Col2+ cells. We found that β-catenin-TCF7 activated the transcription of CCL2, a known critical factor in osteoarthritic pain. Using a lumbar spine instability model, we showed that a β-catenin inhibitor relieved low back pain. Our study indicates that β-catenin plays a critical role in maintaining spine tissue homeostasis, its abnormal upregulation leads to severe spinal degeneration, and its targeting could be an avenue to treat this condition.Spine degeneration is an aging-related disease, but its molecular mechanisms remain unknown, although elevated β-catenin signaling has been reported to be involved in intervertebral disc degeneration. Here, we determined the role of β-catenin signaling in spinal degeneration and in the homeostasis of the functional spinal unit (FSU), which includes the intervertebral disc, vertebra and facet joint and is the smallest physiological motion unit of the spine. We showed that pain sensitivity in patients with spinal degeneration is highly correlated with β-catenin protein levels. We then generated a mouse model of spinal degeneration by transgenic expression of constitutively active β-catenin in  The lumbar facet joints form posterolateral articulations connecting the vertebral arch of one vertebra to the arch of the adjacent vertebra.4 In patients with LBP, recurrent rotational strains lead to changes to the discs and paired facet joints, causing loss of disc height, osteophyte formation, and hypertrophy of the facets.6 Cumulative evidence suggests that changes in any part of the spine tissue could lead to corresponding changes in the other parts. For example, loss of structural integrity of the IVDs has been shown to result in concomitant degenerative changes in the facet joints.9 The reverse is also true. Pathological abnormalities in facet joints accelerate the degenerative process of the IVDs.12 These findings suggest that spinal degeneration is a disease affecting multiple tissues in the spine, including the IVDs, vertebra, and facet joints. These three tissues constitute the structural basis of the functional spinal unit (FSU), the smallest physiological motion unit of the spine. The degenerative changes in these tissues significantly contribute to the development of LBP, the major symptom leading to individuals seeking medical treatment. Therefore, spinal degeneration could be controlled by a common molecular mechanism. Although spinal degeneration often affects the entire FSU, few studies have investigated this disease in an integrative way. Using traditional approaches to analyze disc degeneration, facet joint OA or discogenic pain separately provides an incomplete understanding of how the degenerative process can affect the entire FSU. Therefore, we believe that if a common molecular mechanism can be defined, it will bring about a fundamental breakthrough for the understanding and treatment strategy for degenerative spine disease.Degenerative disc disease is an aging-related disease and is a leading cause of low back pain (LBP). It is marked by metabolic disturbances in the matrix of intervertebral discs (IVDs) and facet joints.13 Inhibition of β-catenin signaling leads to defects in postnatal cartilage development,14 and cartilage-specific activation or inhibition of β-catenin signaling showed that β-catenin regulates chondrocyte maturation, generation of ossification centers, and perichondrial bone formation during skeletal development.15 Abnormal inhibition or upregulation of β-catenin signaling in articular cartilage causes defects in cartilage development and osteoarthritis-like phenotypes.17 Upregulation of β-catenin signaling in IVD tissues also results in dysfunction of disc tissues.18 However, the role of β-catenin signaling in spinal degeneration has not been fully defined. In this study, we determined the role of β-catenin signaling in maintaining the homeostasis of the FSU and in the development of spinal degeneration. We generated transgenic mice that express constitutively active β-catenin in Col2-expressing cells in the spine and analyzed their spinal phenotype. We found multiple spinal degeneration-related phenotypes in these mice, including severe defects in disc tissues, high bone mass in vertebral bone adjacent to the disc, facet joint OA-like alterations and low back pain. We investigated the molecular signaling by which β-catenin activation leads to pain. Using a lumbar spine instability (LSI) model,19 we also demonstrated that a β-catenin inhibitor protected mice from disc degeneration and relieved pain sensation. Together, our findings provide novel insights into the mechanisms of spinal degeneration and low back pain, suggesting that elevated β-catenin signaling acts as a common molecular mechanism to induce defects in all three tissue components of the FSU and that targeting this pathway could be a potential therapeutic intervention for spinal degeneration disease.β-Catenin is the key mediator of the canonical Wnt signaling pathway and plays an important role in skeletal development.20 According to the visual analog scale (VAS), a psychometric pain response scale, 35 patients were recruited and divided into low VAS  and high VAS  groups. We then determined the correlation between β-catenin protein levels and pain experience in patients with spinal degeneration. We found that the patients with high β-catenin protein levels at the growth plate (GP) and endplate (EP) cartilage areas Col2ER , that express a constitutively active form of β-catenin in Col2-expressing cells by breeding β-catenin(ex3)flox/flox mice with Col2-CreER transgenic mice. We found that the size of 3-month-old β-cateninAct mice was smaller than that of their Cre− littermates  cells but not nucleus pulposus (NP) cells.22 To further analyze β-catenin activation in mice, we performed Safranin O/Fast Green staining and found loss of GP and EP cells and cell cluster formation in the GP and EP areas of β-cateninAct mice  scores, a semiquantitative measure of OA, and osteophyte formation in β-catenin activation mice compared to their Cre− littermates. Deletion of Runx2 in β-cateninAct; Runx2KO double mutant mice partially reversed the increase in OARSI scores seen in the β-cateninAct mice  and performed IF and qPCR to detect changes in the expression of neuronal markers. We found that the protein expression of PGP9.5 and TUJ1 and the mRNA expression of ls Figs. , S2a–f. ls Fig. S, suggestCcl2 and Ccr2 mRNA were increased in a dose-dependent manner in chondrocytes after cells were treated with BIO  and other chemokine genes  in a dose-dependent manner in C28/I2 cells treated with BIO  and found that iCRT14 did not affect the expression of Tcf7, Axin2, Dkk1, Ccl2, Ccl3, Ccl5, Ccr1, Ccr2, Ccr5, Cxcr1 and Cxcr2 in C28/I2 cells in a noninflammatory environment   Fig. S. In cont1β Fig. S. In addi1β Fig. S.Fig. 6β-19 Four weeks after surgery, LSI mice were treated with iCRT14 (50 mg·kg−1) or Celebrex (10 mg·kg−1) (an anti-inflammatory factor that acts as a positive control) for 6 weeks. We found that LSI caused LBP (as determined by a reduction in von Frey scores). Treatment with iCRT14  or treatment with Celebrex  significantly reversed LSI-induced mechanical allodynia at 10 and 13 weeks after surgery  to mediate its signaling to activate genes encoding matrix degradation enzymes. Our findings indicate that β-catenin plays a critical role in spinal degeneration and that activation of β-catenin signaling under pathological conditions leads to severe defects in multiple tissues within the FSU.In this study, we found that abnormal upregulation of β-catenin signaling in the spine leads to a degenerative phenotype in multiple tissues of the FSU, including IVD degeneration, vertebral sclerosis, facet joint OA, and low back pain. We investigated the molecular mechanisms involved in the degenerative processes and found that Runx2, MMP13 and CCL2 are key downstream mediators of β-catenin in 29 and circumferentially encloses the NP, which is a highly hydrated structure that is predominantly composed of aggrecan. It is not known whether degeneration of the IVD is initiated from defects in the NP tissue or from tissues outside the NP, such as GP and EP cartilage or the AF. However, as Col2-CreER mice, used in the current study, only target GP and EP cartilage and AF tissues, but not NP tissue,21 our findings suggest that IVD degeneration could be initiated from tissues outside the NP. Changes in NP tissue are secondary to alterations in GP and EP cartilage or AF tissues.The IVD is a complex structure that provides flexibility to the vertebral column and sustains the weight applied through the spine. The vertebrae are covered superiorly and inferiorly by the GP and EP cartilage, which allow for the intake of nutrients and the removal of waste products of the NP. The IVD is formed by the peripheral AF. The inner region of the AF is characterized by a transition from type I to type II collagen and increased proteoglycan content31; however, the source of pain and how nerve signaling is activated by local pain mediators have not been defined. In this study, we found that striking nerve ingrowth occurs after the loss of GP and EP cartilage tissues in β-catenin activation mice, indicating that nerve ingrowth at the region of the GP and EP cartilage could be a potential source for LBP. In addition, we also found that the size of the intervertebral foramina is reduced due to large amounts of osteophyte formation in surrounding tissues, which could repress the nerve tissue inside the intervertebral foramina, resulting in pain sensation. It is well known that β-catenin plays an important role in neuropathic pain.35 However, the role of β-catenin in LBP and related molecular mechanisms have not been determined. In the current study, we found that β-catenin interacts with TCF7 and directly regulates CCL2 gene transcription. CCL2 was shown to mediate OA-related pain in previous studies.36 In the current study, we demonstrated that CCL2 is a direct downstream target gene of β-catenin signaling, suggesting that CCL2 mediates LBP during the development of spinal degeneration.LBP has been recognized as the main symptom of spinal degeneration37 Similar findings were also reported by other studies showing a positive correlation between IVD degeneration and facet joint OA.13 However, clear evidence regarding changes in the entire FSU during spinal degeneration has not been thoroughly investigated. Consistent with these reports, in the current study, we clearly demonstrated that activation of β-catenin signaling in Col2-expressing cells in the spine leads to degenerative changes in all tissues of the FSU as well as increased pain sensation.In a magnetic resonance imaging (MRI) study evaluating the relationship between facet joint OA and IVD degeneration, the former was rarely found in the absence of the latter but tended to be most pronounced at spinal levels associated with advanced IVD degeneration.There are several limitations in the current study. 