Sex-determining region Y-box2 (SOX2), a master regulator of embryonic and induced pluripotent stem cells, drives cancer stem cells (CSCs) properties, fuels tumor initiation, and contributes to tumor aggressiveness. Our previous study has demonstrated the oncogenic role of SOX2 in colorectal cancer (CRC). In this study, we sought to elucidate the underlying mechanisms. Cell function experiments were performed to detect chemoresistance, proliferation, stemness, migration, and invasion in vitro. Chromatin immunoprecipitation, co-immunoprecipitation, luciferase reporter assay, and immunofluorescence were performed to explore the regulation of ABCC2, β-catenin, and Beclin1 by SOX2. The carcinogenic role of SOX2-β-catenin/Beclin1-ABCC2 axis in vivo was analyzed by CRC tissues and xenograft models. Here, we reported that SOX2 sustained chemoresistance by transcriptional activation of ABCC2 expression. Suppressing either β-catenin or autophagy signaling curbed SOX2-driven chemoresistance, stemness, and epithelial–mesenchymal transition (EMT). Mechanistically, SOX2 combined with β-catenin and increased its nuclear expression and transcriptional activity. Transcriptional activation of Beclin1 expression by SOX2 consequently activating autophagy and inducing malignant phenotype. Furthermore, overexpression of β-catenin or Beclin1 facilitated ABCC2 expression. The clinical analyses showed that high expression of ABCC2 and Beclin1 were positively correlated with SOX2 and were associated with poor prognosis in CRC patients. Finally, xenograft models revealed that inhibition of SOX2 expression and autophagy restrained tumor growth and chemoresistance in vivo. Conclusively, we demonstrated a novel mechanism by which the SOX2-β-catenin/Beclin1/autophagy signaling axis regulates chemoresistance, stemness, and EMT in CRC. Our findings provide novel insights into CRC carcinogenesis and may help develop potential therapeutic candidates for CRC. Cancer stem cells (CSCs) represent a minor subpopulation of the tumor bulk that exhibits self-renewal capacity, induces therapy resistance, and initiates metastasis, thereby promoting tumor progression and recurrence3. Sex-determining region Y-box2 (SOX2), a master regulator of embryonic and induced pluripotent stem cells (iPSCs), drives cancer stemness, fuels tumor initiation, and contributes to tumor aggressiveness4. In our recent study, we found that SOX2 promotes chemoresistance, confers CSCs properties, and promotes epithelial–mesenchymal transition (EMT) in colorectal cancer (CRC)5. However, the underlying mechanism of this phenomenon is not well-characterized. Elucidation of the underlying molecular mechanism may help identify novel diagnostic biomarkers and facilitate the development of effective therapeutic interventions against CRC.A large body of evidence indicates that human cancer can be categorized as a stem cell disease9. Several ABC transporters, including ABCB1, ABCC2, ABCG2, and ABCB5, have been identified in CSCs-driven chemoresistance13. Importantly, SOX2 has been implicated in the acquisition of chemoresistance by upregulating the expression of ABC transporters in glioma cells and gastric cancer cells15. However, the role of ABC transporters in SOX2-induced chemoresistance is poorly understood in CRC.It’s reported that ATP-binding cassette transporters (ABC transporters) serve as efflux pumps that extrude anti-cancer drugs from the cancer cells16. SOX2 has been reported to promote EMT in laryngeal cancer and to contribute to tamoxifen resistance in breast cancer through the WNT/β-catenin pathway18. Therefore, we speculated that SOX2 may promote malignant phenotypes via the β-catenin signaling pathway in CRC.Aberrant WNT/β-catenin signaling plays an essential role in the progression of CRC21. Furthermore, SOX2 has been shown to repress mTOR expression and promote cellular reprogramming through induction of autophagy in iPSCs; ectopic SOX2 expression promoted autophagosome formation and autophagic flux in head and neck squamous cell carcinoma23. However, the involvement of autophagy in SOX2-mediated malignant phenotypes in CRC is not well-characterized. Besides, autophagy has been shown to participate in the degradation of β-catenin or to activate the WNT/β-catenin pathway25. The cross-talk between autophagy and β-catenin in SOX2-induced malignant phenotypes in CRC deserves further exploration.Autophagy, which is an essential cellular mechanism for the degradation of cytoplasmic proteins and organelles and the recycling of their components, has been reported to induce resistance against chemotherapy-driven cytotoxicity via maintaining the survival of CSCs through conferring stress tolerance and limiting the damage; autophagy can regulate cell migration through selective degradation of focal adhesion proteinsIn this study, we aimed to investigate the mechanism of how SOX2 induces chemoresistance, CSCs properties, and EMT in CRC. Our findings may provide new insights into CRC carcinogenesis and help identify autophagy-related therapeutic targets for CRC.2 atmosphere at 37°C. Human embryonal kidney 293 cells (HEK293T) were preserved at the Nanfang Hospital, Southern Medical University  and cultivated in a DMEM  medium supplemented with 10% FBS. SN38 , 5-Fu , and oxaliplatin  were used at a concentration of 4 µM, 15 µg/mL, and 60 µM, respectively. Rapamycin  (50 nM) was used to activate autophagy, while 3-methyladenine  (10 mM) was used to block autophagy.Human CRC SW480 and SW620 cell lines were obtained from the American Type Culture Collection (ATCC) and authenticated according to the recommendations of ATCC and grown in RPMI 1640  containing 10% fetal bovine serum  in a humidified 5% COThe SOX2, ABCC2, β-catenin, or Beclin1 construction were generated by sub-cloning PCR-amplified full-length human SOX2 (NM_003106), ABCC2 (NM_000392.5), β-catenin (NM_001098210.2) or Beclin1 (NM_001313998.2) cDNA into plasmid Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin, Ubi-MCS-3FLAG-SV40-Cherry-IRES-neomycin, Ubi-MCS-3FLAG-SV40-Cherry-IRES-neomycin or pLOV-EF1a-mcherry-P2A-Puro-CMV, respectively. To knock down endogenous SOX2 or ABCC2, 2 short hairpin RNA (shRNA)  were seeded in each well of a 6-well plate. Upon attainment of 90–100% confluence, the cell monolayer was scratched in a straight line using a sterile pipette tip, followed by careful washing to remove the detached cells. Subsequently, the cells were cultured at 37 °C with 5% CO2. Photographs of the scratch wound in different samples were recorded at 0 and 48 h.CRC cells , and the purified DNA fragments were subjected to PCR amplification set, as previously describedNuclear and cytoplasmic proteins of SW480 and SW620 cells were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit , according to the manufacturer’s protocol. For Western blot analyses, GAPDH and Histone were used as cytoplasmic and nuclear control markers, respectively.2 μg of the anti-SOX2  or anti-β-catenin antibody  was added to 1 mg of the protein lysates and rotated overnight at 4 °C. An equal amount of protein lysates incubated with a non-specific IgG antibody was used as the negative control. An equal amount of protein lysate was used as the positive control. The immune complexes were precipitated by protein A/G beads  overnight at 4 °C. Beads with extracted proteins were washed 3 times with cold PBS, followed by CelLytic M lysis buffer 1 time . The bound proteins were eluted by boiling at 95 °C for 5 min with 20 μL of SDS protein buffer and then processed for Western blot.The cells grown on coverslips in a 24-well plate were washed twice with cold PBS, and then fixed with 4% paraformaldehyde for 15 min at room temperature (RT). Subsequently, the cells were permeabilized with 0.1% Triton X-100 for 5 min on ice, and stained with following antibodies: SOX2 , β-catenin , anti-mouse IgG/PE  and anti-rabbit IgG/FITC . Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI) dye. Images were obtained with laser scanning confocal microscopy . The cells were transiently transfected with mRFP-GFP-LC3B adenovirus  for 6 h; subsequently, the number of LC3B puncta was analyzed by confocal microscopy .The ABCC2 promoter reporter (Guangzhou Kidan BioTechnology Co. Ltd.), Beclin1 promoter reporter (Guangzhou Kidan BioTechnology Co. Ltd), or TOP/FOP Flash reporter containing TCF/LEF1 binding sites (GeneCopoeia) was introduced into cultured cells using Lipofectamine 3000 , according to the manufacturer’s instructions. After 48 h, cells were lysed, and luciferase activity was assessed with Dual Luciferase Reporter Assay Kit , as per the manufacturer’s instructions.CRC cells were cultured in 10-cm-dishes for 24 h and fixed in 2.5% glutaraldehyde in 100 mM phosphate buffer for 8 h at 25 °C, and re-fixed in 1% osmic acid for 3 h at 4 °C. Subsequently, the cells were embedded in Spurr’s resin , sectioned, doubly stained with uranyl acetate and lead citrate, and analyzed using a transmission electron microscope .28.Tissue microarray with 90 pairs of CRC tissues (Shanghai Outdo Biotech Co. Ltd.) were used to assess the expression of SOX2, ABCC2, and Beclin1 using immunohistochemistry (IHC). The intensity of staining of malignant cells was scored as follows to analyze the levels of protein expression: + (no staining), ++ (weak staining), +++ (moderate staining), and ++++ (strong staining). An intensity score greater than + + was classified as high expression, whereas a score of less than or equal to ++ was considered as low expression, as described elsewhere6 cells in 0.1 mL PBS) were subcutaneously injected into the right dorsal flanks of 4-week-old female Balb/c nude mice (n = 3/group), respectively. Twenty-two days later, mice bearing tumors were administered intraperitoneal injection of PBS, Chloroquine , irinotecan , or combination of irinotecan and CQ every 3 days. Tumor size was measured with a digital caliper every 3 days. Tumor volume was calculated from digital caliper measurements of tumor dimensions in mm using the formula for a prolate ellipsoid: (L × W2)/2, where L is the longer of the 2 measurements. At the endpoint, tumors were harvested and weighed. The excised tissues were either fixed in 10% neutral-buffered formalin or snap frozen in liquid nitrogen. Tumor sections from paraffin-embedded blocks were used for histologic examination. All animal experiments were conducted as per the protocol approved by the Animal Care and Use Committee of the Southern Medical University.SW620 cells stably transfected with SOX2 short hairpin RNA (shRNA) or negative shRNA  indicated that there was a positive correlation between SOX2 high expression and ABC transporters signatures . The results showed that there was a potential SOX2-binding site in the promoter of ABCC2   expression; in contrast, SOX2 knockdown in SW620 cells had the opposite effect  Fig. . Immunofg/) Fig. . Moreoveg/) Fig. . These rect Fig. . Upregulect Fig. . Moreoveect Fig. .These findings demonstrated that SOX2 combined with β-catenin increased β-catenin nuclear expression and transcriptional activity in CRC. Moreover, as a transcriptional partner, β-catenin acted in synergy with SOX2 in the transcriptional regulation of ABCC2 in CRC.n = 465, Supplementary Fig. GSEA plot indicated that “GO_REGULATION_OF_AUTOPHAGY” and “GO_ POSITIVE_REGULATION_OF_AUTOPHAGY” gene signatures enriched in the SOX2 high expression group, suggesting the interaction between SOX2 and autophagy  was marked upregulation in SW620 cells than that in SW480 cells  Fig. . ChIP asRecent studies have reported that autophagy bilaterally regulates β-catenin. Thus, it’s necessary to explore the relationship between autophagy and β-catenin in CRC. However, we found no distinct effect of autophagy on β-catenin expression and transcriptional activity in SW480 and SW620 cells, as well as in SW480 and SW620 cells transfected with SOX2 clone or SOX2 shRNA Fig. . BesidesIn general, these results revealed that SOX2 activated autophagy by transcriptional promoting Beclin1 expression. Moreover, autophagy and β-catenin signaling pathways were not found to interfere with each other in SOX2-induced malignant phenotypes in CRC.P < 0.05), and Beclin1 overexpression in clinical samples was associated with TNM stage (AJCC) (Supplementary Table P < 0.05). The survival duration of patients with high ABCC2 or Beclin1 expression was significantly shorter than that of