Pancreatic cancer is one of the most lethal malignancies and has an extremely poor diagnosis and prognosis. The development of resistance to gemcitabine is still a major challenge. The long noncoding RNA PVT1 was reported to be involved in carcinogenesis and chemoresistance; however, the mechanism by which PVT1 regulates the sensitivity of pancreatic cancer to gemcitabine remains poorly understood.The viability of pancreatic cancer cells was assessed by MTT assay in vitro and xenograft tumor formation assay in vivo. The expression levels of PVT1 and miR-619-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blotting analysis and qRT-PCR were performed to assess the protein and mRNA levels of Pygo2 and ATG14, respectively. Autophagy was explored via autophagic flux detection under confocal microscopy and autophagic vacuole investigation under transmission electron microscopy (TEM). The functional role and mechanism of PVT1 were further investigated by gain- and loss-of-function assays in vitro.In the present study, we demonstrated that PVT1 was up-regulated in gemcitabine-resistant pancreatic cancer cell lines. Gain- and loss-of-function assays revealed that PVT1 impaired sensitivity to gemcitabine in vitro and in vivo. We further found that PVT1 up-regulated the expression of both Pygo2 and ATG14 and thus regulated Wnt/β-catenin signaling and autophagic activity to overcome gemcitabine resistance through sponging miR-619-5p. Moreover, we discovered three TCF/LEF binding elements (TBEs) in the promoter region of PVT1, and activation of Wnt/β-catenin signaling mediated by the up-regulation of Pygo2 increased PVT1 expression by direct binding to the TBE region. Furthermore, PVT1 was discovered to interact with ATG14, thus promoting assembly of the autophagy specific complex I (PtdIns3K-C1) and ATG14-dependent class III PtdIns3K activity.These data indicate that PVT1 plays a critical role in the sensitivity of pancreatic cancer to gemcitabine and highlight its potential as a valuable target for pancreatic cancer therapy. Pancreatic cancer is one of the most lethal malignancies due to its frequently late diagnosis, aggressive tumor growth and high level of metastasis. The overall 5-year survival rate of patients with pancreatic cancer is less than 5% . Due to Long noncoding RNAs (lncRNAs) comprise a heterogeneous family of RNA molecules longer than 200 nucleotides with no or limited protein-coding potential. LncRNAs mainly play their roles through the following mechanisms: 1) LncRNAs competitively adsorb miRNA as competing endogenous RNAs (ceRNAs) through base complementarity, resulting in the loss or reduction of miRNA function and controlling the regulation of miRNAs on target genes. 2) LncRNAs activate or inhibit certain cell activities by binding some epigenetic-related proteins to modify specific protein translation after transcription. 3) LncRNAs inhibit gene transcription by directly binding chromosomal DNA or promote gene transcription by recruiting transcription factors. 4) Finally, lncRNAs function as precursors of miRNAs . The lncMacroautophagy (hereafter referred to as autophagy) is an evolutionarily ancient and highly conserved catabolic process. It begins with the formation of double-membrane vesicles known as autophagosomes that engulf cellular proteins and organelles for delivery to the lysosome . AutophaThe Wnt/β-catenin signaling pathway plays an important role in regulating various cellular activities, such as self-renewal, cell proliferation, embryonic development, tissue homeostasis and cell death . IntraceIn this study, we attempted to investigate the contributions and mechanisms of the lncRNA PVT1 to the sensitivity of pancreatic cancer to gemcitabine. We demonstrated that PVT1 specifically promotes pancreatic cancer cell resistance to gemcitabine via increased Wnt/β-catenin pathway signaling and autophagic activity. We also indicated that PVT1 modulates gemcitabine resistance by the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes in pancreatic cancer cells. Moreover, we found that PVT1 is up-regulated by Pygo2 through increased binding of the β-catenin/TCF4 complex to its promoter region. Furthermore, we also showed that PVT1 interacts with ATG14 in the cytoplasm and promotes assembly of the PtdIns3K-C1 complex and the activation of ATG14-dependent class III PtdIns3K activity. Collectively, our findings highlight the role of PVT1 in the regulation of chemoresistance in pancreatic cancer.Human pancreatic cancer cell lines PANC-1 and ASPC-1 were purchased from the cell center of Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (TCHu 98 and TCHu 8). The human pancreatic cancer cell line SW1990 and gemcitabine resistant cell line SW1990/GEM was purchased from Biobw . HEK293T cell line was maintained in our lab. Gemcitabine-resistant PANC-1 (PANC-1/Gem) cells were established by our laboratory. Briefly, PANC-1 cells were initially exposed to 0.2 μM of gemcitabine for 1 week. When cells returned to normal growth rate after the period of recovery, the concentrations of gemcitabine were gradually increased  until cells became resistant to 40 μM of gemcitabine, approximately for 10 months. Gemcitabine HCl was purchased from APEXBIO (A1402). Nocodazole and autophagy inhibitor chloroquine diphosphate salt  was purchased from Sangon Biotech (A606391 and A506569). Wnt/β-catenin inhibitor XAV-939 was purchased from Selleck (S1180). Wnt/β-catenin activator lithium chloride (LiCl) and recombinant mouse Wnt3a protein were purchased from Sangon Biotech (A100416) and Abcam (ab81484) respectively. Commercially available antibodies and dilutions used are as follows: anti-β-catenin , anti-CyclinD1 , anti-C-myc , anti-Axin2 , anti-Ki67 , anti-GAPDH , anti-LC3 , anti-p62/SQSTM1 , anti-Mouse HA , anti-Rabbit HA , anti-Pygo2 , anti-P-Glycoprotein/P-gp , anti-Ago2 , anti-ATG14/Barkor  , anti-Beclin1/BECN1 , anti-PIK3C3/VPS34 . anti-ATG7 . anti-ATG9 . anti-PIK3C3/VPS34 . anti-ULK1 .DNA fragments encoding LncRNA PVT1 and Pygo2 were amplified by PCR from the genomic DNA of PANC-1 cells, and sub-cloned into the pCDNA3.0 and pCMV-HA plasmid respectively. DNA fragments encoding ATG14 and ZFYVE1 amplified by PCR were gifts from Prof. Jiahuai Han . The PCR product of ATG14 was cloned into pCMV-HA. The PCR products of ZFYVE1 was cloned into pCMV-N1-EGFP . GFP-LC3 was a gift from Prof. QingKenneth Wang . The nucleotide sequences of all constructs were confirmed by DNA sequencing. The sequence of miR-619-5p was 5′ - GCUGGGAUUACAGGCAUGAGCC-3′. Commercially synthesized 2′ -O-methyl-modified antisense oligonucleotide of miR-619-5p was used as a miR-619-5p inhibitor. HEK293T cell line and human pancreatic cancer cells PANC-1, ASPC-1 and SW1990 were cultured in Dulbecco’s modified Eagle’s medium . All culture media were supplemented with 10% fetal bovine serum , 100 U/ml penicillin G and 100 μg/ml  streptomycin at 37 °C in a humidified incubator containing 5% CO2. siRNA for PVT1#1 was 5′- GAGCUGCGAGCAAAGAUGU-3′, #2 was 5′- ACUUUAAGUGGAGGCUGA AUCAUCU − 3′. siRNA for Pygo2 was 5′- CCAUGAUCUCACCCACCAU − 3′. siRNA for ATG14 was 5′- AUCUUCGACGAUCCCAUAUAUUA − 3′. siRNA for ATG7 was 5′- CAGCCUGGCAUUUGAUAAATT − 3′. Lipofectamine 2000 Transfection Reagent  was used to transfect the PANC-1, ASPC-1 and SW1990 cell lines with the miR-619-5p mimics, inhibitors and siRNAs, and the expression vector of PVT1, Pygo2 or ATG14 was transfected using Lipofectamine 3000  according to the manufacturer’s protocols.