1) Although we clearly demonstrated that abnormal activation of β-catenin signaling leads to degenerative changes in all tissues in FSU, we do not know if this is always true for spinal degeneration caused by alterations in other signaling molecules. Further follow-up studies are needed. 2) Although we found a positive correlation between pain sensation and high β-catenin protein levels in disc and facet joint tissues in patients with spinal degeneration, this study needs to be further validated using large numbers of patient samples. Current enrolled patient numbers are relatively small, especially the facet joint samples.In summary, our findings indicate that there is a common signaling pathway controlling the degenerative process, which may represent an effective drug target for the treatment of spine degeneration and its associated pain.The objective of this study was to determine the role of β-catenin signaling in maintaining spine tissue homeostasis and in the development of spine degeneration.We first analyzed β-catenin protein levels by IHC and LBP by analyzing VAS scores in patients with spine degeneration. Correlations between β-catenin expression and VAS scores were assessed using Pearson’s rank correlation test.β-cateninAct) by breeding β-catenin(ex3)flox/flox mice with Col2-CreER transgenic mice. We analyzed the spine degeneration phenotype in 3-month-old β-catenin activation mice using micro-CT, histology, IHC and IF analyses and pain-related behavior tests. Changes in spontaneous activities of β-catenin activation mice were analyzed by distance moved, speed, locomotion, immobility, climbing and rearing using the Laboratory Animal Behavior Observation Registration and Analysis System . Mechanical allodynia was determined by the von Frey test. After behavior tests, all the mice were sacrificed, and lumbar 3–5 and dorsal root ganglion (DRG) tissues were collected. Cortical BMD, cortical thickness, trabecular thickness, and the height of lumbar 4 were quantified using micro-CT reconstructed 3D images. The expression of MMP13, Col-X, Adamts4 and Adamts5 was examined by IHC. The expression of PGP9.5 and TUJ1 was analyzed by IF in DRG tissues. The mRNA levels of TrpA1, Ngf, Calca, Scn9a, TNF-α and IL-1β were detected by qPCR in DRG tissues.We then generated β-catenin conditional activation mice  staining in β-cateninAct and Cre− mice. The expression of nerve cell markers PGP9.5 and TUJ1 and vascular markers CD31, CD34 and VEGF-A in GR/EP areas was detected and quantified.Five-week-old Twelve-week-old C57BL/6 (C57) mice underwent LSI surgery to mimic the process of spine degeneration. Sham surgery was used as a control. After surgery, all C57 mice were allowed to rest for 4 weeks following 6 weeks of iCRT14 (β-catenin inhibitor) treatment. Corn oil was used as a negative control, and celecoxib was used as a positive control to alleviate discogenic pain. After treatment, all mice were examined by mechanical allodynia (von Frey test) and spontaneous activity tests. IHC assays, including β-catenin, MMP13, Col-X, and Adamts5, were performed to evaluate changes in expression in IVD tissues.Axin2 and Dkk1) were detected by qPCR. CCL2 and CCR2 play a key role in pain signaling in OA.26 The expression of a series of chemokines, Ccl2, Ccl3, Ccl5, Ccr1, Ccr2, Ccr5, Cxcr1 and Cxcr2, was also detected by qPCR in C28/I2 cells after treatment with BIO or transfection of Tcf7. Western blotting and IF (CCL2) were performed to examine β-catenin/TCF7-induced CCL2 transcription in chondrocytes. ChIP assays were performed to determine TCF7 binding to the CCL2 promoter in 293 cells treated with IL-1β or transfected with the Tcf7 plasmid.The 293 cell line (HEK293) and C28/I2 cell line were used for in vitro molecular mechanism studies. The chemical compound BIO and TCF7 expression plasmid were used to activate β-catenin signaling in these cell lines. The β-catenin inhibitor iCRT14 was used to block β-catenin signaling in these cell lines. IL-1β was used to promote the expression of inflammation-related genes. To confirm that β-catenin signaling was successfully activated or inhibited, the downstream target genes of β-catenin signaling ;The patient was diagnosed with IVD degeneration based on imaging evidence;The patient had symptoms of LBP;The patient intended to undergo IVD surgery.The patient had autoimmune disease, diabetes, thyroid disease, a nervous system disorder and/or other diseases that may cause abnormal pain;The patient had used hormone drugs in the past three months;The patient currently had symptoms of infection;The patient had previously undergone major spinal surgery.The basic information for the participants is presented in Table β-cateninAct (β-catenin(ex3)Col2ER) mice were generated from Col2a1-CreER transgenic mice (C57BL/6 strain), as previously described,18 and β-catenin(ex3)flox/flox mice (129/Sv strain) were generated as originally reported by Harada et al.38 and used in our previous studies.18 Tamoxifen  was administered to 2-week-old mice by i.p. injection . Runx2flox/flox mice (C57BL/6 strain) were generated as previously described.39 Wild-type (C57BL/6) mice were purchased from GemPharmatech . Rosa26 reporter mice (129/Sv strain) were obtained from Jackson Laboratories .40 Genotyping was performed by PCR analysis of DNA isolated from mouse tails, and Cre− Littermates were used for all experiments.The animal protocol of this study was approved by the Ethics Committee of the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, and all experimental methods and procedures were carried out in accordance with the approved guidelines to comply with all relevant ethical regulations for animal testing and research (IACUC No.: SIAT-IACUC-210201-YYS-LK-A1538). Cre-specific primers: upper primer, 5′-ATCCGAAAAGAAAACGTTGA-3′; lower primer, 5′-ATCCAGGTTACGGATATAGT-3′; β-catenin (ex3). Flox-specific primers: upper primer, 5′-AGGGTACCTGAAGCTCAGCG-3′; lower primer, 5′-CAGTGGCTGACAGCAGCTTT-3′.The following primers were used for genotyping: β-cateninAct mice were used for the detection of vascular invasion and nerve ingrowth. Twelve-week-old C57BL/6 mice were imported for LSI surgery. No criteria or exclusions were used in this study.In this study, male and female mice were kept in a specific pathogen-free facility (SPF) in individually ventilated cages, 5 mice per cage, and the animals were on a 12 h day/night cycle and housed at a controlled temperature of 22 °C. Caging, food, and water bottles were changed weekly. Five-week-old n = 7 per group). The randomization sequence was generated by computer. Surgeries were performed in a random order, with mice being taken from alterative cages to avoid potential confounders, such as the order of treatments and measurements and animal/cage locations. After being anesthetized with ketamine and xylazine, mice were operated on to perform resection of the 3rd to 5th lumbar (L3–5) spine processes along with the supraspinous and interspinous ligaments to induce instability of the lumbar spine, as previously described.19 Sham operations were performed by detachment of only the posterior paravertebral muscles from the L3–5 vertebrae. Operative fields were closed with 4-0 nylon sutures.Twelve-week-old C57BL/6 mice were randomly divided into the sham group, the LSI group, the LSI + celecoxib group and the LSI + iCRT14 group .The assessment of long-term movements of mice was conducted with the Laboratory Animal Behavior Observation Registration and Analysis System (LABORAS)  according to protocols, as previously described.41 Before a von Frey hind paw test, the mice were allowed to acclimate on a wire mesh grid for 15 min. If the mice exhibited any nocifensive behavior, including brisk paw withdrawal, licking, or shaking of the paw, either during the application of the stimulus or immediately after filament removal, it was considered a positive response. A set of von Frey filaments was used to poke from below the hind paw to calculate the 50% force withdrawal threshold using an iterative approach. The tests were performed in a blinded manner such that the investigator was not aware of the identification of the animals, as well as the study groups.To determine changes in mechanical allodynia, von Frey tests were performed using a calibrated set of von Frey filaments , as previously described.th lumbar vertebra and disc height were analyzed by CTAn V1.15.4 software . For long bone analysis, we used a NEMO Micro-CT scanner  with a 90 kV source and 70 μA current for formalin-fixed mouse legs with a resolution of 10 μm. The ROI of the distal femur was reconstructed, and its bone parameters were quantitatively analyzed.We used a Scanco µCT35 scanner  with a 55 kVp source and 200 μAmp current for formalin-fixed mouse spines with a resolution of 6 μm. The scanned images from each group were evaluated at the same thresholds to allow 3-dimensional structural rendering of each sample. Regions of interest (ROI) of L3–5 lumbar spines were reconstructed, and their bone volumes were quantitatively assessed. Cortical BMD, cortical thickness, trabecular thickness (Tb.Th.), the height of the 4For histological staining, slides of mouse disc and facet joint  coronal sections cut 5 μm thick were stained with Safranin O/Fast Green and Alcian blue/Hematoxylin & Orange G (AB/H&OG) for morphologic analysis. The vascular invasion area, EP thickness and growth plate area were analyzed using the ImageJ program.For IHC staining, rehydrated coronal sections of discs and facet joints were heated at 95 °C in Antigen Unmasking Solution for 10 min and then sequentially treated with Endogenous Peroxidase Blocking Buffer , 0.5% Triton X-100, and an Avidin/Biotin Blocking Kit. After blocking with 10% normal goat serum for 1 h, sections were treated with primary antibodies, including anti-MMP13, anti-Col-X, anti-Adamts4, anti-Adamts5, anti-Runx2 and anti-β-catenin antibodies  were incubated with specific primary antibodies, including those against PGP9.5, Tuj1, CD31, VEGF-A, CD34 and CCL2  was used to induce the expression of inflammation-related genes in C28/I2 cells for 12 h. BIO  was used to treat C28/I2 cells for 12 h to activate β-catenin signaling and induce β-catenin target genes. 50 μmol·L−1 iCRT14  was used to treat C28/I2 cells for 12 h to inhibit β-catenin signaling. The TCF7 plasmid  was transfected into C28/I2 cells or 293 cells using Lipofectamine 3000 to overexpress TCF7. qPCR, Western blotting or ChIP assays were performed 48 h after transfection. The cell lines were tested for mycoplasma-free status before they were used.The human chondrocyte cell Line C28/I2  and the human embryonic kidney 293 cell line (HEK293)  were cultured according to the vendor’s instructions. C28/I2 cells were cultured in DMEM/F-12 medium , and HEK293 cells were cultured in DMEM . All media were supplemented with 10% fetal bovine serum at 37 °C under 5% CO−1 PMSF, 1 mmol·L−1 Na3VO4 and protease inhibitor cocktail . After separation, samples were electrotransferred onto PVDF membranes  probed with the antibodies listed in Table After cell culture and treatment, cells were homogenized and incubated in RIPA lysis and extraction buffer supplemented with 1 mmol·L18 Briefly, 293 cells were treated with IL-1β or transfected with TCF7 plasmid for 24 h. After harvesting and centrifugation, the cell pellet was cross-linked and isolated. The cross-linked cells were then lysed and digested by MNase for further immunoprecipitation (IP). A TCF7 antibody  was used for pull-down of proteins or DNA binding with TCF7. After the DNA recovery step, the enriched DNA was subjected to qPCR. The binding region of TCF7 to the CCL2 promoter was predicted by JASPAR, a database of transcription factor binding profiles (https://jaspar.genereg.net/).The ChIP assay was performed as previously described.