Nearly half of orthopaedic trauma patients are intoxicated at the time of injury, and excess alcohol consumption increases the risk for fracture nonunion. Previous studies show alcohol disrupts fracture associated Wnt signaling required for normal bone fracture repair. Intermittent parathyroid hormone (PTH) promotes bone growth through canonical Wnt signaling, however, no studies have investigated the effect of PTH on alcohol‐inhibited bone fracture repair. Male C57BL/6 mice received two‐3 day alcohol binges separated by 4 days before receiving a mid‐shaft tibia fracture. Postoperatively, mice received PTH daily until euthanasia. Wnt/β‐catenin signaling was analyzed at 9 days post‐fracture. As previously observed, acute alcohol exposure resulted in a >2‐fold decrease in total and the active form of β‐catenin and a 2‐fold increase in inactive β‐catenin within the fracture callus. Intermittent PTH abrogated the effect of alcohol on β‐catenin within the fracture callus. Upstream of β‐catenin, alcohol‐treated animals had a 2‐fold decrease in total LRP6, the Wnt co‐receptor, which was restored with PTH treatment. Alcohol nor PTH had any significant effect on GSK‐3β. These data show that intermittent PTH following a tibia fracture restores normal expression of Wnt signaling proteins within the fracture callus of alcohol‐treated mice. We hypothesize that intermittent PTH administration following a fracture would counteract the effect of alcohol on fracture healing by restoring Wnt/β‐catenin signaling during fracture repair.2All animal procedures were approved by the Institutional Animal Care and Use Committee of Loyola University Chicago  and compiled with the US National Research Council's Guide for the Care and Use of Laboratory Animals, the US Public Health Service's Policy on Humane Care and Use of Laboratory Animals, and Guide for the Care and Use of Laboratory Animals.2.1A total of 23 male C57BL/6 mice 6‐7 weeks of age were obtained from Jackson Laboratory  and acclimated to the facility for one week. The animal facility is a ventilated environment with a 12hour:12hour light‐dark cycle at a constant temperature of 21°C and the animals had unrestricted access to food and water. Animals were then randomly assigned to one of four treatment groups: Saline/Saline (n = 6), Alcohol/Saline (n = 5), Saline/PTH (n = 6), and Alcohol/PTH (n = 6). The nomenclature for each group corresponds to the pre‐fracture treatment  followed by the post‐fracture treatment . The treatment paradigm is summarized in Figure 2.2One hour after receiving the final alcohol or saline injection, animals were administered an induction dose of anesthesia  to facilitate hair removal from the left hind limb. Following hair removal, animals received subcutaneous prophylactic gentamycin  and were then placed on 1%‐2% vaporized isoflurane  for the duration of the fracture surgery. Under sterile conditions, a longitudinal incision was made over the left stifle and the patellar tendon exposed Figure . The tib2.32 and tibia specimens collected for molecular analysis on POD 9.All animals received daily intraperitoneal injections of human (1‐34) PTH  or an equivalent volume of saline beginning at postoperative day (POD) one until euthanasia. The dose of 40 μg/kg PTH was selected based on published rodent PTH dosing regimens.2.4Tibia specimens were flash frozen in liquid nitrogen and stored at −80°C until use. Fracture callus tissue was separated from uninjured bone using a Dremel tool  and pulverized in lysis buffer  using a freezer mill . Total protein was measured using a Pierce BCA Protein Assay kit . Twenty micrograms of total protein from each sample was resolved on a 4%‐20% mini‐protean TGX Stain‐Free precast gel . The total protein loaded was visualized using UV activation of the gel and analyzed with Image Lab software , then transferred to PVDF membranes . Membranes were probed with rabbit anti‐mouse total β‐catenin , non‐phospho β‐catenin , phospho‐β‐catenin , total LRP6 , total GSK‐3β , phospho‐GSK‐3β (Ser9)  and phospho‐GSK‐3β (Y219) . Visualization of bound antibodies was possible via secondary goat anti‐rabbit IgG  and developed with SuperSignal West Pico chemiluminescent substrate . Densitometric analysis was carried out utilizing the Image Lab software . Detected protein signals were normalized against the total protein concentration determined by Stain‐Free signal.2.5P‐value ≤.05 was noted as statistically significant.All data is expressed as the mean ± SEM and was analyzed using the SAS Version 9.4  statistical program. A Kruskal‐Wallis test was used to assess for overall variability in the ratio among the four experimental groups for each analysis set. A 33.1P = .017 and P = .016, respectively).Canonical Wnt signaling is propagated through downstream effector protein β‐catenin. We have previously reported that alcohol‐treated animals have reduced fracture callus total β‐catenin compared to saline‐treated animals.P = .008) and a significant increase in hyperphosphorylated β‐catenin as compared to saline‐treated animals .P = .010). In agreement with our past studies, alcohol‐treated animals had a significant increase in callus hyperphosphorylated β‐catenin as compared to saline‐treated animals .β‐catenin is regulated by phosphorylation. Hypophosphorylated β‐catenin is activated, promoting translocation of the protein to the nuclear to activate transcription, where hyperphosphorylated β‐catenin is inactive and targeted for degradation through the proteasome. In agreement with our previous findings, alcohol‐treated animals have a statistically significant reduction in callus hypophosphorylated β‐catenin . Several studies have reported that LRP6 acts as a co‐receptor for the PTH receptor (PTHr).Low‐density lipoprotein receptor‐related protein 6 (LRP6) is a co‐receptor and key component in canonical Wnt receptor activation and signal propagation.3.3Glycogen synthase kinase 3 beta (GSK‐3 β) through substrate‐mediated hyperphosphorylation of β‐catenin, is the principal negative regulator of canonical Wnt signaling.4Alcohol abuse has been implicated as a major risk factor for delayed fracture healing and nonunion in fracture repair.et al. found PTH accelerated pelvic and distal radius fracture healing respectivelyet al. and Johansson studying femoral neck and proximal humerus fractures did not find a benefit to union or improvement in patient reported outcomes, respectively.et al. found weekly PTH injections significantly improved lumbar fusion rates in patients undergoing interbody fusion.Parathyroid hormone acts as an anabolic bone agent when delivered intermittently and several randomized controlled trials have been performed to determine its effect on fracture healing and spinal arthrodesis.Prior studies have shown that PTH activates Wnt signaling by increasing LRP6 receptor activation, β‐catenin activation and Wnt gene transcription,Administration of intermittent PTH has been found to stimulate bone formation.*. in our alcohol‐treated animals as compared to saline‐treated animals. Further studies need to be done to determine if intermittent PTH reduces active GSK‐3β (phospho‐Y216) within the fracture callus of alcohol‐treated animals.β‐catenin is negatively regulated by the APC, axin and GSK‐3β complex by hyperphosphorylation, which targets β‐catenin to the proteasome for degradation.In a process that resembles endochondral ossification, mesenchymal stem cell‐derived chondrocytes and osteoblasts create a fracture callus following bone fracture. Previous studies show that intermittent PTH improves and accelerates fracture healing in part by increasing the size and improving the biomedical strength of the fracture callus,The present study has its limitations. Most notably, this study was meant to serve as a preliminary study to determine whether intermittent PTH improved Wnt/β‐catenin signaling within the fracture callus of alcohol‐treated animals. Further research will be required to determine if the differences seen on the molecular level correlate to functional differences in the histology and biomechanics of the callus that forms during fracture healing. Additionally, this study was limited by the relatively small sample size of mice, leading to some of the variability in the dataset. Finally, the results of all the analyses are at a single time point in the complex process of fracture healing. Previous research show β‐catenin expression remains elevated for weeks after fracture, however, levels in a fracture callus reach maximal expression on post‐injury day 9.In summary, the current work adds new information about the effects of intermittent PTH on Wnt/β‐catenin signaling within the fracture callus of alcohol‐treated animals. Intermittent PTH should continue to be investigated as a useful adjunct to fracture union given the scarcity of other clinically available systemic therapies.5Alcohol exposure inhibits Wnt signaling within the fracture callus. Administering intermittent PTH following a tibia fracture in alcohol‐treated mice restores normal expression of Wnt signaling proteins within the fracture callus.None.EMK and TJR performed and analyzed experiments. EMK, TJR, AT and JME analyzed data and participated in manuscript writing. All authors read and approved the final manuscript. All authors agree to be accountable for all aspects of the work.