patients with low expression  than mice injected with SOX2-silenced SW620 cells (~300 mm3)  Fig. , indicatm3) Fig. . These rm3) Fig. . IHC stam3) Fig. .Fig. 7KnIn summary, these results suggested that SOX2-β-catenin/Beclin1/autophagy signaling axis plays an essential role in CRC growth and chemoresistance in vivo.31. Our previous study has demonstrated that SOX2 promotes chemoresistance, CSCs properties, and EMT in CRC5. In this study, we sought to elucidate the underlying mechanism by which SOX2 regulates malignant phenotypes in CRC. Our findings demonstrated that SOX2-β-catenin/Beclin1/autophagy signaling axis is crucial for chemoresistance, CSCs properties, and EMT in CRC, which may help develop potential therapeutic candidates for CRC treatment.The persistence of CSCs promotes ABC transporters expression and induces EMT, resulting in chemoresistance, metastasis, and poor prognosis in CRC32. Resistance to anti-tumor therapy is associated with SOX2-mediated activation of ABC transporters, which are able to efflux drugs across the cell membrane by utilizing ATP14. However, the role of ABC transporters in SOX2-induced chemoresistance in CRC is poorly understood. Our findings indicated that ABCC2 is a direct functional target of SOX2 in CRC. SOX2 promotes chemoresistance by transcriptionally upregulating ABCC2 expression, as well as via β-catenin and autophagy signaling, respectively.SOX2 is credited with catalyzing pro-survival and anti-apoptotic signaling in a diverse range of cancers, which ultimately lead to therapeutic resistance and clinical relapse16. Consistently, we found that β-catenin serves as an oncogenic factor in CRC. Moreover, β-catenin knockdown partly abrogated the activation of chemoresistance, CSCs properties, and EMT in SOX2-overexpressing SW480 cells; in contrast, β-catenin upregulation partly eliminates the inhibition of chemoresistance, CSCs properties, and EMT in SOX2-silencing SW620 cells. These results indicated the involvement of β-catenin in SOX2-mediated malignant phenotypes in CRC. Subsequently, we investigated the mechanism of how SOX2 regulates β-catenin in CRC. A previous study indicated that β-catenin is a transcription partner for SOX2, and that the combination of SOX2 with β-catenin promotes the transcriptional activity of each other in breast cancer33. Besides, SOX2 has been shown to bind to the promoter of β-catenin to promote the expression of β-catenin34. Similarly, we found that SOX2 combines with β-catenin and increases β-catenin total expression, nuclear accumulation, and transcriptional activity in CRC. Additionally, as a transcriptional partner, β-catenin acts in synergy with SOX2 in the transcriptional regulation of ABCC2 in CRC.Aberrant β-catenin signaling plays an essential role in the progression of CRC35. Emerging evidence indicates that autophagy acts as a “double-edged sword” in the context of cancer. On the one hand, autophagy suppresses tumorigenesis by clearing the damaged proteins and organelles, and perhaps by maintaining energy homeostasis through intracellular recycling; this ultimately decreases the accumulation of deleterious mutations that drives tumorigenesis38. On the other hand, autophagy confers tumor cells with superior stress tolerance that limits damage, maintains viability, sustains dormancy, and facilitates recovery40. Herein, we showed that autophagy activation promotes chemoresistance, proliferation, stemness, and metastasis in CRC cells. Most importantly, SOX2-induced chemoresistance, CSCs properties, and EMT were partially diminished by inhibition of autophagy, which suggested the involvement of autophagy in SOX2-induced malignant phenotypes in CRC. Subsequently, we investigated the mechanism of how SOX2 regulates autophagy. Autophagy is a complex metabolic process regulated by multiple autophagy-related genes. BECN1 is the first identified mammalian autophagy-mediated gene involved in the formation of autophagosomes41. In this study, SOX2 was found to transcriptionally induce Beclin1 expression and activate autophagy in CRC. Recent studies have reported that autophagy participates in the degradation of β-catenin or activation of the WNT/β-catenin pathway25. The inconsistent cross-talk between autophagy and β-catenin may be attributable to carcinoma heterogeneity. Herein, we showed that autophagy and β-catenin signaling pathways do not interfere with each other in SOX2-mediated malignant phenotypes. In other words, SOX2 can promote chemoresistance, CSCs properties, and EMT in CRC via β-catenin and autophagy signaling, respectively.Autophagy is a highly regulated process aimed at the degradation and recycling of cellular components43. Consistently, we found upregulation of SOX2 in CRC tissues compared to the adjacent normal tissues5. ABCC2 is expressed at a high level in tamoxifen-resistant breast cancer, and ABCC2 upregulation suggests a poor prognosis in pancreatic cancer45. Similarly, we found elevated expression of ABCC2 in CRC tissues. However, different studies have shown conflicting roles of Beclin1 in tumors. Decreased expression of Beclin1 is frequently found in human breast, ovarian, and prostate cancers47, while 95% of colon cancer tissues have high expression of Beclin149. This discrepancy is largely attributable to the differences in the cancer cells and tumor models used in various studies. In this study, CRC tissues analysis showed significantly increased expression of Beclin1. Furthermore, ABCC2 or Beclin1 is positively correlated with SOX2 expression and associated with TNM stage (AJCC), predicting poor prognosis in CRC patients. Besides, we verified the effect of SOX2-β-catenin/Beclin1/autophagy signaling-ABCC2 axis in tumor growth and chemoresistance in vivo via mouse models. CQ is a well-established autophagy inhibitor. Our results demonstrated that the combination of irinotecan and CQ therapy increased tumor chemosensitivity to irinotecan. Moreover, the chemotherapy effect of irinotecan combined with CQ treatment on the tumor in SW620 SOX2 KD group is better than that in the SW620 control group, suggesting that SOX2 knockdown may increase tumor chemosensitivity in vivo.According to our in vitro findings, we further assessed the IHC expression of SOX2, ABCC2, and Beclin1 in a microarray with 90 pairs of clinical CRC tissues. Elevated SOX2 expression has been reported in many cancers, such as osteosarcoma, lung and esophageal squamous cell carcinomaIn summary, we demonstrate a novel mechanism whereby the SOX2-β-catenin/Beclin1/autophagy signaling axis regulates chemoresistance, CSC properties, and EMT in CRC Fig. . HopefulSupplementary Figures and Figure LegendsSupplementary Tables


CTNNB1, which encodes β-catenin, and loss-of-function mutation of AXIN1 occur in approximately 35% of human HCC samples. Human HCCs with activation of the Wnt/β-catenin pathway demonstrate unique gene expression patterns and pathological features. Activated Wnt/β-catenin synergizes with multiple signaling cascades to drive HCC formation, and it functions through its downstream effectors. Therefore, strategies targeting Wnt/β-catenin have been pursued as possible therapeutics against HCC. Here, we review the genetic alterations and oncogenic roles of aberrant Wnt/β-catenin signaling during hepatocarcinogenesis. In addition, we discuss the implication of this pathway in HCC diagnosis, classification, and personalized treatment.Deregulated Wnt/β-catenin signaling is one of the main genetic alterations in human hepatocellular carcinoma (HCC). Comprehensive genomic analyses have revealed that gain-of-function mutation of  Hepatocellular carcinoma (HCC) is the most common form of liver cancer, representing the third leading cause of cancer-related death . The incThe Wnt/β-catenin cascade is one of the major signaling pathways regulating liver homeostasis, regeneration, and tumorigenesis , which hCTNNB1, which encodes β-catenin, occur in 27% of HCC patients  staining in these cells , 14. In patients . Most CTactivity . Studiesactivity . Mutatioectively . APC andpressors . Importa and OAT .SFRP1), a Wnt/β-catenin antagonist  and solitary lesions. Typical, albeit non-pathognomonic, microscopic features of CTNNB1-mutated HCCs are microtrabecular and acinar growth, a high degree of differentiation (Edmondson grade G1–G2), homogeneous microscopic appearance, prominent cholestasis, and lack of steatosis and inflammation. Interestingly, they showed that robust and uniform immunohistochemical expression of glutamine synthetase (GS), a target of the Wnt/β-catenin pathway, was more sensitive (90%) than cytoplasmic/nuclear β-catenin positivity (63%) in identifying CTNNB1-mutated HCCs, though with equal specificity (both 98%). Indeed, based on TCGA analysis, the upregulation of GLUL, which encodes GS, and other canonical Wnt/β-catenin target genes is strongly associated with CTNNB1 mutation status in HCC  in the mouse liver triggers HCC formation  or tankyrase (TNKS) have also been developed to block Wnt/β-catenin signaling in cancer cells. PORCN is an ecretion . The PORecretion , and it ecretion . TNKS taecretion . Severalecretion –132. Preecretion . HoweverCtnnb1-mutant mouse HCC model induced by diethylnitrosamine (DEN) and phenobarbital, use of locked nucleic acid (LNA) antisense oligonucleotides against β-catenin strongly impaired HCC progression  AXIN1 mutations promote canonical Wnt pathway activation in HCC  and TBX3, tracks strongly with GOF CTNNB1 mutations in human HCC samples (AXIN1 or CTNNB1 mutations? Most studies so far have relied on HCC cell lines (AXIN1 or CTNNB1 mutations.Based on the published data and the recent genomic studies, such as TCGA analysis, it is clear that Wnt ligands and their receptors are frequently upregulated in human HCC samples. However, one can also clearly see that high expression of canonical Wnt target genes, including  samples . Therefoll lines , 145. Holl lines  or Kupffll lines . The celCtnnb1 mutations are sensitive to mTOR inhibition (CTNNB1 mutations might be helpful to demonstrate the clinical efficacy of this drug. Furthermore, everolimus is a first-generation and partial mTORC1 inhibitor. The second-generation mTOR inhibitors, including mTORC1/mTORC2 inhibitors and mTOR/PI3K inhibitors, might have improved efficacy against HCCs with GOF CTNNB1 mutations (As we discussed above, activated β-catenin leads to mTORC1 activation, and mouse HCCs with GOF hibition . On the hibition . Howeverhibition , includihibition ; in addiutations . AdditioCTNNB1 mutations, especially point mutations, are attractive targets for such a gene editing approach for cancer treatment. One can imagine delivering the proper guide RNA into HCC cells and reversing the mutant form of the CTNNB1 allele into the wild-type sequence. However, small molecules directly targeting Wnt/β-catenin are frequently associated with significant gastrointestinal toxicity, as Wnt/β-catenin is necessary for intestinal stem cell renewal and proliferation. This toxicity substantially limits the clinical application of these small molecules. The gene editing approach has the advantage of not affecting the Wnt/β-catenin pathway in any other cells besides HCC cells that harbor the CTNNB1 mutations. However, we do not know whether conversion into the wild-type CTNNB1 sequence will lead to HCC regression, since wild-type β-catenin may be sufficient to support HCC progression. In addition, an efficient delivery method so that the guide RNAs can target all HCC cells should be developed.Recent progress with CRISPR/Cas9–based gene editing technology opens the door to genetic modification of tumor cells. GOF As we discussed above, animal studies have demonstrated that the activation of Wnt/β-catenin alone is insufficient to promote HCC development. Instead, a second oncogenic signal is required for liver tumor formation . TherefoIn summary, in the era of precision medicine, we can readily detect HCCs harboring activated Wnt/β-catenin signaling. These HCCs have peculiar molecular and pathological features and might be treated with effective and specific targeted therapies. However, our understanding of how the Wnt/β-catenin pathway contributes to HCC molecular pathogenesis remains incomplete. Therefore, additional molecular and biochemical studies are required to investigate this vital issue to identify novel targeted therapies against HCC with aberrant Wnt/β-catenin activation.