−1 primers . DNAse I-treated total RNA was reverse transcribed using an RT reagent kit . The cDNA was amplified by PCR in a total volume of 20 μL reaction solution containing 10 pmol·L−1 body weight) and administered every 3 days for 6 weeks by i.p. injection. Injection of corn oil  was used as a negative control, and celecoxib  was used as a positive control. Celecoxib was also first dissolved in DMSO and then dissolved in corn oil (10 mg·kg−1 body weight) and delivered by i.p. injection on the same schedule as iCRT-14.After surgery, all mice were allowed to rest for 4 weeks and then treated with the β-catenin inhibitor iCRT14 for 6 weeks. iCRT-14 (S8704) was purchased from Selleck  and was first dissolved in DMSO and then further dissolved in corn oil 2/d2 to calculate sample numbers, where d = (u1 − u2)/sigma (sigma = SD). We assumed a probability of type I error, or alpha of 0.05, and a probability of type II error, or beta of 0.20, and thus obtained a value of Zalpha = 1.645, and Zbeta = 0.842, so n = 2(1.645 + 0.842)2/d2. Statistical analyses were conducted using Prism GraphPad Prim 8.0 software. All the data are expressed as the mean ± S.D., as indicated in the figure legends. Unpaired Student’s t test (for two groups), one-way ANOVA (for multiple groups with one variable factor) and two-way ANOVA (for multiple groups with two variable factors) were used followed by the Tukey‒Kramer test. Correlations were assessed using Pearson’s rank correlation test. All experiments were performed and analyzed using 3 individual samples unless otherwise mentioned. P < 0.05 was considered statistically significant and is denoted in the figures. The experiments were randomized, and the investigators were blinded to allocation during experiments and outcome assessment.The sample size for each experiment was determined based on our previous studySUPPLEMENTAL MATERIAL


C9orf72, SOD1 and TARDBP genes, the three main ALS genetic causes. We show that in all mutant MNs, light NF (NF-L) chains rapidly accumulate in MN soma, while the phosphorylated heavy/medium NF (pNF-M/H) chains pile up in axonal proximal regions of only C9orf72 and SOD1 MNs. Excitability abnormalities were also only observed in these latter MNs. We demonstrate that the integrity of the MN axonal initial segment (AIS), the region of action potential initiation and responsible for maintaining axonal integrity, is impaired in the presence of pNF-M/H accumulations in C9orf72 and SOD1 MNs. We establish a strong correlation between these pNF-M/H accumulations, an AIS distal shift, increased axonal calibers and modified repartition of sodium channels. The results expand our understanding of how NF accumulation could dysregulate components of the axonal cytoskeleton and disrupt MN homeostasis. With recent cumulative evidence that AIS alterations are implicated in different brain diseases, preserving AIS integrity could have important therapeutic implications for ALS.Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease in adults with no curative treatment. Neurofilament (NF) level in patient’ fluids have recently emerged as the prime biomarker of ALS disease progression, while NF accumulation in MNs of patients is the oldest and one of the best pathological hallmarks. However, the way NF accumulations could lead to MN degeneration remains unknown. To assess NF accumulations and study the impact on MNs, we compared MNs derived from induced pluripotent stem cells (iPSC) of patients carrying mutations in The online version contains supplementary material available at 10.1007/s00018-023-04797-6. C9orf72 gene responsible for 30–46% of familial ALS (fALS), mutations in the superoxide dismutase 1 (SOD1) gene accounting for 12–20% of all fALS and mutations in the TARDBP gene (encoding for TDP-43) responsible for ~ 5% of fALS cases [Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease of the adult, leading to paralysis and death within 2–5 years after diagnosis. The most common genetic causes of ALS are hexanucleotide expansions in the LS cases . As fundG93A showed that NFs accumulated at the pre-symptomatic disease stage, in soma and proximal axonal regions [In ALS patients, NF deposits in degenerating MNs have been described since the 1980s. NF accumulations were first suggested by electron microscopy in proximal axon swellings in spinal cord tissues –13 and t regions , with pa regions . Strikin regions . While,  regions –26. Know regions  and that regions , 29, we C9orf72, SOD1 and TARDBP—as well as isogenic or age and sex-matched control iPSC clones. Our objective was to compare different ALS forms in the same experimental context to identify common alterations, with a specific focus on NF accumulations and AIS alterations. We therefore differentiated iPSC into spinal MNs and showed that the vast majority of produced MNs expressed markers of skeletal MNs able to innervate limb muscles, those primarily affected in ALS patients. Interestingly, our results showed that mutant C9orf72, SOD1 and TARDBP MNs accumulated NF-L in their soma and had fragmented NF-L-positive neurite networks compared to controls. Importantly, when pNF-M/H accumulated specifically in the AIS region of C9orf72 and SOD1 MNs, we could correlate this with strongly altered structural components of the AIS, leading to molecular and geometric disorganizations of the axonal proximal region. Taken together, our results highlight a novel pathway by which NF accumulations disrupt MN homeostasis.To achieve this, we generated human induced pluripotent stem cells (iPSC) from fibroblasts of patients carrying mutations in the three main genes responsible for ALS—ALS patients were enrolled in a brain donation program declared by the Ministry of Research and Universities, as requested by French laws. An explicit consent was signed by the patient himself or by the next of kin, in the name of the patient, in accordance with the French Bioethical Laws. Patients are described in the Supplementary Table 1. Three levels of spinal cord  were taken. Immunostainings with anti-pNF-H antibodies (SMI31) were performed after deparaffinization of 5 μm-thick sections by an automatic slide stainer (Benchmark XT Ventana staining system). Slides were pre-treated at 95 °C in CC1 (pH 8) proprietary retrieval buffer . Antibodies were targeted with a biotin-free detection system .Comité de Protection des Personnes Île de France VI”). Fibroblasts carrying the TARDBPG348C mutation were obtained from the Erasmus Hospital . The study was conducted in accordance with the Declaration of Helsinki and was approved by an independent ethics committee . Written informed consent was obtained from each patient. For control subjects, skin fibroblast cultures were obtained from the Centre de Ressources Biologiques (CRB) of Lyon (France). A statement of all biological samples was made according to French laws formulated by the Research Ministry.To establish a human fibroblast collection to generate iPSC, skin biopsies were proposed for French ALS patients, in collaboration with the French National Referral Center for ALS at the Pitié-Salpétriêre hospital  and the Pasteur Institute (project promotor) . The study was conducted in accordance with the Declaration of Helsinki and was approved by an independent ethics committee . For the patient with the G348C mutation in the TARDBP gene, reprogramming of fibroblasts was done with integrative retroviral vectors as previously described [Fibroblasts of a control subject (69 years old), of the patient carrying the N139D mutation in the escribed , except escribed . The conSOD1 and TARDBP mutations as well as analysis of hexanucleotide repeat expansions in the C9orf72 gene was performed on genomic DNA isolated from fibroblasts, iPSC clones and MNs, as previously described  [For characterization, iPSC cultured as embryoid bodies (EBs) for 10 days were plated on Geltrex and cultured for another 10 days in fibroblast medium  to induce spontaneous differentiation. Pluripotency and differentiation potential of iPSC and their EB derivatives were analyzed by immunostaining and PCR with reverse transcription (RT-PCR), as described previously  , 31. Whe Fig. 4) .N139D (clone SOD1-2) and TARDBPG348C (clone TARDBP-1) mutated genes, the CRISPR/Cas9 technology was used [To correct point mutations in iPSC carrying SOD1was used . Briefly5 cells/ml are suspended into a Neuronal Basic Medium (NBM) supplemented with SB431542 , LDN-193189  and CHIR99021 . NBM is composed of 1:1 DMEM/F-12/Neurobasal™, N2 supplement, B27™ supplement minus vitamin A  and l-ascorbic acid . EBs form within 1 day, and 2 days later the NBM is supplemented with retinoic acid  and SAG smoothened ligand  until day 14. CHIR99021 is removed on day 2, while SB431542 and LDN-193189 are removed on day 4. BDNF and GDNF  are added from day 7. On day 9, DAPT  is added and EBs are dissociated the next day with Trypsin–EDTA (Thermo Fisher Scientific)  and fixed with 4% paraformaldehyde (PFA) (Euromedex) for 10 min at room temperature (RT). For ion channel staining, cells were fixed for 10 min with PFA 1% (RT) and 10 min in methanol at − 20 °C. Cells were then incubated in a blocking solution , 2% normal goat serum (NGS), 0.1% Triton X-00 for 1 h (RT). Primary antibodies were added, and cells were incubated either for 2 h at RT or overnight at 4 °C. Secondary antibodies were added for 1 h at RT in PBS supplemented with BSA and NGS. Antibodies are listed in Supplementary Tables 2 and 3. Nuclei were stained with Hoechst H33342 (Thermo Fisher Scientific). Slides were mounted with Fluoromount G (Southern Biotech).Whole-cell patch-clamp recordings were performed on iPSC-derived MNs, derived from SOD1-1, C9orf72-1, TARDBP-1 iPSC clones and control clones. Three days after pMNs dissociation and plating, neurons were incubated (90 min at 37 °C) with lentiviral vectors carrying the cDNA encoding the red fluorescent protein (RFP) under the control of the HB9 promoter . Lentivi2, 0.1 mM CaCl2, 4 mM ATP-Mg, 0.3 mM GTP, 10 mM Na phosphocreatine, 1 mM EGTA and 10 mM HEPES, adjusted to pH 7.2 with KOH (Sigma). The extracellular solution contained 140 mM NaCl, 3 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 10 mM HEPES adjusted to pH 7.3 with NaOH. Capacitance transients were cancelled out. Series resistance was typically between 5 and 7 MΩ and was compensated by at least 70% for voltage-clamp experiments. Only cells with an Rs ≤ 10 MΩ and a resting membrane potential (RMP) more hyperporlarized than − 35 mV were included