-ΔΔct method . Experimental mice were euthanized at the end of the observation period, and then the tumors were excised and weighed.The subcutaneous xenograft mouse model was used to assess the tumor formation ability of PANC-1 cells stably expressing PVT1 treated with or without gemcitabine. All animal experiments were carried out in compliance with a protocol specifically approved for the use of laboratory animals by the Hubei University of Technology Animal Care and Use Committee. Four-week-old (18–22 g) male BALB/c nude mice were purchased from Vital River Laboratory Animal Technology . PANC-1 cells . All data are presented as the mean ± SD (standard deviation) from triplicates. Differences with a To address the potential role of PVT1 in the gemcitabine sensitivity of pancreatic cancer cells, we first stably overexpressed PVT1 in the PANC-1 and SW1990 human pancreatic cancer cell lines and knocked down PVT1 expression in the gemcitabine (Gem) resistant PANC-1/Gem and SW1990/Gem cell lines by using lentiviral transfection. The expression of PVT1 was remarkably increased or decreased as expected  and chloroquine  or small interfering RNA targeting β-catenin and ATG7, which are critical proteins in Wnt/β-catenin signaling and autophagy, respectively. The sensitivity of PANC-1 cells to gemcitabine was significantly restored after XAV-939 or CQ treatment Fig. f. SimilaPygopus2 (Pygo2), a newly identified components of the Wnt/β-catenin signaling pathway, was reported to activate MDR1 expression and mediate chemoresistance in human brain glioma and breast cancer via the Wnt/β-catenin signaling pathway by our team and other researchers , 26. To MDR1 gene), increasing the energy-dependent efflux of cytotoxic drugs from cancer cells [MDR1, the results of which indicated that PVT1 specifically increased the mRNA level of MDR1. Moreover, the increase in MDR1 mRNA was inhibited by either treatment with XAV-939 or depletion of β-catenin and Pygo2  [Through sequence alignment using the JASPAR website  , we wereOne of the mechanisms by which lncRNAs function in cell regulation is competitively adsorbing miRNA as a ceRNA, which results in the loss or reduction of miRNA function and controls the regulatory effect of miRNA on target genes , 29. By To further determine whether miR-619-5p can down-regulate Pygo2 expression, we performed dual-luciferase reporter assays in PANC-1 cells transfected with a luciferase plasmid harboring the predicted miR-619-5p binding sequence in the 3’UTR of Pygo2 and miR-619-5p mimics, miR-619-5p inhibitor or control sequences. The results revealed that miR-619-5p significantly inhibited luciferase activity driven by the luciferase reporter containing the WT Pygo2 3’UTR sequence but not the reporter containing the MT Pygo2 3’UTR sequence, but the miR-619-5p inhibitor had the observed opposite effect on luciferase activity . We first analyzed the expression of some autophagy-related genes after PVT1 knockdown, the results of which showed that the expression of ATG14 was significantly decreased  [LncRNAs have been reported to exert their biological functions by interacting with certain proteins . InteresRPISeq/) . To confRPISeq/) . TherefoRPISeq/) . To deteRPISeq/) . ImmunofTo investigate the biological role of the PVT1/miR-619-5p axis in human pancreatic cancer gemcitabine resistance, we assessed the viability of PANC-1 cells treated with gemcitabine at different concentrations for different durations. The results showed that co-transfection of PVT1 and miR-619-5p decreased the gemcitabine IC50 value compared with PVT1 overexpression alone  that plays an essential role in the initiation of autophagosomal particles . MoreoveBoth Wnt/β-catenin signaling and autophagy were found to benefit from overcoming chemo-resistance in multiple cancers. However, the reported regulatory feedback mechanism between Wnt/β-catenin and autophagy had a suppressive effect –55. The In summary, it is our novel discovery of a positive feedback loop between the lncRNA PVT1 and Wnt/β-catenin signaling involved in human pancreatic cancer gemcitabine resistance. We also demonstrated the mechanism by which lncRNAs regulate autophagy, which occurs through their binding to autophagy-related proteins. We also determined that cooperation between autophagy and Wnt/β-catenin signaling overcomes chemotherapeutic stress.Our findings provide insight into PVT1 to be a prognostic marker for pancreatic cancer chemosensitivity, as well as in the development of novel treatment against human pancreatic cancer.Additional file 1: Figure S1. PVT1 promotes pancreatic cancer cell viability and inhibit cell apoptosis. FigureS2. PVT1 enhances the expression of Wnt/β-catenin target gene. FigureS3. PVT1 transcription was enhanced by Wnt/β-catenin signaling pathway. FigureS4. The distribution of PVT1 in PANC-1 and ASPC-1 cells


X-ray dark-field (XDF) imaging accesses information on the small-angle scattering properties of the sample. With grating interferometry, the measured scattering signal is related to the sample’s autocorrelation function, which was previously demonstrated for simple samples, such as mono-dispersed microspheres for which the autocorrelation function is mathematically given. However, in potential clinical applications of XDF imaging, complex microstructures, such as lung parenchyma are under investigation. Their bahaviour in XDF imaging is not yet known and no mathematical description of the autocorrelation function is derived so far. In this work we demonstrate the previously established correlation of the XDF data of complex sample structures with their autocorrelation function to be impractical. Furthermore, we propose an applicable correlation between XDF and the sample’s structural parameter on the basis of mean chord length, a medically-approved measure for alveolar structure, known to be affected by structural lung diseases. Our findings reveal a correlation between energy-dependent XDF imaging and the sample’s mean chord length. By that, a connection between a medical measure for alveoli and XDF is achieved, which is particularly important regarding potential future XDF lung imaging applications for the assessment of alveoli size in diagnostic lung imaging. They are able to access information not only about the attenuation properties of the observed object, but also of phase and scattering properties. Thus, information about the sample’s microstructure and refractive properties, related to ultra-small-angle X-ray scattering, can be accessed6. Furthermore, previous studies have demonstrated a relation between the scattering signal and the size of structural features for the prior mentioned imaging modalities8.The main application of X-rays is in diagnostic imaging, which is based on their ability to penetrate matter and show the body’s internal structure noninvasively. Various modalities, such as radiography, computed tomography (CT) and mammography are based on the interaction of X-rays with matter and are highly developed. Up to now, image formation in clinical state-of-the-art X-ray applications is based on attenuation. In recent years, several X-ray imaging techniques, such as analyzer-based imaging9. In recent years, previous studies aimed to provide a quantitative correlation between XDF signal at a distinct sampled correlation length and the autocorrelation function of the sample’s microstructure. It was derived theoretically and subsequently shown experimentally by Lynch et al.10 and Yashiro et al.11 with monochromatic X-ray sources. Further, Prade et al.8 extented the experimental proof of the correlation to a polychromatic laboratory grating-based XDF setup by reference to an adequately simple problem formulation with mono-dispersed microspheres.In terms of clinical applicability, grating-based X-ray dark-field (XDF) imaging with a three grating Talbot-Lau interferometer shows promise as it is feasible with conventional X-ray sources and does not require high spatial or temporal coherence12. Among the most important structural lung pathologies are pulmonary emphysema and fibrosis, which differ in microstructure. Emphysema presents as a destruction of alveolar structure, whereas fibrosis is linked to the formation of excess tissue and densification of alveoli13. A widely used quantity for the characterization of the lung parenchyma is the mean chrod length (MCL). It is a mean intercept measure, well-established in histological analysis of lung tissue and was first introduced by Campbell and Tomkeieff14, based on calculations for convex shapes presented by Tomkeieff15. The MCL describes the entire acinar airspace complex16 and is known to be affected by several structural lung diseases. On the one hand, emphysema leads to an increased MCL17, on the other hand, fibrotic lung tissue is demonstrated to show smaller MCLs compared to healthy lung tissue18.As XDF imaging is highly depending on the structure of the sample on the micometer scale, diagnostic lung imaging is a highly promising application of grating-based XDF imaging. The air-tissue interfaces formed by alveolar walls cause small-angle X-ray scattering and thus a strong XDF signal, especially in comparison to the surrounding soft tissue in the thorax19 and fibrosis20, which suggests a similar behaviour for humans and would enable a diagnosis of potential pathological changes.The diagnostic