Furthermore, pharmacological inhibitors of this pathway generated a robust antiviral state against multiple laboratory and clinical strains of HSV-1. Collectively, our findings assign a novel regulatory role to HPSE as a driver of β-catenin signaling in HSV-1 infection.Under pathological conditions like herpes simplex virus 1 (HSV-1) infection, host-pathogen interactions lead to major reconstruction of the host protein network, which contributes to the dysregulation of signaling pathways and disease onset. Of note is the upregulation of a multifunctional host protein, heparanase (HPSE), following infection, which serves as a mediator in HSV-1 replication. In this study, we identify a novel function of HPSE and highlight it as a key regulator of β-catenin signal transduction. The regulatory role of HPSE on the activation, nuclear translocation, and signal transduction of β-catenin disrupts cellular homeostasis and establishes a pathogenic environment that promotes viral replication. Under normal physiological conditions, β-catenin is bound to a group of proteins, referred to as the destruction complex, and targeted for ubiquitination and, ultimately, degradation. We show that virus-induced upregulation of HPSE leads to the activation of Akt and subsequent stabilization and activation of β-catenin through (i) the release of β-catenin from the destruction complex, and (ii) direct phosphorylation of β-catenin at Ser552. This study also provides an in-depth characterization of the proviral role of β-catenin signaling during HSV-1 replication using physiologically relevant cell lines and  Herpes simplex virus 1 (HSV-1) commonly infects roughly 3 billion people worldwide and is the leading cause of infection-related blindness in the developed world. HSV-1 infection induces a chronic immune-inflammatory response resulting in corneal scarring, thinning, and neovascularization. This leads to development of various ocular diseases such as herpes stromal keratitis. HSV-1 can also invade the central nervous system and lead to encephalitis, a relatively common cause of sporadic fetal encephalitis worldwide , a key mediator in HSV-1 replication. HPSE is the only known mammalian enzyme that can degrade heparan sulfate, an essential polysaccharide in the extracellular matrix. Our previous work highlights HPSE as a key driver of HSV pathogenesis primarily through the degradation of HS and promotion of virion release and viral spread –5. HPSE –6–More recently, we demonstrated the multifunctional properties of HPSE and established important connections between its function and cellular proliferation and defense in response to viral infection . CuriousUnder normal physiological conditions, β-catenin is either bound to the intracellular domain of E-cadherin to maintain structural integrity of the cell or is recognized by the destruction complex and targeted for degradation . Wnt proWe had recently reported that β-catenin is a driver of proviral signaling and that inhibition of β-catenin resulted in robust antiviral activity against HSV-1 infection . HoweverFinally, we delve into the therapeutic potential of targeting the β-catenin pathway. We show the robust antiviral properties of PRI-724, iCRT14, and KYA1797k in multiple cell lines and against various laboratory and clinical strains of HSV-1. With drugs targeting the β-catenin signal transduction pathway for therapeutic intervention already undergoing clinical trials for diseases like cancer and hepatitis B virus (HBV), there is great potential for its use as an antiviral against herpes infection –19.–in vitro infection with the clinical isolates resulted in increased protein levels of p-GSK-3β (Ser9) and p-β-catenin (Ser33/37/Thr41) over the time course of infection  Western blot analysis of HEK293 cells (A) and MEFs (B) infected with of HSV-1 KOS strain at MOI of 0.1 and collected at the indicated time points. (C) Western blot analysis of HCE cells infected with indicated time points of HSV-1 clinical strain U or B at MOI of 0.1. (D) Confocal immunofluorescence microscopy of HSV-1 in mouse corneal sections following 1 and 2 dpi with HSV-1 McKrae strain at 4 × 10Copyright © 2021 Koujah et al.2021Koujah et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro, we sought to confirm these findings in vivo. C57BL/6 mice were infected with 4 × 105 PFU of HSV-1 McKrae strain, and eyes were collected and fixed at 1 and 2 days postinfection (dpi). Starting at 1 dpi, upregulation of β-catenin protein was observed compared to mock-infected corneas. By 2 dpi, significant upregulation of β-catenin was observed, along with nuclear translocation in the superficial-most layer of the corneal epithelium, where tight and adherens junctions are present to provide a permeability barrier between cells . Thi. Thi5). 10.1128/mBio.02792-21.2FIG S25 PFU/ml. Download FIG S2, PDF file, 2.1 MB.Hpse-KO cells have endogenously lower levels of β-catenin protein expression. (A) Confocal immunofluorescence microscopy of WT and Hpse-KO MEFs stained for β-catenin. (B) Confocal immunofluorescence microscopy of β-catenin in Hpse-KO mouse corneal sections following 1 and 2 dpi with HSV-1 McKrae strain at 4 × 10Copyright © 2021 Koujah et al.2021Koujah et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The HPSE gene encodes a proenzyme whose activation involved proteolytic cleavage by cathepsin L CatL), yielding an enzymatically active heterodimer . To undeatL, yielAfter observing the direct role of HPSE on regulating β-catenin activity through phosphorylation of upstream targets, we sought to verify these findings by examining the phosphorylation of GSK-3β in wild-type and Hpse-KO MEFs. Following infection with a KOS HSV-1 strain with an MOI of 0.1, a significant increase in phosphorylation of GSK-3β was observed in the wild-type cells. However, the opposite trend was observed in HPSE knockout cells where the expression level of p-GSK-3β (Ser9) was reduced following infection . In orde10.1128/mBio.02792-21.3FIG S3FIG S3, PDF file, 1.9 MB.CHIR-99021 enhances β-catenin nuclear localization. (A) Confocal immunofluorescence microscopy of cells treated with CHIR-99021 for 24 h prior to HSV-1 infection with MOI of 0.1 of HSV-1 infection in HCE cells. Download Copyright © 2021 Koujah et al.2021Koujah et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the After establishing a key role for HPSE in phosphorylation of upstream targets of β-catenin, Akt, and GSK-3β, we next explored the downstream repercussions of HPSE activity on phosphorylation of β-catenin at Ser552. Previous findings have indicated that phosphorylation of β-catenin at Ser552 by Akt promotes β-catenin transcriptional activity. Prior reports have also shown that HPSE is involved in the induction of phosphorylation of Akt. To investigate this relationship further, wild-type and Hpse-KO cells were treated for 24 h with independent or combinatorial administration of lithium chloride (LiCl), an inhibitor of GSK-3β, or Wnt3a-conditioned medium (Wnt3a-CM) . As expeTo further understand the consequences of HPSE regulation of β-catenin activation on HSV-1 infection, wild-type and Hpse-KO cells were primed with Wnt3a-CM for 12 or 24 h prior to infection with a KOS strain with an MOI of 0.1 for 24 h . Treatmein vitro, in vivo, and in humans and show limited adverse effects when used at the therapeutic concentrations used in this study  Immunofluorescence micrographs of HCE cells infected with HSV-1 K26-GFP at MOI of 0.1 and therapeutically treated with the indicated concentration of mock (0.1% DMSO), PRI-724. or iCRT14. (B) Immunofluorescence micrographs of HCE cells infected with HSV-1 K26-RFP at MOI of 0.1 and therapeutically treated with indicated concentrations of KYA1797k. (C and D) Intracellular (C) and extracellular (D) infectious particles collected from HCE cells therapeutically treated with various concentrations of KYA1797k following infection were assessed by plaque assay. (E) Percent cell viability of cells following treatment with KYA1797k at increasing concentrations in HCE cells. (F and G) Western blot analysis of viral protein gB following therapeutic treatment with PRI-724 and iCRT14 after infection with HSV-1 laboratory KOS strain (F) and clinical strain BLS or ULS (G) in HCE cells. (H) Western blot analysis of viral protein production in HCE cells lacking β-catenin protein expression. Cells were transfected with si-β-catenin for 48 h prior to infection with HSV-1 KOS strain at MOI of 0.1 for the indicated time points. Download Copyright © 2021 Koujah et al.2021Koujah et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Furthermore, gene silencing of β-catenin through the use of small interfering RNA (siRNA) was used to confirm that the absence of β-catenin results in reduction of HSV-1 infection  and G. TThe effect of β-catenin activation by CHIR-99021 was also assessed in HCE cells  to L. CHAfter having observed the dependence of β-catenin activation on HPSE to promote viral replication during HSV-1 infection, we sought to determine whether expression of transcriptionally active β-catenin would promote HSV infection in the absence of HPSE. Three plasmid constructs were used expressing wild-type (WT), constitutively active (S33Y), and a truncated and transcriptionally active (ΔN89) β-catenin . After tDysregulation of the Wnt/β-catenin pathway has been implicated in the onset of diseases, including various cancers as well as viral infections such as influenza virus, hepatitis C virus (HCV), hepatitis B virus (HBV), herpesviruses, and human immunodeficiency virus (HIV), among others 20–, 30–36. 