in data analysis. Access resistance was monitored at the end of the experiment and cells were discarded if the series resistance changed by more than 20%. In current clamp mode, holding potentials were corrected for a calculated liquid junction potential of 15 mV. RMP was determined by averaging for 30 s of recording and afterward a holding current was used to clamp the resting membrane potential as close as possible to − 70 mV. Whole-cell recordings were performed on control and ALS groups of neurons in parallel on the same day. Electrophysiological recordings were carried out at room temperature (22–24 °C). Data were analyzed off-line using Clampfit 10 software (Molecular Devices) and OriginPro (Origin Lab Corporation). The first AP from the first trace with evoked AP was used to determine the delay, threshold, amplitude and half-width of AP. Firing frequency was obtained by measuring the maximum number of spikes during the current steps protocol (1 s).Recordings were performed using an Axopatch 200B amplifier and a Digidata 1440A Analog/Digital interface (Molecular Devices). Data were low-pass filtered at 2 kHz and digitized at a sampling rate of 50 kHz. Patch pipettes were pulled from thin wall borosilicate glass capillaries (Harvard Apparatus) on a Zeitz DMZ puller and had a resistance of 2–3 MΩ when filled with the intracellular solution, containing 10 mM KCl, 130 mM K-gluconate, 2 mM MgClOptical sectioning was obtained using an Imager.M2 equipped with AxioCAM MRm, Apotom.2 module, HXP 120C light source, objectives/NA: 20×/0.8; 40×/1.3; 63×/1.4. Confocal images were obtained with SP8 X WLL, DMI8 stand (Leica). All images were taken at the same laser intensity.Z stacks was used and analysis done with the Fiji software. MN cell bodies were circled by hand on β3-tubulin-labeled images and ROI registered. Second, intensity threshold was set up on NF random images of control MNs. Finally, positive area fraction per cell was calculated. For neurite quantification, we measured on MIP of Z stacks the percentage of the total area stained by β3-tubulin, allowing us to compare percentages of β3-tubulin positive area occupied by control neurite networks to those occupied by mutant neurite networks. On each image, MN cell bodies were circled and deleted to measure only the neurite networks.The maximum intensity projection of Z stacks acquired at 63 × was used to measure neuron characteristics with a semi-automatic Fiji Macro. Only cells with a single soma-derived AIS were included in the quantitative analysis. To measure the length or area of the AIS and of the region between the soma and the AIS (the GAP), we used the methods described in Senol et al. . The MAPTotal RNA was extracted using the RNeasy Plus Micro kit (Qiagen) according to the manufacturer’s protocol. RNA concentrations were determined with high-sensitivity RNA ScreenTape on TapeStation (Agilent). cDNA librairies were prepared using KAPA mRNA Hyper Prep kit (Roche) according to the manufacturer’s protocol (150 ng RNA input). Samples were run with an ILLUMINA short-reads sequencer to obtain an average coverage of 30 million of reads per sample . Library preparations and sequencing were performed by the ICM iGeneSeq core facility.www.genosplice.com). Sequencing, data quality, reads repartition  and insert size estimation were performed using FastQC, Picard-Tools, Samtools and rseqc. Reads were mapped using STARv2.4.0 [p values ≤ 0.05 (P-Adj) after false discovery rate correction and fold changes ≥ 1.5. First, we compared controls to each ALS form individually  and calculated differential of gene expressions between yMNs and mMNs. But due to the limited number of patients in the study, we also pooled all samples at each time point to compare all ALS samples (from the 4 patients) to all control samples to identify genes and/or pathways that could be commonly deregulated in the three ALS forms.RNA-seq data analyses were performed by GenoSplice technology . Quantitative reverse transcription PCR was then performed in a Roche LightCycler480 using 100 ng of equivalent cDNA and the LightCycler probes master according to Roche procedures. Primers and Taqman probes were from Thermo Fisher Scientific: Hs00544069_m1 and Hs02786624-g1 for CHL1 and GAPDH, respectively.A minimum of three independent experiments, considered as three differentiation batches from iPSC, was always done. Statistical analysis was performed with GraphPad Prism 8. D’Agostino and Pearson normality test was used to assess Gaussian distribution for large data. For data passing the normality test, one-way ANOVA with Tukey’s multiple comparisons test was performed. For data not passing the normality test, Mann–Whitney test or Kruskal–Wallis with Dunn’s multiple comparisons test were used. When the normality test was not possible, a Gaussian distribution was assumed.SOD1 (SOD1N139D), C9orf72  and TARDBP (TARDBPG348C) genes. Four non-related age- and sex-matched control iPSC clones were included in our analysis (Supplementary Table 4), as well as SOD1 and TARDBP iPSC isogenic controls generated with the CRISPR–Cas9 technology (SOD1-ISO and TARDBP-ISO) (see Methods). iPSC clones were fully characterized at the molecular and functional levels . Genome integrity was checked for each iPSC clone .iPSC clones were generated from four patients carrying mutations in Each iPSC clone was differentiated into spinal MNs within 17 days , whereas markers of interneuron progenitors were not or barely detected   [ISLET1 and HB9 transcription factors  and markers of ISLET1-positive interneurons (expressing TLX3 and BRN3A) [ACHE) , the enzyme known to produce glutamate. This result agrees with previous work showing that a single MN can release both acetylcholine and glutamate at the mixed MN and Renshaw cell synapse [GFAP, ALDHL1, S100β, EAAT1), oligodendrocytes  and microglia  , was detected in all control and ALS MNs  and with time in culture these accumulations increased significantly in all 3 mutant ALS forms compared to controls  and pNF-M/H, to identify MAP2-negative axons, the AIS and NFs, respectively. Results showed that the pNF-M/H intensity staining was higher in proximal axons where the AIS-specific AnkG staining was also localized Fig. a, b. IntSoma areas, without pNF-M/H accumulations, were similar in size between control and mutant SOD1, C9orf72 and TARDBP MNs  channel repartitions along the axonal proximal region of MNs. Indeed, Nav channels could be affected by pNF-M/H accumulations, as the AIS is highly enriched in these channels that are anchored to AnkG , 63. ImmOverall, our data show that the molecular organization and the geometry of the axonal proximal region were strongly modified in the presence of pNF-M/H accumulations, with a distal delocalization of the AIS, a GAP increase of Nav channels, as well as increased axonal calibers and axonal areas of the entire region GAP/AIS region in iPSCs-derived SOD1 and C9orf72 MNs Fig. i. In TARINa/IK density ratio significantly increased with time in SOD1 and C9orf72 MNs, while the ratio had a trend to decrease in TARDBP MNs  and AIS molecular and geometric organizations. Neither pNF-M/H accumulations nor AIS modifications were observed in TARDBP MNs. Moreover, at late time points, SOD1 and C9orf72 MNs progressively acquired excitability alterations. Taken together, these results suggest a strong correlation between pNF-M/H accumulations and AIS alterations. These results expand our understanding of how NF accumulations could dysregulate components of the axonal cytoskeleton, and in particular of the AIS, an axonal sub-compartment crucial for polarity and excitability of MNs.The RNA sequencing analysis we performed on iPSC-derived MNs allowed to characterize MN subtypes present in our cultures, to confirm that they were representative models of ALS vulnerable MNs. We showed that progenitors expressed markers of the pMN domain of the embryonic spinal cord  and thatTo our knowledge, our study is the first one showing ALS phenotypic defects in iPSC-derived MNs characterized by their specific expression of LMC and alpha MN markers. This is an important step forward in the field of ALS modeling with human iPSC, as generation and analysis of both affected and not affected MNs in the same culture could mask disease-specific phenotypic differences. The mechanisms underlying the different MN death susceptibilities in ALS remain not fully understood, but it is highly likely that the vulnerability of certain MNs to degeneration is related to anatomical specificities and their transcriptome profiles , 66. TheA hallmark of many neurodegenerative diseases is the presence of protein inclusions in post-mortem tissues of patients. Despite years of research there is still a debate on whether aggregates are protective or deleterious to MNs. In ALS iPSC-derived MN models, abnormal protein accumulations were rarely described , 68 and NFs abnormalities have been so far only described in few studies of iPSC-derived MNs from two SOD1 patients , two TARWe also show that pNF-M/H accumulated in axonal proximal region of SOD1 and C9orf72 MNs. As it is well known that NF-M and NF-H phosphorylation is crucial for NF stability , other cMoreover, we show that pNF-M/H accumulation is strongly correlated with several alterations in molecular and geometric organizations of both AH and AIS, suggesting that all the axonal trafficking, before and after the AIS, could be disturbed. Indeed, the AIS plays an essential role in the axo-dendritic polarity regulating protein mobility and vesicular trafficking between the soma and the axon, with both a surface and cytoplasmic diffusion barrier , 58, 96.G127X mouse model [The AIS is a highly structured axonal segment that has crucial roles in neuron homeostasis. An essential role of the AIS in neurons is that it is the unique axonal region where APs are generated and shaped before their propagation along the axon , 98. MNsse model , 105, anse model . HoweverAt the molecular level, we were interested to look for common deregulated genes between ALS forms and controls. When we analyzed each ALS form separately and compared differences between yMNs and mMNs, only few deregulated genes were identified and none was in common. However, when comparing all different ALS mMNs as a pool to control mMNs, we identified upregulation of the CHL1 gene. Interestingly, CHL1 encodes a protein able to bind and to recruit AnkG to the plasma membrane , 59. A h. showed that the frontotemporal dementia (FTD)-causing V337M mutation in MAPT impaired structural AIS plasticity, leading to excitability modifications in iPSC-derived neurons from FTD patients [Interestingly, during the past decade, several studies have assessed if AIS plasticity and alterations of its components could be implicated in neurodegenerative and psychiatric diseases, as well as in brain trauma , 109–111patients . Here, wpatients , and recpatients . WhetherSupplementary file1 (PDF 3223 KB)Supplementary file2 (XLSX 92 KB)Supplementary file3 (XLSX 65 KB)Supplementary file4 (DOCX 46 KB)Below is the link to the electronic supplementary material.