Amyotrophic lateral sclerosis (ALS) is a progressive multifactorial disease characterized by the loss of motor neurons (MNs). Not all MNs undergo degeneration: neurons of the oculomotor nucleus, which regulate eye movements, are less vulnerable compared to hypoglossal nucleus MNs. Several molecular studies have been performed to understand the different vulnerability of these MNs. By analyzing postmortem samples from ALS patients to other unrelated decedents, the differential genomic pattern between the two nuclei has been profiled. Among identified genes, adenylate cyclase activating polypeptide 1 (ADCYAP1) gene, encoding for pituitary adenylate cyclase-activating polypeptide (PACAP), was found significantly up-regulated in the oculomotor versus hypoglossal nucleus suggesting that it could play a trophic effect on MNs in ALS. In the present review, some aspects regarding the different vulnerability of oculomotor and hypoglossal nucleus to degeneration will be summarized. The distribution and potential role of PACAP on these MNs as studied largely in an animal model of ALS compared to controls, will be discussed. Amyotrophic lateral sclerosis (ALS) is an incurable and multifactorial neurodegenerative disease induced by the synergistic action of genetic and environmental factors. The ALS cases are classified into sporadic (SALS) and familial (FALS) forms. Mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene account for approximately 20% of FALS cases . For theThrough microarray technology, The involvement of PACAP in ALS has been described in several papers. Comparison of microarray datasets of sporadic ALS motor cortex and mutated SOD1 (mSOD1) G93A mice brains revealed that some genes, including those of PACAP and its receptor PAC1R, are deregulated in the same direction in both human and transgenic animals .The cellular and regional distribution of PACAP and its receptors, known as PAC1R, VPAC1, and VPAC2 receptors, has been extensively investigated in central and peripheral nervous system. In response to neurodegenerative insult, the peptide is released from MNs and exerts a neuroprotective role through autocrine and/or paracrine mechanisms mediated by cAMP/PKA-(cyclic adenosine monophosphate/protein kinase A) or PI3K (phosphoinositol three kinase) pathways . MoreoveThe present review aims to summarize findings on the role played by PACAP in the neurodegenerative process affecting lower MNs during ALS on postmortem samples of decedents with ALS and in an animal model of ALS, mainly focusing on the oculomotor and hypoglossal nuclei.ALS is a motor neuron disease, a degenerative, progressive and paralytic disorder targeting MNs of the brain and spinal cord . The earThe ALS cases are classified in sporadic (SALS) and familial (FALS) forms. The first one, without genetic inherited component, occurs in 90% of cases, while FALS cases (10%) are associated with mutations identified in distinctive genes. Among these, superoxide dismutase 1 (SOD1) was the first mutated gene to be discovered about two decades ago and it accounts for 20% of FALS cases . More thDuring the last 15 years, other FALS mutated genes have been identified, including TAR DNA binding protein (TARDBP), Fused in Sarcoma (FUS) and Chromosome 9 open reading frame 72 genes (C9orf72). TDP43, encoded by the most common mutated ALS gene TARDBP, and FUS proteins regulate RNAs expression and maturation . AdditioBased on brain localization, MNs are classified as upper MNs located in the motor cortex and lower MNs situated in brainstem motor nuclei and in spinal cord ventral horns .Although upper and lower MNs are both affected during the disease, lower MNs show a different susceptibility to degeneration. From disease onset, MNs localized in ventral horns of spinal cord and hypoglossal nucleus begin to degenerate, whereas MNs of oculomotor, trochlear and abducens nuclei controlling eye movements are spared . In accoTypical clinical symptoms linked to upper MN degeneration comprise spasticity, uncontrolled movement and significant reduction of sensitivity . Lower MIn the present paper, we are focusing on data regarding the different vulnerability of lower MNs in oculomotor versus hypoglossal nucleus. The mechanism underlying their different death susceptibility is not fully understood, but it is clear that the resistance of some MNs to degeneration is related to anatomical specificity and their transcriptome profile.The oculomotor nucleus, adjoining to trochlear nucleus, is situated at the level of the superior colliculus in the midbrain and it extends rostrally up to the posterior commissure. By examining its ultrastructure in rat brains, it has been demonstrated that it comprises either large MNs with abundant cytoplasm and well-developed organelles as well as small MNs with a low amount of cytoplasm. Although axo-somatic and axo-dendritic synapses are the most prevalent, axo-axonic synapses have also been identified. Fibers of the oculomotor nucleus as well as autonomic fibers of the accessory parasympathetic nucleus  converge into the oculomotor nerve . Somatic nerve fibers are bundled inside the nerve and are surrounded by the autonomic ones .In mammals, conjugate eye movement results from combined contraction of six striated muscles inserted on the external surface of the eye bulb. Four of the six extraocular muscles (EOMs), known as medial rectus, inferior rectus, superior rectus and inferior oblique muscles are innervated by MNs of oculomotor nucleus. The superior oblique muscle is innervated by trochlear nerve MNs whereas the abducens nucleus MNs innervate the lateral rectus muscle.EOMs comprise six different types of fibers, including those with very high mitochondrial content and strong fatigue resistance. They show a more complex pattern of innervation than almost all skeletal muscles. In particular, they have neuromuscular junctions extensively dispersed along their length from origin to muscle insertion . FurtherHypoglossal nucleus is localized on the floor of the fourth ventricle in the dorsal part of the medulla oblongata. It contains a high number of MNs and inhibitory interneurons. MNs of the hypoglossal nucleus show large soma containing high levels of Nissl substance . Their aG93A rat model of ALS, In addition to the anatomical and functional differences, oculomotor and hypoglossal nuclei MNs have also a different genes expression pattern. By using laser capture microdissection and microarray analysis in a SOD1In a previous paper, By comparing gene expression profile data, we have found that majority of genes deregulated in oculomotor/hypoglossal nuclei 127/188;  are alsoPACAP is a neuropeptide identified for the first time in the ovine hypothalamus and belonging to VIP/secretin/glucagon family . There aPACAP and its receptors are expressed in different tissues and organs, where they are involved in several biological processes including cell division and survival . PACAP iTissue distribution studies have demonstrated that PACAP mRNA precursor is present in cerebral cortex, olfactory bulb, cingulate cortex, dentate gyrus, CA1 and CA4 subregions of the hippocampus and cerebellum in the developing and adult rat brain . PACAP-iA review paper has highlighted that PAC1R has greater expression than VPAC1-R and VPAC2-R receptors in the CNS . In partin vitro model of MNs-derived from human induced pluripotent stem cells (iPSC) exposed to neurodegenerative insult (The analysis of genome expression profile ALS patients motor cortex has highlighted a significant deregulation either in PACAP and PAC1R mRNAs. In particular, PACAP is over-expressed in SALS1 and down-regulated in SALS2 motor-cortex samples, whereas PAC1R is down-regulated in both subgroups as compared to controls. To evaluate the role of PACAP in MNs degeneration, the effect of peptide has been tested in an e insult . The rese insult . In accoe insult . Particue insult . The ende insult . After ne insult . Moreovee insult . On the e insult .Therefore, it is possible to speculate that over-expression of PACAP encoding gene, ADCYAP1, in oculomotor nucleus may be implicated in its less vulnerability during ALS degeneration. In accord to this hypothesis, PACAP was found up-regulated in the axotomized facial motor nuclei of transgenic SOD1 G93A mice . FurtherDespite these evidences, Apparently, conflicting data exist regarding PAC1R expression. Indeed, it is highly expressed in hypoglossal nucleus compared to oculomotor nucleus in healthy rat brains or after a moderate traumatic brain injury . Howeverin vitro model of ALS (It has been hypothesized that PACAP, binding to PAC1R, activates the MAPK survival pathway directly or indirectly through EGFR trans-activation , b. In al of ALS . This nel of ALS . Accordil of ALS .The data shown in the present review present several limitations considering that most of the studies were performed in SOD1 G93A mice model at different disease phases  and in bAlthough SOD1 G93A mice is the widely used animal model, reproducing motor defects similar to that observed in ALS-affected patients, there are important caveats to acknowledge. Firstly, the mSOD1 mouse model has a propensity to spontaneously delete copy number which can alter the severity of disease presentation ; secondlMoreover, many other genes are involved in sporadic forms of ALS in addition to SOD1 G93A mutation. Therefore, to better characterize the role of PACAP and its receptor on MNs survival in ALS, new studies should be performed in other transgenic animal models.The involvement of PACAP has been demonstrated in different neurodegenerative diseases, including MNs damage occurring in ALS. The evidences, above described, suggest that the different vulnerability of some cranial nerve motor nuclei could be also related to differential expression of PACAP in MNs. Moreover, PACAP’s protective effects in MNs during ALS progression could be direct through PAC1R activation or mediated by EGFR trans-activation promoting MAPK or PI3K signaling survival cascade .However, too much is still unknown. To better characterize the pathogenetic mechanisms involved in this progressive neurodegenerative disease, it is advisable that the research community extends studies of gene expression and transcriptomes on samples from ALS decedents obtained at autopsy and from stereotactic biopsies on living patients when available.GMa and VD’A designed the research projects and wrote the manuscript. AD’A, DR, and SC contributed to the manuscript preparation. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HOMER family scaffolding proteins (HOMER1-3) play critical roles in the development and progression of human disease by regulating the assembly of signal transduction complexes in response to extrinsic stimuli. However, the role of HOMER protein in breast cancer remains unclear.HOMER3 expression was examined by immunohistochemistry in breast cancer patient specimens, and its significance in prognosis was assessed by Kaplan–Meier survival analysis. The effects of HOMER3 in growth factor-induced β-Catenin activation were analyzed by assays such as TOP/FOP flash reporter, tyrosine phosphorylation assay and reciprocal immunoprecipitation (IP) assay. Role of HOMER3 in breast cancer metastasis was determined by cell function assays and mice tumor models.Herein, we find that, among the three HOMER proteins, HOMER3 is selectively overexpressed in the most aggressive triple negative breast cancer (TNBC) subtype, and significantly correlates with earlier tumor metastasis and shorter patient survival. Mechanismly, HOMER3 interacts with both c-Src and β-Catenin, thus providing a scaffolding platform to facilitate c-Src-induced β-Catenin tyrosine phosphorylation under growth factor stimulation. HOMER3 promotes β-Catenin nuclear translocation and activation, and this axis is clinically relevant. HOMER3 promotes and is essential for EGF-induced aggressiveness and metastasis of TNBC cells both in vitro and in vivo.These findings identify a novel role of HOMER3 in the transduction of growth factor-mediated β-Catenin activation and suggest that HOMER3 might be a targetable vulnerability of TNBC. Breast cancer incidence has increased steadily during the past decades and is currently the most common malignancies in females worldwide . There aβ-Catenin, central effector of Wnt signaling pathway, mainly serves as a transcriptional co-activator of T-cell factors/lymphoid