30–in vitro and in vivo contexts. We report, for the first time, β-catenin activation, nuclear translocation, and signal transduction in HCE cells and the murine cornea following HSV-1 infection. Furthermore, pharmacological inhibition of β-catenin establishes an antiviral cellular environment at therapeutically safe concentrations. We also study the mechanisms leading to β-catenin activation and show that HPSE is a key regulator of β-catenin signaling. HPSE and β-catenin have independently been implicated in regulating key processes in pathological conditions; however, the relationship between the two proteins has never been examined.Host signaling pathways are altered during pathological conditions like HSV-1 infection, and better understanding of the vast host interactome reconfiguration will conceptually advance the field and lead to the identification of novel drug targets. In this study, we examine the role of Wnt/β-catenin pathway in physiologically relevant Previous studies by our lab examined the effect of virus-induced upregulation of HPSE during HSV-1 infection, which, in turn, generates a proviral environment through the cleavage of heparan sulfate proteoglycans, permitting the release of virus particles and reinfection of surrounding cells –4. In mo–Several studies have shown that Akt activation and phosphorylation of GSK-3β lead to the stabilization of β-catenin and release from the destruction complex . This isWe next examined the differential contributions of HPSE on phosphorylation of β-catenin. The N terminus of cytosolic β-catenin is constitutively phosphorylated by a multiprotein complex commonly referred to as the destruction complex, which is bound to the scaffold protein Axin. Among other proteins, Axin has binding sites for β-catenin, GSK-3β, and casein kinase 1 (CK1). CK1 primes β-catenin by phosphorylating it at Ser45 prior to GSK-3β phosphorylating β-catenin at Ser33, Ser37, and Thr41. When β-catenin is phosphorylated at these sites, it is ultimately recognized by the β-TrCP E3 ubiquitin-ligase complex, ubiquitinylated, and rapidly degraded by the 26S proteasome. β-Catenin encompasses other phosphorylation sites that, rather than tagging it for degradation, enhance the signaling activity. Of note is the phosphorylation of β-catenin at Ser552 by Akt, which enhances β-catenin protein levels and nuclear signaling . Since HWe next sought to investigate the levels of β-catenin phosphorylation in response to HSV-1 infection in the presence and absence of HPSE. We pretreated the cells with Wnt3a-CM prior to infection to prevent the degradation of β-catenin. As expected, in the wild-type cells, treatment with Wnt3a-CM prior to infecting caused a robust increase in p-β-catenin (Ser552) only in the presence of HPSE. This increase in active β-catenin caused an increase in viral protein production, further proving the proviral role of β-catenin. Furthermore, introduction of β-catenin plasmids expressing wild-type constitutively active S33Y mutation and constitutively active deltaN89 truncation showed that, following Wnt3a-CM treatment and HSV-1 infection, expression of constitutively active β-catenin resulted in enhanced viral protein production in wild-type cells. This correlated with increased protein expression of p-β-catenin Ser552 and a decrease in p-β-catenin S33/S37/Thr41. However, in the absence of HPSE, expression of β-catenin plasmids did not result in any changes to HSV-1 infection, and phosphorylation of β-catenin remained constant under all conditions. These studies confirm that β-catenin activation is dependent on a kinase cascade beginning with HPSE-induced phosphorylation of Akt, leading to the phosphorylation of β-catenin at S552.In summary, our findings assign a novel regulatory role to HPSE as a driver of β-catenin signaling during HSV-1 infection. While further investigation is required to determine the mechanism of HPSE-induced Akt activation, this study provides key insights on the cross talk of cell signaling pathways that are conducive to pathogenic milieu and viral replication. Our findings also support further development of therapeutic agents targeting HPSE and/or β-catenin signaling pathway to combat herpesvirus infection and, quite possibly, many other pathological conditions.Cellular proteins were extracted using radioimmunoprecipitation  buffer and Halt protease inhibitor cocktail (Thermo Scientific). Lysis was performed on ice with agitation for 1 h, followed by 10 min centrifugation at 13,000 rpm. After removing cellular debris, lysates were then denatured at 95°C for 8 min in the presence of 4× LDS sample loading buffer (Life Technologies) and 5% β-mercaptoethanol  and electrophoresed by SDS-PAGE with NuPAGE 4 to 12% Bis-Tris 1.5-mm 15-well gels (Thermo Scientific). Proteins were then transferred to nitrocellulose using iBlot2 system (Thermo Scientific), and membranes were blocked in 5% milk/Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature, followed by incubation with primary antibody overnight at 4°C. After washes with TBST and incubation with respective horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, protein bands were visualized using SuperSignal West Femto substrate (Thermo Scientific) with Image-Quant LAS 4000 biomolecular imager .in vitro overexpression experiments according to the manufacturer’s specifications. The siRNA of β-catenin was obtained from Dharmacon .For Western blotting, the housekeeping gene GAPDH  was used for normalization as a loading control. Antibodies used in this study are GAPDH  at dilution 1:1,000, gB  at dilution 1:10,000, gD  at dilution 1:10,000, β-catenin  at dilution 1:1,000, phospho-β-catenin (Ser33/37/Thr41)  at dilution 1:1,000, phospho-β-catenin (Ser552)  at dilution 1:1,000, phospho-GSK-3β  at dilution 1:1,000, GSK-3β  at dilution 1:1,000, E-cadherin  at dilution 1:1,000, HPSE  at dilution 1:1,000, and phospho-Akt (Ser473)  at dilution 1:1,000. HPSE expression constructs, including the Myc-GS3 plasmid, were provided by Israel Vlodavsky . The following plasmids were created by Eric Fearon lab and acquired through Addgene: human β-catenin pcDNA3 (plasmid no. 16828), pcDNA3 deltaN89 β-catenin (plasmid no. 19288), and pcDNA3-S33Y β-catenin (plasmid no. 19286). Lipofectamine 2000 transfection reagent  was used for all in vivo experiments, mouse tissue was extracted and incubated in 50 μl of 2 mg/ml collagenase D  in phosphate-buffered saline (PBS) for 1 h at 37°C. Tissues were then triturated with a pipette tip and resuspended in TRIzol, and extraction of RNA and cDNA was performed as described above. Real-time quantitative PCR (qPCR) was performed using Fast SYBR green master mix (Life Technologies) on QuantStudio 7 Flex system (Life Technologies).RNA was extracted from cultured cells using TRIzol  following the manufacturer’s protocol, and cDNA was produced using high-capacity cDNA reverse transcription kit (Life Technologies). For HCE cells or WT and Hpse-KO MEFs were cultured in glass-bottom dishes (MatTek Corporation). Cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton-X  for 10 min at room temperature for intracellular labeling. This was followed by incubation with primary antibody for 1 h at room temperature. When a secondary antibody was needed, cells were incubated with respective fluorescein isothiocyanate (FITC)- or Alexa Fluor 647-conjugated secondary antibody  at a dilution of 1:100 for 1 h at room temperature. NucBlue Live ReadyProbes Hoechst stain  was included with secondary antibody stains when applicable, according to manufacturer specifications.Anesthetized mouse corneas were scarified in a 3 by 3 grid using a 30-gauge needle and infected as previously described .Collected mouse tissue was first fixed with 4% paraformaldehyde (PFA) for 24 h and then incubated in 70% ethanol. Sample processing and paraffin embedding was performed by the University of Illinois at Chicago core facilities. We took 10-μm section thickness using Microm HM 340. Following deparafinization, antigen retrieval was performed by incubating the slides in subboiling temperatures of 10 mM sodium citrate buffer with 0.05% Tween 20 at pH 6.0 for 15 min. Slides were then blocked in 3% bovine serum albumin (BSA) in TBST for 1 h at room temperature. Primary antibody was incubated overnight at 4°C using 1:100 concentration of β-catenin . Slides were then washed for 15 min prior to incubation with Alexa Fluor 647-labeled anti-rabbit secondary antibody and DAPI  to label nuclei for 1 h at room temperature. ProLong Gold antifade mountant with DAPI was used to mount the slides.The HCE cell line was obtained from Kozaburo Hayashi , and the cells were cultured in minimum essential media  supplemented with 1% penicillin-streptomycin (Thermo Fisher Scientific) and 10% fetal bovine serum . African green monkey fetal kidney epithelial (Vero) cells were provided from P. Spear (Northwestern University) and cultured in Dulbecco’s modified Eagle medium  with 10% FBS and 1% penicillin-streptomycin. Wild-type and Hpse-knockout mouse embryonic fibroblasts were provided by Israel Vlodavsky (Rappaport Institute). Virus strains used in these studies, HSV-1 (KOS-WT), green fluorescent protein (GFP)-HSV-1 (K26-GFP) with KOS strain background, and McKrae were provided by Patricia G. Spear . Acquisition of clinical strains used in this study is as previously described. All virus stocks were propagated in Vero cells and stored at −80°C. Wnt-3A-expressing cells were acquired from ATCC (CRL-2647) to culture Wnt3A-conditioned media according to the manufacturer’s protocol.Pharmacological inhibitors of the β-catenin system were purchased from Selleckchem  and Tocris . GSK-3 inhibitors were purchased from Selleckchem  and Sigma-Aldrich .in vitro experiments. Vero cell monolayers were inoculated for 2 h with virus-containing sample and incubated at 37°C and 5% CO2. The viral suspension was aspirated and replaced with complete DMEM containing 0.5% methyl cellulose (Fisher Scientific) for 48 to 72 h. Cells were then fixed with 100% methanol and finally stained with crystal violet solution to visualize plaques.Plaque assay was performed with tear samples from infected mice as well as with cell supernatant from Propidium iodide  and Hoechst nucleic acid stain were added to complete DMEM for 30 min prior to imaging. The Biotek Lionheart FX system was used to detect fluorescence intensity on PI, DAPI, and bright-field channels.4 per well and left overnight until they were 80% confluent. Compounds to be tested and dimethyl sulfoxide  were added to the designated wells and incubated for a period of 24 h at 37°C and 5% CO2. Postincubation, wells were washed with PBS twice before 100 μl of 0.5 mg/ml MTT  was added to each well and incubated for 4 h. Formazan crystals formed due to the mitochondrial activity were dissolved using acidified isopropanol (0.1% Tris-HCl in isopropanol) and transferred to a new 96-well plate. Genesis Pro Plate reader at 562 nm was used to analyze samples. Experiments were conducted in triplicates and individually repeated 5 times to compensate for pipetting errors.Cellular viability was analyzed following treatment with β-catenin inhibitors. Briefly, HCEs were plated in a 96-well plate at a density of 1 × 10GraphPad Prism software was used for statistical analysis, with bar heights representing the mean and error bars representing standard deviation. Statistical analysis in each figure is described in the legend, as well as explanations of dot plot representation. For images requiring measurement of total cell fluorescence, ImageJ was used. Briefly, the drawing/selection tool was used to select the cell of interest. From the analyze menu, “set measurements” was used to acquire area-integrated intensity and mean gray value. Next, “measure” was selected from the analyze menu where values indicating area, mean, and integrated density of fluorescence are given. An area with no fluorescence was used as background for the calculation of corrected total cell fluorescence (CTCF). The calculation used is as follows: CTCF = integrated density − (area of selected cell × mean fluorescence of background readings).All animal care and procedures were performed in accordance with the institutional and NIH guidelines and were approved by the Animal Care Committee at University of Illinois at Chicago (ACC protocol nos. 17-077 and 20-065).