To elucidate the reinforcing mechanism, we fabricated and extensively analyzed composites with varying weight percentages of GrF. The GrF-based SMP epoxy composite exhibits a 57% increase in thermal conductivity, measuring 0.296 W mK−1 at 70 °C, due to the interconnected 3D graphene network within the matrix. Notably, this composite also demonstrates remarkable electrical conductivity, making it suitable for dual-triggering applications. The GrF-SMP epoxy composite achieves a maximum shape recovery ratio and a significant 23% improvement in the recovery rate, effectively addressing the issue of slow recovery associated with SMPs. We investigated the effect of switching temperatures on the shape recovery rate. We identified the optimal triggering temperature to initiate shape recovery for epoxy SMP and GrF-epoxy SMP as thermal energy equivalent to Tg + 20 °C. Additionally, we fabricated a bird-shaped composite using GrF reinforcement, which showcases self-healing capabilities through the crack opening and closure and serves as a tangible demonstration of the transformative potential of the composite. These GrF-epoxy SMP composites, responsive to stimuli, hold immense promise for diverse applications, such as mechanical systems, wearable sensors, morphing wings, foldable robots, and antennas.Shape memory polymer (SMP) epoxy composites have attracted significant attention due to their easy processing, lightweight nature, and ability to recover strain. However, their limited recovery rate and inferior mechanical properties have hindered their functional applications. This research explores the potential of three-dimensional (3D) graphene foam (GrF) as a highly efficient reinforcement for SMP epoxy composites. We demonstrated that the incorporation of a mere 0.13 wt.% GrF into mold-cast SMP epoxy leads to a 19% increase in the glass transition temperature (T The increasing need for smart devices for biomedical , electro2 bonded carbon atoms, is a good filler in polymers. Its remarkable electrical [Graphene, a two-dimensional (2D) monolayer of spectrical , thermalectrical , and mecectrical ,34 have ectrical . This isectrical ,37. The 3, which is highly desirable for lightweight reinforcement. High surface energy and large specific surface area (850 m2/g) [A few studies have examined graphene’s influence on the shape memory behavior of epoxy SMPs. Wang et al. revealed that the shape recovery ratio and shape memory H-EP recovery speed increased as the graphene content increased from 1 wt.% to 3 wt.% . Another50 m2/g)  of GrF c50 m2/g) . This woHigher thermal conductivity (0.26–1.7 W/m-K)  and elecg) and correlated with thermal properties. Furthermore, the GrF-reinforced epoxy SMP composite is shaped into a bird-like structure, mimicking natural biological designs to investigate the crack opening and closure behavior during simulated wing flapping. Utilizing these advanced composites can revolutionize the design of smart materials and structures, introducing possibilities for innovative technologies and significantly improve performance across various industries.Despite the progress in utilizing GrF to enhance the performance of epoxy-based SMPs, there remains a critical need for a comprehensive scientific understanding of the underlying mechanisms, particularly in the context of dual-triggering mechanisms involving heat and electricity. While adding GrF has demonstrated remarkable improvements in SMPs, such as increased thermal conductivity, enhanced mechanical strength, and superior shape memory effects, the specific interactions and structural changes responsible for these enhancements have not been fully elucidated. The primary objective of this study is to investigate the potential of GrF as an additive to shape memory epoxy, enabling the development of multifunctional structures, which can be thermally and electrically activated. To achieve this goal, epoxy SMP composites reinforced with varying weight percentages of GrF are fabricated using a mold-casting technique, forming different geometric shapes to exemplify their concept. To ensure the practical applicability of GrF-reinforced SMP composites as actuators, the samples underwent comprehensive analysis to assess the SME repeatability across multiple actuation cycles and temperatures, employing quantitative imaging techniques. The recovery rate behavior is also investigated near the glass transition temperature  (Mw = 340 g/mol) and neopentyl glycol diglycidyl ether (NGDE) (Mw = 216 g/mol) were obtained from Hexion Specialty Chemicals  and Sigma-Aldrich , respectively. Curing agent poly(propylene glycol)bis(2-aminopropyl) ether (Jeffamine D230) (Mw = 230 g/mol) was purchased from Huntsman . The epoxy SMP components were used as received. Three-dimensional macro-porous, freestanding GrF with a pore size of 580 µm and 1–2 mm thickness was obtained from Graphene Supermarket .EPON 826 was weighed into a ceramic cup and melted in an oven preset at 70 °C. This was followed by pouring the melted EPON 826 and a predetermined amount of Jeffamine D230 and NGDE into a cylindrical plastic container. The components were hand-mixed vigorously for 15 s. Freestanding GrF of 40 mm in length, 2 mm in width, and 1.2 mm in thickness was laid in an aluminum pan. The epoxy SMP mixture was poured into an aluminum pan, infiltrating the GrF. It is important to acknowledge that GrF is a delicate material, and cutting it to the desired shape and size presents significant challenges, as the foam can easily disintegrate under applied pressure. Therefore, the wt.%. of GrF varies depending on the shape and size of the samples required for specific tests. The GrF-epoxy SMP sample was cured at 100 °C for 1.5 h and post-cured at 130 °C for 1 h. Neat epoxy SMP samples were prepared similarly by casting as a control sample. After the curing process, the GrF-epoxy samples were demolded and prepared into different shapes for shape recovery testing. As the samples are cut into different shapes for various tests, the wt.% of GrF in the SMP matrix varies. However, the morphology of GrF remains the same throughout, which is the prime determinant for thermal and electrical conduction.−1 to obtain the chemical signature.A JEOL JSM-6330F field emission scanning electron microscope  was used to characterize the microstructure and interface of GrF-epoxy SMP composite. At an operating voltage of 20 kV and a working distance of 15 mm, the SEM imaged the macro-porous structure of GrF and the fracture surfaces of the composite specimen. Micro-Raman spectroscopy was conducted using a Spectra-Physics  equipped with Ti-sapphire crystal (514 nm) as the target. The Spectra-Physics also consists of a detector with 4:1 cm spectral resolution from Kaiser Optical Systems, Inc. , with a laser power of 18 mW, and a spot size of 2 μm. A micro-Raman spectroscopy study was conducted on composites to examine the graphene sheets’ interaction with SMP epoxy chain molecules. The FTIR spectra of SMP epoxy-based composites were recorded using a 4000-type spectrophotometer  from 4000 to 400 cmg values of epoxy SMP and GrF-epoxy SMP composites were determined from the curves’ inflection temperatures. Thermal diffusivities and specific heat capacities of the epoxy SMP and GrF-epoxy SMP composites were measured by the flash method using NETZSCH LFA 467 HT HyperFlash  within the temperature range of 25 to 70 °C. A circular sample of 11 mm diameter and 3 mm thickness was used for thermal diffusivity measurements. The GrF-epoxy SMP, when cut to the dimension mentioned above, had 0.13 wt.%. of GrF. Thermal conductivity was calculated by measuring the diffusivities, specific heats, and bulk densities of at least 5 samples.SDT Q600  was used to obtain a glass transition temperature of neat SMP epoxy and GrF-SMP composite. The specimens were heated to 200 °C at a heating rate of 100 °C/min in the argon environment. The Tg + 28 °C for 5 s. The GrF-epoxy SMP had 0.75 wt.% of GrF. After removing it from the hot plate, the sample was manually deformed into a temporary ‘U’ shape with a radius of 3 mm in its rubbery state. Shape retention was allowed by promptly dipping the sample in a cold-water bath (20 °C) to lock the elastic deformation energy while maintaining the external constraint. The deformed, retained ‘U’ shape was ‘thermally’ actuated for shape recovery by placing it on the hot plate and using a hot air gun. The shape recovery was recorded using a digital camera. The images were extracted from the recorded video at different time intervals  and analyzed in terms of the change in the angle to quantify shape recovery.Shape memory recovery measurements were performed using (i) thermal and (ii) electrical actuation methods. Rectangular samples of epoxy SMP and GrF-epoxy SMP measuring 50 × 3 × 1 mm were placed on a hot plate at Tt-test method.For electrical stimulation, GrF was connected to a platinum wire (Pt) of 0.1 mm diameter  using a conductive Pelco colloidal silver paste . The silver paste’s addition between Pt wire and GrF allows contact resistance to be minimized during the current-induced heating of GrF. The silver paste was cured at 100 °C for 30 min. SMP epoxy was poured into GrF and cured as described in 2 carbon–carbon bonds’ in-plane vibrations (G band), out-of-plane vibrations due to structural defects (D band), and second disorder band (2D band). The D peak’s significant low intensity compared with the G peak indicates that the graphene sheets are primarily defect-free [−1, respectively. However, impregnation of GrF with epoxy SMP shifted the D and 2D peaks to the right with an assigned value of 1343.24 and 2709.32 cm−1, respectively. This implies the physical interaction of the backbone chain of epoxy SMP with a network of graphene sheets. Such interaction may result in bond length adjustment of the epoxy SMP molecules.A micro-Raman spectroscopy study was conducted on GrF-epoxy composites to examine graphene sheets’ interaction with epoxy SMP chain molecules. ect-free ,50,51,52−1, attributed to stretching and bending vibrations from O-H, C-H, N-H, and C-O, respectively [−1, C-H at 2970 cm−1, and O-H at 3348 cm−1 [ectively . The GrF348 cm−1 . The GrF348 cm−1 . Thus, sg of the epoxy SMP is measured at 42 °C. Adding GrF filler to the epoxy SMP composite, specifically at 0.13 wt.%, increases the Tg to 50 °C, indicating a 19% improvement. GrF has a high aspect ratio and large surface area, providing numerous opportunities for strong interfacial interactions with the epoxy matrix. The functional groups on the surface of GrF can form hydrogen bonds with the epoxy resin, as shown in g. Moreover, the 3D interconnected framework provided by GrF within the epoxy SMP matrix promotes the entanglement of terminal hydroxyl groups in the side chains of epoxy SMP. This entanglement immobilizes the surrounding chains, preventing cross-linking and leading to a higher Tg. The enhanced physical interaction between the epoxy SMP’s terminal groups and the 3D graphene sheets network causes the epoxy SMP chain to wrap around the branches and nodes of GrF. Consequently, moving confined epoxy SMP chains from a glassy to a rubbery state requires more thermal energy. The increased Tg is advantageous, as it prevents shape recovery degradation of epoxy SMP after thermal cycling and expands the temperature range for structural aerospace applications.g transition, indicating restricted chain conformations and reduced relaxation time distributions. This narrower transition temperature in the GrF-epoxy SMP composite promotes faster shape recovery rates. In comparison, a broader transition temperature in SMP epoxy can lead to incomplete shape fixing and slower recovery.Additionally, the addition of 0.13 wt.% GrF into SMP epoxy significantly reduces the width of the Tg), resulting in the elastic deformation of NGDE chains and energy storage within the EPON stiff phase.The shape recovery of epoxy SMP and GrF-epoxy SMP composites is evaluated through direct heating. Direct heating with a hot plate is a widely recognized method for thermally activating SMP epoxy, while a heat gun can also be used. The shape memory behavior of SMP epoxy under direct heating is governed by its dual-component mechanism. g (T3 < Tg), and the stress is removed, causing the NGDE polymer segments to cool and freeze. This freezing allows the SMP epoxy to retain its temporary shape. The shape memory effect (SME) is triggered when T3 > Tg, where reheating reactivates the frozen micro-Brownian motion, fully recovering the distorted polymer molecules. The SMP epoxy is cooled below TThe SMP epoxy samples’ temporal gradient is qualitatively assessed by subjecting them to direct heating via the hot plate. The SMP epoxy composite exhibits a significantly higher temporal gradient than SMP epoxy. The temporal gradient of the SMP epoxy samples is quantified in terms of their recovery angle, as determined by the shape recovery ratio equation (Equation (1)). The recovery angle can be expressed as follows:g. Based on The EPON phase of epoxy SMP plays a crucial role in its shape recovery, providing the necessary energy for the material to return to its permanent shape. The covalent interactions between the amine and epoxide groups in the EPON segment contribute to epoxy SMP’s high shape recovery rate of 97.35%. However, adding GrF to epoxy SMP enhances the shape recovery performance, achieving 100% shape recovery in the composite. The GrF framework, composed of high-modulus graphene sheets, adds stiffness to the net points within the epoxy SMP. The uniform dispersion of GrF throughout the SMP matrix enables the distribution of stiffness, thanks to strong interfacial interactions between the graphene sheets and epoxy SMP. The attractive van der Waals forces between the graphene layers also contribute to the energy storage capacity of the composite, allowing for maximum shape recovery. The combined influence of the EPON