soil containing different ratios of bamboo fungus culture substrates were measured at 1, 2, and 3 mpt . At 1 mpAt 2 mpt, the average plant height of ZJ3 seedlings followed the order CK > 1/4 > 1/2 > ZS. The leaf length and width of ZJ3 in CK group showed no significant difference with the 1/4 group, but seedlings in these two groups were both significantly higher than ZS group seedlings. The leaf length and width of ZJ4 seedlings in CK group showed no significant difference with 1/4 group, while their average plant height, leaf length, and leaf width were significantly larger than those of the ZS group. At 3 mpt, the plant height, leaf length, and leaf width of ZJ3 and ZJ4 both followed the order CK > 1/4 > 1/2 > ZS, and these parameters of CK group were all significantly higher than other groups. The plant height and leaf length of ZS group were significantly lower than those of other groups, and the leaf width of ZS group was not significantly different from that of 1/2 group, but significantly lower than that of CK group and 1/4 group.At 3 mpt, the biomass and root development parameters of banana seedlings were also measured . The rooAt 3 mpt, the relative chlorophyll content and nitrogen content of banana seedlings were also measured. It was found that the relative chlorophyll content and nitrogen content of ZJ3 and ZJ4 both followed the order: CK > 1/4 > 1/2 > ZS . The relFocTR4. To accelerate the banana wilt symptom appearance of S. indica colonized banana seedlings, roots were wounded before FocTR4 inoculation. In SF group, the corms of ZJ3 and ZJ4 both showed a large area of browning, which accounted for about 35.50% and 27.68% of their whole corm, respectively. The symptoms in ZJ3 and ZJ4 corms in SZF group were much milder than those of SF group. This indicated that the addition of bamboo fungus culture substrates to the nutrient soil could also improve the Fusarium resistance of S. indica colonized banana (The disease symptoms in banana were observed at 5 weeks post-pathogen inoculation . Resultsd banana . MoreoveFusarium oxysporum f.sp. cubense (Foc) for a long time [S. indica is an arbuscular mycorrhizal-like fungus that can colonize plant roots extensively and form a symbiotic relationship with its host. Accumulated evidence has shown that the colonization of S. indica could improve the nutrient absorption, promote plant growth and development, and enhance the resistance or tolerance of banana to abiotic and biotic stresses [D. echinovolvata could inhibit the FocTR4 growth on PDA medium [FocTR4 resistance. Results obtained in this study were as follows.The banana industry has been threatened by banana wilt disease caused by ong time ,21. Up tstresses ,6,7,8. BA medium . HoweverS. indica has a growth-promoting effect similar to arbuscular mycorrhizal fungi [S. indica could advance seed germination time, promote plant height, leaf number, leaf length, leaf width, root number, and root length, and improve the quality and yield of Dendrobium officinale [S. indica showed significantly increased plant height, stem diameter, leaf area, net photosynthetic rate, and increased drought resistance [S. indica in the fungal colonized ‘Zhongjiao No.3’ and ‘Zhongjiao No.4’ banana cultivars was also proved, indicating that the fungus’s growth promoting ability is not strictly cultivar-dependent. Li et al. reported that S. indica colonization could significantly enhance the chlorophyll accumulations in banana leaves [al fungi . It can al fungi . Inoculaficinale . Cotton sistance . The grosistance ,5. In thD. echinovolvata [Our study revealed that bamboo fungus culture substrates had a certain inhibitory effect on banana growth. By determining the growth-related parameters, we found that almost all the parameters of banana seedlings in different groups followed the order: CK > 1/4 > 1/2 > ZS. This indicated that bamboo fungus culture substrates inhibited the growth of banana seedlings, and the higher bamboo fungus culture substrates proportion in the nutrient soil, the more obvious inhibitory effect. This might be caused by the antioxidant and anti-microbial activities of ovolvata . MoreoveAs the growth of banana seedlings grown in the nutrient soil containing 1/4 bamboo fungus culture substrates showed no significant difference compared with seedlings grown in nutrient soil at 1 mpt, we further investigated the influence of bamboo fungus culture substrates addition on the banana Fusarium wilt disease resistance. Results showed that both ‘Zhongjiao No.3’ and ‘Zhongjiao No.4’ banana seedlings grown in the nutrient soil containing 1/4 bamboo fungus culture substrates showed much milder symptoms than their control groups, indicating that bamboo fungus can alleviate the symptoms of banana Fusarium wilt to a certain extent and improve the Fusarium wilt resistance of banana. Thus, it can be concluded that the edible bamboo fungus intercropping or applying bamboo fungus fermentation solution might be a much easier and more favorable way for banana wilt disease control ,28.S. indica colonization can improve the resistance of many plants to pathogenic fungi [S. indica enhanced the resistance of wheat to Fusarium head blight [Foc is a soil-borne fungus with a long incubation time and low spore concentration [S. indica can delay the onset of Fusarium wilt symptoms by increasing antioxidant enzyme activities [FocTR4 induced fatty acid accumulation in banana root [S. indica colonized banana seedlings and compared the symptom appearance of seedlings grown in normal nutrient soil and in nutrient soil containing 1/4 bamboo fungus culture substrates. As S. indica colonized banana seedlings showed much delayed symptom onset, injury treatment was applied [S. indica non-colonized seedlings, the symptoms in corms of S. indica colonized ZJ3 and ZJ4 seedlings grown in nutrient soil containing 1/4 bamboo fungus culture substrates also showed much milder symptom than those of grown in normal nutrient soil, which again indicated that bamboo fungus culture substrates addition can alleviate the Fusarium wilt symptoms and improve the resistance of banana to Fusarium wilt disease.ic fungi ,30,31. Fd blight , Arabidod blight , tomato d blight , chickped blight , and so ntration ,37, makitivities  and by sana root . In our  applied ,40. ResuS. indica on banana hosts and the anti-microbial activities of bamboo fungus, the application of them in banana may be considered as an eco-friendly strategy for Fusarium wilt disease control in the future [The development of banana Fusarium wilt epidemics could be influenced by many factors, including banana genetic resistance, soil physical and chemical properties, soil microbial populations, crop management, and so on ,42. The e future .S. indica colonization significantly improved the growth of banana seedlings, while the growth of seedlings grown in nutrient soil containing bamboo fungus culture substrates was suppressed. The suppression effect increased as the proportion of the bamboo fungus substrates increased. S. indica colonization could alleviate the symptoms caused by FocTR4, and both the S. indica colonized and non-colonized banana seedlings grown in nutrient soil containing 1/4 D. echinovolvata culture substrates showed obvious milder symptoms compared with the seedlings grown in normal nutrient soil after FocTR4 infection, indicating that the addition of D. echinovolvata culture substrates can alleviate the Fusarium wilt symptoms. The results obtained in this study can provide technical support and a theoretical basis for the application of S. indica and bamboo fungus intercropping in the prevention and control of banana Fusarium wilt disease (S. indica and bamboo fungus on banana Fusarium wilt resistance needs to be further studied in future research.Our study revealed that the  disease . The und