potential of XDF iamging is given due to the correlation of structure size and XDF signal. Thus, the difference in microstructure of the lung pathologies results in a variation of XDF signal. Recent small-animal studies revealed a decrease in XDF signal for both emphysemaHowever, the quantitative correlation between XDF and the autocorrelation function was only demonstrated for spheres. But lung parenchyma can not be approximated as sphere-like structures, but rather are a complex system of alveoli walls and air-filled spaces. The purpose of the present work is to demonstrate the difficulty to relate the autocorrelation function and XDF imaging of complex sample structures quantitatively. Furthermore, we present an alternative correlation between energy dependent XDF imaging and the sample’s MCL, which is shown to be more practicable for fututre XDF imaging applications on samples with complex microstructure, such as lung parenchyma.21, an intensity curve is acquired for each pixel. The visibility of this curve is defined by its maximum and minimum intensity. Based on the visibility acquired with and without the sample, the dark-field signal D can be calculated as the reduction of visibility due to scattering3.In grating-based XDF imaging, three contrast channels are retrieved, namely transmission, differential phase and dark-field. With the phase-stepping approach10,The length scale on which correlations within the sample can be measured in grating-based XDF imaging is given by the autocorrelation length t22,With a grating interferometer, the differential scattering cross-section is transformed back into the real-space. Thus, in the case of isotropic multiple scattering, the XDF signal can be directly related to the autocorrelation length 23,The scattering cross-section x denotes the beam direction. The z-direction corresponds to the sensitivity direction of the setup, which is perpendicular to the grating lamellae as well as x. The autocorrelation function 24, the normalized autocorrelation function 23. F denotes the three-dimensional Fourier transform and its inverse is termed In terms of XDF imaging, 8 on the example of mono-dispersed microspheres and the use of the mathematical discription of E  were aquired.For non-spherical structures, the autocorrelation function is mathematically not known. Therefore, we calculated mentclass2pt{minimmentclass2pt{minimV(E) of the setup and the effective spectra 29 and tuned via calibration measurements for the specific detector model used in our experiments. The effectively sampled autocorrelation lengths D by photons with energies of low flux is minor, compared to photon energies of higher flux. The same applies for photon energies for which the visibility is low. The visibility spectrum gives the visibility of the XDF setup depending on the X-ray energy and is independent of the accelaration voltage of the X-ray source. Typically, the peak visibility is located near the design energy of the grating interferometer. We measured the visibility spectrum V(E) with an X-ray spectrometer by Amptek, Inc. . The photon spectrum was simulated based on the applied accelaration voltage of 120 kV. Additionally, the absorption due to the gold gratings of the interferometer was taken into account. Figure The XDF signal measured with a polychromatic source can be interpreted as the summation over all dark-field signals for each individual sampled autocorrelation length of different photon energies. We assume that for a polychromatic X-ray beam, the effectively sampled autocorrelation length mentclass2pt{minimmentclass2pt{minimIn this work, we investigated samples with complex micro structure, such as various types of closed-cell plastic foams, namely: neoprene (CR-L), ethylene propylene diene monomer rubber (EPDM) , and polyurethane (PU) rubber . Addtionally, non-suspended hollow glass microspheres, namely K1 and S60  were investigated.An example CT slice for the three different types of closed-cell foams  as well as the two types of hollow glass microspheres (K1 and S60) are depicted in Fig. Due to the differences in structure size among the sample materials under investigation, we expect them to appear differently in XDF imaging. In our experiments, energy-dependent XDF signals were measured at a polychromatic setup with a two-threshold photon-counting detector.For both energy intervals, the measured XDF signals for the sample materials under investigation are provided in Table According to Eq. , the twol by Eq. . For theentclass1pt{minimaentclass1pt{minimaG in Fig. G are measured. Due to assumed isotropy of the samples under investigation, better statistics is obtained by averaging over all angular radial profiles, which yields the unit-less projected real-space autocorrelation function G depending on In a next step, we caluclated the real-space autocorrelation function of the  attenuation coefficients, from the microCT data sets. Figure ying Eq. , the autn in Eq. ). After z)] Fig. b, the prFor increasing r to Eq. . The locBased on Eq.  and the entclass1pt{minimaAlthough the trend in both the XDF data provided in Table 31. An example slice of EPDM after segmentation is depicted in Fig. As the autocorrelation function was demonstrated to be unsuitable for a correlation between the sample’s structure size and XDF imaging, especially in view of large scale clinical applications, we demonstrate a correlation between XDF and an alternative structural parameter, namely MCL. The measurement procedure of MCL can be easily transferred from two-dimensional histological sections to two-dimensional sectional images from microCT. For that purpose, the microCT scans were binary segmented using the interactive learning and segmentation toolkit ILASTIK16. Both was constituted in our case. The MCL was measured for all sample materials under investigation.If a cavity extends beyond the boundaries of the observed region of interest, it is excluded, as it would cause an underestimation of MCL. This method relies on the use of test lines, thus, isotropy is required, which can be given by the sample itself. Moreover, by randomizing the spatial orientation of the line set, isotropy can be achievedAs we expect differences in energy-dependency of the XDF signal among the sample materials, the ratio of both energy bins represents the differences qualitatively and is used for a correlation with the MCL. Dividing both energy bin images is possible as they are spatially registered by the photon-counting detector. Therefore, a finite volume element within the sample is represented in the identical pixel in both energy bins. In Fig. 32, resulting in an overlap of the spectra of both intervals, which may be higher than presumed. As a consequence, the effectively sampled autocorrelation lengths lie more closely together than calculated. To minimize uncertainties induced by the detector, an integrated charge-sharing correction was used. But also other effects, such as fluorescence of the sensor material, can cause an imprecise bin separation. No further corrections for such effects were implemented in this work. Apart from that, the determination of an effective autocorrelation length for each energy bin neglects the extension of the sample in beam direction. The correlation length decreases along the beam direction thoughout the sample. Consequently, the larger the sample thickness, the larger the range of sampled correlation lengths gets. In view of potential applications of correlations between XDF and microstructure size, this is a disadvantage. In diagnostic imaging for instance, the lung diameter of an average person is 15 cm, for which a difference in autocorrelation length from one to the other patient side is expected. Another important point is the limited number of data points given by the two avaiable energy bins which is disadvantageuos for a correlation. Hence, a higher number of smaller energy intervals could lead to more precise results by providing additional sampled correlation lengths. This could be achieved by using a photon-counting detector with more adjustable thresholds. However, photon-counting detectors with a higher number of thresholds are rarley available and a higher number of thresholds is currently not available in combination with a charge-sharing correction. The overlap of the effective spectra without charge-sharing correction will be increased and the difference in effectively sampled