net points and the stiffness and energy storage of GrF enables 100% shape recovery in the GrF-epoxy SMP composite.g and temperatures around the Tg region. They are an essential parameter in the thermal actuation of epoxy SMP and GrF-epoxy SMP composite.Switching temperatures for shape recovery refers to Tg + 20 °C. This suggests that supplying thermal energy at Tg + 20 °C ensures sufficient heat distribution to initiate shape recovery in both epoxy SMP and GrF-epoxy SMP composite, making Tg + 20°C the optimal triggering temperature for shape recovery.g of epoxy SMP composite. Additionally, the T-M cycling process influences the Tg behavior of the GrF-epoxy SMP composite, as revealed in g. However, the 47th T-M cycle exhibits a significant increase in Tg from 50 °C to 58 °C, corresponding to a 16% Tg increase in the graphene-foam-epoxy SMP composite. The constant Tg observed during the initial T-M cycles may be attributed to the constrained entanglement of GrF with the side chains of epoxy SMP. As the number of T-M cycles increases, the physical interaction between GrF and the backbone chain of epoxy SMP intensifies, resulting in the confinement of the backbone chain’s internal rotation. This confinement leads to a 16% Tg increase in the GrF-epoxy SMP composite during the 47th T-M cycle. Consequently, T-M cycling provides a means to tailor the Tg of the graphene-foam-epoxy SMP composite.A direct thermal source for the actuation of shape change in epoxy SMP limits its practical applications. The induction of heat through electricity (indirect heating) as an alternative trigger method in epoxy SMP is essential. Graphene as a carbon-based filler has been considered to trigger epoxy SMP electrically . HoweverIncreasing the applied current from 200 mA to 400 mA intensifies the heat radiation through the GrF, emphasizing its role as an efficient resistive heating element in the epoxy SMP. This demonstrates that GrF effectively conducts current-induced heat into the epoxy SMP, making it a suitable conductive filler for triggering shape memory behavior. A 90% shape recovery is achieved with a maximum applied current of 400 mA, which can be attributed to the interfacial thermal resistance (ITR) between the GrF and epoxy SMP. A simple thermal equation (Equation (2)) can be used to determine the ITR between GrF and epoxy SMP, as shown below :(2)R=∆TADetermining parameter R, which represents the resistance at the interface, plays a crucial role in controlling the heat transport across the GrF–epoxy SMP interface. This determination is influenced by factors such as the temperature drop (∆T) across the interface, the cross-sectional area (A), and the total heat transfer (Q) across the interface. The ITR can lead to phonon vibrational mismatch and scattering at the GrF–epoxy SMP interface during phonon transfer. This implies that the nano-scale contact area between the GrF and epoxy SMP is responsible for the shape recovery observed in electrically actuated GrF-epoxy SMP composites.Epoxy SMP and its composites possess remarkable structural versatility, making them highly sought-after smart polymers in engineering applications. For instance, epoxy SMP and GrF-epoxy SMP composites were engineered into S, L, inverted U, and accordion shapes. By utilizing hot water or hot air, these shapes could be temporarily deformed and triggered back to their original shape see . The accg of 70 °C. When cooled to room temperature, the wings retain their deformed shape. To trigger the recovery of the wings to their original shape, hot water at 70 °C is used as a stimulus. Furthermore, heat-induced dynamic factors cause adjustments in the chain conformations of the EPON phase around the crack region of the GrF-epoxy SMP bird wing. These conformation adjustments unlock energy stored within the EPON phase, contributing to recovering the wing’s original shape. Additionally, the release of stored energy within the GrF aids in closing the cracks in the GrF-epoxy SMP bird wing. The concept demonstrated by the GrF-epoxy SMP bird highlights the potential of GrF-epoxy SMP composites in designing morphable aircraft wings. These composites can serve as multifunctional hierarchical materials in aircraft components, particularly for morphing wings  and deicg. Furthermore, the GrF-epoxy SMP composite exhibited remarkable electrical conductivity, enabling dual-triggering capabilities. A 90% shape recovery is achieved with a maximum applied current of 400 mA due to the interfacial thermal resistance (ITR) between the GrF and epoxy SMP, visualized by thermal imaging. The design and programming of biomimetic bird-shaped composites demonstrated a faster recovery time and crack opening and closure behavior after multiple thermo-mechanical cycles. This behavior highlights the potential of the GrF-epoxy SMP composite as a self-healing material. The GrF-based SMP epoxy composites presented in this research offers promising opportunities for various applications, including mechanical systems, wearable sensors, morphing wings, foldable robots, and antennas. These composites, responsive to stimuli and featuring enhanced properties, have the potential to revolutionize the field of smart polymeric materials and inspire further advancements in the design of functional structures.The findings of this study contribute to addressing the limitations of slow recovery due to inferior thermal properties associated with SMPs. The incorporation of GrF within the epoxy SMP matrix facilitated enhanced heat conduction through its interconnected 3D network, leading to a significant 57% increase in thermal conductivity. As a result, the GrF-epoxy SMP composite exhibited a 23% faster recovery rate than the pure SMP epoxy. The GrF-epoxy SMP composites exhibited enhanced shape recovery behavior, with shape recovery of ~97% for thermally triggered samples. The study also established that as the number of thermo-mechanical cycles increases, the physical interaction between GrF and the backbone chain of epoxy SMP intensifies, resulting in a 16% increase in the TAgarwal, A., Thomas, T., Idowu, A., and Boesl, B., Florida International University FIU, 2021. Shape memory polymer inks and methods of printing the same. U.S. Patent 10,906,238.


There was an error in the original publication . In the A correction has been made to Discussion, Paragraph 5:CORR measures the similarity between two signals. CORR ML-AP and CORR AP-V showed decreased values during the COG condition and similar values for POST and PRE conditions. Decreased CORR means the signals were less coupled during the COG condition but they returned to baseline during the POST condition. KURT is a statistical metric that quantifies how spread out signal amplitudes are from the mean. KURT in the AP direction was significantly higher during the COG condition compared to the PRE condition. Higher values mean more peaked distributions (fewer outliers) and indicate less variable sway. LZ measures the predictability of the signal, and higher values indicate less predictable, more complicated signals [25]. LZ in the V direction was significantly higher during COG condition with a return to baseline during the POST condition, pointing to more complex postural control while under cognitive load.A correction has been made to Discussion, Paragraph 7:We are unable to determine whether the changes we observe represent maladaptive effects on balance control  or other adaptive strategies. For example, higher complexity and randomness in the signal may reflect better online adjustments, allowing the individual to adapt to perturbations more easily. LZ V points to higher complexity, ENTR ML and AP point to higher local regularity, and WE ML points to higher global randomness. Different explanations for changes in postural control performance in older adults, cognitive task difficulty, stiffening method and signal-to-noise ratio, may support our varied results.In the original publication , there wThe authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original article has been updated.


As a new and promising reinforcing filler, electrospun glass nanofibers (EGNFs) have attracted attention in the field of polymer composite materials. However, the reinforcing effectiveness of surface-modified EGNFs using different silane coupling agents in epoxy resin is still not quite clear. In this research, a series of silane coupling agents with increasing chain lengths in the order of methyl trimethoxysilane (MTMS), (3-aminopropyl) triethoxysilane (APTES), (3-glycidyloxypropyl) trimethoxysilane (GPTMS), and dual silane coupling agent APTES–GPTMS were employed to carry out surface treatment on the EGNFs. The pristine and silane functionalized EGNFs were then incorporated into epoxy resin as reinforcing fillers at low loading levels, i.e., 0.25 wt.%, 0.5 wt.%, and 1 wt.%, and the mechanical properties of the resultant epoxy nanocomposites, including strength, stiffness, ductility, and toughness, were evaluated. A commercial product of glass nanoparticles (GNPs) was used as a control to compare the reinforcing effectiveness of the EGNFs and the GNPs. This study revealed that the EGNFs could provide significant reinforcing and toughening effects at ultra-low loading (0.25 wt.%) in epoxy nanocomposite materials. Furthermore, surface modification of the EGNFs with silane coupling agents with long chain lengths, e.g., by using dual silane coupling agents, APTES–GPTMS, could enhance the interfacial bonding between the EGNFs and the epoxy matrix and further increase the mechanical performance of the EGNF-reinforced epoxy nanocomposite materials. Through this research, we realized epoxy nanocomposite materials with much-improved mechanical properties, i.e., 37%, 24%, 18%, 57% improvement in strength, stiffness, ductility, and toughness, respectively, with respect to those of the cured neat epoxy material with an ultra-low loading (0.25 wt.%) of APTES–GPTMS–EGNFs. Our research paves the road for developing lighter and stronger epoxy nanocomposite materials with EGNFs. In ications ,9,10,11.ications  and can,ications . EGNFs hications ,14. In aications ,16,17.Epoxy resins are a popular class of thermoset prepolymers, and they are extensively used as matrices in fiber-reinforced polymer composite materials, coatings, and adhesives in a wide range of industries, such as aerospace, construction, transportation, and electronics, due to their excellent physical properties after curing ,19,20. IIn this study, we addressed the abovementioned research gap and aimed to comprehend the role of grafted silane molecular chain length as a contributing factor in the mechanical performance of the resultant epoxy-based nanocomposite materials. We performed surface modification to the EGNFs using silane coupling agents with increasing chain lengths, including methyl trimethoxysilane (MTMS), (3-aminopropyl) triethoxysilane (APTES), (3-glycidyloxypropyl) trimethoxysilane (GPTMS), and dual silane APTES–GPTMS, and evaluated the performance of these surface-modified EGNFs in reinforcing epoxy resin, including the mechanical properties of strength, stiffness, ductility, and toughness at low loading levels (0.25–1 wt.%). A commercial product of glass nanoparticles (GNPs) was used as a control to compare the reinforcing effects of EGNFs and GNPs in the epoxy matrix. Our research findings provide in-depth information to understand the reinforcing effect of EGNFs in epoxy-based nanocomposite materials, as well as directions for further enhancing their reinforcing effect in future EGNF-reinforced high-performance epoxy nanocomposite materials.2 nanoparticles, average diameter 20 nm, and BET surface area 200 m2/g) were purchased from Sigma-Aldrich . Ethanol and anhydrous acetone were purchased from Fisher Scientific . All the chemicals were used as received without further purification.The epoxy resin (EPON 862) and corresponding curing agent of EPIKURE W were purchased from Miller Stephenson . Tetraethyl orthosilicate  was purchased from Acros Organics . Polyvinylpyrrolidone , N, N-dimethylformamide , dimethyl sulfoxide , methyl trimethoxysilane (MTMS), (3-aminopropyl) triethoxysilane (APTES), (3-glycidyloxypropyl) trimethoxysilane (GPTMS), glass nanoparticles  at 60 °C for 10 min. Then, the EGNF-acetone dispersion was mixed with the epoxy resin upon stirring at 600 rpm and 60 °C for 2 h to homogenously distribute the EGNFs in the epoxy resin and, simultaneously, to remove acetone. Subsequently, the epoxy resin with the dispersed EGNFs was further sonicated for another 10 min at 200 W to improve the distribution of the EGNFs in the epoxy matrix. Afterward, the curing agent (EPIKURE W) was added into the system at 26.4 wt.% of epoxy, and the mixture was further stirred at 600 rpm and 60 °C for 10 min to obtain a uniform mixture. The mixture was next degassed under a vacuum at 60 °C for 10 min to remove air bubbles. After being degassed, the epoxy resin system was poured into a 5 in. × 5 in. mold and cured at 350 °F for 4 h. Pristine and surface-modified GNPs were also used to fabricate the GNP-reinforced epoxy nanocomposites at loadings of 0.25 wt.%, 0.5 wt.%, 1 wt.%, 1.5 wt.%, and 2 wt.%, following the same procedure as described for the EGNFs, along with the neat epoxy (without any nanofillers), for comparison purpose.−1. Dynamic light scattering  Zetasizer Nano Range) was used to determine the zeta potential and the hydrodynamic diameter of all the EGNF and GNP samples. For each sample, specifically, the nanofiller was mixed in DI water with a weight ratio of 1:10,000 and sonicated for 5 min for uniform dispersion. 