enhancer factor (TCFs/LEF) to dictate downstream genes transcription, and plays crucial roles in development and progression of human diseases . UnsurprCrosstalk between growth-factor signal transduction and canonical Wnt pathway allows β-Catenin activation in Wnt-independent ways. As a common joint of multiple signaling pathways, β-Catenin can be phosphorylated on tyrosine residues upon the effective stimulation of growth factors, resulting in excessive β-Catenin nuclear translocation and transcriptional activation of targeted genes in the context of malignant disease –14. NeveHOMER family scaffolding proteins comprise three members (HOMER1-3) in mammals , 16. WheIn the present study, we find that, among the HOMER family members, HOMER3 is selectively overexpressed in TNBC and correlates with poor prognosis. Notably, HOMER3 plays a scaffolding function to simultaneously interact with c-Src and β-Catenin and promotes their efficient interactions, thus facilitating the growth factor-induced β-Catenin tyrosine phosphorylation and activation. HOMER3 is essential for EGF-mediated aggressiveness and metastasis both in vitro and in vivo. These findings uncover the role of HOMER3 in growth-factor-induced β-Catenin activation and TNBC metastasis, and open new avenues to prevent or overcome TNBC by targeting HOMER3.Breast cancer cell lines were obtained from the ATCC cultured following the provider’s recommendations. MCF-7, T47D, MDA-MB-231 were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS). ZR-75–1, BT-549 and 4T1 cells were maintained in RPMI-1640 medium with 10% FBS. SUM159PT cells were maintained in Ham's F-12 with 10% FBS. All cells were supplemented with penicillin/streptomycin, hydrocortisone, insulin, HEPES and L-glutamine, and maintained at 37 °C in 5% CO2. Cell lines were authenticated by short tandem repeat (STR) fingerprinting.This study was conducted on a total of 347 paraffin-embedded breast cancer samples including 256 non-TNBCs and 91 TNBCs, which had been histopathologically and clinically diagnosed at the Sun Yat-sen University Cancer Center from 2005 to 2013. Clinicopathological characteristics are summarized in Additional file IHC staining was performed on the 347 paraffin-embedded breast cancer tissue sections using HOMER3 and β-Catenin antibodies as previously reported . The res5) with or without HOMER3 knockdown were orthotopically injected into the mammary fat pads of BALB/c mice and inoculated for 5 weeks. Metastasis was detected using the IVIS imagining system  by blocking the orthotopic tumor signals.Breast cancer spontaneous metastasis model and lung colonization model were performed as previously described . Female 6, n = 8/group). Lung metastasis burden of animals was monitored weekly using bioluminescent imaging (BLI). Mice were euthanized 9 weeks after inoculation, and lung metastases were evaluated.In the lung colonization model, BALB/c-nude mice were intravenously injected with control or HOMER3 silencing MDA-MB-231-luc cells  every other day as previously reported . For valCell lysates were prepared from the indicated cells using lysis buffer . Lysates were then incubated with anti-HOMER3 , or anti-c-Src, or anti-β-Catenin  antibody, and protein G-conjugated agarose, or Flag, Myc, HA affinity agarose (Sigma-Aldrich), at 4 °C overnight. Beads containing affinity-bound proteins were washed 6 times by IP wash buffer , followed by eluting using 1 M glycine (pH 3.0). The eluates were then mixed with sample buffer and denatured and used for the western blot analysis. Target proteins were blot with primary antibodies derived from biological sources different from those used in IP to avoid high background. For the detection of β-Catenin Tyr phosphorylation levels, cell lysates were first pulldown with anti-β-Catenin antibody  and subjected for western blot, followed by staining with p-Tyr specific antibody .7 293 T cells transfected with 200 μg Flag-HOMER3, or Flag-c-Src, or Flag-β-Catenin expressing plasmid was lysed using RIPA buffer (0.25% SDS). Lysates were then incubated with 200-μl Flag affinity agarose (Sigma-Aldrich) overnight at 4 °C. Beads containing affinity-bound proteins were washed six times by 5-ml RIPA buffer, followed by elution with 500 μl of Flag competing peptides  twice. The elutes were pooled and washed with 5 ml PBS using 3-kDa MW cutoff filter units (Millipore) to remove the competing peptides. The purities of HOMER3, c-Src and β-Catenin were examined by SDS-PAGE and Coomassie blue staining and then subjected for in vitro binding assays.Protein purification was performed as previously reported . BrieflyRecombinant proteins including Wnt3A, EGF and TGFα were purchased from R&D system  and SinoBiological . Before treatment, cells were changed to 1% FBS medium culturing for 1 h; cytokines were then added to a final concentration of 20 ng/ml for Wnt3A, 20 ng/ml for EGF, and 10 ng/ml for TGFα, respectively. For the matrigel 3-D spheroid formation assay, cells were replenished with 50-μl serum-free or EGF-supplemented medium every other day.The wild-type (TOP) and mutant (FOP) LEF/TCF reporters were cloned into pGL3 luciferase constructs (Promega). Twenty thousand cells were seeded in triplicate in 48-well plates and allowed to settle for 24 h. One hundred nanograms of TOP or FOP flash, plus 1 ng of pRL-TK Renilla plasmid (Promega), was transfected into cells using the Lipofectamine 3000 reagent according to the manufacturer's recommendation. Luciferase and Renilla signals were measured 24 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega) according to a protocol provided by the manufacturer. The results were calculated as the ratio of specific TOP-Flash over non-specific FOP-Flash relative renilla luciferase units (RLU).2 test, Spearman-rank correlation test and Student’s t test (two-tailed). Multivariate statistical analysis was performed using a Cox regression model. P < 0.05 was considered statistically significant.Statistical analyses were performed using the SPSS version 19.0 statistical software package. Statistical tests for data analysis included the log-rank test, χAdditional information is provided in Supplementary Materials and Methods.HOMER scaffolding proteins mediate cellular signaling transduction and emerge as important regulators in development and disease , 16. To http://kmplot.com/analysis) indicated that high expression of HOMER3 was significantly associated with tumor metastasis, relapse and patient survival in breast cancer  of the TCGA breast cancer samples according to HOMER3 expression to identify its regulated oncogenic signals. Interestingly, the GSEA analysis showed consistent enrichment of β-Catenin-upregulated gene signature (BCAT_BILD_ET_AL_UP) in HOMER3-high subgroup, and β-Catenin-repressed gene signature (BCAT_BILD_ET_AL_DN) in HOMER3-low samples, suggesting that HOMER3 may regulate the β-Catenin activity Fig. a, b.Fig.In fact, β-Catenin can be activated by cytokines including the Wnt family proteins and growth factors –14. SurpGrowth factors increase tyrosine phosphorylation of β-Catenin to promote its nuclear translocation and transcriptional activation , 13. IndPrevious studies have identified c-Src as a direct upstream tyrosine-protein kinase of β-Catenin , 30. NotTo further understand the details of the interactions between HOMER3 and c-Src, truncated forms of HOMER3 and c-Src were expressed and subjected for IP assays. We found that only those HOMER3 truncations with EVH1 domain could bind to c-Src, suggesting that this domain was responsible for its interaction with c-Src  for efficient interaction with substrates . To furtWe further investigated the role of HOMER3 in the aggressiveness of breast cancer cells. As HOMER3 is substantially increased in TNBC, we first tested the effects by silencing the endogenous HOMER3 in human TNBC cell lines SUM159PT and MDA-MB-231, as well as a metastatic mouse TNBC cell line 4T1  with or without HOMER3 downregulation were implanted into the mammary fat pads of BALB/c female mice. Knockdown of HOMER3 showed an approximate 20% repression on the growth of 4T1 tumors  is the most challenging subtype of human breast cancer, demanding new biomarkers and therapeutic targets. HOMER family proteins (HOMER1-3) are scaffolding proteins that regulate the assembly of signal transduction complexes in response to extrinsic stimuli. However, the role of HOMER protein in breast cancer, especially the potential in TNBC metastasis remains unclear. In this study, we identified that HOMER3 was selectively overexpressed in TNBC and associated with poor prognosis. Intriguingly, HOMER3 was essential for growth factor-mediated but not canonical Wnt-induced β-Catenin activation. HOMER3 used its EVH1 domain to recognize and interact with both c-Src and β-Catenin, and utilized the coiled-coil domain to provide a multimeric scaffolding platform to facilitate c-Src-induced β-Catenin tyrosine phosphorylation under growth factor stimulation.Importantly, silencing of HOMER3 robustly inhibited TNBC metastasis in vivo. These findings uncover a novel role of HOMER3 in growth factor-mediated constitutive activation β-Catenin in TNBC and suggest that HOMER3 might be a targetable vulnerability of TNBC.Additional file 1.Figure 1. (A) Expression levels of HOMER family genes in normal, non-TNBC and TNBC samples from The Cancer Genome Atlas (TCGA) dataset. (B) Similar to (A), analysis was compared in different molecular subtypes. (C) Real-time PCR analysis of HOMER family genes in 5 normal breast tissues, 10 non-TNBC and 10 TNBC tissues. (D) The prognostic values of HOMER3 in DMFS, relapse-free survival (RFS), and OS of breast cancer patients were further assessed by a public Kaplan-Meier Plotter program . All settings were default except the following ones: probe (204647_at), survival , and auto select best cutoff (on).Additional file 2.Figure 2. (A) Normalized luciferase activities of specific TOP-Flash over non-specific FOP-Flash relative renilla luciferase units (RLU) in MDA-MB-231 and SUM159PT cells cultured with 1% or 10% Fetal Bovine Serum (FBS). ns, not significant. (B) Total and phosphorylation levels of EGFR and c-Src in MDA-MB-231, SUM159PT, MCF-7 and T47D cells treated with or without EGF. (C) Western blot analysis of p-EGFR, EGFR, p-c-Src, c-Src in control and HOMER-3 silencing MDA-MB-231 cells treated with PBS, EGF, or TGFα. (D) Real-time PCR analysis of HOMER3 in MDA-MB-231 cells treated with PBS, EGF, or TGFα.Additional file 3.Figure 3. (A) Western blot analysis of HOMER3, c-Src and β-Catenin in indicated cells. α-Tubulin was used as a loading control. (B) Normalized luciferase activities of specific TOP-Flash over non-specific FOP-Flash relative renilla luciferase units (RLU) in control or HOMER3-overexpressing TNBC cells with or without c-Src silencing.Additional file 4.Figure 4. (A) Western blot analysis of HOMER3 in MDA-MB-231, SUM159PT and 4T1 cells that stably transduced with HOMER3 shRNA#1 or shRNA#2. (B) Tyr phosphorylation levels of β-Catenin in indicated cells. (C) Western blot analysis in control and HOMER3-overexpressing MCF-7 and T47D cells with or without EGF treatment. (D) Normalized luciferase activities of specific TOP-Flash over non-specific FOP-Flash relative renilla luciferase units (RLU) in MCF-7 and T47D with PBS or EGF treatment. (E) Knockdown of c-Src in control or HOMER3 overexpressing MCF-7 and T47D cells was validated by western blot analysis. (F) Representative images and quantification of invading MCF-7 and T47D cells in the transwell matrix penetration assays. (G) 3-D spheroid cultured in matrigel was used to determine the invasive capacity of indicated cells.Additional file 5.Figure 5. (A) Western blot analysis of indicated proteins in control or c-Src-P307L overexpressing MDA-MB-231 and MCF-7 cells with or without EGF treatment. (B) Tyr phosphorylation levels of β-Catenin in indicated cells. (C) Representative images and quantification of invading MDA-MB-231 and MCF-7 in the transwell matrix penetration assays.SAdditional file 6.Figure 6. (A) The proliferation rate of control or HOMER3 silencing 4T1, MDA-MB-231 and SUM159PT cells was examined by MTT assay. (B) The metastatic index (ratio of nodule number/tumor volume) in each group was calculated by the ratio of nodule number to tumor volume. (C) IHC staining of p-β-Catenin-Y333 in MDA-MB-231 lung metastases.Additional file 7. Supplementary materials, methods, and tables.