autcorrelation lengths decreased. Furthermore, the problems regarding the detector response and overlapping energy intervals still remain and might be enhanced.Although the trend of measured XDF signals depending on mentclass2pt{minim18 up to 30. As our method provides a qualitative but not quantitative correlation, no assumptions of the detector’s energy response, the source spectrum, nor the visibility spectrum have to be made. However, the choice of the threshold settings is expected to influence the results quantitatively and should preferrably be done by considering the source and visibility spectrum. The approach of accessing the sample’s structure based on the MCL with spectral XDF imaging can be performed at any grating-based interferometric setup. To enable a qualitative ranging of structure sizes based on XDF imaging, a calibration with known sample structures, for instance the sample materials used in this work, would have to be performed.The overall difficulty of relating the autocorrelation function and XDF signal for samples with complex and non-spherical microstructure makes its appliation impracticle. Therefore, we have demonstrated an alternative approach to correlate spectral XDF imaging with the sample’s microstructure size on the basis of MCL. Due to a difference in energy-dependent scattering properties of the sample materials. The MCL is an established medical quantity, which is known for common pathologies in the lung. In addition, the determination of MCL is robust and can be done equally for many types of porous materials. Furthermore, the MCLs of the samples under investigation were distributed over a broad range between Conclusively, our findings suggest the possibility to grade objects with various structural sizes by accessing information about their structural properties in energy-dependent grating-based XDF imaging with a spectral detector. Furthermore, the demonstrated correlation between XDF imaging and a medical measure for alveolar structure implies a potential increase of diagnostic power of XDF lung imaging. With our results an attempt to directly distinguish samples with structural properties comparable to human lung tissue in spectral grating-based XDF imaging was presented. Hence, information about destruction or densification of lung tissue may be directly accessible and precise statements about structural changes in lung tissue could be made. For patients with lung diseases in early stages, XDF chest imaging could consequently allow a differentiation between pathological structural changes. In comparison to state-of-the-art chest imaging modalities, such as conventional chest radiography which typically lacks sensitivity in terms of diagnostic lung imaging, our method has the potential to increase the sensitivity in XDF applications. To further investigate the applicability of the demonstrated correlation between MCL and spectral grating-based XDF imaging, research should focus especially on the impact of additional absorbers which can cause beam-hardening and therefore falsify the measured XDF signal.The spectral XDF measurements were performed at a polychromatic cone-beam microfocus setup. The source was the XWT-160-CT  X-ray tube with a tungsten reflexion target. The tube window consisted of 2 mm aluminum. The source was operated at 120 kV and 50 W. The Talbot-Lau grating interferometer consisted of three gratings and was arranged horizontally in symmetric geometry. The distance between the source and reference grating as well as the distance between reference and analyzer grating were 0.925 m, which corresponds to the first Talbot order. The total length of the grating interferometer was 1.85 m. The periods of source V(E).The photon-counting detector XCounter Flite X1 was used, which consists of a 0.75 mm CdTe layer and its pixel size is 21 with seven phase steps. For each phase step, 20 frames with D were obtained from the raw phase-stepping by an expectation maximization algorithm as described by Marschner et al.33.Data acquisition was performed with a phase-stepping approachTo access information about the structure of sample materials, microCT datasets were acquired with the ZEISS Xradia VersaXRM-500 , a microCT system developed for non-destructive industrial testing. The tube was operated with 45 kV and 4 W. An objective with a 4


Upper endoscopy is an essential tool for diagnosing pediatric gastrointestinal issues. This study aimed to assess the indications, diagnostic yields, concordance between histopathological and endoscopic findings and suitability of upper endoscopies performed at a tertiary university hospital in Jordan.Hospital records of children who underwent upper endoscopy were retrospectively reviewed. Demographics, endoscopic details , and histopathological findings were collected. The relationship between endoscopic findings and histopathological abnormalities was reported.The study included 778 patients . The most common age group was children younger than 60 months . The most common indication for endoscopy was abdominal pain, followed by vomiting and failure to thrive or weight loss. Normal upper endoscopy was reported in 411 patients (52.8%). Age below 60 months, abdominal pain, dysphagia/odynophagia, and heartburn were predictive of abnormal endoscopy in multivariate analysis with p-value 0.000, 0.048, 0.001 and 0.01 respectively. Abnormal endoscopy showed 67.3% sensitivity and 69.9% specificity to predict histopathological abnormalities. Of those performed, 13.6% endoscopies were described as inappropriate indication. The suitability of the procedure was a sensitive predictor for abnormal endoscopic and histopathological findings.Abdominal pain is the most common indication for upper endoscopy in our population. It is associated with a higher chance of abnormal endoscopy. Concordance between endoscopic and histopathological findings is not high. Normal endoscopic findings shouldn`t discourage the endoscopist from obtaining tissue biopsies. Considering more biopsies may improve pathological detection rates. Compliance with established endoscopy guidelines may reduce unnecessary procedures. With the widespread availability of upper endoscopy, an increasing number of children are referred for endoscopic evaluation. Upper endoscopy allows for the direct examination and obtaining tissue biopsies.  CouplingThe European Society of Gastrointestinal Endoscopy (ESGE) and European Society for Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) have provided evidence-based guidelines with clear indications and timing of diagnostic and therapeutic endoscopy in pediatric patients.  MultipleThis study focused on reviewing our pediatric patients who underwent upper endoscopy in King Abdullah University Hospital (KAUH) during the study period, and it aimed at describing the patients’ characteristics, endoscopic and histopathological findings, the discrepancy between normal endoscopic finding and abnormalities in histology as well as evaluating the suitability of the procedures performed.This is a retrospective, chart-based study conducted in the Pediatric Gastroenterology Division of the Pediatrics Department at Jordan University of Science and Technology (JUST) and King Abdullah University Hospital (KAUH) . The study was approved by the institutional review board of the Faculty of Medicine and the Research Committee at JUST .All procedures were completed by senior pediatric gastroenterologists  in the pediatric endoscopy unit at KAUH. Patients underwent the procedures under light sedation with intravenous midazolam and local lidocaine spray. Heavier sedation (ketamine/fentanyl) was used on a case-specific basis.Histopathological assessments were performed in the histopathology department at KAUH by a senior pathologist, pediatric pathologist, or gastrointestinal pathologist.For this study, all patients who underwent upper endoscopy by a pediatric gastroenterologist at KAUH between January 2014 and January 2020 were identified. Electronic files, including clinical/inpatient files, endoscopic reports, and histopathological reports, were reviewed. For each patient, the following data were retrieved: age, sex, presenting symptoms, examination findings, laboratory test results, endoscopy abnormalities, histopathology findings, and adverse events (sedation or procedure related). The endoscopy procedure list contains both patients evaluated by the pediatric