This paper presents a computational analysis on the mechanical and damage behavior of novel hybrid polymer composites with graphene and MXene nano-reinforcements targeted for flexible electronics and advanced high-strength structural applications with additional functions, such as real-time monitoring of structural integrity. Geometrical models of three-dimensional representative volume elements of various configurations were generated, and a computational model based on the micromechanical finite element method was developed and solved using an explicit dynamic solver. The influence of the geometrical orientation, aspect ratio, and volume fractions of the inclusions, as well as the interface properties between the nano-reinforcements and the matrix on the mechanical behavior, was determined. The results of the presented research give initial insights about the mechanical and damage behavior of the proposed composites and provide insight for future design iterations of similar multifunctional materials. These nanofillers drew attention due to their flexible properties, allowing us to create multi-functional composite materials that can be used for a range of applications such as aerospace, energy storage, and electromagnetic interference shielding ,2,3. TwoD nanomatsolvents ,18. Ti3Cuctivity ,20,21, auctivity  and mechuctivity  of polymuctivity . Furtheructivity .3C2Tz flake is 0.8 nm, while a thickness of 1 nm is characteristic for the flake with surface functional groups, and the mean lateral size is usually around 500 nm [The mechanical properties of the hierarchical composite greatly depend on 2D nanofiller geometry . Depending on the synthesis method of the MXene, varying thickness and number of layers can be obtained ,26. The d 500 nm . As multd 500 nm ,29,30,31Recently, the electrical properties of polymer composites have gained great interest. The conductivity properties of hybrid composites with carbon nanotubes and carbon black can by utilized for energy storage . Carbon The finite element-based approach was proven to be a very valuable tool for studying the mechanical and damage behavior of various composite materials. In the field of finite element-based analysis on nanocomposites, the majority of published works are focused on graphene for improving the mechanical characteristics of nanocomposites ,50,51,52The tensile response and the damage mechanism of MXene/epoxy composites MXene/polyvinyl alcohol composites were investigated  by develThe aim of this research is to study the mechanical and damage behavior of hybrid polymer composites with MXene and graphene nano-reinforcements by developing a computational model based on the micromechanical finite element method, which would allow initial insights on the mechanical and damage behavior of such hybrid composites to be presented and estimations to be made about the influence of the geometrical orientation, aspect ratio and volume fractions of the inclusions as well as the interface properties between the nano-reinforcements and the matrix.The investigated composite materials are composed of an epoxy matrix and 2D nanosheets of graphene and MXenes. The mechanical properties of the materials are presented in Gf and MXf, respectively) of graphene and MXene inclusions, various aspect ratios (Gρ and MXρ), and different alignment configurations were created using Digimat-FE . In practice, the alignment of nano-reinforcements can be achieved through electrical methods [To investigate the mechanical behavior of such hybrid polymer composites reinforced with graphene and MXene nanosheets, a computational model was developed on the basis of the micromechanical finite element method. Geometrical models of three-dimensional representative volume elements (RVEs) with various volume fractions . These values were in the range determined in the previous research [The created RVEs were imported to the commercial finite element software Abaqus FEA , which was used to develop computational model and carry out the simulation tasks. The periodic boundary conditions  were optresearch . The maxresearch . The MXeStress distributions in RVEs subjected to tension along the x-axis direction are shown in At the beginning, the MXene/epoxy interfaces start to fail e. High-sGρ = 500, MXρ = 125, Gf = 0.1%, MXf = 1.6%, and MXE = 0.5mE, the effective Young’s modulus was 3.4 GPa, and the tensile strength was 40.1 MPa with randomly placed inclusions, while the effective Young’s modulus along the alignment (x-axis) direction was 4.6 GPa and the tensile strength was 58.4 MPa with aligned inclusions. The influence of the aspect ratio of aligned inclusions is shown in Gρ = 500, Gf = 0.1%, MXf = 3.2%, and MXE = 0.5mE. At MXene aspect ratio values of 60, 125, and 250, the effective Young’s moduli were 4.7 GPa, 6.17 GPa, and 8.1 GPa, respectively. Additionally, higher values of elongation at break were observed at higher values of aspect ratio.The influence of geometrical orientation of the inclusions is shown in Gρ = 500, MXρ = 125, Gf = 0.1%, and MXf = 1.6%, the tensile strength was 40.1 MPa, 48.6 MPa, and 63.5 MPa, at MXE = 0.5mE, MXE = 0.75mE, and MXE = 1.5mE, respectively (Gρ = 500, MXρ = 125, Gf = 0.1%, and MXf = 6.4%, where MXenes aligned, the tensile strength was 69.2 MPa, 82.6 MPa, and 116.1 MPa, at MXE = 0.5mE, MXE = 0.75mE, and MXE = 1.5mE, respectively (MXE = 1.5mE compared to the case when MXE = 0.5mE.The MXene/epoxy interface did not have a significant influence on the effective Young’s modulus of the composite; however, higher properties of the interface result in significantly higher values of the tensile strength . In the ectively a. In RVEectively b. MoreovThese results confirm the previous findings  that theGρ = 500, MXρ = 125, Gf = 0.1%, and MXE = 0.5mE, the obtained effective Young’s moduli were 2.8 GPa and 3.4 GPa at MXf = 0.8% and MXf = 1.6%, respectively. For the composites with aligned MXenes, containing Gρ = 500, MXρ = 125, Gf = 0.1%, and MXE = 0.5mE, effective Young’s modulus values of 3.5 GPa, 4.6 GPa, 6.1 GPa, and 10.1 GPa were obtained at MXf = 0.8%, MXf = 1.6%, MXf = 3.2%, and MXf = 6.4%, respectively.The influence of the MXene volume fraction is shown in Summarizing the results in Besides load carrying capabilities, the proposed composite can utilize the electrical properties of MXene. For example, when a structure made of such composite loses its structural integrity, the electrical conductivity is also deteriorated. This can be easily measured in real-time and give a reference about the structural integrity state.Novel hybrid polymer composites with graphene and MXene nano-reinforcements were proposed, and a computational model based on the micromechanical finite element method was developed for studying the mechanical and damage behavior of hybrid polymer composites with MXene and graphene nano-reinforcements.The influence of the geometrical orientation, aspect ratio and volume fractions of the inclusions, as well as the interface properties between the nano-reinforcements and the matrix, on the mechanical behavior was studied. The modelling demonstrated that both the effective Young’s modulus and the tensile strength were higher in the composites with aligned MXene and graphene inclusions in comparison to the composites with randomly placed inclusions. An increase in the volume fracture of the nano-reinforcements results in a significant increase in the effective Young’s modulus of the analyzed composites. The effective Young’s modulus of the composite with aligned nano-reinforcements containing 0.1% and 6.4% volume fractions of graphene and MXene, respectively, was 3.65 times higher compared to the Young’s modulus of the matrix. Moreover, the tensile strength increased as the volume fraction of MXene increased in the composites with aligned nano-reinforcements. However, in the composites with randomly placed nano-reinforcements, an increase in the volume fraction resulted in only an increase in the effective Young’s modulus. The MXene/epoxy interface did not have a significant influence on the effective Young’s modulus of the composite; however, higher properties of the interface resulted in significantly higher values of the tensile strength. Further, higher mechanical properties were observed at higher aspect ratios of nano-reinforcements. Crack pinning and deflection of the epoxy matrix were observed in the fractured RVEs.Graphene nano-reinforcements may be used for additional strengthening of multifunctional composites with MXenes and further expand the scope of application of such materials by utilizing the great mechanical properties and electrical conductivity of MXene. The proposed novel hybrid polymer composites with graphene and MXene nano-reinforcements can be applied for flexible electronics and advanced high-strength structural applications with additional functions as real-time monitoring of structural integrity.The results of the computational analysis revealed that MXene and graphene nano-reinforcements demonstrate considerable promise in the development of novel multifunctional composites, exhibiting excellent mechanical properties.