While nanoparticles from the carbon family have been incorporated effectively for polymer matrixes, there is no clear information available for understanding the impacts of the morphology of different carbon nanoparticles on the performance of carbon-based nanocomposites. Therefore, this study aimed to provide a comprehensive, comparative investigation to systematically assess the impacts of nanoparticles on the tribological, mechanical, and electrochemical properties of the epoxy coatings using three representative 0D, 1D, and 2D nanoparticles: Fullerene-C60 (C60), graphene nanoplatelets (GNPs), and carbon nanotubes (CNTs). The anti-corrosion performance of the nanocomposites in both the short and long term was characterized. The mechanical properties were examined by abrasion, adhesion, and tensile tests. Fourier-transform infrared spectroscopy (FTIR) was conducted to determine their chemical structures, while scanning electron microscopy (SEM) was used to determine their surface texture. The electrochemical impedance spectroscopy (EIS) results revealed that the coatings reinforced by C60 and GNP had better anti-corrosion performance than that of the CNT/epoxy samples. The incorporation of C60 and CNT led to a considerable improvement in tensile properties, while improved abrasion resistance was observed in all types of nanofiller/epoxy groups. C60-loaded composites exhibited a significant enhancement in tensile properties as compared to CNT or GNP composites. Nanomaterials, due to their excellent physical, electrical, and other proprieties, are widely accepted as a building block for broader applications . Among tThe inclusion of CNTs, GNPs, or C60 for synthesizing nanocomposite coatings has recently received wide attention ,21,22,23Meanwhile, fullerene-C60 (C60) nanoparticles served as nanofillers due to the unique spherical structure with a nanosized diameter to reinforce the polymer matrix ,26. CompTo the best of the authors knowledge, there is still a lack of research to comparatively evaluate the influence of geometric shape for the carbon-based nanofillers on nano-reinforcement, especially an experimental investigation of the tribological, mechanical, and electrochemical properties. Three representative carbon nanoparticles, CNT, GNP, and C60, were selected as additives for reinforcing the epoxy coating, and the major focus was on their enhancements on the tribological, mechanical, and electrochemical properties. The nanocomposites were fabricated using an identical mixture design and an identical dispersion procedure to generate a comparative situation to better determine the effects of different nanoparticles on their performance. The influence of the inclusion of the nanofillers in the polymeric coating was investigated in detail by: (i) Potentiostatic EIS test, (ii) Taber abraser test, and (iii) coupon tensile test. SEM and transmission electron microscopy (TEM) were used to characterize micro-scale information of the nanofillers and the nanocomposites.This section describes the detailed experimental investigation for providing a comprehensive comparative study of incorporating three different nanomaterials into the coating. Detailed information of the materials and characterization methods are presented in detail below.As stated, three different carbon-based nanofillers were selected, and the atom structures of CNT, GNP, and fullerene-C60 are illustrated in The fabrication procedure of coating systems is illustrated in The corrosion barrier performance of the coatings was evaluated by EIS test. The coating films were cast on S-36 steel panels  and a three-electrode system was used in EIS measurement. The steel panel of the coating specimen was employed as the working electrode, while a platinum net and saturated calomel electrode were used as the counter electrode and reference electrode, respectively. A glass tube was clamped on the panel and 1.0% NaCl solution was added to immerse the electrodes. A Gamry Reference 600 Spectroscope  was used to collect the EIS data and was presented as impedance vs. frequency curves. In addition, salt spray exposure (ASTM B117) was employed as an accelerated durability test to evaluate the long-term durability of the nanofiller-reinforced coatings.2 on the coating as the surface layer was removed.To examine the wear resistance of the developed coatings, a Taber abraser test was conducted in accordance with the ASTM D4060 standard. The abrasion resistance was characterized as the mass loss in a certain number of abrasive cycles under applied loads. In this study, each specimen was abraded by two CS-10 wheels with a rotational speed of 72 rpm for 1000 cycles while under a load of 1000 g during the test. During the test, the abrading wheels created a circular abrasion mark with an area of 30 cmA coupon test was employed to examine the tensile properties of the developed nanocomposites. The tensile test was operated by a Shimadzu EZ-X tester  by following the ASTM D638 standard. Tensile strength was applied to pull the specimen until the specimen broke in the narrow section. During the test, the loading rate was maintained at 1 mm/min, while real-time tensile strength and strain were recorded.Transmission electron microscopy (TEM) photographs of the as-received raw CNT, GNP, and C60 nanoparticles is shown in As shown in The nanofillers and nanocomposites were characterized using FTIR analysis. FTIR spectra of nanofillers and nanocomposites are illustrated in −1 indicates the presence of an O–H bond, which was contributed by an unbound or free hydroxyl group (moisture) attached to the nanofiller surfaces [−1 [To examine the potential of hydrogen bonding between the nanofiller and epoxy resin during the dispersion process, FTIR spectra were used to examine the existence of hydroxyl groups on each of the nanofillers’ surfaces. As presented in surfaces ,28. On taces [−1 .−1 was due to the C–H stretch of the polymer backbone, and the peaks at 1607 and 1456 cm−1 were used to characterize the DGEBA epoxy resin that corresponds to the aromatic ring stretching of C=C. The disappearance of a peak around 913 cm−1 for all the tested samples indicates the ring opening polymerization during crosslinking, which reflects the curing process [As illustrated in  process ,31,32. C process .One of the most important factors in the nanoparticle’s reinforcement is the dispersion state of nanofillers. The size of nanoparticles can affect the reinforcement in the nanocomposites as larger aggregates create more severe defects, which leads to longer micro-cracks . To exam3 to 105, 102 to 104, and 102 to 103 for CNT, GNP, and fullerene-C60, respectively  have the highest tendency to form agglomeration, followed by the 2D materials (GNP), where the nanoplatelets stack up after 1.0 wt.%. The 0D particles (fullerene-C60) have the least tendency to form agglomeration, as no significant increases were observed in the particle size.This section discusses the impacts of different carbon-based nanofillers as a reinforcement on the corrosion barrier performance of the nanocomposites in both the short term and long term, as summarized and shown in (a) Neat epoxy as the references: At the onset of exposure, the obtained results suggested that the pure epoxy had good corrosion resistance. However, the bending in the low-frequency region of the Bode diagram indicated that the coating film could not act as an intact layer to protect the substrate. Researchers ,34,35 su(b) CNT/epoxy: mod value began to decrease in composites with 1.5% by weight of GNP. Unlike pure epoxy and CNT/epoxy, 0.1%, 0.5%, and 1.0% GNP-epoxies had a slope of −1 in the impedance diagram, while the values reached 90° in the phase angle plot. This result suggests that the GNPs worked as barriers to the corrosive media; moreover, the existence of well-distributed graphene nanosheets in these groups effectively improved the barrier properties of the polymeric films. In this case, these GNP/epoxy coatings offered excellent corrosion protection to the substrate, and this observation was consistent with the other researchers’ studies [(c) GNP/epoxy: As illustrated in  studies ,38. Howe0.01Hz values in the impedance plot, suggesting the coating films had excellent barrier performance and provided perfect resistance to corrosion.(d) C60/epoxy: 0.01Hz) dropped about two and a half orders, indicating that the barrier properties of the coating had been decreased. Thus, the minimum region of the phase angle curve approached the high-frequency region, suggesting that the neat epoxy coating degraded during the exposure, which led to reduced corrosion protection properties, as also confirmed by other researchers [(a) Neat epoxy after 100/200 h accelerated durability tests: The long-term performance of the neat epoxy coating after 100/200 h exposure of salt spray, illustrated by the Bode plots earchers .(b) CNT/epoxy after 100/200 h accelerated durability tests: (c) GNP/epoxy after 100/200 h accelerated durability tests: As clearly illustrated in 0.01Hz in the 3.0%C60-epoxy group was reduced, suggesting that the coating was damaged and delaminated during the salt fog exposure. This observation pointed out that a higher loading of C60 nanoparticles could offer a short-term improvement in anti-corrosion properties; however, they would not be able to provide enough durability against long-term exposure. When the exposure time increased up to 200 h, it is clear that 1.0%C60-epoxy and 1.5%C60-epoxy groups were damaged as the |Z|0.01Hz values were significantly decreased. Meanwhile, the breakpoint of the phase angle shifted from low frequencies toward intermediate frequencies. On the contrary, the 0.5% and 1.0%C60-epoxy samples exhibited outstanding corrosion protection properties, despite the exposure duration. The impedance values of 0.5% and 1.0%C60-epoxy samples were maintained over 1010 Ω/cm2 after salt, while a straight line was observed in the impedance plot. Thus, these observations confirmed that the coating behaved as intact films to protect the substrate during the exposure, suggesting that the 0.5 and 1.0 wt.% of C60 nanoparticles significantly enhanced the anti-corrosion performance of the epoxy coating, with improved durability as well.