gastroenterologists and referrals.Therapeutic endoscopy cases  were excluded. For patients who underwent multiple endoscopic procedures (whether as a follow-up or due to the persistence of the complaint), only the first diagnostic procedure was included. Patients who underwent endoscopy outside our facility were not considered. In addition, patients who underwent prior surgical procedures leading to expected abnormal endoscopy  were excluded. Procedures with insufficient data  were excluded.We opted to use the endoscopy reports for comparison because not all patients undergoing endoscopy procedures had records of a histopathological examination.An endoscopy was categorized as normal when the upper endoscopy report mentioned either no abnormality or only a minor abnormality that did not contribute to the patient’s illness. It was categorized as abnormal when the upper endoscopy report mentioned an abnormality in any of the areas examined. Histopathology was classified as negative when a histopathological report described no abnormalities or only a mild nonspecific abnormality and positive when the report described abnormal findings.Indication for the procedure was described as appropriate when compliant with the 2016 Pediatric Gastroenterology Endoscopy Guidelines by the ESGE and ESPGHAN, whereas inappropriate indication described noncompliance.[2 and fisher exact test were used as appropriate to compare categories, p-value of < 0.05 considered significant. Variables identified to be significant in the univariate analysis were used as predictors of the probability of abnormal EGD findings or abnormal histopathology in the multivariate analysis.Data were analyzed using IBM SPSS Statistics20.0 software . Descriptive data were reported as percentages of the total for categorical data, as means ± standard deviation (SD). T-test, xDuring the study period, 1,072 upper endoscopy procedures (as some patients had more than one procedures) were identified. After 313 were excluded according to the predetermined criteria , 787 patients were included in the study . As shown in Table n = 359; 45.1%), followed by vomiting  and failure to thrive or weight loss  . Neither sex nor the number of identified indications for endoscopy showed any statistical difference between the normal and abnormal upper endoscopy groups.  patients. Children with normal endoscopy were significantly younger . By contrast, abdominal pain, dysphagia/odynophagia, and heartburn were significantly associated with abnormal endoscopy   Table .p = 0.0001, 0.001, 0.044 and 0.020 respectively  of the examined tissue biopsies. The stomach was the most common site of reported endoscopic abnormalities  and the highest yielding site of histopathological biopsies , followed by the esophageal and duodenal sites, respectively . This has resulted in a change in therapeutic plan in 56 cases. H.pylori gastritis was the most common finding followed by celiac disease and reflux esophagitis.  of the procedures were considered unsuitable. Appropriate indication was a sensitive indicator for abnormal endoscopic and histopathological abnormalities (85.2% and 90.1% respectively), but not specific (13.1% and 23.2% respectively) Table . SeriousPediatric endoscopy is a safe and effective diagnostic tool for children with symptomatic upper gastrointestinal lesions. Although the utility of upper endoscopy in children is on the rise, data on patients from the Middle East are scarce. This study describes the indications as well as the endoscopic and histopathological yields of upper endoscopy in patients treated at our center in Jordan. In addition, this study assesses the appropriate indication of upper endoscopic procedures according to published guidelines. Abdominal pain is one of the most common complaints of patients who present to a pediatric gastroenterology clinic. A significant portion of these children undergo upper endoscopy. In our cohort, abdominal pain is the most common indication for upper endoscopy. Sheiko et al. reported that more than one-third of children underwent endoscopy at their facility due to abdominal and epigastric pain. In additA previous study from northern Jordan by Rawashdeh et al. published in 1996 reported that small intestinal biopsies surpassed abdominal pain as an indication for upper endoscopy. [Endoscopic examination provides a specific morphological description of upper intestinal pathologies. Sheiko et al.  found 37Indications for endoscopy influenced the chance of having abnormal endoscopic findings. Consistent with previous reports , 8, abdoIn our cohort, abdominal pain is associated with a higher chance of abnormal endoscopic findings. The diagnostic yield of upper endoscopy in abdominal pain is highly affected by patient selection. A previous study from Iran reported that 84% of patients with recurrent abdominal pain had abnormal endoscopic findings, whereas another study from Kuwait reported 68% . In a stWang et al. reported a 28% rate of abnormal histological findings in children with newly presenting gastrointestinal symptoms, which is consistent with other reports from the developed world. , 6, 12. Diagnostic concordance between endoscopic and histological findings may not be highly reliable.  In our sThe utility of endoscopic biopsies in otherwise normal endoscopic exam has not been well studied A previous study from Canadian Adult Endoscopy Unit evaluated utility of endoscopic biopsies in adult patients with normal upper endoscopy. The authors concluded that the yield is highly affected by the site of the biopsies. The added expenses mandate high selectivity to augment the yield . Due to In our study; the biopsy yield was affected by the biopsy site. The stomach represented the most common site followed by the duodenum and the esophagus. Reflux esophagitis, H.pylori gastritis and celiac disease where the most common diagnoses. In multivariate analysis of features that could predict abnormal histopathology in our children with normal endoscopic exam; younger age group and abdominal pain were positive predictors. The small size of the group and the absence of cost-effectiveness evaluation limit generalization of our results and warrant further evaluation of such group.The classification of suitability of endoscopic procedures would help practitioners identify which patients should undergo endoscopic evaluation. In our situation, financial resources are limited and the small endoscopy team can become overwhelmed with referrals. Jantchou et al.  found thFurther studies addressing the effect of upper endoscopy results on the patient management and reporting milder post-procedure complications are needed to improve the diagnostic yield and the procedure outcome. Evaluating patients referred for endoscopy at the pediatric gastroenterology clinic prior to endoscopy enlisting might decrease unnecessary procedures and costs.Our study provides valuable data on the epidemiology, endoscopic, and pathological findings of upper endoscopy and the correlation between endoscopic and histological findings. However, the study has some weaknesses. First, this study uses a retrospective design. Second, endoscopic description is sometimes subjective and depends on the endoscopist, which may lead to the misclassification of the findings. Third, this study did not collect data about any pre-endoscopic treatments, which could have led to discordance between endoscopic and histopathological findings. Fourth, the inconsistent number and sites of endoscopic biopsies, which cannot be fully explained due to the retrospective nature of the study, limit the generalization of these study results. And lastly, this study didn`t quantify the effect of the procedure on the patients` management plan. Even normal endoscopy provides significant input by refuting a diagnosis or modifies treatment.Pediatric upper endoscopy is a valuable diagnostic tool. Older children are associated with a higher chance of abnormal endoscopic findings. Abdominal pain is the most common indication for upper endoscopy and is associated with a higher chance of abnormal endoscopy. Concordance between endoscopic and histopathological findings is not high, so routine biopsies need to be considered. Compliance with the published guidelines could improve pathology detection rates and reduce unnecessary referrals. Further studies on the effect of the endoscopy results on the management decisions are needed.