Although thousands of long noncoding RNAs (lncRNAs) have been annotated, only a few lncRNAs have been characterized functionally. In this study, we aimed to identify novel lncRNAs involved in the progression of gastric carcinoma (GC) and explore their regulatory mechanisms and clinical significance in GC.A lncRNA expression microarray was used to identify differential lncRNA expression profiles between paired GCs and adjacent normal mucosal tissues. Using the above method, the lncRNA SGO1-AS1 was selected for further study. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) were performed to detect SGO1-AS1 expression in GC tissues. Gain-of-function and loss-of-function analyses were performed to investigate the functions of SGO1-AS1 and its upstream and downstream regulatory mechanisms in vitro and in vivo.SGO1-AS1 was downregulated in gastric carcinoma tissues compared to adjacent normal tissues, and its downregulation was positively correlated with advanced clinical stage, metastasis status and poor patient prognosis. The functional experiments revealed that SGO1-AS1 inhibited GC cell invasion and metastasis in vitro and in vivo. Mechanistically, SGO1-AS1 facilitated TGFB1/2 mRNA decay by competitively binding the PTBP1 protein, resulting in reduced TGFβ production and, thus, preventing the epithelial-to-mesenchymal transition (EMT) and metastasis. In addition, in turn, TGFβ inhibited SGO1-AS1 transcription by inducing ZEB1. Thus, SGO1-AS1 and TGFβ form a double-negative feedback loop via ZEB1 to regulate the EMT and metastasis.SGO1-AS1 functions as an endogenous inhibitor of the TGFβ pathway and suppresses gastric carcinoma metastasis, indicating a novel potential target for GC treatment.The online version contains supplementary material available at 10.1186/s13046-021-02140-0. Gastric carcinoma is one of the most common malignancies worldwide and the fourth leading cause of cancer death . ApproxiLncRNAs are transcripts longer than 200 nucleotides without protein-coding potential . Tens ofHere, we demonstrate that the lncRNA SGO1-AS1 , which is downregulated in gastric carcinoma and associated with tumor progression and patient prognosis, prevents gastric carcinoma EMT, invasion and metastasis in vitro and in vivo. Mechanistically, SGO1-AS1 reduces the stability of TGFB1/2 mRNA by competitively binding the PTBP1 protein, resulting in reduced TGFβ production. In turn, TGFβ inhibits SGO1-AS1 transcription by inducing ZEB1. Thus, in this study, we identified a novel metastasis-suppressive lncRNA, i.e., SGO1-AS1, with crucial biological, mechanistic and clinical impacts on GC that mediates a double-negative feedback loop with TGFβ via ZEB1.Five pairs of snap-frozen GC tissues and matched adjacent normal mucosa tissues were obtained for the lncRNA microarray analysis. Furthermore, the following two cohorts of frozen samples were collected for the qRT-PCR assay: a small GC cohort Cohort 1) containing 18 pairs of GC tissues and corresponding adjacent normal mucosa tissues to confirm 13 lncRNAs with more than a 4-fold difference in the microarray analysis and a large GC cohort (Cohort 2) including 92 pairs of GC tissues and matched adjacent normal samples to detect the expression levels of SGO1-AS1, TGFB1/2 and ZEB1. Additionally, GC tissue microarrays containing 95 GC tissues and 80 adjacent tissues (Cohort 3) were included in this study for the ISH analysis. All tissues were collected immediately after surgery from the Affiliated Cancer Hospital of Guangzhou Medical University . All procedures carried out in this research involving human participants were performed in accordance with the ethical standards of the Institutional Review Board of the Affiliated Cancer Hospital of Guangzhou Medical University. The clinical and histopathological characteristics of the patients are described in Additional file  containiThe total RNA was extracted from 5 paired GC tissues and corresponding adjacent normal mucosa tissues using TRIzol reagent  according to the manufacturer’s instructions. The total RNA was amplified and reverse-transcribed into fluorescent cDNA. Then, the labeled cDNA was hybridized onto the LncRNA+mRNA Human Gene Expression Microarray V4.0 , and after washing, the arrays were scanned with an Agilent Scanner G2565CA (Agilent). Agilent Feature Extraction software (version 10.7.3.1) was used to analyze the acquired array images and the Agilent qRT-PCR results. The data are available via Gene Expression Omnibus (GEO) under accession number GSE157289.-∆∆Ct. The ∆Ct was calculated by subtracting the Ct of β-actin from the Ct of the gene of interest. The ∆∆Ct was calculated by subtracting the ∆Ct of the control sample from the ∆Ct of the treatment sample. The primer sequences for each gene are provided in Additional file The total RNA was isolated from patient tissues and cultured cells using TRIzol reagent (Invitrogen), and cDNA was synthesized using a PrimeScript RT Reagent Kit . Subsequently, quantitative polymerase chain reaction (qPCR) analyses were performed using a SYBR Premix Ex Taq Kit . β-actin was used as the endogenous control to normalize gene expression. The mRNA expression of SGO1-AS1, TGFB1, TGFB2 and PTBP1 in the human tissues is presented as -∆Ct, and the gene expression in cells with different treatments is presented as 2The ISH analysis was performed using a kit from Boster . Tissue microarray slides were deparaffinized, digested with proteinase K, hybridized with DIG-labeled probes for SGO1-AS1 and U6 (positive control) at 52 °C overnight and subsequently visualized with an anti-DIG-POD antibody and DAB complex. The SGO1-AS1 probe was 5′-CCGCCTCCCAGCCAACCAATGGAGGAGCGAGGCG-3′. The results were evaluated by two individuals in a blinded fashion, and the SGO-AS1 expression levels were quantified according to its positive percentage and staining intensity.We used 5′-RACE and 3′-RACE analyses to determine the transcriptional initiation and termination sites of SGO1-AS1 using a SMARTer™ RACE cDNA Amplification Kit  following the manufacturer’s instructions. Nested PCR products were cloned into the pMD20-T vector and then sequenced. The sequences of the SGO1-AS1-specific primers used in the nested PCR of the RACE assay are shown in Additional file Nuclear and cytoplasmic separation was performed using a PARIS Kit  according to the manufacturer’s instructions, and then, a qRT-PCR analysis was conducted.2.The GC cell lines SGC7901, BGC823, AGS, MGC803, MKN45 and MKN28 were obtained from the Chinese Academy of Medical Science , and the gastric epithelial cell line GES-1 was obtained from the Beijing Institute for Cancer Research . The GC cell line NCI-N87 and the HEK293T cell line were obtained from the American Type Culture Collection . The cell lines involved in our experiments were reauthenticated by a short tandem repeat analysis every 6 months after resuscitation in our laboratory. These cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (Gibco) at 37 °C in 5% COThe recombinant lentiviral vectors used for SGO1-AS1 overexpression or knockdown were purchased from RiboBio , and the PTBP1 short hairpin RNA (shRNA) lentiviral vectors were obtained from GeneChem . The target sequences for SGO1-AS1 and PTBP1 were as follows: shAS1#1, 5′-GCTATCTTCCTCCTCCTCACA-3′; shAS1#2, 5′-CTACCGCCGCCACATTCGAAA-3′; shAS1#3, 5′-GCCTCCCTCTTGTGAGAAGAA-3′; shAS1#4, 5′-AGCTTGCAACGCGGAAGCAGC-3′; and shPTBP1, 5′-GCGGCCAGCC CATCTACATC-3′. To establish the cell lines that stably overexpress or deplete SGO1-AS1, SGC-7901 cells were infected with recombinant SGO1-AS1 lentiviruses, while MKN28 cells were infected with SGO1-AS1 shRNA lentiviruses. Then, the infected cells were selected with 1 mg/L puromycin  for 2 weeks to obtain cells with stable overexpression or knockdown of SGO1-AS1. siRNAs targeting ZEB1 or AGO2 were designed and synthesized by Sangon Biotech , and their sequences are as follows: siZEB1#1 sense, GGCAAGUGUUGGAGAAUAAUC, antisense, UUAUUCUCCAACACUUGCCUU; siZEB1#2 sense, GGACAGCACAGUAAAUCUACA, antisense, UAGAUUUACUGUGCUGUCCUG; siAGO2 