(d) C60/epoxy after 100/200 h accelerated durability tests: As shown in To evaluate the wear resistance of the prepared carbon-based nanofiller-reinforced epoxy coatings, the abrasion resistance test was performed, and wear mass loss was measured during the test. To clarify, less mass loss after the test indicates that the coating has stronger wear resistance. As displayed in In general, the CNT/epoxy groups exhibited a remarkable improvement in abrasion resistance in all the tested concentrations, as reduced mass loss was observed in all the tested concentrations. It is apparent that the CNT/epoxy system showed a maximum enhancement with a content of 1.0 wt.%, as the mass loss was reduced by around 40%. The values of the CNT/epoxy composite with 1.5 and 1.0 wt.% were very close, indicating that the 1.0 to 1.5 wt.% content of CNT provided strong reinforcement in the epoxy matrix. However, material degradation was observed at samples with a higher concentration of CNT. One reasonable suggestion is that the degradation is caused by the severe agglomeration of CNT, which was confirmed in the particle size distribution test.An improvement in abrasion resistance was obtained in all prepared C60-reinforced nanocomposites. The maximum improvement in abrasion resistance was obtained in 1.0%C60-epoxy, and the weight of mass loss was 60 mg, which was reduced by 47%. For the composites containing other contents of fullerene-C60, the mass loss was generally higher than that of the coating with 1.0 wt.% of fullerene-C60. However, improved abrasion resistance was observed by comparing to neat epoxy; the mass loss of the other nanocomposites ranged from 80 to 90 mg. Generally, regardless of the types of nanofillers, degradation of wear resistance performance was obtained in the nanocomposites containing a high amount of nanofillers.Compared to other nanofillers, a higher mass loss was observed in the GNP/epoxy coatings, regardless of the concentrations of nanoparticles. Close to the CNT/epoxy and C60/epoxy, a similar tendency was observed in GNP/epoxy, where the lowest mass loss was observed in 1.0 and 1.5 wt.% groups, and yet, the mass loss increased with the higher-content GNP, revealing that material degradation was obtained.SEM images of the abraded surface were utilized to identify the surface properties of the coatings after the Taber abraser test; thus, the neat epoxy and nanocomposite with 1.0 wt.% of nanofillers were used as examples in Studying tensile properties could be used to evaluate the damage tolerance of the nanofiller-reinforced epoxy. The coupon tensile test was utilized to examine the tensile properties of the nanofiller/epoxy composites. The analysis was performed by discussing the tensile strength, maximum strain, and Young’s modulus of specimens, by following ASTM D638.The failure strain of the nanocomposite coating is presented in However, the results of Young’s modulus were different from the max tensile strength and strain see c. The hiThe morphologies of fracture surfaces for specimens under tension are illustrated in mod value started to decrease after 100 h of exposure. The lowest reinforcement effect was found in the CNT/epoxy coating in both short-term and long-term cases.By comparing the EIS data before and after exposure, the highest corrosion resistance was obtained in the fullerene-C60-reinforced epoxy coatings. Strong corrosion resistance was also observed in the GNP/epoxy coating at the fresh stage, but the ZStrong reinforcement of abrasion resistance was obtained with the presence of C60 and CNT nanoparticles, and the mass loss of these two composites was very close. The addition of GNP particles also increased the abrasion resistance, but their reinforcement effects were weaker than those of CNT and fullerene-C60.A significant improvement in adhesion between the coating and substrate was only observed in the CNT/epoxy group.The highest tensile strength was obtained in the C60/epoxy composite, while the GNP/epoxy had the lowest reinforcement, and the CNT/epoxy was in between these two coatings. A substantial improvement in failure strain was only observed in the fullerene-C60/epoxy group, and the highest Young’s modulus was observed in the CNT/epoxy group.The results of this study suggested that the geometric shape of nanofillers plays a vital role in nano-reinforcement, as the linearly shaped CNT results in a strong improvement in mechanical properties, while the 2D nanosheet (GNP) significantly increased the barrier properties. In addition, the spherically shaped nanofiller (fullerene) has the least potential to form agglomeration, which leads to an overall enhancement in nanocomposites. Meanwhile, the addition of 0.5–1.5 wt.% nanofillers will potentially result in the highest improvement in both electrical behaviors and mechanical properties.Epoxy coatings with differently shaped carbon nanofillers  were successfully fabricated and characterized. The tribological, mechanical, and electrical properties were studied to investigate its potential as a high-performance coating for protecting metal structures. Nano-reinforced epoxy coating systems were fabricated with various amounts of nanofillers . Based on the experimental results, the following main findings were summarized:


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by gradual degeneration and elimination of motor neurons (MNs) in the motor cortex, brainstem, and spinal cord. Some familial forms of ALS are caused by genetic mutations in superoxide dismutase 1 (SOD1) but the mechanisms driving MN disease are unclear. Identifying the naturally occurring pathology and understanding how this mutant SOD1 can affect MNs in translationally meaningful ways in a valid and reliable human cell model remains to be established. Here, using CRISPR/Cas9 genome editing system and human induced pluripotent stem cells (iPSCs), we generated highly pure, iPSC-derived MNs with a SOD1-G93A missense mutation. With the wild-type cell line serving as an isogenic control and MNs from a patient-derived iPSC line with an SOD1-A4V mutation as a comparator, we identified pathological phenotypes relevant to ALS. The mutant MNs accumulated misfolded and aggregated forms of SOD1 in cell bodies and processes, including axons. They also developed distinctive axonal pathologies. Mutants had axonal swellings with shorter axon length and less numbers of branch points. Moreover, structural and molecular abnormalities in presynaptic and postsynaptic size and density were found in the mutants. Finally, functional studies with microelectrode array demonstrated that the individual mutant MNs exhibited decreased number of spikes and diminished network bursting, but increased burst duration. Taken together, we identified spontaneous disease phenotypes relevant to ALS in mutant SOD1 MNs from genome-edited and patient-derived iPSCs. Our findings demonstrate that SOD1 mutations in human MNs cause cell-autonomous proteinopathy, axonopathy, synaptic pathology, and aberrant neurotransmission. While mthe body .SOD1 gene , promising sources of vulnerable and disease-relevant cell types, have enabled an opportunity to understand human disease modeling and mechanisms, and to explore human disease-relevant therapeutic development . In addiHere, we hypothesized that introduction of a disease-causing SOD1-G93A mutation to human iPSCs will phenocopy spontaneously occurring human ALS neuropathology after their differentiation into MNs. To test the hypothesis, we utilized CRISPR/Cas9 genome editing technology on iPSCs and introduced a SOD1-G93A missense mutation by performing knock-in into the genome . By combThe institutional biosafety committee (JHU registration B1011021110) approved the use of human cells. The protocols met all ethical and safety standards for work on human cells. All iPSC lines used in this study are listed in Mouse cortical astrocytes were used to enhance attachment of iPSC-derived MNs for subsequent experiments including patch clamp recording. They were isolated from P3 to P4 CD1 mouse pups as described  and cultFAST BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) solution  for 10–20 min at room temperature until the color disclosure. Solution was then removed and the cells were washed with PBS. Following the wash, cells were imaged by microscopy.An alkaline phosphatase stain (Sigma) was used to identify undifferentiated, pluripotent iPSCs . BrieflyMseI restriction enzyme, and H2O (Introduction of SOD1-G93A mutation using CRISPR/Cas9  was done and H2O . This waTaq reaction buffer (NEB), 0.2 μM each primer, 200 μM dNTP, and 1.25 unit of Taq DNA polymerase (NEB). The PCR program started with an initial denaturation step at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 45 s. The final extension step was at 72°C for 2 min. This protocol allowed simultaneous genotyping of all STRs and AMEL in a single PCR amplification. To acquire better resolution for separation of the PCR products, they were loaded onto a 3.5% NuSieve GTG Agarose gels (Lonza). The size of the gels was 14 cm x 12 cm x 1 cm. DNA electrophoresis was performed for 6 h at 110 V and visualized by ethidium bromide staining and ChemiDoc Imager (Bio-Rad).The verification of isogenicity of the mutant and the wild-type iPSC lines after the genome editing was done using short tandem repeat (STR) profiling analysis. Thirteen STP loci  and amelogenin (AMEL) for sex determination were interrogated. Patient-derived iPSCs with an A4V mutation served as a control. Genomic DNA was extracted from all cell lines (Qiagen) according to the manufacturer’s instructions and the STR loci and the amelogenin locus were subsequently amplified using PCR primer pairs which were assessed previously . The 50 For off-target analysis, top seven candidates were selected based on COSMID web tool  The listDirected differentiation of MNs from human iPSCs  was carrTM Cholinergic—SeV Kit . The protocol was used because it is overall more rapid in generating differentiated MNs. This differentiation of human iPSCs into cholinergic neurons is driven by a Sendai virus (SeV)-based vector  which allows forced overexpression of neural inducing factors , after being mixed with human primary astrocytes (Thermo Fisher Scientific). This protocol has been shown to yield cholinergic MNs as identified