1 mL of liquid from the prepared mixture was then used for the DLS measurement. Dog-bone-shaped specimens were cut from the cured epoxy nanocomposite panels, using a water jet cutting machine -MACH 2). The tensile test of all the cured epoxy nanocomposite materials was carried out using a universal testing machine  3384) as per ASTM D638-Type IV, at room temperature. Five specimens of each sample were prepared and tested. The average measurement values and the standard deviations were calculated. The thermal stability test of the epoxy nanocomposites was performed by using a thermogravimetric analyzer ). The TGA analysis was carried out by heating a sample of ~10 mg, from room temperature to 800 °C, at a heat rate of 10 °C/min in a nitrogen environment, with a flow rate of 50 mL/min.A Zeiss  Auriga Crossbeam FIB field emission scanning electron microscope (FESEM) was used to examine morphology of the EGNFs and the GNPs, and fracture surfaces of the epoxy nanocomposites. Prior to SEM, all the samples were sputter coated with gold–palladium in a thickness of 7 nm to avoid charge accumulation. The average fiber diameter of the respective nanofiber sample was obtained by measuring the diameters of at least 30 randomly selected nanofibers in corresponding SEM images, using Image J software . A Varian  670 FTIR spectrometer was used to confirm the surface modification of the EGNFs and the GNPs with various silane coupling agents over the wavenumber range of 4000–500 cmThe EGNFs were collected in the form of non-woven mat and exhibited a good cylindrical fiber shape A. The diThe mechanical properties, including tensile strength, Young’s modulus, elongation at break, and work of fracture, of the epoxy nanocomposites reinforced by pristine EGNFs at loading levels of 0.25 wt.%, 0.5 wt.%, and 1 wt.%, respectively, were evaluated by tensile test. For comparison, the counterparts with the pristine GNPs at loading levels of 0.25 wt.%, 0.5 wt.%, 1 wt.%, 1.5 wt.% and 2 wt.%, respectively, were also investigated. The neat epoxy resin with only the hardener was cured and tested as a control.Tensile strength indicates the maximum stress that a material can withstand prior to failure. The inclusion of nanofillers, both EGNFs and GNPs, resulted in significant improvements in the strength of epoxy nanocomposites, except at 1 wt.% of the EGNFs A. Among 2. Compared to the modulus of the neat epoxy (2.92 GPa), the moduli of the GNP-reinforced epoxy nanocomposites were observed in the range of 3.10–3.22 GPa (an improvement of up to 10%), while the moduli of the EGNF-reinforced epoxy nanocomposites were observed in the range of 3.27–3.39 GPa (an improvement of up to 16%).Young’s modulus indicates the stiffness of a material. Incorporation of the glass nanofillers showed a positive effect on stiffness of the resultant epoxy nanocomposites B. All thElongation at break is an important parameter for depicting the rupture behavior of a composite material, and it denotes the ductility of the material to undergo significant deformation before its failure . The nea3, Work of fracture (WOF) is a parameter that characterizes the toughness of a material, and it denotes the energy absorbed by the specimen before its failure. The WOF is achieved by the area under the tensile stress–strain curve. This is a direct and efficient method for assessing the static toughness of materials, and it has gained widespread acceptance among researchers in the field of polymer composites ,25. CompOverall, the EGNF-reinforced epoxy nanocomposite at 0.25 wt.% loading and the GNP-reinforced epoxy nanocomposite at 1 wt.% loading demonstrated the best comprehensive mechanical properties in the respective group. In particular, the EGNF-reinforced epoxy nanocomposite at an ultra-low loading level (0.25 wt.%) not only outperformed the GNP counterpart and the neat epoxy for all of the mechanical properties, but also outperformed all of the GNP-reinforced epoxy nanocomposites in strength and stiffness, indicating the great potential of EGNFs in reinforcing epoxy-based nanocomposite materials. However, EGNFs have demonstrated much worse agglomeration than GNPs at higher loading, which might be due to fiber entanglement, and need to be further studied in future research.In order to study the effect of the surface-grafted silane chain length of EGNFs after silane treatment on the mechanical performance of the resultant epoxy nanocomposite, four types of silane coupling agents with increasing chain lengths, including MTMS, APTES, GPTMS, and APTES–GPTMS, were employed, respectively, to carry out the surface treatment of the EGNFs. According to the reaction mechanisms, as demonstrated in 3, –CH2–, and –CH < peaks in the range of 2800 cm−1–3000 cm−1, while the pristine EGNFs did not show any peaks in that range, indicating the successful graft of silane molecules on the surface of the EGNFs.FTIR was used to characterize the surface-modified EGNFs to confirm the graft of various silane molecules on the nanofiber surface. The FTIR spectra of the pristine EGNFs, MTMS–EGNFs, APTES–EGNFs, GPTMS–EGNFs, and APTES–GPTMS–EGNFs are compared in To investigate the physical stability of the silane-treated EGNFs, DLS was used to characterize the zeta potential and the average size of the surface-modified EGNFs . SurfaceIn DLS measurement, the particle size of sample is not measured directly, but is based on the movement of the particles. The obtained particle size is called the hydrodynamic diameter, which refers to the size of the smooth and spherical particles that diffuse at the same speed as the sample particles. EGNFs can be treated as nanorods  and theiIn terms of physical stability, as indicated by a higher absolute value of the zeta potential, as well as a smaller average size, the dual silane treatment involving APTES and GPTMS exhibited the most favorable overall outcome for the surface-modified EGNFs and GNPs. Grafting longer silane chains on the surfaces of EGNFs and GNPs tends to improve their stability with less agglomeration, which could be beneficial in further improving the mechanical properties of the corresponding EGNF- and GNP-reinforced epoxy nanocomposites.Based on the overall good comprehensive mechanical properties, 0.25 wt.% loading was used to study the effect of surface-modified EGNFs from silane treatment on the mechanical properties of the surface-modified EGNF-reinforced epoxy nanocomposites. For a similar reason, 1 wt.% loading was used to study the surface-modified GNP-reinforced epoxy nanocomposites, for comparison.3, which were improvements of 44% and 110%, respectively, with respect to those of the neat epoxy.We observed that, in most cases, the surface modification of EGNFs and GNPs with silane coupling agents showed significant improvement in all of the mechanical properties of the resultant epoxy nanocomposites with respect to those of the respective pristine EGNF and GNP counterparts . Among aThe mechanical property improvement of the epoxy nanocomposites from the integration of surface-modified EGNFs (or GNPs) can first be attributed to the interaction between the surface-modified EGNFs  and the epoxy molecules (the matrix). MTMS–EGNFs have the shortest grafted molecular chain length on their surface and, thus, the weakest entanglement with surrounding epoxy molecules. APTES–GPTMS-EGNFs have the longest grafted molecular chain length on their surface and, thus, the strongest entanglement with surrounding epoxy molecules. APTES and GPTMS have molecular chain lengths between those of MTMS and APTES–GPTMS, while GPTMS possesses a slightly longer chain length than APTES. From the point of view of chain length and entanglement, GPTMS should outperform APTES in terms of reinforcing effect. However, APTES and GPTMS both contain reactive end groups, which could have chemical reactions with the epoxy molecules during the curing process. The amine functional groups of APTES could react with the epoxide functional groups of the epoxy resin during the mixing/curing process, while the epoxide functional groups of GPTMS could react with the epoxy curing agent in the process of curing, both of which complicate the APTES and GPTMS chain length effects. Overall, GPTMS and APTES showed close reinforcing effects on the mechanical properties of the surface-modified EGNF- (or GNP-) reinforced epoxy nanocomposites, while GPTMS demonstrated greater contribution to the strength of the resultant epoxy nanocomposites and APTES demonstrated greater contribution to the stiffness and toughness of the resultant epoxy nanocomposites.Furthermore, the distribution of nanofillers in the epoxy matrix also played an important role. APTES–GPTMS–EGNFs had the smallest average size, indicating the least agglomeration and the most homogeneous distribution of APTES–GPTMS–EGNFs in the epoxy matrix compared to other silane-treated EGNFs. This was also applicable in the case of APTES–GPTMS–GNPs. Based on the longest graft chain length on the EGNF surface and the least agglomeration, the epoxy nanocomposite with APTES–GPTMS–EGNFs at 0.25 wt.% loading demonstrated the highest strength and the highest stiffness among all the studied epoxy nanocomposites . The epoTo better understand the reinforcing effect imparted by the dual silane-treated EGNFs (APTES–GPTM–EGNFs) in the epoxy nanocomposite, fracture surfaces of the specimens of the APTES–GPTMS–EGNF-reinforced epoxy nanocomposite at 0.25 wt.% loading after the tensile test were examined by using SEM to link the interfacial bonding between the surface-modified EGNFs and the epoxy matrix . The fraWe observed that the neat epoxy A exhibitIt is important to highlight that the EGNFs could exhibit the advantage of “bridging” effect, due to their large aspect ratios . If a crOverall, the surface-modified EGNFs provided great potential to reinforce epoxy resin. Longer grafted silane molecular chains on the surface of EGNFs could lead to stronger interfacial bonding between EGNFs and epoxy matrix, a result of stronger molecular entanglement . The whoTo assess the thermal stability of the pristine and surface-modified EGNF-reinforced epoxy nanocomposites, TGA was used to investigate decomposition temperatures of the EGNF-reinforced epoxy nanocomposites at 0.25 wt.% loading, corresponding to 10%, 20%, 60%, and 80% weight loss. The pristine and surface-modified GNP-reinforced epoxy nanocomposites at 1 wt.% loading were also examined, for comparison.It was observed that the incorporation of the glass nanofillers in the epoxy resin could improve its thermal stability . However3, improvements of 44% and 110%, respectively, with respect to those of the neat epoxy. The superior mechanical performance of epoxy nanocomposite with APTES–GPTMS–EGNFs at ultra-low loading (0.25 wt.%), i.e., 37%, 24%, 18%, 57% improvement in strength, stiffness, ductility, and toughness, respectively, with respect to those of the neat epoxy could be attributed to the longest grafted silane molecular chains on the EGNF surface and, consequently, the strongest interfacial adhesion, due to the greatest entanglement with epoxy matrix molecules, as well as the best distribution of the APTES–GPTMS–EGNFs in the epoxy matrix. However, a challenge of employing EGNFs in the epoxy matrix for further mechanical property improvement is their severe and increasing agglomeration, especially at higher loadings (>0.5 wt.%), which was a reason why the EGNFs demonstrated the best reinforcing effect at an ultra-low loading level (0.25 wt.%). In this research, glass nanofibers with an average diameter of ~90 nm were successfully prepared through electrospinning and, subsequently, surface-modified with a variety of silane coupling agents that had increasing chain lengths in the order of methyl trimethoxysilane (MTMS), (3-aminopropyl) triethoxysilane (APTES), (3-glycidyloxypropyl) trimethoxysilane (GPTMS), and dual silane APTES–GPTMS. The pristine and surface-modified electrospun glass nanofibers (EGNFs) were employed as reinforcing fillers in the epoxy resin at low loading levels , while a commercial product of glass nanoparticles (GNPs) was used as a control for comparison purposes. Compared to the neat epoxy, the incorporation of 0.25 wt.% pristine EGNFs increased the strength, stiffness, ductility, and toughness of the resultant epoxy nanocomposite by 24%, 16%, 7%, and 32%, respectively, and outperformed the pristine GNP counterpart. Further increasing the EGNF loading in the epoxy nanocomposite resulted in a significant decrease of its mechanical performance, due to EGNF agglomeration, which was much worse than that of the GNP counterparts at higher loadings. The surface modification of the EGNFs with silane coupling agents further improved the mechanical properties of the resultant epoxy nanocomposites. The surface-modified EGNFs with the longest grafted molecular chain on the surface via APTES–GPTMS treatment demonstrated the most effectiveness in mechanical property improvement of the resultant epoxy nanocomposite. The epoxy nanocomposites with 0.25 wt.% of APTES–GPTMS–EGNFs and 1 wt.% of APTES–GPTMS–GNPs showed the best comprehensive mechanical properties among all the studied epoxy nanocomposites. The former exhibited the highest strength and stiffness, i.e., tensile strength of 79.2 MPa and Young’s modulus of 3.62 GPa, improvements of 37% and 24%, respectively, with respect to those of the neat epoxy, while the latter showed the highest ductility and toughness, i.e., elongation at break of 10.8% and work of fracture of 2052 kJ/mThe overall reinforcing performance of the surface-modified EGNFs is a result of comprehensive and balanced interactions with the epoxy matrix from the chain length of surface-grafted silane molecule, the “bridging” effect from the fiber shape, and the agglomeration of EGNFs. Reducing the agglomeration of EGNFs is the key to successful employment of EGNFs for reinforcing purposes. The scalability of EGNFs production, surface modification, and integration with epoxy resin is worthy of further investigation for future commercialization. This study pointed out directions for effectively designing and conducting surface modifications on nanofillers for high-performance polymer nanocomposite materials.