As-received and acid-treated CNTs were added to aerospace grade epoxy (CYCOM® 977-3), and the composites were characterized. The epoxy resin is expected to infiltrate the network of the CNTs and could improve mechanical properties. Epoxy composites were tested for flexural and viscoelastic properties and the as-received and acid treated CNTs were characterized using Field-Emission Scanning and Transmission Electron Microscopy, X-Ray Photoelectron Spectroscopy, and Thermogravimetric Analysis. Composites containing 0.4 wt% as-received CNTs showed an increase in flexural strength, from 136.9 MPa for neat epoxy to 147.5 MPa. In addition, the flexural modulus increased from 3.88 GPa for the neat epoxy to 4.24 GPa and 4.49 GPa for the 2.0 wt% and 3.0 wt% as-received CNT/epoxy composites, respectively. FE-SEM micrographs indicated good dispersion of the CNTs in the as-received CNT/epoxy composites and the 10 M nitric acid 6 h treatment at 120 °C CNT/epoxy composites. CNTs treated with 10 M nitric acid for 6 h at 120 °C added oxygen containing functional groups  and removed iron catalyst present on the as-received CNTs, but the flexural properties were not improved compared to the as-received CNT/epoxy composites.Huntsman–Merrimack MIRALON MIRALON® CNTs are networked, highly entangled, commercially available, and scalable with very high aspect ratios. Due to the scalability of these unique CNTs, there is considerable interest from the aerospace community in their use on a commercial scale. These highly networked CNTs are anticipated to infiltrate the aerospace grade toughened epoxy resin which could improve mechanical properties.The carbon nanotube (CNT) is an ultra-strong and ultra-stiff material due to its covalent sp500 GPa) . Due to 500 GPa) ,5,6,7,8.The realization of the reinforcing effect of high strength/modulus nanotubes relies on the ability to transfer load between epoxy and nanotube phases of the composite, due to van der Waals forces or, in the case of some functionalized nanotubes, covalent linkages. Many methods for promoting covalent functionalization of the CNT have been explored in the literature  ,10,11,12® 977-3) composites containing various amounts of as-received CNTs (MIRALON® pulp) and acid treated CNTs. The CYCOM® 977-3 resin is a high-performance aerospace grade resin toughened with a thermoplastic co-polymer and is used in state-of-the-art aerospace polymer composite structures. Composites were tested for flexural properties as well as dynamic mechanical analysis (DMA) to study the compatibility of the matrix-filler material and changes in glass transition temperature, respectively. In addition, MIRALON® CNT pulp was treated in a variety of acid treatment methods and characterized for surface morphology, graphitic character, surface chemistry, and thermal stability using Field Emission Scanning Electron Microscopy (FE-SEM), Transmission Electron Microscopy (TEM), Raman spectroscopy, X-Ray Photoelectron Spectroscopy (XPS), and Thermogravimetric Analysis (TGA). Per the authors knowledge, these material properties have never been previously reported in the open literature.In this work, researchers fabricated aerospace grade epoxy . The CYCOM® 977-3 resin is a high-performance toughened epoxy system with a proprietary blend of epoxy and a thermoplastic copolymer with a co-continuous morphology. When ramping 2 °C/min, the viscosity of the resin decreases to a minimum of 600 cP at 155 °C and begins climbing again at 170 °C due to a crosslinking reaction. The density of the cured resin at 25 °C is 1.29 g/cm−3 [The epoxy matrix used was Solvay CYCOM9 g/cm−3 .® CNT pulp . The MIRALON® pulp is manufactured by chopping non-woven MIRALON® CNT sheets into small porous bundles that are approximately 0.05 mm (50 µm) in diameter and 1 mm in length. CNTs are unraveled when dispersed in resin, with individual fiber diameters of 0.01 µm (10 nm). The MIRALON® pulp used was manufactured using high growth rate (HGR) grade MIRALON® CNTs. HGR MIRALON® pulp contains approximately 30 wt% iron carbide and a 5 wt% amorphous carbon content [The CNT filler was a Huntsman–Merrimack MIRALON content . The sto® pulp was treated with a sulfuric/nitric acid mixture (0.5 L) in a 1 L glass Erlenmeyer flask. The acid mixture was 3:1 (by volume) of 10.0 M sulfuric acid (0.33 L) and 4.0 M nitric acid (0.17 L), respectively. CNT pulp (2.5 g) was added to the acid solution and the CNT suspension was mixed at 23 °C with a PTFE magnetic stir bar (13 mm diameter and 76 mm long) on a magnetic stir plate, for either 6, 12, or 18 h.MIRALONThe CNTs were separated using a vacuum filtration assembly with a 55 mm diameter filter with 2.5 µm pore size (Whatman Grade 42). Iterations of washing the CNTs with water in the vacuum filtration assembly were performed until pH probe detected a near-neutral pH (between 6 and 7). The CNT cake was retrieved from the filtration apparatus and dispersed in methylethylketone (MEK) . The suspension was poured into glass petri dishes and placed in an oven at 95 kPa vacuum at 23 °C for 3 h with a liquid nitrogen cold trap to collect the MEK. CNTs were retrieved from the bottom of the petri dishes. CNTs were then placed in an oven to dry for 6 h at 120 °C at 95 kPa vacuum. After drying, the acid treated pulp was stored in a moisture barrier bag.® pulp was treated with aqueous nitric acid at 2.5 M and 10.0 M concentrations (0.5 L) in a 1 L glass Erlenmeyer flask. CNT pulp (2.5 g) was added to the solution, and the CNT suspension was sonicated for 30 min in a Branson sonicator  at 40 Hz and refluxed for 3 h at 90 °C or for 6 h at 120 °C. The same method was used to process the treated CNTs as was described in the nitric/sulfuric acid treatment discussed above in MIRALON® 977-3 epoxy were fabricated by mixing epoxy in a FlackTek SpeedMixer DAC 150.1 FVZ , which is a planetary centrifugal mixer and is shown in Plaques of CYCOMNeat resin was then degassed in the vacuum oven at 100 °C and held for 10 min at 101 kPa vacuum. The resin was degassed for 30 min at 100 °C by alternating between atmospheric pressure and 101 kPa vacuum. Then, 101 kPa vacuum was applied to a vacuum chamber for an additional 2 h. Then, the resin was removed from the oven to cool at 23 °C and was then placed in the freezer at −18 °C until the plaques were manufactured.Neat epoxy plaques were manufactured using a Wabash Vantage Series Model V75H-18-CLX  compression molding press. All steel from the tooling assembly was sprayed with Mann Ease 2300  release compound. Frozen pieces of degassed resin were placed in a 20 cm × 20 cm steel chase that sat on the bottom half of the steel assembly. The steel chase generated 15 cm × 25 cm plaques that are 3.2 mm thick. The tooling assembly was vacuum bagged at 101 kPa vacuum and placed in the compression molding machine and pressed with 10 kN force. The compression molding machine was heated to 100 °C at 101 kPa vacuum pressure and held for 20 min. Platens were then ramped to 130 °C and held for 30 min at 130 °C. The platen temperature was then ramped to 177 °C, and 25 kN was applied and held at 177 °C for 6 h. Platens were cooled with air and water from 177 °C to 150 °C. Cooling air was turned off, and water cooling continued from 150 °C to 30 °C. The pressure was released, and the assembly was removed. Test samples were cut from the plaque.® pulp with epoxy in a SpeedMixer to achieve target CNT concentrations . Each of 2 SpeedMixer cups were charged with 70 g of neat 977-3 resin. The appropriate amount of MIRALON® pulp was added to each cup to produce the target formulation of CNT/epoxy. The same method was used to mix, degas, and compression mold as-received CNT/epoxy composite plaques as was described above for the neat epoxy plaques.Plaques of as-received CNT/epoxy were fabricated by mixing MIRALONPlaques of epoxy composites made with CNTs treated with nitric/sulfuric acid mixture at 23 °C and with 2.5 M nitric acid at 90 °C were fabricated using the same method as described for as-received CNT/epoxy composites described above. Plaques of epoxy composites made with CNT pulp treated with 10.0 M nitric acid at 120 °C were fabricated by sonicating 30 g of MEK and 0.28 g of treated CNTs in each of two test tubes in the Branson sonicator for 2 h at 40 Hz. Then 70 g of epoxy resin was combined with the MEK/CNT suspension in each of 2 SpeedMixer cups and mixed in the SpeedMixer at 23 °C for 10 min at 1750 rpm and 2 min at 2500 rpm. The mixture was put in an oven at 100 °C and 101 kPa of vacuum for 1 h with a liquid nitrogen cold trap to collect the MEK. Three zirconium mixing cylinders  were added to each cup, and the mixing cycle  was repeated once at 100 °C. The same method was used to degas and compression-mold the acid treated CNT/epoxy composites as was described above for the neat epoxy plaques.® 977-3 epoxy plaques and MIRALON® CNT/epoxy plaques. ASTM D790 3-point bend test method was used with an Instron 4206 screw driven mechanical testing machine  [Flexural properties were obtained using a 3-point bend testing method at 23 °C on neat CYCOMMA, USA) . Specimeg) for neat epoxy and CNT/epoxy composites were obtained according to ASTM D7028 using a TA Instruments Q800 DMA  with a single cantilever clamp [g for the material (150 °C). The sample was then air cooled to 30 °C.Glass transition temperatures (Ter clamp . DMA speg) tests, two temperature ramps were performed for each specimen. The first temperature ramp was to fully cure the resin system and to erase the thermal history of the sample. A second temperature ramp was used to locate the Tg. Once the test was completed, the data were analyzed to determine the Tg by plotting the tanδ curve against temperature. The Tg was determined to be at the peak of the tanδ curve.To perform the glass transition temperature   was used to view as-received and acid treated MIRALONThe FE-SEM was used to view the flexural fracture surfaces of CNT/epoxy composite samples. The samples were cut to 3.2 mm × 2 mm × 13 mm (thickness × width × length) and placed on carbon tape on an aluminum sample stud. Then, the samples were sputter coated with a 2-nm thick platinum/palladium coating using a Cressington 208HR sputter coater. The film thickness was measured with a Cressington MTM-20 High Resolution Thickness Controller . Samples were viewed using 10 kV accelerating voltage and 10 µA emission current. The upper secondary electron detector was used to collect the images.