sense, GGUUGAUACUUAAGCUCUAUU, antisense, UAGAGCUUAAGUAUCAACCUG. To construct the reporter vectors for SGO1-AS1 promoter activity, the wild-type SGO1-AS1 promoter sequence (1 kb sequence upstream of the transcription start site) and its ZEB1-binding site mutated sequences were chemosynthesized by Huada  and inserted into the vector pGL3 basic (Promega) upstream of the firefly luciferase gene.A small guide RNA (sgRNA) targeting the genome sequence of PTBP1 was cloned into LentiCRISPRv2 (Addgene), and lentivirus particles were generated by cotransfecting the recombinant vector and packaging plasmids into HEK293T packaging cells. MKN28 cells were infected with lentiviruses, and single cells were isolated 48 h after infection by FACS (BD FACS Aria III) into 96-well plates. Independent clones were allowed to grow for 3 weeks. The PTBP1 knockout cells were identified by Western blotting and targeted Sanger sequencing. The sgRNA targeting PTBP1 was 5′-CAGAGCAGACCCGCGGGGGA-3′.The Western blotting analysis was performed using standard procedures. The following primary antibodies were used in the experiments: anti-PTBP1 antibody , anti-PTBP2 antibody , anti-PTBP3 antibody , anti-HNRNPK antibody (Abcam), anti-HNRNPM antibody (Sigma-Aldrich), anti-FUBP3 antibody (Abcam), anti-CPSF2 antibody (Abcam), anti-G3BP2 antibody (Atlas Antibodies), anti-TGFβ1 antibody (Proteintech Group), anti-TGFβ2 antibody (Abcam), anti-p-SMAD2 antibody , anti-SMAD2 antibody , anti-p-SMAD3 antibody , anti-SMAD3 antibody , anti-SMAD5 antibody (Abcam), anti-ID2 antibody (Abcam), anti-ZEB1 antibody (Abcam), anti-SNAI antibody (Abcam), anti-E-cadherin antibody (Proteintech Group), anti-Vimentin antibody , anti-N-cadherin antibody  and anti-GAPDH antibody (Sigma-Aldrich). The blots were incubated with a goat anti-rabbit or anti-mouse secondary antibody (Sigma-Aldrich) and visualized with a commercial ECL kit .The RNA pull-down assays were carried out as previously described. Briefly, the SGO1-AS1 sequences were cloned into the pMD20-T vector with the T7 promoter and transcribed in vitro with biotin RNA labeling mix and T7 RNA polymerase (Invitrogen) according to the manufacturer’s instructions. The RNA pulldown assay was performed using a Pierce Magnetic RNA-Protein Pull-Down Kit  according to the manufacturer’s instructions. Finally, the retrieved proteins were measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) gels for mass spectrometry or a Western blot analysis.The RIP assays were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit  according to the manufacturer’s instructions. Briefly, 100 μL of cell extract were incubated with magnetic bead-antibody complex. Antibodies were used for RIP, and IgG served as a negative control. The precipitated RNAs were isolated using TRIzol (Invitrogen) for the RNA sequencing (RNA-seq) and qRT-PCR analyses.The ChIP assays were performed using a Chromatin Immunoprecipitation Assay Kit . MKN28 cells were exposed to TGFβ1 or vehicle for 24 h and then crosslinked, lysed and sonicated. Immunoprecipitation was performed using an anti-ZEB1 antibody  and IgG. The precipitated DNA was quantified using qPCR and normalized to the respective 2% input.P-value < 0.05 were selected. To reveal the PTBP1-bound mRNAs, RIP experiments were conducted using a PTBP1 antibody  or IgG. The total RNA was isolated with TRIzol (Invitrogen), and ribosomal RNA was removed from the total RNA. RNA-seq was performed at CLOUDSEQ  using HiSeq3000 . The data are available via GEO under accession numbers GSE157582 and GSE157941.To identify the differentially expressed genes upon PTBP1 knockout, the total RNA was isolated from the PTBP1 knockout or control MKN28 cells using TRIzol reagent, and PolyA RNA was subsequently purified from the total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module. RNA-seq was performed to detect the mRNA expression profiles at GENTED  using HiSeq3000 . The differentially expressed genes with a fold change > 2 and a HEK293T cells were seeded in 24-well plates and transfected with SGO1-AS1 promoter reporter constructs with wild-type or mutated ZEB1 binding sites. The pTK-Cluc vector was used as an internal transfection control. The transfected cells were treated with TGFβ1 (5 ng/mL) or vehicle control for 48 h, and firefly and Renilla luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions. The SBE4 promoter luciferase reporter vector (Addgene) was transfected into the PTBP1 knockout or control MKN28 cells. In addition, HEK293T cells were transfected with SBE4 promoter reporter vectors and then treated with conditioned medium from cells with SGO1-AS1 knockdown or overexpression. The firefly and Renilla luciferase activities were measured 48 h after the transfection using a dual luciferase system.For the cell invasion assay, starved cells suspended in serum-free DMEM were seeded into the upper chamber with Matrigel in the insert of a 24-well culture plate (Corning Costar). Medium containing 15% fetal bovine serum was added to the lower compartment as a chemoattractant. After incubation for 48 h, the invasive cells adhering to the lower membrane of the inserts were fixed, stained, counted and imaged. The cell migration ability was measured using a wound-healing assay. The cells were placed in 6-well plates and cultured until reaching 90% confluence. An artificial scratch was created using a 10 μL pipette tip, and the cells were cultured in serum-free medium for 36 h or 48 h. Wound closure images were captured in the same field under magnification. Cell proliferation was examined using cell counting. The cells were seeded into 6-well plates, and the cell numbers were counted after 1, 2, 3, 4, 5, 6 and 7 days of culture in DMEM supplemented with 10% fetal bovine serum using a Coulter Counter.Cells were seeded into ultralow attachment 6-well plates (Corning Costar) and cultured in DMEM/F12 medium (Gibco) supplemented with 2% B27 (Life Technologies), 20 ng/ml FGF , 20 ng/ml EGF (R&D Systems) and 5 μg/ml insulin (R&D Systems). Two weeks later, sphere pictures were obtained, and the sphere formation ratios were calculated.6 cells per mouse) were injected into the tail vein or stomach of the BALB/c nude mice. The luciferase signal intensity was monitored in vivo using an In Vivo Imaging System . Then, the mice were sacrificed, and the metastatic foci in the abdominal cavity and lung were evaluated. In addition, SGC7901 cells with SGO1-AS1 overexpression or control cells were subcutaneously injected into nude mice The mice were sacrificed 28 days after implantation, and the tumors were excised and weighed.Subsequently, 6- to 8-week-old female BALB/c nude mice were purchased from the Experimental Animal Center of Guangdong . To investigate the role of SGO1-AS1 in tumor metastasis and growth in vivo, luciferase-labeled SGC7901 cells overexpressing SGO1-AS1 or the control vector  three times per week for 3 weeks. The luciferase signal intensity was monitored in vivo by bioluminescence imaging. All animal studies were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University, and the animals were treated ethically and humanely.P-values < 0.05 were considered statistically significant.A Student’s t-test or chi-square test was used for the two-sample comparisons. The differences among three or more groups were analyzed with a two-way analysis of variance. The overall survival curves were plotted using the Kaplan-Meier method, and the survival differences were evaluated with a log-rank test. A Cox regression was utilized to estimate the hazard ratio and 95% confidence intervals of survival. The pairwise expression correlations were analyzed using Pearson correlation tests. P < 0.05, Fig. To identify GC-relevant lncRNAs, we examined the lncRNA expression profiles in five paired GC and adjacent normal mucosa tissues using a microarray. We found that 185 lncRNAs were differentially expressed in GCs compared to those in the adjacent tissues , we further confirmed the downregulation of SGO1-AS1 in GC tissues via a qRT-PCR