by islet1 and Hb9 staining in combination with choline acetyltransferase (ChAT) staining . To exclude the possibility of the MEA signals being artifacts, tetrodotoxin (TTX) was used as a negative control to block action potentials. Following baseline recording, vehicle (H2O) and two different concentrations of TTX  were added sequentially to the wells and the activity was recorded for 600 s after each treatment. Additionally, recordings were made on wells with only astrocytes plated to determine if astrocytes are contributing to activities. All the collected data were digitized and analyzed with NeuralMetric Tool and AxIS Metric Plotting Tool (Axion Biosystems).Approximately 60,000–80,000 wild-type control or ALS mutant MNs along with 15,000–20,000 human primary astrocytes in 10 μL differentiation medium were plated on each well of a poly-l-ornithine-coated CytoView MEA 48-well plate. Glia-MN co-culture was used to allow for better uniform dispersal of MNs throughout the cultures for improved global recording within the wells. After 1–3 h of allowing the cells to settle, additional 300 μL of the medium was added to each well and further incubated at 37°C, 5% COin vitro (DIV) 28 were placed in a submersion recording chamber on an upright microscope  fitted for infrared differential interference contrast (IR-DIC) microscopy. The recording chamber was continuously superfused (2–4 ml/min) with artificial cerebrospinal fluid (aCSF) composed of (in mM): 125 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 1 MgSO4, 20 glucose, 2 CaCl2, 0.4 ascorbic acid, 2 pyruvic acid, and 4 L-(+)-lactic acid; pH 7.3, 315 mOsm, continuously bubbled with 95% O2/5% CO2. Neurons were visualized with a digital camera . Glass recording electrodes (2–4 MΩ) were filled with an internal solution containing (in mM): 2.7 KCl, 120 KMeSO4, 9 HEPES, 0.18 EGTA, 4 MgATP, 0.3 NaGTP, 20 phosphocreatine (Na), pH 7.3, 295 mOsm. Cell electrical activities were obtained using a Multiclamp 700B amplifier (Molecular Devices) and an ITC-18 interface unit (Instrutech), both controlled using a customized routine written by Igor Pro (Wavemetrics). Action potentials were evoked by 1 s-long 10–100 pA current injections into cells. Hyperpolarizing responses were investigated by −20 pA current injections into cells. In a subset of current-step test, cell membranes were held at ∼80 mV to see action potential initiation more clearly. All signals were low-pass filtered at 10 kHz and sampled at 20 kHz.Differentiated MN cultures on round coverslips (12 mm diameter) at days 6 cultured MNs for each line were used. Cells were first washed with PBS, collected by using a cell scraper, and harvested by centrifugation for 5 min at 200 g, 4°C. RIPA buffer with protease inhibitors was added to the pellet and the cells were incubated for 20 min on ice. During the incubation, probe sonication was performed briefly for 2–3 times to disrupt cell membranes and homogenize the samples. The lysates were centrifuged for 10 min at 14,000 g, 4°C and the supernatant (RIPA-soluble fraction) was collected. The pellet was washed with PBS and centrifuged again with the same condition. Urea buffer  with protease inhibitors was then added to the pellet. After incubation for 30 min at room temperature, the samples were centrifuged for 10 min at 14,000 g, 4°C and supernatant (Urea-soluble fraction) was collected for SDS-PAGE and western blotting.Approximately 6 × 10MNs spheroids, derived from treatment with small molecules , on day MNs on day 60, a time at which synapses are well developed and prominent , were fiTotal cellular levels of ATP of iPSC-derived MNs were measured using ViaLight Plus kit (Lonza) according to manufacturer’s instruction. Dissociated 20,000 cells were plated in each well of Matrigel-coated 96-well plates. On the day of measurement, cells were first washed with PBS and lysed for 20 min in cell lysis reagent on a shaker to extract ATP. The cell lysates were then transferred to a 96-well clear bottom white polystyrene microplate (Corning) and ATP monitoring reagent plus was added to generate luminescent signal. After 1–2 min, the plate was placed in LMax II luminometer (Molecular Devices) and the ATP levels were measured with SoftMax Pro software (Molecular Devices).Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and washed three times with PBS. Following fixation, cells were permeabilized with 0.2% Triton X-100 for 10 min, blocked in 10% donkey serum in PBS for 1 h, and incubated in primary antibodies overnight at 4°C. Cells were then washed in PBS, incubated with secondary antibodies, and stained with Hoechst 33258 DNA dye for nuclear visualization. See Protein concentrations were first determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and samples were loaded and separated by SDS-PAGE. For RIPA- and UREA-soluble fractions, 25 μg of proteins from RIPA-soluble fractions and equivalent volumes of proteins from UREA-soluble fractions were used. Proteins were transferred onto nitrocellulose membranes, washed with a blocking buffer, and probed with primary antibodies overnight at 4°C. The primary antibodies used are listed in Cells on coverslips were mounted in ProLong Gold Antifade Mountant  and confocal images were taken and analyzed using Leica TCS SP8 microscope and LAS X software . Fluorescence images were obtained by Keyence BZ-X700 fluorescence microscope. For phase-contrast images, Nikon Eclipse TS100 was used.t-test and one-way analysis of variance using GraphPad Prism. All data are represented as mean ± SEM. Statistical significance was considered when p-value was less than 0.05 .Statistical analyses were performed by using Student’s As G93A is the most widely studied SOD1 mutation in animal models including transgenic mice  and A4V SOD1 allele along with a single-stranded donor oligonucleotide (ssODN) (We introduced the SOD1-G93A missense mutation by CRISPR/Cas9 genome editing  into a h (ssODN) . After i (ssODN)  and off- (ssODN) . To furt (ssODN) . As show (ssODN) . As a co (ssODN) . The SOD+ presynaptic puncta and PSD95+ postsynaptic puncta became more prominent in sizes and numbers over time , indicating maturation of synapses in the MN culture  with either SOD1-A4V or SOD1-G93A mutation were positive for a well-characterized misfolded SOD1 antibody . MNs with wild-type SOD1 also showed a low level of reactivity and barely perceptible inclusions , representing basal endogenous misfolding of wild-type SOD1 . Defects in axonal branching were also observed. MNs with SOD1-A4V and SOD1-G93A mutations had significantly fewer axonal branch points when compared to the wild-type . Moreover, SOD1 mutant MNs formed neuritic swellings seen as focal enlargement of processes with a beaded or fragmented morphology .As we observed misfolded and aggregated SOD1 in MN processes including axons, we assessed axonal pathology, which is a common feature of ALS , in the rphology . In addiSOD1A4V+/ MNs was smaller than the wild-type whereas that of SOD1G93A+/ MNs was larger . For the size of postsynaptic puncta labeled by PSD95, however, both SOD1A4V+/ and SOD1G93A+/ MNs showed significantly larger puncta size than the wild-type . The synaptic puncta density (number of puncta per 100 μm of dendritic length), was also quantified. There was a trend for pre- and postsynaptic puncta densities being relatively higher in mutant MN cultures compared to wild-type . We also carefully examined the density of synapses, specifically quantifying sites at which both synapsin and PSD95 colocalized. The numbers of SYN+PSD95+ puncta per 100 μm of dendritic surface were significantly greater in the mutants compared to wild-type MNs .Synaptic dysregulation has been studied as an early pathological event contributing to motor deficits in ALS . We lookild-type , though SOD1A4V+/, SOD1G93A+/, and +/+SOD1 MNs, respectively, p < 0.01, one-way ANOVA) with lower mean firing rates  in the mutants during the recording episode. Network bursting of these MNs was also interrogated. Network bursting, an intermittent and synchronized network-wide bursts of action potentials characterized by brief periods of intense and multiple spikes  and their bursting did not occur as frequently as the wild-type control  that allow repeated and simultaneous extracellular recordings of cells in a non-invasive manner . MNs dife spikes , is espeSOD1A4V+/, SOD1G93A+/, and +/+SOD1 MNs, respectively, p < 0.01, one-way ANOVA), which corresponded to the less number of spikes and lower mean firing rate detected from individual MN pools . Although not reaching significance, the average number of spikes per network burst in the mutants was also higher than the wild-type . To confirm that the signals detected are not due to artifacts, we treated the cells with TTX and recorded the plate. After the baseline recording, cells were treated with vehicle (H2O) and then with two different concentrations of TTX  sequentially. The activities of MNs diminished drastically at 2.5 nM TTX treatment and with 25 nM TTX, no activities were detected, suggesting that the signals are solely from the neuron Na+ channel-driven action potentials (0.0067 ± 0.0013 Hz, and 0.0159 ± 0.0023 Hz for MN pools . These rtentials . In addiWe tested the hypothesis that human iPSCs expressing a heterozygous SOD1-G93A mutation generated by CRISPR/Cas9 genome editing and their differentiation into MNs could be used as a faithful cell system that develops autonomous ALS-relevant pathological phenotypes. The evidence indicates that this hypothesis was correct. The human genome-edited SOD1-G93A MNs, as well as MNs generated from a patient-derived iPSC line with SOD1-A4V, developed robust disease phenotypes including proteinopathy, structural attrition, axonopathy, synaptic pathology, and functional defects.in vivo (The human iPSCs harboring a SOD1-G93A missense mutation were generated by us using CRISPR/Cas9 for the first time . The celin vivo  and in pin vivo . Howeverin vivo . From thAs with primary neural cell cultures , modelinA prominent phenotype discovered in our mutant MNs was misfolded and aggregated SOD1. It was seen diffusely in the cell body and processes, including axons, and as discrete cytoplasmic inclusions . This moSOD1A4V+/ and SOD1G93A+/ MNs develop prominent axonopathies identified