® CNT pulp were dispersed in isopropanol using an ultrasonic homogenizer  for 2 min. Dispersed solution was dropped on the Lacey carbon grid and dried overnight. CNT structures were observed at high resolution transmission electron microscope  with 80 kV acceleration voltage.As-received and acid treated MIRALONA TA Instruments Q50 thermogravimetric analyzer  was used to conduct thermal analysis experiments of the as-received and treated CNT pulp per ASTM E2550 to determine the residual catalyst wt% and to detect changes in weight . In addi−1. The laser was calibrated using an internal silicon standard before data were taken. Laser power was set at 1% with an exposure time of 20 s. The measurement was generated and analyzed using WiRE3 software.The Raman spectrographs of the nanotube pulp samples were taken with a Reinshaw inVia Raman microscope system . A 785 nm laser was used to analyze a spectral range from 897 to 1955 shift/cm2 squares and held on to the sample puck with a spring clip. Powder samples were placed on a piece of double-sided copper tape that was adhered to the puck. The samples were placed in a transfer chamber that was pumped down to high vacuum prior to being transferred into the ultra-high vacuum (UHV) analysis chamber, with a base pressure better than 1 × 10−7 Pa. Mg X-rays (photon energy = 1254.6 eV) were used to analyze the nanotube samples. The X-ray power was 400 W for sheets and 100 W for the powder samples.The XPS instrument used was a Phi 5800 XPS  equipped with a dual source (Mg/Al) anode for generating X-rays. The nanotube samples for XPS were placed on a 25 mm diameter sample puck. Sheet samples were cut to 10 mmThe XPS survey spectra were collected using a pass energy of 187.85 eV, a data spacing of 0.8 eV/step, a dwell time of 20 ms/step and a range from 0 to 1175 eV. The survey spectra were collected for 5 min (10 scans). The high-resolution C1s spectra were collected using a pass energy of 23.50 eV, a data spacing of 0.1 eV/step, and a dwell time of 100 ms/step. The high-resolution C1s spectra were collected for 5 min (15 scans).Jeong Tai et al.  reportedSince the flexural strength increased for the 0.4 wt% CNT/epoxy composite, this formulation was investigated further by various acid treatments. It is possible that adding additional oxygen containing functional groups to the CNT surface could increase CNT/epoxy composite flexural properties. g) is the temperature during heating at which an amorphous solid becomes soft as it transitions from a glassy state to a rubbery state. Glass transition temperatures (Tg) of neat 977-3 and as-received MIRALON® CNTs/977-3 composites are shown in g for neat 977-3 was found to be 249.55 ± 0.60 °C. At the 95% confidence level, there is no statistical difference between the neat epoxy and ≤2.0 wt% CNT/epoxy composites. Seyler [g of 261 °C using DMA testing on neat 977-3 epoxy specimens while testing at 1 Hz frequency and ramp rate of 10 °C/min. Koo et al. [g of 237 °C using DMA testing on neat 977-3 epoxy specimens while testing at 1 Hz frequency and ramp rate of 5 ° C/min. Our neat epoxy Tg is between those of these two researchers. Valentini et al. and Kumar et al. [g for composites containing ≤0.2 wt% CNT in epoxy.The glass transition temperature  than the as-received CNT pulp. ® CNT pulp/epoxy. Good dispersion is observed in all epoxy composites containing up to 1.5 wt% CNTs. ® CNT pulp/epoxy and 0.4 wt% acid treated pulp (6 h in 10 M nitric at 120 °C)/epoxy. In both cases, it appears that there is good dispersion of CNT in the composite flexural fracture surface.® pulp and MIRALON® pulp acid treated for 6 h in 10 M nitric acid at 120 °C. The images show double and multi walled CNTs with 10–20 nm diameters. ® CNTs plotted against temperature. −1 and the D-band at 1350 cm−1. The G-band represents the sp2 structure of the graphitic surface, and the D-band represents disorder in the crystal structure. −1, which is another indication of disorder in the graphitic structure. The D’ band becomes most pronounced for the nitric acid treatment at 120 °C for 6 h. An increased D/G ratio is evidence of a more disordered structure due to added functional groups from the acid treatment. 2 C-C peaks. A gaussian line shape was used for peak fitting the rest of the high-resolution C1s. 2 C-C at 284.5 eV, sp3 C-C at 285.5 eV, C–O at 286.0 eV, C=O at 287.0 eV, O=C–O at 288.5 eV, and a π-π* shake-up at 290.5 eV.High resolution C1s spectra were deconvoluted using CasaXPS software. A Tougaard background subtraction was used for the C1s region. Doniach Sunjic lineshape was used for sp2 aromatic content (84.8%) than the as-received CNTs (92.5%). The oxygen containing functional groups all increased for the acid treated CNTs. For example, the C–O percentage increased from 3.2 to 6.4%. For C=O, the percentage increased from 0.4 to 2.5 %. For O=C–O, the percentage increased from 0.9 to 1.9%. These results show that the acid treatment resulted in the addition of functional groups containing oxygen  and the decrease in sp2 character of sample surface [ surface ,34,35,36® CNT pulp to CYCOM® 977-3 epoxy on flexural mechanical properties and viscoelastic properties. It was determined, at the 95% confidence level, that epoxy composites containing 0.4 wt% CNT demonstrated no change in flexural modulus and a 9% increase in the flexural strength when compared to neat epoxy. Composites containing 2 wt% CNT demonstrated a 9% increase in flexural modulus when compared to neat epoxy. Composites containing 3 wt% CNT demonstrated a 16% increase in flexural modulus and a 15% decrease in flexural strength when compared to neat epoxy. Various acidic treatments were applied to the CNTs, and none of them caused an increase in flexural properties. FE-SEM micrographs appeared to show good dispersion of the as-received and treated CNTs in epoxy. FE-SEM micrographs also showed significant densification of the CNTs after acid treatment. At the 95% confidence level, DMA experiments demonstrated that epoxy composites containing ≤2 wt% as-received CNTs show no change in tanδ Tg when compared to neat epoxy.The goal of this research was to explore the addition of a unique and commercially scalable CNT product to an aerospace grade resin. An objective of this research was to determine the effects of adding as-received MIRALON® CNTs/Solvay CYCOM® 977-3 epoxy have never been previously reported in the open literature.The as-received CNTs and acid- treated CNTs were further characterized. TEM, TGA, and XPS results showed that the iron catalyst was removed from the as-received CNTs as a result of the 10 M 120 °C 6 h nitric acid treatment. Raman results showed that the D/G ratio increased from 0.88 for the as-received CNTs to 1.58 for the CNTs treated with 10 M nitric acid for 6 h at 120 °C. This increased D/G ratio shows an increase in the disorder  present in the acid treated CNTs. XPS results also noted a decrease in the crystalline structure for the CNTs treated with 10 M nitric acid for 6 h at 120 °C as compared to the as-received CNTs. XPS results reported that the carbon atom% decreased from 98.5 for the as-received CNTs to 91.6 for the CNTs treated with 10 M nitric acid for 6 h at 120 °C. XPS results also showed an increase in oxygen atomic% from 1.1 for the as-received CNTs to 8.4 for the CNTs treated with 10 M nitric acid for 6 h at 120 °C. Further investigation of the XPS data showed that the oxygen containing functional groups  all increased for the CNTs treated with 10 M nitric acid for 6 h at 120 °C as compared to the as-received CNTs. Apparently, the 10 M nitric acid CNT treatment for 6 h at 120 °C did increase the amount of oxygen containing functional groups to the CNT surface; however, this did not translate into improved flexural properties. Per the authors’ knowledge, these material properties on as-received and acid-treated Huntsman–Merrimack MIRALON


Soleus long-latency reflexes increased more than 100% from feedforward muscle activity regardless of trunk condition. Feedforward muscle activity differentially predicted long-latency reflex responses depending on the muscle (R2: 0.47–0.97). These findings support the concept that trunk positioning influences motor control responses to perturbation and that feedback responses may be invariant to the feedforward control strategy.Trunk positioning and unexpected perturbations are high-risk conditions at the time of anterior cruciate ligament injury. The influence of trunk positioning on motor control responses to perturbation during dynamic performance is not known. We tested the influence of trunk position on feedforward and feedback control during unexpected perturbations while performing a novel single-limb squatting task. We also assessed the degree that feedforward control was predictive of feedback responses. In the flexed trunk condition, there were increased quadriceps ( The US population continues to be burdened by anterior cruciate ligament (ACL) injuries and the high costs associated with recovery. From 2005–2013 the average cost in healthcare utilization after ACL reconstruction was $13,403.38 with a total of 229,446 surgeries performed , and recA perturbation elicits a rapid update to a pre-planned movement strategy in order to accommodate altered task demands. For example, when an athlete encounters an unexpected perturbation , the pre-planned (feedforward) movement strategy is modified by reflex and volitional (feedback) responses. In most cases these corrective feedback responses facilitate successful completion of the altered motor task. In other cases, such as in non-contact ACL injury, feedback activation of the athlete’s own muscle forces  may contFeedback responses to perturbations include the short-latency reflex (0–50 ms), the long-latency reflex (51–200 ms), and volitional reaction time (>200 ms) . The lonFeedforward factors such as biomechanical position ,20,21,22A flexed trunk position increases hamstrings activation and posterior shear force at the knee  which maTwenty healthy participants  tasks, once with an upright trunk (<15°) and once with a flexed trunk (≥30°) on a custom-built device . The tesThe SLS device consists of a knee pad attached to a rack and pinion system with horizontal resistance administered by an electro-magnetic brake ,18 Figu. ParticiThe 9 conditions of the SLS test varied by levels of resistance, ), and target signal velocities  which corresponds to approximately 10°/s, 20°/s, and 30°/s knee joint flexion velocities. Once per condition, the resistance of the brake was randomly released to 0% BW during knee flexion (never during the first cycle) causing a perturbation or a freefall into flexion. The perturbation was delivered during the initial 1/3 of knee flexion based on the participant’s horizontal knee position. The duration of brake release was 500, 250, and 200 ms for the 0.2, 0.4, and 0.6 Hz velocities, respectively. SLS signals  were collected at 2000 Hz using an analog to digital data acquisition (DAQ) board . Up to 60 s of rest was allowed between each of the 9 conditions. A 5-min seated rest was mandated between two trunk-position conditions.Performance was assessed via coherence, absolute error, and user error rate. Coherence is the ability of the user to match the velocity of the sinusoidal task with 0 representing a completely non-related signal and 1 representing a linearly matched signal. Absolute error was calculated as the peak absolute difference between the target and user signals. It was