analysis, and 63% (58/92) of the GC cases showed more than 2-fold downregulation of SGO1-AS1 relative to the corresponding normal tissues . Therefore, we examined whether SGO1-AS1 could affect the expression of the sense gene SGO1 and found that although knockdown of SGO1-AS1 moderately induced SGO1 expression, overexpression of SGO1-AS1 did not affect the expression of SGO1  family and a critical regulator of mRNA splicing , 16, RNAhttps://cancergenome.nih.gov) stomach adenocarcinoma RNA-seq dataset and demonstrated that TGFβ1 target genes were significantly enriched in GC patients with low SGO1-AS1 expression  that plays a role in mRNA metabolism by binding target mRNAs . We hypoion Fig. f, implyiGiven that PTBP1 is an RNA binding protein , we perfSince our above results indicate that depletion of PTBP1 reduces the TGFB mRNA levels, we investigated whether PTBP1 and SGO1-AS1 may influence TGFB mRNA stability. Actinomycin D (ACD) was used to block de novo transcription in PTBP1-knockout and control cells infected with SGO1-AS1 shRNA. As expected, the depletion of PTBP1 increased TGFB mRNA degradation following ACD treatment in MKN28 cells. However, silencing SGO1-AS1 stabilized TGFB mRNAs but had no significant effect on TGFB mRNA stability under conditions in which PTBP1 was depleted Fig. k. FurtheNext, we explored the mechanism by which PTBP1 promotes the stability of TGFB1/2 mRNA. Numerous reports indicate that the RNA-induced silencing complex (RISC) mediates mRNA decay , 26, andAs SGO1-AS1 reduces TGFβ production, we evaluated whether SGO1-AS1 may affect TGFβ downstream signaling. As expected, silencing SGO1-AS1 enhanced the phosphorylation of SMADs and induced the transcriptional activity of SBE4 (SMAD-binding element) in control cells but showed little effect in the PTBP1-deficient cells, and the activation of TGFβ signaling by SGO1-AS1 knockdown was reversed by the treatment with the TGFβ type I receptor (TβRI) inhibitor SB431542  Fig. a, b. To http://jaspar.genereg.net/) to identify potential regulatory transcription factors. The bioinformatics analysis revealed four ZEB1 binding motifs at − 222 to − 214 (site A), − 731 to − 721 (site B), − 822 to − 814 (site C) and − 947 to − 940 (site D) inside the SGO1-AS1 promoter  , 30 wereWe sought to determine whether our findings could be extended to patients with gastric carcinoma. The expression levels of TGFB1, TGFB2, ZEB1 and PTBP1 were detected by qRT-PCR in 92 pairs of gastric carcinoma specimens and adjacent normal tissues (Cohort 2). The expression levels of these four genes were significantly increased in the tumors compared to those in the normal tissues, and 51.1% (47/92), 58.7% (54/92) 39.1% (36/92), and 52.2% (48/92) of the GC samples showed more than a 2-fold upregulation of TGFB1, TGFB2, ZEB1 and PTBP1, respectively . The results are expressed as -∆Ct. **P < 0.05, **P < 0.01, ***P < 0.001. Fig. S2 Characterization of human SGO1-AS1 as a long noncoding RNA. a. Schematic diagram of the genomic locus and isoforms of SGO1-AS1 in the UCSC Genome Browser (http://genome.ucsc.edu/). b. Identification of full-length SGO1-AS1 by 5′ and 3′ RACE. Left: Representative images of the PCR products from 5′ RACE and 3′ RACE. Right: Nucleotide sequence of full-length human SGO1-AS1. c. Coding potential of SGO1-AS1 as predicted by the CPAT and CPC tools. The lncRNA MALAT1 and the protein-coding genes GAPDH and ACTB are also shown. d. Relative distribution of SGO1-AS1 in gastric cell lines as determined by RT-PCR. SGO1-AS1 was mainly expressed in the cytoplasm of epithelial cells. Fig. S3 SGO1-AS1 inhibits GC cell migration and growth. a. qRT-PCR showed the relative levels of SGO1-AS1 expression in human gastric cancer cell lines compared to those in the immortalized human gastric epithelial cell line GES1. b. qRT-PCR analysis was used to confirm the level of SGO1-AS1 in cells with stable overexpression of SGO1-AS1. c. qRT-PCR analysis was performed to assess the inhibition efficiency in MKN28 cells infected with SGO1-AS1 shRNA lentiviruses targeting different regions of SGO1-AS1. d. Cell migration in BGC823 cells stably expressing SGO1-AS1 and control cells was examined by a wound healing assay. Scale bar, 150 μm. e. Cell proliferation assay in SGC7901 and BGC823 cells stably expressing SGO1-AS1, MKN28 cells stably silencing SGO1-AS1 and the respective control cells. f-g. A soft agar colony formation assay was carried out in the indicated cells. Scale bars, 100 μm. h. Overexpression of SGO1-AS1 inhibits GC tumor growth in a nude mouse model. SGC-7901 cells with stable overexpression of SGO1-AS1 or the control were inoculated subcutaneously into nude mice (n = 6 mice/group). The mice were sacrificed at 28 days postinoculation, and the tumors were weighed. Error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001. Fig. S4 Effects of SGO1-AS1 on the expression of the sense gene SGO1. a-b. qRT-PCR and Western blotting analyses of the SGO1 levels in SGC7901 and BGC823 cells stably expressing SGO1-AS1, MKN-28 cells stably silencing SGO1-AS1 and the related control cells. Error bars indicate SDs from three independent experiments. *P < 0.05, **P < 0.01. Fig. S5 SGO1-AS1 reduces TGFB1/2 mRNA stability. a. Western blot analysis was used to evaluate PTBP1 expression in human gastric cancer cell lines and normalgastric epithelial cell line. b. qRT-PCR analysis was performed to examine the expression levels of genes related to the TGFβ pathway in SGC7901 cells transfected with PTBP1 shRNA or the control. c. PTBP1 RIP assay was performed to analyze the interactions between PTBP1 and TGFB1/2 or ID3 in SGC7901 cells. The relative fold enrichment of these mRNAs compared to IgG was determined by qRT-PCR. SMAD5 and GAPDH were used as negative controls. d. RIP-qPCR assay was used to evaluate the interaction between PTBP1 and ID2 mRNA in MKN28 cells with SGO1-AS1 knockdown or SGC7901 cells with SGO1-AS1 overexpression. e. Deletion mapping was performed to determine the binding domain of PTBP1 to TGFB1/2 mRNA using full-length or truncated PTBP1. f-g. mRNA (f) and protein (g) expression levels of TGFβ1/2 in xenograft tumor tissues recovered from nude mice. #1–3 denote individual tumors grown in different mice. h. Western blot analysis of AGO2, TGFβ1 and TGFβ2 in control and PTBP1-KO MKN28 cells transfected with siAGO2 or control siRNA. i. RIP-qPCR assay of the interaction between the AGO2 protein and TGFB1/2 mRNA in MKN28 cells with PTBP1 knockout or SGO1-AS1 knockdown. Error bars are SDs from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant. Fig. S6 SGO1-AS1 changes cell morphology and suppresses cell invasion and stemness. a-c. Representative phase-contract (a), cell invasion (b) and tumor spheroid images (c) of the indicated cells are shown. a, Scale bars,100 μm; b-c, Scale bars, 150 μm. Fig. S7 Expression level of PTBP1 and its correlation with SGO1-AS1, TGFB1/2 and ZEB1 expression in GC tissues. a. Relative expression levels of PTBP1 in 92 paired GC and normal tissues from Cohort 2 were quantified by qRT-PCR. The pie chart shows the proportions of samples in the downregulation (blue), upregulation (red) and no change (yellow) categories. b. The expression level of PTBP1 in GCs according to their clinical stage and status of lymph node or distant metastasis. c. Correlation between PTBP1 expression and SGO-AS1, TGFB1, TGFB2 and ZEB1 expression in GC specimens. a-c, Error bars indicate SDs. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant. Table S1. Clinicopathological characteristics of Cohort 2 samples. Table S2. Clinicopathological characteristics of Cohort 3 samples. Table S3. Primers used for quantitative PCR. Table S4. Primers for rapid amplification of cDNA ends analysis. Table S5. Correlation between clinicopathological parameters and SGO1-AS1 levels in 95 cases of GC tissues (Cohort 3). Table S6. Univariate and multivariate analyses of factors associated with overall survival. Table S7. Mass spectrometry protein identification results for biotinylated SGO1-AS1 RNA pull down.