as axonal length truncation, smaller number of branch points, and axonal swelling in the mutants when compared to the wild-type MNs (Because we observed misfolded and aggregated SOD1 in MN processes including axons, we examined our cell cultures for axonal pathology, which is a dominant feature of ALS neuropathology . We fountype MNs . These ftype MNs . Also, itype MNs . As axontype MNs , its deftype MNs . Axonopatype MNs . Shortertype MNs . As dismtype MNs , once reper se, the synapse has been receiving great attention as a promising therapeutic target for the disease (Dysregulation of synapses is thought to play a role in ALS . By inte disease .TA315 in mouse cortical neurons also showed elevated levels of GluR1 (+PSD95+synaptic puncta density was higher in the mutants. This finding mirrors results in which glutamatergic excitatory synaptic inputs and dendritic spine densities were increased in presymptomatic TDP-43KQ331 mice, consistent with the concept of excitotoxicity mechanisms in ALS (The larger postsynaptic puncta size was demonstrated by PSD95 which is a major member of the membrane-associated guanylate kinase family and known for regulating glutamate receptors at the synapse. The size and intensity of PSD95 puncta correlates with synaptic development and maturity of glutamatergic excitatory synapses . In iPSCof GluR1 . We alsos in ALS . Our resLastly, our MEA results could be related to defects that we observed in the axons and synapses of the mutant MNs. Network bursting of the mutant MNs has possible relevance in generation of rhythmic motor patterns . CentralALS has aging as a key risk factor, with the symptoms usually developing between the ages of 55–70 . In ParkIn conclusion, our findings demonstrate that genome edited iPSCs using CRISPR/Cas9-mediated targeted gene editing and their differentiation into MNs provide important cellular tools to study mechanisms of disease in human ALS, including proteinopathy, axonopathy, synaptic pathology, and electrophysiological defects. This work can provide needed new insight into human cell-relevant therapeutic targets.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.BK and LM conceived and designed the experiments, analyzed the data, and wrote the manuscript. JR assisted in stem cell maintenance and motor neuron differentiation. YJ took fluorescence images and performed ATP assay. JK conducted whole-cell patch clamp recordings and analyzed the data. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wnt/β-catenin signaling is involved in various biological processes, including the development of the central nervous system. The dysfunction of mitochondria has been shown to participate in the progress of subarachnoid hemorrhage (SAH). Traumatic subarachnoid hemorrhage (tSAH) is a serious complication in acute craniocerebral trauma. Opioids can activate the canonical Wnt/β-catenin signaling pathway. c-Myc, a downstream protein of Wnt/β-catenin signaling, contributes to the fusion of mitochondria. Here, we investigated the protective roles of Propoxyphene (Pro) against Oxyhemoglobin (OxyHb)-induced primary cultured neuron apoptosis. The data indicated that Pro rescued active-β-catenin from OxyHb-induced decline. Furthermore, Pro attenuated OxyHb-induced apoptosis and fission of mitochondria in primary cortical neurons. However, the protective effects were abrogated under active-β-catenin-deficient conditions. Together, the data presented here showed that Pro, a weak opioid analgesic drug, attenuates OxyHb-induced mitochondria-dependent apoptosis in an active-β-catenin-c-Myc-dependent manner. Subarachnoid hemorrhage (SAH), which accounts for 5% of all stroke cases, is a severe neurological disorder with extremely high rates of mortality and morbidity . The cauWnt/β-catenin signaling is involved in various biological processes, including proliferation and apoptosis . The canPropoxyphene (Pro) is an opioid analgesic drug intended for the treatment of mild pain . BecauseMitochondria are vital organelles in eukaryotes and supply energy for all cellular activities . MitochoOpioids can activate the canonical Wnt/β-catenin signaling pathway . c-Myc, in vitro model of SAH, the cortical neurons were exposed to 10 μM OxyHb  based on a previous study . The digested tissues were filtered through a sterile mesh filter and centrifuged at 1000 rpm for 5 min. The cells were cultured with neurobasal medium . The primary cortical neurons were cultured to day 7 for different treatments. To establish the us study .2, Bax, and c-Myc were purchased from Abcam . Anti-mouse-HRP IgG and anti-rabbit -HRP IgG were obtained from Biosynthesis Biotechnology .DMEM and fetal bovine serum (FBS) were purchased from Gibco . Propoxyphene  and MTT (purity≥98%) were obtained from Sigma-Aldrich . Oxyhemoglobin  was obtained from Bomei Biotechnology Co., Ltd. . MitoTracker Red was purchased from Life Technologies . Antibodies to active-β-catenin, actin, and cleaved caspase-8 were purchased from Cell Signaling . Antibodies to β-catenin, BclPrimary rat cortical neurons were seeded in 6-well plates. The cells were then transfected with β-catenin siRNA or negative control siRNA  using Lipofectamine 2000 for 48 h . The finPrimary rat cortical neurons were suspended in lysis buffer after treatment. Equivalent amounts of protein were then loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to immobilon nitrocellulose (NC) membranes . The NC membranes were blocked with 5% BSA in TBST at room temperature for 2 h and incubated with primary antibodies overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The protein bands were detected using a Bio-Rad imaging system .Primary neuron apoptosis was analyzed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Briefly, after being fixed in 4% formaldehyde solution in PBS for 20 min and permeabilized with 0.2% Triton X-100 for 10 min, the cortical neurons were incubated with TUNEL reaction mixture at 37°C for 1 h. After being washed three times with PBS, the slides were incubated with DAPI for 5 min in the dark. Finally, the TUNEL-positive cells were examined with a fluorescence microscope .Cortical neurons were grown on the slides and fixed in 4% formaldehyde solution in PBS for 10 min. After permeabilization with 0.2% Triton X-100 for 30 min and blocking with 5% BSA for 2 h, the primary cortical neurons were incubated with primary antibodies at 4°C overnight. The cells were then washed with PBS three times and labeled with secondary antibodies. The nuclei were labeled with DAPI. Fluorescence images were obtained with a fluorescence microscope .The morphology of mitochondria was detected by MitoTracker Red according to the manufacturer’s instructions. Briefly, cortical neurons were grown on slides and stained for 30 min with 10 nM MitoTracker Red at room temperature. Mitochondria were imaged under a fluorescence microscope .Cell viability was determined by MTT assay. Primary cortical neurons were cultured in 9-well plates. After the designed treatment, the medium was replaced with MTT solution for 2-4 h at 37°C. The MTT solution was then discarded, and 150 μl DMSO was added. Absorbance was read at 490 nm using a microplate reader . Cell viability was calculated using the following formula: cell viability (%) = optical density in test well per experiment/optical density in control well per experiment×100%.All results are expressed as mean ± SEM. Prism 5  was used to analyze the data in our study. Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by Bonferroni test for multiple groups. The value of P < 0.05 was considered to be statistically significant.A cell tSAH model was established by adding OxyHb to the rat cortical neurons. The Western blots indicated that the levels of active-β-catenin were gradually decreased in the rat cortical neurons at 12, 24, and 48 h following OxyHb treatment and that the relative protein level of total β-catenin was stable E. FurthRat cortical neurons exposed to OxyHb were pretreated with 10 μM Pro for 6, 12, 24, and 48 h. The results indicated that the level of active-β-catenin reached a maximum concentration after 24 h of Pro treatment before adding OxyHb compared with cells treated with OxyHb alone and that the level of total β-catenin was stable .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This paper presents an experimental evaluation of the fire resistance of glued-in rod timber joints using epoxy resin, with and without modification. A heat-resistant modified resin was designed by adding inorganic additives into the epoxy resin, aiming to improve the heat resistance. Joints that were made using the modified epoxy resin at room temperature showed a bearing capacity comparable to those with commercial epoxy resin. Twenty-one joint specimens with the modified epoxy resin and six with a commercial epoxy resin were tested in a fire furnace to evaluate the fire resistance. The main failure mode was the pull-out of the rod, which is typical in fire tests of this type of joints. As to the effects of the test parameters, this study considered the effects of adhesive types, sectional sizes, stress levels, and fireproof coatings. The test results showed that the fire resistance period of a joint can be evidently improved by modifying the resin and using the fireproof coating, as the improvements reached 73% and 35%, respectively, compared with the joint specimens with commercial epoxy resin. It was also found that, for all specimens, the fire resistance period decreased with an increase in the stress level and increased with an increase in the sectional sizes. In recent years, timber structures have experienced considerable development throughout the world, owing to their ‘green’ and low-carbon properties. Research on timber structures has become a significant focus worldwide, especially in North America and Europe, and even in China .As is well-known, joints or connections are important aspects of timber structures ,3. AmongMany experimental and theoretical studies on glued-in rebar timber joints have been carried out. Broughton and Hutchinson  investigHence, research on glued-in rod joints at room temperature is comprehensive and detailed. However, considering the importance of the structural behaviour of timber joints under fire conditions, it