calculated for the entire task (averaged absolute error for each condition) and for the feedforward time bin for perturbed cycles (−50–0 ms prior to perturbation). User error rate, defined as the best-fit line from the target signal minus the user signal, was calculated after the perturbation during the short-latency reflex (0–50 ms post-perturbation) and the long-latency reflex (51–200 ms post-perturbation) time bins.Surface EMG data were collected for the vastus medialis (VM), vastus lateralis (VL), medial hamstrings (MH), lateral hamstrings (LH), gluteus medius (GMed), and soleus (SOL) muscles at 2000 Hz using wireless electrodes  placed according to SENIAM guidelines . Prior tEMG and SLS signals were analyzed using a custom DIAdem script . Root mean square of the EMG signals were calculated every 10 ms and peak activity was reported as a percentage of the peak MVIC for the feedforward (−50–0 ms), short-latency reflex (0–50 ms), and long-latency reflex (51–200 ms) time bins during the SLS task.Sagittal kinematics were calculated for the trunk , hip, knee, and ankle joints during the SLS task using eight motion capture markers and a ten-camera system . Kinematics were collected at 100 Hz and synced to the SLS and EMG signals via a DAQ board . Frontal and transverse plane motions were not analyzed as the SLS device confines movement to the sagittal plane.p < 0.05. Significant findings were analyzed using Tukey’s post hoc tests.Distinct motor control strategies exist between the sexes ,31,32. Tp’s > 0.05) with the exception of knee flexion velocity during the short-latency reflex  in which males flexed the knee 44.3°/s and 29.8°/s and females flexed the knee 36.7°/s and 32.6°/s during the flexed and upright trunk conditions, respectively. Analysis of the trunk conditions showed that maximum trunk flexion differed significantly between the two trunk condition tasks   =  693.457, p < 0.001). At the time the perturbation was delivered, there was 19.3°, 22.8°, and 3.5° greater flexion at the trunk (F(1.39)  =  203.190, p < 0.001), hip (F(1.39)  =  114.014, p < 0.001), and knee (F(1.39)  =  6.081, p = 0.023) in the flexed condition compared to the upright condition. Ankle dorsiflexion at the time of perturbation did not differ between the two trunk conditions (F(1.39)  =  0.811, p = 0.379). There were no differences between male and female kinematics of the trunk, hip, knee, or ankle at the moment when the perturbation was delivered, or during feedforward, short-latency reflex, or long-latency reflex time bins  suggesting participants did not anticipate the perturbation. Females demonstrated greater feedforward muscle activity than males by 14% (%MVIC) in VM  and by 7.8% (%MVIC) in GMed . There were increases in feedforward muscle activity in the flexed condition for GMed (p < 0.001) and MH (p = 0.002) compared to the upright condition, but no feedforward muscle activity difference for VM (p = 0.051), VL (p = 0.267), LH (p = 0.274), or SOL (p = 0.165). The quadriceps to hamstrings muscle activity ratio was 45 percent greater in the upright condition versus the flexed condition (p = 0.017). The ratios of VM to MH (p = 0.054) and VL to LH (p = 0.146) muscle activity were not different between trunk conditions.There were no differences in feedforward muscle activity between perturbed and unperturbed squat cycles within the same trunk condition for any muscle  and GMed , respectively. The short-latency reflex was 6–8 percent greater in the flexed versus the upright condition for GMed (p = 0.010) and MH (p < 0.001). There were no differences in the short-latency reflex between trunk conditions for VM (p = 0.182), VL (p = 0.609), LH (p = 0.115), or SOL (p = 0.261). The quadriceps to hamstrings muscle activity ratio was 52 percent greater (p = 0.002), the VM to MH ratio was 85 percent greater (p = 0.011), and the VL to LH ratio was 33 percent greater (p = 0.004) in the upright condition versus the flexed condition. When feedforward muscle activity (−50–0 ms) was used as a co-variate, the MH remained 3 percent greater (F(1.37)  =  5.290, p = 0.027) and the VL to LH ratio was 53 percent lower (F(1.37)  =  4.508, p = 0.04) in the flexed condition versus the upright condition. Feedforward EMG predicted the short-latency reflex response with R2 values between 0.75–0.98 for the VM, MH, VL, GMed, LH, SOL, quadriceps to hamstrings, VM to MH, and VL to LH ratios .During the short-latency reflex, females demonstrated 15.1% (%MVIC) and 7.7% (%MVIC) greater muscle activity in the VM  and 7.6% greater GMed activation  during the long-latency reflex. Based on the trunk condition, the long-latency reflex was 9–13.5 percent greater in the VM (p = 0.004), GMed (p < 0.001), MH (p < 0.001), and VL (p = 0.026), during the flexed condition versus the upright condition. There were no differences in the long-latency reflex between conditions for LH (p = 0.109) or SOL (p = 0.305). The time to peak long-latency reflex was shorter during the upright trunk condition compared to the flexed trunk condition in GMed . Time to peak long-latency reflexes did not differ between males and females or between trunk conditions for any other muscle  with latencies between 106–154 ms. The quadriceps to hamstrings ratio was 47 percent greater (p = 0.007), the VM to MH ratio was 60 percent greater (p = 0.003), and the VL to LH ratio was 36 percent greater (p = 0.014) in the upright condition versus the flexed condition. Although the SOL long-latency reflex response did not differ between trunk conditions, muscle activity increased more than 100 percent from feedforward background activity (F(1.19)  =  77.734, p < 0.001). Using feedforward muscle activity as a co-variate, MH and GMed remained 3.4 (F(1.37)  =  5.251, p = 0.028) and 4 percent (F(1.37)  =  5.651, p = 0.023) increased in the flexed condition versus upright condition. The VM to MH ratio also remained 27 percent (F(1.37)  =  5.119, p = 0.03) greater in the upright condition than the flexed condition. Feedforward EMG predicted the long latency reflex response with R2 values between 0.47–0.97 for the VM (p’s < 0.001).Compared to males, females demonstrated 22.7% greater VM activation  with no other effects of sex on SLS performance (All p’s > 0.075). Absolute error was greater throughout the SLS task during the flexed condition versus the upright condition for both the perturbed (F(1.39)  =  12.077, p = 0.003) and unperturbed squat cycles (F(1.39)  =  15.832, p < 0.001). Coherence was 9 percent greater in the upright condition than in the flexed condition (F(1.39)  =  11.518, p = 0.003). During squat cycles with a perturbation, absolute error during the feedforward time bin was no different between trunk conditions (F(1.39)  =  1.364, p = 0.257), suggesting participants were performing similarly on the task in both trunk conditions at the time the perturbation was delivered. After the perturbation, the user error rate did not differ between trunk conditions during the short-latency reflex (F(1.39)  =  2.19, p = 0.155), but was greater during the long-latency reflex in the flexed condition than the upright condition (F(1.39)  =  11.401, p = 0.003). The flexed condition also resulted in greater trunk, hip, knee, and ankle flexion velocity than the upright condition during the feedforward, short-latency reflex, and long-latency reflex time bins .Females demonstrated 35.9% greater absolute error during the feedforward time bin than males  depended upon sex of the participant. These findings support sex distinct responses to unexpected perturbations which coincides with findings from other perturbation studies ,33. It iFeedforward muscle activity (−50–0 ms) predicted feedback reflex responses  after the perturbation in both trunk conditions. Short-Latency reflexes were more strongly predicted by feedforward activity than long-latency reflexes, consistent with the task dependent nature of long-latency reflexes . CompareA novel finding was the triggering of a long-latency reflex in the gluteus medius when squatting with a flexed trunk. This burst in the gluteus medius, a muscle with a major role in frontal plane stability of the pelvis, persisted even after we adjusted for changes in feedforward control. It is conceivable that the reflex was triggered by a stretch of the posterior gluteus medius muscle fibers or by activation of the vestibular system after the perturbation. Others noted that the vestibular system initiates a hip correction strategy to regain balance ; thus, tA new finding was the substantial increase (>100%) in the soleus long-latency reflex that was independent of trunk position and the associated change in feedforward control. This finding indicates that the soleus, a powerful postural muscle, has the capacity to contribute a significant response to unexpected perturbations. Even though the soleus is a single joint ankle plantar flexor, it has been shown to exert significant posterior tibial shear force at the knee during single-leg landing  and unanShort-latency reflexes (0–50 ms) are considered to have little functional relevance  when resCo-contraction of the quadriceps and hamstrings was lower (higher Q/H ratio) in the upright trunk condition for both the feedforward and feedback phases surrounding the perturbation. Primary reliance on the quadriceps to control knee movement, termed quadriceps dominance, is a movement strategy associated with ACL injury . This stThe single limb squatting task in our study was associated with better performance in the upright trunk condition. Participants were better able to match the velocity of the task (coherence) and minimize the disruption of their knee position after the perturbation (error rate) in the upright trunk condition. The findings of improved single-leg squatting performance coupled with greater injury risk factors (decreased co-contraction) is similar to work by others who showed performance and injury mechanisms were inversely related . PerhapsIt is important to consider that knee injury is often the result of multi-planar forces ,22, thusIn summary, feedforward muscle activation predicted feedback reflex responses after unexpected perturbations during a dynamic single-leg squatting task, independent of biomechanical trunk position. Flexing the trunk while squatting resulted in greater co-contraction around the knee, increased multi-segmental lower extremity joint flexion, and increased feedforward activation and long-latency reflexes in the quadriceps, gluteus medius, and medial hamstrings during unexpected perturbations. Task performance such as velocity matching and knee control were improved when squatting with an upright trunk. Together, these findings suggest performing single-leg squats with concomitant trunk flexion may decrease risk factors associated with ACL injury but at the cost of performance. Clinical interventions to improve feedforward control during movement tasks may be important in modulating responses to unexpected perturbations.