Simultaneous in-situ exfoliation and functionalization of MoS2 were achieved in the presence of cetyltrimethylammonium bromide (CTAB) via sonication. The aim was to improve the dispersion of MoS2 nanoplatelets in epoxy and enhance the interfacial interaction between nanoplatelets and epoxy matrix. Epoxy nanocomposites with CTAB functionalized MoS2 (f-MoS2) nanoplatelets, ranging in content from 0.1 wt% up to 1 wt%, were fabricated. Modified MoS2 improved the fracture properties (81%) of tetrafunctional epoxy nanocomposites. The flexural strength and compressive strength improved by 64% and 47%, respectively, with 0.25 wt% loading of f-MoS2 nanoplatelets compared to neat epoxy. The addition of f-MoS2 nanoplatelets enhanced the thermomechanical properties of epoxy. This work demonstrated the potential of organically modified MoS2 nanoplatelets for improving the fracture and thermal behavior of tetrafunctional epoxy nanocomposites.In this work, improved fracture toughness of tetra-functional epoxy polymer was obtained using two-dimensional (2H polytype) molybdenum disulfide (MoS Tetrafunctional epoxy resins are among the most critical high-performance thermosetting polymers. These epoxy resins offer excellent strength and heat resistance for advanced structural and high-temperature applications in aerospace industries. The last decade has seen the increasing use of epoxy-based composites in the aerospace industry, especially in the Boeing 787 and Airbus A350. In these aircrafts, the fuselage, wings, and empennage, which are major structural parts, are manufactured using epoxy-based fiber reinforced composites. Due to their excellent resistance to fatigue and corrosion as well as enabling of a reduction in the number of components, more and more structural parts in aircraft are being designed using epoxy-based composites. However, the high crosslink density of epoxy polymers is low, which leads to decreased resistance to crack propagation. To address this problem, toughening agents that enhance the fracture properties of tetrafunctional epoxy without compromising modulus and thermal properties are required. There is little work reported on improving the fracture properties of tetrafunctional epoxy polymer. Most of the published work is on bifunctional epoxy polymers for low-temperature applications. Graphene and its derivatives provide excellent reinforcement for epoxy polymers and enhance their thermal properties at low concentrations ,2. Howev2) has gained special interest due to its applications in the field of electronics [2 nanosheets exhibit excellent mechanical properties, such as extremely high Young’s modulus of 0.3 TPa [−1 (23 GPa) [2 nanosheets is their high bandgap that does not impart conductivity to the insulating polymer matrices. These properties make the 2H polytype of MoS2 nanosheets an ideal complementary filler to graphene. MoS2 has been explored by many researchers for improving the thermal and fire-retardant properties of thermoplastic polymers, such as polypropylene [2, covalent functionalization is achieved by the intercalation method. One of the most used methods involves the Li-ion intercalation step followed by treatment with functional molecules [Apart from graphene, two-dimensional (2D) transition metal chalcogenides (TMD) ,7,8,9,10ctronics ,18,19, cctronics ,22,23,24ctronics ,26,27, ectronics , and polctronics ,30. Few  0.3 TPa  and brea(23 GPa) , which aropylene ,34, polyropylene . To overropylene ,36,37,38olecules ,39,40. H2 for thermosetting polymers such as epoxy [2 platelets leading to the formation of microparticles by self-assembly of nanosheets. The use of these functionalized MoS2 demonstrated a 23% increase in the tensile strength [2 nanosheets [2 nanosheets [2 nanosheets. Chen et al. covalently modified MoS2 using amine-containing melamine for improving the chemical interaction of filler with epoxy for improved tensile properties [2 showed a maximum improvement in properties at 0.8 wt% of filler. The literature available focusing on the effect of MoS2 nanosheets on the fracture, compression, and flexural properties of high-performance tetrafunctional epoxy polymers is still inadequate. Eksik et al. [2 nanosheets. They concluded that the surface modification of MoS2 is required to improve the interaction of the filler with the epoxy matrix. We have reported the effect of surface-functionalized tungsten disulfide (WS2) on the fracture, compression, flexural properties, and thermal behavior of epoxy in our previous study. This study explores MoS2 as an economically favorable alternative to WS2 for enhanced mechanical andthermal properties of epoxy nanocomposites [A few studies have reported using functionalized MoSas epoxy . Chhetristrength  of bifunnosheets . Howevernosheets . The throperties . Similaroperties , covalenk et al.  reportedmposites .2 nanosheets on the fracture, compression, flexural, and thermal behavior of tetrafunctional epoxy polymers. The work involves the one-pot exfoliation and modification of MoS2 nanoplatelets at room temperature using cetyltrimethylammonium bromide (CTAB) by bath sonication. The f-MoS2 has been incorporated in the range 0.1 wt% to 1 wt% to study and compare the properties of epoxy nanocomposites with neat epoxy polymers.In this work, we report the effect of surface modified MoS2) was purchased from Sigma Aldrich, Bengaluru, India. Cetyl trimethyl ammonium bromide (CTAB) was purchased from SRL Chemicals, Bengaluru, India. N,N,N′,N′-tetraglycidyl-4,4′-methylenebisbenzenamine resin   as well as 4,4′-diaminodiphenylsulfone  powder  were procured from Huntsman Advanced Materials, The Woodlands, TX, USA. In all the experiments, double autoclaved MilliQ water  was used.Powdered 2H polytype molybdenum disulfide  for 10 min. The supernatant containing f-MoS2  was separated, further washed  with ethanol and water several times, and dried for a duration of 24 h at 50 °C.In a typical experiment, 900 mg of 2H polytype—MoS2 nanoplatelets were dispersed in acetone (10 mg/mL) using a bath sonicator for 2 h. Epoxy was heated to 80 °C, and the required amount of f-MoS2 nanoplatelets suspension was added and stirred for 2 h using an overhead stirrer  followed by mixing using a homogenizer for 10 min. Post mixing, the modified epoxy resin was degassed in a vacuum oven for 12 h at 90 °C to remove the residual solvent molecules. A stoichiometric amount of hardener was added to the mixture at 120 °C assisted with stirring at high speed for 20 min to ensure complete mixing of the epoxy and the hardener. The mixture was again degassed for 2 h at 90 °C to remove the bubbles introduced during the mixing process. Finally, the mixture was cast in a mold (preheated to avoid gas entrapment) and cured at 150 °C for 2 h followed by 180 °C for 2 h followed by 200 °C for 2 h. Nanocomposites containing 0.1, 0.25, 0.5, and 1 wt% of f-MoS2 nanoplatelets were fabricated, and for comparison, neat epoxy composites were also fabricated using similar processing conditions.Dried f-MoS2 and fractured epoxy nanocomposites were analyzed by scanning electron microscopy  Bulk MoS2 and f-MoS2 nanoplatelets suspensions were prepared in an aqueous medium. Suspensions were diluted and drop cast on a freshly cleaned silicon substrate. Each sample was sputtered to build a 10 nm thick Au layer to prevent charge accumulation during analysis.Surface morphology of bulk exfoliated MoS2 nanoplatelets were analyzed by transmission electron microscopy . Nanoplatelets were sonicated in an aqueous medium for 30 min, drop-cast on a Cu grid (carbon-coated), dried and analyzed.The structure and morphology of f-MoS2 nanoplatelets. Samples were prepared by sonicating f-MoS2 nanoplatelets in deionized water for 30 min, followed by drop-casting on a freshly cleaned silica wafer. Samples were dried and desiccated before conducting AFM analysis.Atomic force microscopy was performed in tapping mode using a NanoWizard 3 AFM  to determine the thickness of f-MoS2 nanoplatelets were evaluated by confocal microscopy . The powdered samples of the bulk and exfoliated f-MoS2 nanoplatelets were mounted on a glass slide and imaged, covering the wavelength from 400 to 700 nm.The photoluminescence properties of the bulk and exfoliated f-MoS2 and f-MoS2 nanoplatelets were performed on Thermo-Nicolet 6700 Fourier transform infrared spectrophotometer  in the 400–4000 cm−1 spectral range. Thermogravimetric analysis was performed on NETZSCH-STA-409  in the range of 30 °C to 800 °C with a