Aiming to gain knowledge in the epoxy-bitumen modification mechanisms, this work explores the effects that epoxy concentration and ambient curing exert on the physico-chemistry and thermo-rheological properties of epoxy-modified binders. Process rheokinetics of epoxy-bitumen blends indicates that binder short-term modification  is accelerated by epoxy concentration. Furthermore, a synergistic effect of epoxy concentration and ambient curing is found during long-term modification . As a result, viscous and viscoelastic rheological properties of binders are enhanced at medium/high in-service temperatures, at least, after one month of curing. FTIR (Fourier Transform Infrared spectroscopy) tests and SARAs  analysis confirm the existence of esterification/etherification reactions between epoxy oxirane groups and the carbonyl groups available in aromatic and resin molecules. Thus, the new high molecular weight compounds increase the asphaltenic fraction of modified bitumen. Likewise, nonreversing heat flow curves obtained by modulated calorimetry corroborate the formation of such highly structured domains responsible for the final binder performance. The drastic increase in vehicle traffic, load, and speed, along with weathering, may result in a shortening of the in-service life of asphaltic pavements . RelatedWith all this in mind, current pavement engineering research focuses on the exploration of new modification approaches in order to develop bituminous materials with enhanced processability, workability, and long-lasting characteristics . In thisOn these grounds, although some experiences using epoxy modifiers were reported in the 1950/1960s , the modAdditionally, other works ,22,23,24Therefore, although some attempts have been made to use epoxies for bitumen modification, a general lack of knowledge still exists. Thus, the novelty of this paper is to study the effects that epoxy addition to neat bitumen (together with ambient curing) exerts on the modification mechanisms, microstructure, and physicochemical characteristics of the resulting bituminous binders. With this aim, different amounts (from 0.5 to 4 wt %) of an epoxy modifier (bisphenol A diglycidyl ether) were added to neat bitumen. These low concentrations of epoxies were selected to avoid the gelation phenomena. Bitumen-epoxy modification kinetics during the mixing process (short-term modification) and curing stage (long-term modification) were studied by rheokinetics tests, rheological measurements, modulated calorimetry (MDSC), thin-layer chromatography (TLC-FID), and infrared spectroscopy (FTIR).−1 and epoxy value of 0.52 mol/100 g, supplied by Panreac, S.A. .A bitumen with 61 dmm penetration and 50.2 °C ring-and-ball softening temperature (EN 1426:2015 and EN 1427:2015) was used as base material for the bitumen modification. The selected epoxy modifier was Bisphenol A diglycidyl ether (hereinafter referred to as BPA), a bifunctional epoxy with a molecular weight of 340 g·molThe modification kinetics during the mixing process was studied by recording binder torque evolution (related to its viscosity) with time, in a controlled-stress rheometer RS-600 . An open batch mixer was used, which consisted of a cylindrical vessel  and a helical ribbon impeller (38 mm diameter). To that end, 90 g of base bitumen was added to the vessel and introduced in an oven for 20 min at the processing temperature (180 °C). Afterwards, the vessel was placed in the rheometer and the helical ribbon impeller was moved to 3 mm above the bottom of the vessel, allowing an agitation speed of 90 rpm. After 10 min mixing, epoxy was added at concentrations between 0.5 and 4 wt %, and the blend was stirred for 24 h. After the 24 h processing, the resulting binder was divided into two parts: one was tested as such (these samples will be referred to as noncured binders), and the other was exposed to ambient conditions for one month . 5 and 8.98 × 104 g·mol−1) was prepared with a Silverson L5M homogenizer at 180 °C for 2 h.For the sake of comparison, base bitumen was also processed under the same mixing conditions , obtaining the so-called blank sample. In addition, a portion of the blank sample was also subjected to the same ambient curing. Finally, a reference bituminous sample containing 3 wt % SBS triblock copolymer , performed in oscillatory shear at 10 rad/s and a strain within the linear viscoelastic range. In order to ensure accurate rheological results, every sample was measured at least twice. The rheological characterization was conducted on bituminous binders using a controlled-stress rheometer Physica MCR-301  and a plate-plate geometry (1 mm gap and 25 mm diameter). Different rheological measurements were carried out: (a) on the one hand, viscous flow tests at 60 and 135 °C; and (b) on the other hand, temperature sweep tests from 30 to 100 °C (1 °C·minThin layer chromatography coupled with a flame ionization detector (TLC/FID) was used for the determination of SARAs fractions , aromatics (A), resins (R), and asphaltenes (As)) of blank sample and epoxy-modified binders. Tests were conducted in an Iatroscan MK-6 analyzer . Elutions were performed with hexane, toluene, and dichloromethane/methanol (95/5) .−1 at 4 cm−1 resolution in the transmission mode.Fourier transform infrared spectroscopy (FTIR) spectra of binders were obtained with a Jasco FT/IR 4200 spectrometer . Firstly, a weight of 0.7 g of blank sample or epoxy-modified binders was dissolved in 25 mL dichloromethane. Afterwards, the solution was always placed on a potassium bromide disk (32 mm × 3 mm) and exposed to ambient for solvent evaporation. Finally, the KBr disk was laid into the sample holder, and the FTIR spectra were obtained in a wavenumber window of 400–4000 cm−1, amplitude of modulation of ±0.5 °C, a period of 60 s, and a flow of 50 mL/min N2 as purge gas. The characteristic temperatures and enthalpies were determined during the second heating sequence.Modulated differential scanning calorimetry (MDSC) tests were conducted on bituminous binders with a TA Q-100 . Samples of 5–10 mg were always subjected to the following procedure: (a) a first heating scan from room temperature to 210 °C was carried out with the goal to remove the thermal history of the sample; (b) a cooling ramp, to −60 °C; and (c) a second heating ramp, to the same final temperature used for the first heating scan. The selected conditions were: heating/cooling rate of 10 °C·min0) with mixing time. The value of M0 corresponds to the constant torque obtained before epoxy addition (which takes places after 10 min of mixing), and M is the actual torque for each mixing time. Thus, M/M0 allows us to quantify torque changes only due to the epoxy modification, regardless of the torque value just before epoxy addition . 