Capsule endoscopy of the gastrointestinal tract is an innovative technology that serves to replace conventional endoscopy. Wireless capsule endoscopy, which is mainly used for small bowel examination, has recently been used to examine the entire gastrointestinal tract. This method is promising for its usefulness and development potential and enhances convenience by reducing the side effects and discomfort that may occur during conventional endoscopy. However, capsule endoscopy has fundamental limitations, including passive movement via bowel peristalsis and space restriction. This article reviews the current scientific aspects of capsule endoscopy and discusses the pitfalls and approaches to overcome its limitations. This review includes the latest research results on the role and potential of capsule endoscopy as a non-invasive diagnostic and therapeutic device. Since capsule endoscopy was introduced in the early 2000s, capsule endoscopy has played an important role in evaluating small intestinal lesions . CapsuleCompared to conventional endoscopy, capsule endoscopy is a less invasive examination method that does not require sedation during the examination process and reduces the patient’s discomfort. In addition, it enables easy access to structures such as the small intestine that were previously difficult to access.Capsule endoscopy has continuously improved since it has been applied in clinical practice ,7,8,9,10One of the disadvantages of capsule endoscopy is the impossibility of operating the device; therefore, it is difficult to observe by adjusting the field of view as desired. This affects the overall miss rate of capsule endoscopy. The miss rates for small bowel tumors, vascular disease, and ulcers were 18.9%, 5.9%, and 0.5%, respectively . EffortsThe application of magnetic fields in the medical field has long been practiced. Recently, it has been applied and used in neurosurgery and the treatment of cardiac arrhythmias ,13. MagnIn 2010, Swain et al. conducted a study to observe the esophagus and stomach by administering MACE by modifying colon-type capsule endoscopy   to the hIn 2010, Siemens Healthcare and Olympus Medical Corp modified the capsule endoscope and developed a 31 × 11 mm MACE with a magnet inside . The magThis system consists of a capsule endoscope, a magnet-controllable robot, a data storage device, and a computer workstation capable of real-time observation and steering  17]. Th. Th17]. MiroCam Navi is a simple system that uses a capsule endoscope and a hand-held magnet to adjust the capsule . The capAlthough capsule endoscopy technology has made remarkable progress, numerous unavoidable shortcomings prevent the expansion of capsule endoscopy applications. For example, capsule endoscopy cannot be observed by stopping at a certain area for diagnostic or therapeutic purposes. It is also difficult to return to an area and re-observe. These shortcomings can be overcome by adding a locomotion system to the capsule endoscope. Several research groups have tried to develop a different type of locomotion system for capsule endoscopy. However, it is still in the experimental laboratory stage because of the complexity of the gastrointestinal tract and limited power ,22,23,24Researchers prefer the friction-based locomotion method because it is based on a simple principle. The inchworm-like method works through three basic movements: anchoring, elongating, and contraction . This acAnother commonly used friction force-based locomotion method is paddle-based motion. This type of locomotion mechanism originates from canoe paddling. Several paddles or legs are included in the capsule endoscope body, controlled by the actuator and pushed backward on the gastrointestinal tract wall so that the capsule endoscope moves forward. Kim et al. presented a paddling-based capsule endoscope and tested the locomotion of active capsule endoscopy in vitro and in vivo  22,24].,24.22,24Hydrodynamic force-based systems are widely used in the design and manufacture of swimming robots. Therefore, several researchers have used this simple mechanism for the locomotion of the capsule endoscope. Chen et al. worked on a swimming robot capsule endoscope consisting of a steering head and rotational body  25], di, di25], 2 insufflation system for use in colorectal capsule endoscopy  an optical dome, (2) lens, (3) light source, (4) image sensor, (5) battery, (6) transmitter, and (7) antenna.The optical dome is made of hemispherical plastic in front of the capsule endoscope. There is an appropriate distance between the dome and the internal camera to observe the digestive tract with the mounted camera. The lens uses a single-focal lens with a small aperture. Light-emitting diodes are used as light sources to illuminate the inside of the digestive tract. Metal oxide silicon imagers use micropower to operate in low light and have many circuits integrated into a small chip.The first capsule endoscopy system was produced by Given Imaging . The third-generation model PillCam SB3  is currently used as a capsule endoscope for the small intestine. In addition, Mirocam , CapsoCam , EndoCapsule , OMOM capsule  were used . VariousThe first-generation PillCam SB capsule endoscope measures 11 × 26 mm, and both the second and third generations have the same size  31,32].,32.31,32Intromedic  released the MiroCam. Other capsule endoscope models use radiofrequency transmission for image transmission, but MiroCam uses human body communication to transmit information. This technology can record videos for a longer period through finer power consumption. MiroCam measures 10.8 × 25.4 mm, has a viewing angle of 170 degrees, takes 3–6 pictures per second, and has a battery life of 12 h.Introduced by CapsoVision (USA) in 2013, CapsoCam can store captured images in the capsule endoscope, eliminating the need to carry external receivers and storage devices during the examination period. In addition, CapsoCam provides a 360-degree panoramic view with four cameras arranged at 90° in the middle of the capsule on the side, unlike the existing capsule endoscope with a camera at the end of the capsule. CapsoCam SV-1 can take 12–20 photos per second and store the images in the capsule itself. However, there is a disadvantage in that it is necessary to find a capsule endoscope in the patient’s stool after examination and check the image taken.White light imaging (WLI) is currently used as the principal technology in the clinical practice of capsule endoscopy. Despite recent advances in endoscopic imaging technology, capsule endoscopy has limitations in using these technological improvements. The clinical usefulness of WLI capsule endoscopy has motivated research groups to improve integrated diagnostic capability with other sensing methods. These advancements have made it possible to overcome the drawbacks of WLI capsule endoscopy through enhanced detection of gastrointestinal pathologic lesions, such as microlesions, subepithelial lesions, and transmural pathology.To overcome the limitations of WLI, alternative imaging technologies, such as chromoendoscopy and narrow-band imaging (NBI), have been developed ,34. WithResearch to integrate autofluorescence imaging into capsule endoscopy has been conducted by developing several prototype imaging devices ,43,44, aOne of the major drawbacks of capsule endoscopy is that it is difficult to accurately measure the lesion’s size. In addition, since capsule endoscopy is difficult to manipulate and inflate air, it is problematic to identify polyps or excavated lesions. To solve this problem, 3D reconstruction of capsule endoscope images has been attempted to obtain more information using current capsule endoscope images ,56,57,58Software methods, including shape-form-shading, have been used to reconstruct the 3D structure of the small bowel ,60. The ® capsule endoscopes. The census-based Hamming distance and absolute difference of intensities were used for stereo matching [Nam et al. presented a new stereo camera-based capsule endoscope  61]. Th. Th61]. matching . The autDuring capsule endoscopy, the diagnostic yield is reduced when food remains or air bubbles are present in the small intestine. Therefore, it is important to perform proper bowel preparation and reduce bubbles in the intestine before performing capsule endoscopy . Studiesp = 0.00) . A . A 72]. Leung et al. proposed an inflatable prototype capsule for hemostasis of the gastrointestinal tract via the balloon tamponade effect . The devCapsule retention is a rare but important complication of capsule endoscopy. Capsule retention is a case in which capsules cannot be ejected for 2 weeks after administration or an intestinal obstruction occurs and surgery is required. To prevent the risk of capsule retention, a patency capsule was developed . PatencyCapsule endoscopy is a convenient examination method for patients, but checking more than 50,000 images is time-consuming. After completing the capsule endoscopy, the image records stored by connecting an external storage device to the computer were analyzed and read using the software program provided by the company. The image reading program tracks the location of the intestinal tract and has a function to view continuous images from multiple sides at the same time, an enlargement function, and a function to detect bleeding and vascular suspicious lesions automatically. New functions are continuously being added . The reaRecently, with the development of artificial intelligence, several researchers have reported the potential application of convolutional neural network (CNN) systems to diagnose various small bowel lesions. The application of this artificial intelligence capsule endoscope will reduce the time required for capsule endoscope interpretation. With the development of deep learning algorithms, the CNN, which obtains specific characteristics by polling and convolutional layers and performs back-propagation to create the best feature map, has become the main deep learning algorithm for image analysis ,76. AokiKlang et al. presented a CNN model for detecting ulcers in Crohn’s disease using 17,000 capsule endoscopy images . This CNSaito et al. developed a CNN model with 30,584 images to detect and classify protruding lesions . This moWireless capsule endoscopy has become a routine examination in clinical practice for investigating the gastrointestinal tract. Although capsule endoscopy has several limitations, studies by several researchers to overcome these shortcomings are ongoing. For more effective technological advancement, both engineers and clinicians need to participate.The usefulness of capsule endoscopy is emphasized because it is less invasive and is convenient during the procedure. Such usefulness will become more prominent over time.Studies to overcome the limitations of capsule endoscopy are meaningful separately; however, they will eventually be integrated into one to bring about greater synergy. However, most of the technological advances described above are still in their infancy, and thus it is necessary to verify the efficacy of technological advances through the human application.