Bacillus subtilis, the Gram-positive model species, there exist two homologous oligopeptide permeases named Opp and App. Previous work showed that the App system is mutated in lab strain 168 and its recovery partially complements an Opp mutation for several developmental processes. Yet, the nature of the impact of App on signalling and development in wild-type strains, where both permeases are active was not studied. Here we re-examine the impact of the two permease systems. We find that App has a minor contribution to biofilm formation, surfactin production and phage infection compared to the effect of Opp. This reduced effect is also reflected in its lower ability to import the signals of four different Rap-Phr RRNPP systems. Further analysis of the App system revealed that, unlike Opp, some App genes have undergone horizontal transfer, resulting in two distinct divergent alleles of this system in B. subtilis strains. We found that both alleles were substantially better adapted than the Opp system to import an exogenous RRNPP signal of the Bacillus cereus group PlcR-PapR system. In summary, we find that the App system has only a minor role in signalling but may still be crucial for the import of other peptides.Oligopeptide-permeases (Opps) are used by bacteria to import short peptides. In addition to their metabolic benefit, imported short peptides are used in many Gram-positive bacteria as signalling molecules of the RRNPP super-family of quorum-sensing systems, making Opps an integral part of cell–cell communication. In some Gram-positive bacteria there exist multiple Opps and the relative importance of those to RRNPP quorum sensing are not fully clear. Specifically, in  In Gramp operon . The subEnterococcus faecalis and B. subtilis)  had a similar growth rate in monoculture and in co-culture.To this aim, ntration . Therefore assay . We usedsrfA . We used the PsrfA-3 ×yfp transcriptional reporter to follow the response to extracellularly added peptides [-;A+) marginally increased with increasing peptide concentrations, remaining similar to that of background levels. This was especially true for PhrC and PhrF, while for the two PhrP peptide variants a modest increase was recorded for peptide concentrations higher than 1 µM  when each signaling-peptide was added to the relevant strain   the fraction of sporulating cells at maximal concentration was 0.2 %. Altogether, these results suggest that the App permease has a strongly reduced ability to import the tested endogenous signalling peptides.RapA indirectly represses the activity of the response regulator Spo0A, the master regulator of sporulation and biofilm formation . To monie levels . The sams [+;A-) , reachinapp and opp operon gene expression under growth in minimal medium. It was previously reported that the expression of the app promoter showed a sharp increase only in the beginning of the stationary phase, while the opp promoter was expressed already 90 min prior to the stationary phase in Schaeffer’s sporulation medium [yfp reporter gene fused to the promoter of each operon. B. subtilis 3610 plasmid free containing either the construct PoppA-3 ×yfp or PappD-3 ×yfp were grown in Spizizen minimal medium . We monitored the expression levels and the optical density of each strain for several time points. YFP expression levels of both permeases were significantly higher than that of the background during the exponential phase, which indicates that both permeases are expressed under our conditions. Interestingly, and in accordance with previous observation, opp expression seems to be constant, while app expression increases with cell density . Surprisingly, we found that the App system displayed a pattern of allelic variation that was not congruent with the phylogenetic tree of the core genome. Specifically, we find that B. subtilis strains contain one of two significantly different alleles of appAB. A phylogenetic analysis of the app operon points to a phylogenetic tree, which is non-congruent with the species tree in appA,B,C  to compB. subtilis development. We wondered whether the App system would enable the import of signalling molecules of exogenous systems. To this end we used the exogenous PlcR-PapR quorum-sensing system from B. thuringiensis st. 407 [). Notably, this signal is longer and much more hydrophobic than the endogenous Phr and arbitrium peptides . These differences may be reflected in differential affinity to the App system. It was previously shown that this system works in the B. subtilis lab strain 168 and responds well to either the native peptide or a shorter pentapeptide, LPFEF [Our results so far indicate that App has a limited activity with respect to endogenous RRNPP signalling and its impact on  st. 407 . This RRe, LPFEF .plcR, regulated by a constitutive promoter, and the reporter gene PplcA-3 ×yfp into either App or Opp single-permease expressing strain. Co-culture experiments were performed as described previously with separate addition of the two PapR peptide variants. We find that, in contrast to the Phrs, both permeases allowed for strong uptake of the PapR signal with similar maximal expression levels for the native heptapeptide signal (Emax=109±6 AU for Opp-only and Emax=79±3 for App-only with the native peptide) and the shorter pentapeptide (K=0.12 µM) than for the Opp system (K=1.5 µM) (K=0.65 µM), while App’s was increased (K=0.29)  . This di(K=0.29) .-;A-) after adding 10 µM of PapR pentapeptide. This mutant shows YFP levels equal to background levels, which precludes the existence of an additional permease that is capable of transporting the PapR peptide .To verify that PapR is not imported through a third, uncharacterized, permease, we checked YFP expression levels of a mutant deleted for both permeases (OB. subtilis isolate spizizenii TU-B-10T (3610 operon with that of spizizenii isolate TU-B-10T (Methods), in a background deleted for the Opp operon . We introduced the plcR reporting system into this strain, which was co-cultured with a strain expressing the 3610 App allele . We then monitored their response as described before for several concentrations of hepta-PapR. We find that both strains showed similar maximal expression levels   had a five-fold lower half-maximal concentration (0.053±0.004 µM)  . The str.004 µM) , suggestBacillus subtilis and many related Bacilli code for two oligopeptide permease systems, termed Opp and App in B. subtilis. The well-studied Opp system has been shown to import signaling-peptides involved in many social behaviours [appA gene of the lab strain 168 and its derivatives [haviours . Howeverivatives , less isivatives .B. cereus group species in mixed communities of B. subtilis group and B. cereus group species.Our results demonstrate that the App system is not well-suited for the import of native RRNPP signalling peptides and correspondingly, have only a minor effect on many of the related phenotypes, including sporulation, surfactin enzymes expression, biofilm formation and phage infection. In contrast, our results suggest that App is superior to Opp in the uptake of the PapR heptamer and as effective in the uptake of the shorter pentapeptide form of PapR. Notably, peptide uptake by oligopeptide permeases can have a dramatic effect on the extracellular concentration of the peptides , and theWith respect to sporulation, previous analysis suggested that Opp deletion in the lab strain JH642 led to a large reduction in sporulation efficiency, which was complemented by restoring the function of the App system in this strain .B. subtilis has been implied to play a role in nutrition acquisition [Bacillus subtilis physiology and the hydrophobicity hypothesis is therefore not further supported by a specific biological function.It has been suggested that App specificity might be skewed towards longer peptides , which auisition , osmoprouisition , sensitiuisition  and signEnterococcus faecalis, codes for the Prg RRNPP quorum-sensing system, which controls its conjugation. This plasmid also expresses PrgZ, a OppA homolog, which has a much higher specificity to the hydrophobic signalling peptides of the Prg system than the native OppA homologue of the bacterium [Selection for hydrophobic residues has been shown to occur in other Opp homologues. For example, the plasmid pCF10 of acterium .appAB genes have two divergent alleles, which were moved by horizontal gene transfer (most likely more than once) within the B. subtilis group. This seems to have occurred through recombination over the appC gene and at the end of the app operon. Such recombination can occur through transformation or by other means and have been shown to occur in other systems such as the ComQXP quorum-sensing system [appA codes for the peptide-binding protein, while appB is probably in direct interaction with it, making the change in this pair sufficient. Our experimental analysis of the two App allelic variants suggests that they are more similar to each other than to the Opp system, but still differ in their affinity to the PapR peptide (appAB alleles is most likely unrelated to signalling and remains unknown.The notion that App has a signalling independent function is further strengthened by its unique phylogeny. While Opp phylogeny follows the general housekeeping phylogeny, our analysis shows that the App system and specifically the g system . Such hog system . From a  peptide . The selB. thuringiensis st. 407, from which the PapR system was cloned [Bacillus genus, but that the effect of this permease diversity on signalling may vary between species.Interestingly, recent analysis identified two functioning Oligopeptide permease systems in s cloned . In conts cloned . In agreClick here for additional data file.