heating rate of 10 °C per minute. X-ray powder diffraction was performed for 2θ angles from 8° to 60° using X’Pert PRO .FTIR analysis of pure CTAB, bulk MoS3. The temperature was gradually changed from 30 to 330 °C at 3 °C min−1 with a frequency of 1 Hz.Dynamic mechanical (DMA) study of nanocomposites was performed on Gabo Eplexor 500N  using samples of three-point bend geometry with measurements 5 × 2 × 45 mmCompression tests were performed using a Zwick/Rowell Z100  per ASTM D695. Five parallels of each sample with a cylindrical geometry  were tested, and the average value is reported here. The cross-sectional area of samples was polished using P4000 grade emery paper to ensure no friction exists between the sample surface and the compression clamps.3 were cast molded. A sharp natural crack was induced in the samples by applying sawing motion using a razor blade dipped in liquid N2 over the machined notch. The fracture toughness KIC was calculated using the below-mentioned formula:max represents the max load on the load-displacement graph, W is the width of the flexural sample, B is the depth of the sample, and a is the length of pre-crack. The geometry dependent function, f (a/W), is calculated using the following equation:The fracture toughness of epoxy nanocomposites was calculated by conducting single edge notch bend tests per ASTM D5045, using a Zwick/Rowell Z100 machine. Samples with dimensions 6 × 12 × 48 mmTests were conducted on six replicas per composition, and the average of the obtained values is reported here. The samples were loaded until a complete fracture occurred.−1, and the average of the values is reported here. Samples with span length 48 mm, thickness 3 mm, and width 12 mm were employed.The flexural strength of neat epoxy and modified nanocomposites were determined using a Zwick/Rowell Z100 according to ASTM D790. Five replicas per composition were tested at a strain rate of 2 mm min2 flakes were exfoliated by sonication in an aqueous solution containing CTAB. The long hydrocarbon chains of the surfactant help in exfoliating the stacked layers of MoS2 when subjected to bath sonication. The presence of the surfactant molecules also provides the appropriate surface tension to overcome the van der Waals forces between the exfoliated layers. A schematic of the process for the simultaneous noncovalent modification of MoS2 with CTAB and exfoliation of f-MoS2 nanoplatelets followed by the fabrication of f-MoS2 modified epoxy nanocomposites is shown in 2 nanoplatelets disperse quickly in a weakly polar solvent such as acetone due to the polar nature of the CTAB molecules.MoS2. The particles were a few microns in size. The exfoliated layers of f-MoS2 are visible in the TEM micrographs  [2 transforms to molybdenum oxide. In the case of f-MoS2, the observed weight loss was much higher in the temperature range of 100–700 °C when compared to bulk MoS2. For example, the temperature for 5 percent weight loss (T−5%) was 114 °C for f-MoS2, whereas T−5% was much higher at 497 °C for bulk MoS2 due to the absence of organic moieties. The residual mass for bulk MoS2 was 95 wt%, whereas f-MoS2 showed 27 wt% residual mass at 700 °C. The increased weight loss can be ascribed to the increased diffusion of heat in exfoliated layers and decomposition of CTAB moieties, thus confirming the successful functionalization of MoS2 nanoplatelets.Thermogravimetric analysis was performed to further confirm the noncovalent functionalization of CTAB molecules on MoS(10 wt%) . Hence t2 was studied by X-ray diffraction (XRD) analysis. 2 and f-MoS2 nanoplatelets. The sharp and intense characteristic peak of bulk MoS2 appeared at 14.35°, which indicated interplanar spacing of 0.62 nm and was attributed to the (002) plane of the bulk MoS2. Other peaks appeared at 28.98, 32.63, 33.46, 39.50, 44.08, and 49.75°, which corresponded to the (004), (100), (101), (103), (006), and (105) planes, respectively. All the peaks were in good agreement with the JCPDS data for the bulk MoS2 (card no. 24-0513). In the case of f-MoS2, the diffraction peaks for the planes (104), (106), and (108) disappeared. The intensity of peak (002) of exfoliated MoS2 reduced dramatically compared to the bulk MoS2. This might be attributed to the reduced crystallite size, which reduces the probability of several (002) planes to satisfy Bragg’s condition. Additionally, peaks representing (004), (100), (101), (103), (006), and (105) also showed a drastic reduction in intensity compared to bulk MoS2. The reduced intensity of the peak (002) indicates the successful exfoliation of MoS2.The effect of exfoliation and surface functionalization on the crystalline structure of MoS2 nanocomposites, and the crucial data are shown in 2 nanoplatelets increased the T−5% of the epoxy nanocomposites by 20–30 °C. For example, the nanocomposite with 0.25 wt.% of f-MoS2 shows T−5% at 332°C, which is greater than T−5% of neat epoxy at 302 °C. This improvement in thermal properties can be ascribed to the barrier effect of well dispersed and exfoliated f-MoS2 nanoplatelets, which restrict the mobility of the volatile products across the matrix. The presence of CTAB molecules enhances the affinity of the MoS2 nanoplatelets towards the epoxy resulting in better dispersion of the f-MoS2 nanoplatelets in the epoxy matrix. The large surface area of MoS2 nanoplatelets, along with CTAB assisted dispersion, paves the way for the improved interlocking of filler with epoxy matrix.The inorganic layered nanofillers are expected to affect the thermal performance of nanocomposites to a significant extent due to their excellent thermal resistance. The thermal performance of nanocomposites was measured by thermogravimetric analysis. 2 nanoplatelets delay the degradation process by restricting the diffusion of degradation products and heat due to enhanced interface. The char yield is an essential factor for assessing the thermal stability of the polymer nanocomposite as the char residue resists heat flow during combustion and helps improve thermal stability. Hence, the higher the char yield, the better the thermal stability of nanocomposites [2 nanoplatelets enhance the thermal stability of epoxy nanocomposites. For example, the nanocomposite with 0.5 wt% filler yielded 29% char, which is much larger than the char yield of 12% for the neat epoxy. It can be noted that epoxy nanocomposites with 1.0 wt% show the least improvement in thermal stability that can be ascribed to the poor dispersion of f-MoS2 nanosheets at high weight % loading. The presence of agglomerates also hinders the nanofiller from forming a stronger interface and imparting improved thermal properties.Hence, the f-MoSmposites ,51. The 2 nanoplatelets on the thermomechanical properties of nanocomposites. The storage modulus and tan δ curves of the nanocomposites and the neat epoxy are shown in g), the epoxy nanocomposites must retain enhanced mechanical properties at elevated temperatures. g of the nanocomposites. The storage modulus is a measure of the stiffness of the nanocomposites. The storage modulus of the nanocomposite Epoxy/f-MoS2-0.25 wt% showed a maximum increase of 28% at 200 °C and 39% at 250 °C in comparison to neat epoxy. Tg of the polymer nanocomposites is taken as the temperature at the maximum of the tan δ curve. 2 nanoplatelets. The epoxy nanocomposites modified with low levels (0.1 and 0.25 wt%) of f-MoS2 nanoplatelets showed a higher Tg than the neat epoxy (281.1 °C) with a higher value of storage modulus. The increase in Tg suggests improvement in the thermo-mechanical properties of the final nanocomposites. The increase in storage modulus values and Tg for epoxy with the loading of f-MoS2 nanoplatelets should be attributed to the robust nature and high intrinsic stiffness of the MoS2 nanoplatelets. The segmental motional is restricted by the large aspect ratio of f-MoS2 platelets. The presence of CTAB molecules over the surface of the MoS2 nanoplatelets provides enhanced compatibility with the epoxy matrix due to its long hydrocarbon chains. Better exfoliation and distribution of the f-MoS2 in the epoxy matrix lead to enhanced effective volume of the filler, which is also one of the factors for the excellent reinforcing effect of f-MoS2 nanoplatelets.The dynamic mechanical study was employed to study the effect of f-MoS2 nanoplatelets with loading up to 1 wt%. All the samples showed similar deformation behavior under the applied load with an initial elastic region up to the diffused yield zone followed by the strain hardening region until the sample fractures. The strain hardening region is an indication of the energy absorbed by the sample before fracture. Unlike in instances of rubber toughened epoxy, a strain-softening region was not observed for any of the epoxy samples. Strain softening causes enhancement in the local cross-sectional area, which stabilizes further local strain [2 nanoplatelets, which leads to stronger composites. It can be noted in 2 nanoplatelets. However, the extent of variation was different for different loading of filler. Nanocomposites with 0.25 wt% of f-MoS2 nanoplatelets showed the most significant increase in the strain hardening region and are, therefore, most effective in absorbing the stress during the compressive loading. 