0 for the base bitumen (which results in the blank sample after completing its processing) and their corresponding epoxy-modified binders containing different epoxy concentrations. The kinetics of the short-term bitumen modification at 180 °C was assessed by monitoring the evolution of the normalized torque  does not produce a significant hardening in the binder due to oxidation. As for the epoxy-modified binders, all of them display the same pattern: (a) a first region with a constant value of M/M0  before epoxy addition; (b) a marked decrease in M/M0 due to the plasticizing effect caused by the addition of a liquid with low viscosity, which becomes more pronounced as epoxy concentration increases; and (c) a monotonous increase in M/M0 with no trend to level off. This behavior is ascribed to an epoxy-induced bitumen hardening, or the chemical crosslinking, since the effect of bitumen oxidation was discarded with the result observed for the base bitumen. In addition, the value of M/M0 after completing processing, which corresponds to the final binder hardening, is proportional to the epoxy concentration. Thus, the highest value was observed for the binder prepared with 4 wt % BPA. First of all, it can be observed that base bitumen experienced negligible evolution of M/M0 curves shown in Interestingly, the continuous increase of M/MAs a whole, short- and long-term epoxy modifications involve changes in binder rheological properties that affect its application in pavements. A rheological characterization based on viscous flow curves, at 60 and 135 °C, and temperature sweep tests were conducted on the binders just after 24 h processing (noncured binders) and on those exposed to ambient curing conditions for one month (cured binders). s’ is a parameter related to the slope of this region. Carreau model parameters for all binders are gathered in As can be seen, all binders show the same viscous response characterized by a Newtonian zone at low shear rates, with constant values of viscosity, followed by a shear-thinning region above a critical shear rate. This viscous behavior can be fitted to the Carreau model:0 after 24 h processing) observed in 60 °C) has been defined as follows: As deduced from 60°C differences between noncured and cured binders allow us to state that even after 24 h of processing there are epoxy groups available for further reaction with bitumen compounds during the ambient curing, this difference being more evident for the binder prepared with 4 wt % BPA. Moreover, As expected, a higher viscosity enhancement is noticed for the noncured binders as epoxy concentration rises, which is favored after their subsequent ambient curing process. Likewise, 3 wt % cured binder presents a higher modification degree than that found for the reference SBS-binder, which is also surpassed even for 4 wt % binder without curing process. Once again, the M.I.eratures ,28. ConsAdditionally, binder viscosity at 135 °C gives valuable information about its pumpability, mixability, and mix workability . Figure According to AASHTO MP320 standard, if binder viscosity at 135 °C exceeds 3 Pa·s, the asphalt mixtures become too hard to be compacted, and it is difficult to form a suitable pavement surface . As seenFinally, oscillatory shear temperature sweep tests, from 30 to 80 °C, were conducted on the reference samples (blank sample and rubber SBS-binder) and two selected epoxy binders (noncured 0.5 wt % BPA and cured 4 wt % BPA). Thus, the evolution with the testing temperature of elastic (G’) and viscous (G’’) moduli and loss tangent (tan δ = G”/G’) are plotted in All bituminous binders show a monotonous decrease of both moduli with increasing testing temperature, prevailing a viscous character . As expected, the 0.5 wt % BPA binder, just after 24 h processing, presents a slight increase in G’ and G’’ with respect to the blank sample. However, 4 wt % BPA cured at ambient conditions displays a marked increase, with both moduli clearly higher than those observed for the SBS-binder (the difference being more evident at low testing temperature). This result is in line with the previous viscous flow response at 60 °C. In all cases, tan δ curves show the elastic binder characteristics decrease with testing temperature. Conversely, the epoxy bitumen modification with 0.5 wt %, without ambient curing, produces a decrease in tan δ values in the entire range of testing temperature (enhancing binder elastic properties). This effect is more pronounced for the cured binder prepared with 4 wt.% BPA. On the other hand, the slope of the tan δ vs. T curve is related with the binder thermal susceptibility at medium/high in-service temperatures. Thus, SBS-binder displays a reduced susceptibility to temperature in the interval of ca. 30–45 °C, with nearly constant tan δ values, similarly to that reported by Martin-Alfonso et al. [The above-commented changes in rheological performance of epoxy modified binders at high in-service temperatures are related to significant changes in their physicochemical characteristics and microstructure. In order to clarify this, FTIR, TLC/FID, and MDSC tests were performed on selected samples.2H4O) groups, which are generated after epoxy ring opening, react with the carbonyl acid groups (RCOOH) present in the bituminous phase forming ester (RCOOR’’) and ether (ROR´) compounds [−1, to evaluate carbonyl acid and ester groups at 1716 and 1735 cm−1, respectively; (B) 1100–950 cm−1, and (C) 1000–830 cm−1 to evaluate the ether and oxirane groups at 1035 and 917 cm−1, respectively. It has been reported that the oxirane  and 1035 cm−1 (ROR’) together with a decrease of peaks at 917 cm−1 (C2H4O) and 1716 cm−1 (RCOOH). Accordingly, epoxy binder prepared with the highest epoxy concentration and subjected to ambient exhibits the largest marked areas for the reaction products  into the so-called “SARAs” fractions , according to their solubility and polarity. Although still a matter of debate, different models have been proposed to describe how these fractions are arranged to constitute the bitumen’s microstructure . Among tAs seen in Delving deeper into the microstructure, MDSC conducted on these selected binders also provides valuable information. As reported by Masson et al. ,39, fourMDSC), being greater after its subsequent ambient curing. This effect is much more pronounced for those binders prepared with the highest epoxy concentration . Lower values of TMDSC shifts chain mobility to lower temperatures  [MDSC gathered in On the other hand, the second event is provoked by a time-dependent cold crystallization of low molecular weight saturated segments ,39 and atallize) ,39,40. CRheokinetic curves and rheological tests have shown epoxy binders present an enhanced behavior after 24 h mixing at 180 °C . Moreover, this improvement is able to continue under ambient curing for at least one month. As a result, the cured 4 wt % epoxy binder presents viscosity values at 60 °C, much higher than those obtained for the SBS binder.Temperature sweep tests in oscillatory shear reveal that, above 50 °C, this epoxy binder displays elastic properties similar to the reference rubbery binder.The improvement in the thermo-rheological properties occurred during either the mixing stage or the ambient curing. In this regards, FTIR and SARAs tests confirm a chemical modification through esterification/etherification reactions between the oxirane groups of BPA and the carbonyl groups available in aromatics and resins.New bitumen compounds are quantified in the asphaltenic fraction. Likewise, these high molecular weight compounds are more structured and ordered, as confirmed by the increase in the area corresponding to the fourth endothermic event in the nonreversing heat flow curves.Interestingly, in addition to the improved rheological response observed at 60 °C, all epoxy binders exhibit viscosity values, at 135 °C, lower than 3 Pa·s. According to AASHTO MP320, the compaction of their resultant asphalt mixes would be carried out successfully.