is extremely urgent to investigate their fire resistance . Some stTo improve the fireproof ratings of timber members, surface impregnation treatment  and encaHowever, only a few research studies have focused on the fire resistance of glued-in rod/rebar joints. Lartigau et al.  studied According to the available literatures, the existing researches on the fire-resistance of glued-in rod joints has mainly focused on those with a traditional and commercial epoxy resin. Only a few heat resistant modified adhesives have been proposed ,45. For 3, respectively, in accordance with EN 13183-1 [The wood species used in this work is Douglas fir glued-laminated (glulam) timber. Its mechanical properties are listed in  13183-1 .The threaded rods adopted in this study were grade 8.8, which were manufactured in accordance with GB/T 15389 . The thrThe adopted adhesives included the heat resistant modified epoxy adhesive proposed by the authors and a type of commercial epoxy resin. The material properties of the commercial epoxy resin, as provided by the manufacturer MKT123  brand, are shown in The modified epoxy adhesive is an innovative epoxy resin created by adding an additive. The additive mainly includes an inorganic adhesive and a refractory filler. Therefore, the modified epoxy adhesive used in this study includes two components: an epoxy resin base material and the inorganic adhesive. The material ratios of each component are listed in To evaluate the improvement of modified epoxy adhesive proposed by the authors in fire resistant performance and the synergistic effect between modified epoxy adhesive and fire-retardant coatings, two types of fire-retardant coatings were employed to the some specimens in fire tests. The adopted fire-retardant coatings include ZN-1 fire-retardant paint and ZOAN200 waterborne fireproof paint.ZN-1 fire-retardant paint was a type of white coating, consisting of inorganic flame-retardant agent, foaming agent, carbonizing agent, synthetic emulsion, titanium dioxide, additives, etc. According to GB 12441 , its firZOAN200 waterborne fireproof paint was a type of colourless coating, which can be applied to the surface of the timber to provide fire protection and retain the grain of the wood. Its flame-retardant performance conforms to grade B1 (difficult-flammability) in accordance with GB 12441 . Its maiDh was equal to 22 mm and represented the hole diameter at the centre of the timber block for the tested end. For all specimens, the supporting ends were designed with a greater capacity when compared with the tested end. For the supporting end, the anchorage length was equal to 20 d, and it was also protected by bulk ceramic fibre in the fire furnace, to ensure the anchorage reliability of the supporting end before the shear-bond failure of the tested end in the fire testing process.As shown in To investigate the bonding strength of the modified epoxy adhesive, pull-out tests were conducted at room temperature. The modified epoxy adhesive and commercial epoxy resin were adopted separately by the pull-pull specimens. Three samples were prepared for each type of adhesive. For all specimens, the anchorage length was 240 mm, whereas the thickness of the glue line was 3 mm. The anchorage length and glue line thickness were consistent with that of the specimens in the fire testing. The results of the pull-out tests at room temperature are depicted in The average bearing capacity for the specimens with traditional commercial epoxy resin (E240-3) is 106.8 kN, whereas that for specimens with the modified epoxy adhesive (M240-3) is 114.2 kN. In addition, two groups of slip curves showed similar slopes denoting that the slip stiffness for the glued-in rod joint specimens with the two types of adhesives adopted has no obvious difference. As shown in For each group of specimens, three duplications were constructed and employed in the fire experiments. The influences of coatings and section dimensions were also investigated. Two types of fire-retardant coatings were employed, including ZN-1 (white coating) and ZOAN200 (colourless coating). In particular, holes of 3 mm in diameter were pre-drilled in designed positions of thermocouples for each specimen, and then the thermocouples were reliably banded into the target depth of the holes. From Fire tests for the glued-in rod timber joints were carried out in a small multipurpose fire-test furnace, as shown in According to the furnace size and specimen installation requirements, a loading steel frame was used to apply a constant load during the fire experiments. The supporting end of each specimen was anchored on the steel frame by bolts, and the tested end was connected to the load jack. The designed shape and dimensions of the loading steel frame are illustrated in Clamp the specimen in the furnace, and cover the supporting end with fireproof cotton.Install the thermocouples, the loading system, and the guyed displacement meters.Apply the load at the designed stress level for more than 10 min.Ignite, and then maintain the load level until failure.Shut down the burners quickly after failure.Halt the wood charring by spraying water onto the specimens.To measure the relative slip between the rod and timber when a specimen was exposed to fire, a guyed displacement meter was adopted in the tests, and was firmly fixed on the tested rod. The specimen was deemed a failure when a target value for a certain stress level could not be maintained, or when an abrupt excess displacement occurred. The charring rate was calculated on the basis of experimental data from fire tests. The procedure for conducting the fire-resistance test for the glued-in rod timber joint was as follows:The failure time of fire tests for glued-in rod joints are presented in At the stress level of 30%, when the modified epoxy resin was used for group Z160-3, the average failure time reached 64 min. As a comparison, the average failure time was 37 min for group S160-3 with a traditional epoxy resin, indicating an improvement of 73.0%.Comparing group Z160-2 with group Z160-5, the failure time declined from 70 min to 57 min, whereas the stress level increased from 20% to 50%.At the stress level of 30%, the introduction of the white coating showed improvements of the fire resistance period by 54.1% and 20.3% for the traditional and modified epoxy resins, respectively.As the joint edge distance changed from 5 d (see Group Z160-3) to 7.5 d (see Group Z240-3), the failure time increased from 64 min to 83 min, i.e., an improvement of 29.7%.It can be seen from Notably, for the specimens Z200-3-3 and SF160-3-3, an unanticipated breakage of the rod occurred. Therefore, they were not considered when calculating the average values of the fire resistance period within those groups.The glued-in rod joint specimens mainly failed as a pull-out failure of the rod, owing to the high temperature at the rod/adhesive interface. During the first 20 min, the relative slip and inner temperature rose slowly. Then, the temperature increased moderately over time, with slight fluctuations. The timber members began to burn with the evaporation of the moisture in the specimens. Once the interfacial temperature reached a certain value, a large slip immediately appeared.As shown in The relative slips at the loading end were recorded, and the slip-time curves are presented in With the increase of the distance from the loading end, the measured temperature decreased gradually; this is mainly owing to the heat transfer of the glued-in rod. Therefore, the fire protection and thermal insulation of exposed metal components, such as exposed rods in glued-in rod timber joints, is extremely significant in practical engineering, due to the relatively large thermal conductivity of metal components National Design Specifications (NDS) for wood construction (NDS-2018)  also staIt can be seen from The edge distance is changed by designing different sectional sizes, as shown in The temperature curves for different fireproof coating conditions are illustrated in As far as mechanical behaviours are concerned, there is little difference between a glued-in rod timber joint with the heat-resistant modified epoxy resin and that with the commercial epoxy resin at room temperature.The fire resistance period of the specimens with the modified epoxy resin can be improved by 73.0% when compared with the specimens with the commercial epoxy resin.The fire resistance time declined to 57 min from 70 min, while the loading level in fire tests increased to 50% from 20%. The loading levels sustained by the stress component should be emphasized.When the specimens were protected by a white coating, the fire resistance period increased by 54.1% and 20.3% for the traditional and modified epoxy resin cases, respectively.As the joint edge distance changed from 5d to 7.5d, the failure time increased from 64 min to 83 min, an improvement of 29.7%.Under the same load level and sectional size, the specimens in group ZF160-3, which adopted the modified epoxy adhesive and white fire coating, showed the best fire resistance performance.In this work, an innovative modified fireproof epoxy resin adhesive was proposed, based on introducing some inorganic additives. Through tests both at room temperature and in fire, the effects on the fire resistance of joints from several influential factors were analysed in detail. The main conclusions of this study are summarised as follows: In conclusion, the modified epoxy adhesive showed the significant improvement in fire resistance compared with the ordinary commercial epoxy resin, without any decline in mechanical performance at room temperatures. Therefore, the modified epoxy adhesive proposed in this article exhibits superior application prospect in glued-in rod timber joints thanks to the advantage of heat resistant.Further investigations will focus on the theoretical analysis about determining the temperature tendency of adhesive based on the charring rate and thermal conductivity of timber, as well as the stiffness reduction factor of glued-in rod joints with different adhesives in fires. Thus, the relationship among fire time, adhesive temperature, and joint stiffness can be established to guide the fire design of glued-in rods joints of timber structures.