2 increased from 0.1 wt% to 0.25 wt%. The addition of 0.25 wt% of f-MoS2 nanoplatelets showed the maximum improvement in compressive strength compared to epoxy nanocomposites loaded with higher wt% of f-MoS2 nanoplatelets (0.5 to 1.0 wt%). For example, the incorporation of 0.25 wt% of f-MoS2 nanoplatelets in epoxy leads to a 46% improvement in compressive strength of the nanocomposite in comparison to the neat one. The enhancement in the compressive properties could be ascribed to the high intrinsic strength and good dispersion of f-MoS2 nanoplatelets caused by organic CTAB moieties, which promote enhanced interaction with the epoxy network and reduce agglomeration. The effective concentration of f-MoS2 improves due to uniform dispersion of the filler owing to organic modification of its surface. Uniform dispersion gives rise to better load transfer from epoxy matrix to f-MoS2 nanoplatelets during the compressive loading. Nanocomposites with 1 wt% of f-MoS2 nanoplatelets show a reduced level of improvement with 26% enhancement in the compressive properties. This reduction could be ascribed to the reduced degree of distribution of f-MoS2 nanoplatelets at higher loading levels.l strain . The abs2 nanocomposites loaded with 0.1, 0.25, 0.5, and 1 wt% of filler, obtained by performing a three-point bend test on notched samples, are shown in 1/2. It is evident that the presence of f-MoS2 nanoplatelets significantly improves the fracture properties of the nanocomposites. The maximum improvement in the fracture properties was observed with an f-MoS2 nanoplatelets loading of 0.25 wt% as it shows KIC value of 1.52 MPa m1/2. This value corresponds to an improvement of 81% when compared to neat epoxy. The extremely high Young’s modulus of MoS2 nanoplatelets [2 modified epoxy nanocomposites. MoS2 nanosheets are intrinsically stiff and provide excellent barrier properties for crack propagation. The toughening effect is further improved by the enhanced compatibility of organically modified MoS2 nanoplatelets with the epoxy matrix. The long chains of CTAB cause hindrance between the layers due to its bulky nature, promoting exfoliation and avoiding the agglomeration of layers. CTAB modification also makes MoS2 layers more compatible with the epoxy matrix. This enhanced affinity of the MoS2 nanoplatelets leads to an even stronger interface which helps in better stress transfer from the epoxy matrix to f-MoS2 nanoplatelets during mechanical loading. The large surface area of MoS2 nanoplatelets enables interlocking of the MoS2 nanoplatelets with epoxy over a much larger interfacial area. The modification of the nanoplatelets with the organic molecules of CTAB helps realize the full potential of the MoS2 nanoplatelets as a toughening agent for epoxy. As the content of f-MoS2 nanoplatelets reaches 1 wt%, the degree of improvement dips to 56%. Thus, f-MoS2 nanoplatelets provide maximum enhancement in fracture properties at lower loading levels of 0.25 wt%. The decrease in toughening effect with loading beyond 0.25 wt% could be ascribed to the increased agglomeration of nanoplatelets in the epoxy at a higher concentration level. Agglomeration leads to a reduction in the interfacial area, thus limiting the interaction of the f-MoS2 nanoplatelets with the epoxy matrix. The presence of agglomerates also increases defects, which reduces the intrinsic cured properties of the epoxy composite. Further analysis of fractured samples obtained from the fracture toughness test was done by scanning electron microscopy.The fracture properties of neat epoxy and Epoxy/f-MoSlatelets , which i2 nanoplatelets in the epoxy matrix was analyzed using SEM. 2 nanocomposites of different compositions. The neat epoxy had a smoother and featureless surface (2 layers showed an irregular surface (2 nanoplatelets cause less agglomeration in the epoxy nanocomposites owing to the lower filler content. The improved dispersion of f-MoS2 nanoplatelets leads to a stronger interface between filler and the matrix, thus enhancing the nanocomposites’ mechanical properties. The enhanced interfacial characteristics of epoxy modified with 0.25 wt% of f-MoS2 may also be attributed to the thin structure of exfoliated nanoplatelets resulting in wrinkled morphology, thus enabling better interlocking between the reinforcing agent and the epoxy polymer chains. The micrographs show that the filler has excellent adhesion to the matrix as no pullout or loose nanoplatelets were observed. The enhanced compatibility of the f-MoS2 nanoplatelets could be ascribed to the presence of organic CTAB molecules on the surface of the MoS2 nanoplatelets. However, as the f-MoS2 content reached 1 wt%, it is observed that the distribution of the filler in the matrix becomes non-uniform (2 nanoplatelets and epoxy, reducing the efficiency of the filler as a reinforcing agent. The agglomerated MoS2 also act as a defect hindering the crosslinking of epoxy and hardener. The agglomeration does not significantly improve the mechanical properties of epoxy nanocomposites with 1 wt% of the filler.The homogeneous dispersion of the nano-reinforcements in the polymer composites provides a maximum enhancement in the mechanical properties of polymer composites. The dispersion state of f-MoS surface a, while  surface b–e. It c-uniform e. The ag2 nanocomposites are compared in 2 nanoplatelets improved the flexural properties of epoxy nanocomposites for the entire range of loading percentage, although the degree of improvement varied with the loading level. Like the trend observed in the compressive and the fracture properties, nanocomposites modified with 0.25 wt% of f-MoS2 nanoplatelets give rise to a maximum increase of 64% in flexural strength compared to the neat epoxy. The flexural strength of the Epoxy/f-MoS2-0.25 wt% corresponds to 143 MPa, which is significantly high compared to the flexural strength of 87 MPa observed for the neat epoxy. The results are consistent with the fracture and compressive properties of the nanocomposites. The improvement in flexural properties is minimal beyond 0.25 wt% of platelets. The least improvement is observed with 1 wt% of f-MoS2 nanoplatelets, which can be attributed to the poor dispersion of the nanoplatelets in the matrix. The results confirm that f-MoS2 nanoplatelets show better improvement in the flexural properties at low loading levels because of the better dispersion and stronger interface facilitated by the presence of CTAB molecules.The flexural properties of neat epoxy and Epoxy/f-MoS2 nanoplatelets were successfully modified using a noncovalent method for the enhanced fracture and thermal properties of tetrafunctional epoxy nanocomposites. Organic modification supports better compatibility, dispersion, and interaction of the MoS2 nanoplatelets with the epoxy matrix. f-MoS2 nanoplatelets were prepared by the one-pot sonication method. The inclusion of f-MoS2 nanoplatelets in relatively low amounts induced an excellent improvement in fracture, flexural, compression, and thermal properties. The remarkable improvement in the mechanical properties was attributed to better interfacial adhesion between f-MoS2 nanoplatelets and the epoxy matrix and a better distribution of the layers due to the presence of the bulky organic groups on the nanoplatelets surface. The loading of f-MoS2 nanoplatelets beyond 0.25 wt% leads to a relatively non-homogeneous distribution of the nanoplatelets in the epoxy matrix, causing a reduction in the extent of interaction and the interfacial area between the filler and the matrix. This work demonstrates the potential of organically modified MoS2 nanosheets as a toughening agent for tetrafunctional epoxy nanocomposites.In conclusion, 2D MoS