Ovarian cancer (OC) is one of the leading lethal gynecologic cancers of women around the world. More than 70% of patients are diagnosed with stage III or IV with poor outcome. This is partly because of lacking early effective screening techniques and potential biomarkers of OC. CXC chemokines in tumor microenvironment (TME) and their interaction with relative receptors can excite the downstream signaling pathways to influence tumor progression. However, the role of CXC chemokines in OC has not been identified.  ONCOMINE, GEPIA, Kaplan–Meier plotter, cBioPortal, TIMER, Metascape, and LinkedOmics were applied in our study.  The transcriptional levels of CXCL1/8/9/10/11/12/13/14/16/17 were significantly elevated while CXCL3 was obviously reduced in OC vs normal ovarian tissue. CXCL8/9/11/13 were correlated with clinic pathological stage. Patients with low expression of CXCL8/9/11/13 were associated with better prognosis. We also found that CXCL3 and CXC12 could be used as potential prognostic markers of OC through Kaplan–Meier plotter. Patients with high expression of CXCL3/12 had a significantly better prognosis. Their functions focus on locomotion, signaling, response to stimulus, undergoing the process of multiorganism, immune system, biological regulation, etc. The differentiated CXC chemokines mainly participate in cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway, and toll-like receptor signaling pathway. Our results showed that CXC chemokines were highly correlated with infiltration of immune cells. The kinase targets of differentially expressed CXC chemokines are mainly in ATM, LYN, LCK, PLK1, FYN, CDK2, and ATR.  Our results may provide a new insight for selecting precision biomarkers of targeted therapy of OC. Ovarian cancer is the most leading lethal gynecological cancer around the world. Patients with epithelial ovarian cancer (EOC), the most common pathological type of OC, are always diagnosed at late stages with poor five-year survival rate (FIGO stage III 51%) or IV 29%)) p=0.004)  Hendrix , respectp=0.018), CXCL9 (p=0.020), CXCL11 (p=0.039), and CXCL13 (p=0.029) were highly correlated with clinic pathological stages . The expression of CXCL8, CXCL9, CXCL11, and CXCL13 was increased as the tumor stage increased. Our results suggested that CXC chemokines play a key role in the progression of ovarian cancer.We then assessed the transcriptional levels of CXC chemokines and their correlation with pathological stage of clinic in the database of GEPIA. Our results showed that CXCL1, CXCL8, CXCL9, CXCL11, CXCL12, CXCL16, and CXCL17 were remarkably elevated in ovarian cancer compared to those in normal tissue . Also, wl stages . The expp=0.02), CXCL3 (p=0.0034), CXCL9 (p=0.0017), CXCL11 (p=0.004), and CXCL13 (p=0.0012) were significantly correlated with long OS. However, low expression of CXCL12 (p=3e − 7) and CXCL14 (p=0.0052) was associated with long OS. The differential expressions of CXC chemokines of progression-free survival (PFS) and post-progression survival (PPS) were also assessed  was significantly correlated with long PPS. However, low expression of CXC12 (1.8e − 05) was relevant to long PPS. Our research suggested that CXCL3 and CXC12 could be used as potential prognostic markers of OC.We then analyzed the prognostic value of differentially expressed CXC chemokines in OC through the Kaplan–Meier plotter database. Overall survival (OS) curves are shown in 14 p=0.002 was ass Figures . We founThe TCGA and CBioPortal were used to analyze the gene alteration rate of CXC chemokines in OC. Among those differentially expressed CXC chemokines, our results showed that 5% of CXCL3, CXCL8, and CXCL11 were altered, while 4% of CXCL12 and CXCL14 were mutated. 3% of CXCL16 and 7% of CXCL17 suffered with gene alteration . We thenGEPIA and Metascape were utilized to analyze the functions, pathway enrichment, and their neighboring genes of differentially expressed CXC chemokines in OC . We delvp=6.42e − 01) (p=5.82e − 01) and CXCL14  were negatively associated with the infiltration of dendritic cells  . The lev2e − 01) . The inf2e − 01) . CXCL3  were highly expressed, while CXC3 was reduced in OC. These results were in accordance with previous researches; CXCL14 and CXCLL16 were highly expressed in OC and they were associated with poor survival outcomes and promoted OCC proliferation , 33. AlsThen, we analyzed the genetic alterations and coexpression of differentially expressed CXC chemokines via TCGA and CBioPortal database. The differentially expressed CXC chemokines all contained a certain proportion of genetic alteration while the altered genes did not affect the prognosis of OC. We found a low-to-high correlation among these differentially expressed genes; it suggested that these CXC chemokines may play a synergistic role in OC progression.Furthermore, we used GO and KEGG pathway enrichment analysis to identify the main functions and pathways of differentially expressed CXC chemokines in OC. Their functions focused on locomotion, signaling, response to stimulus, undergoing the process of multiorganism, immune system, biological regulation, etc. The cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway, and toll-like receptor signaling pathway were the vital pathways that participated in OC. For instance, researchers found that cancer-associated fibroblasts (CAFs) in TME induced epithelial-mesenchymal transition (EMT) and cisplatin resistance in OC via CXCL12/CXCR4 axis . In brea+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells) that were correlated with differentiated CXC chemokines. The results indicated that CXC chemokines could be immune regulators in OC.Recently emerging data suggested that immune cell infiltration plays a key role in tumor progression . Our resAll in all, our research took advantages of public databases to systematically delve into data of CXC chemokines in OC. However, our results may need to be verified by in-depth experiments in vivo and in vitro. Multiple clinical trials are needed to validate potential biomarkers of CXC chemokines. We hope our results can provide novel insights for our researchers and these potential targets could be applied in clinic someday.


Expression of chemokine receptor CX3CR1 is reportedly restricted to several cell types including natural killer cells, cytotoxic T cells, monocytes, and macrophages. However, its expression and function on exosomes, which are nanosized extracellular vesicles known to act as mediators of intercellular communications, remain unclear. Here, we investigated CX3CR1 expression on exosomes isolated from various cell types. Although we found that all the exosomes tested in our study highly expressed CX3CR1, this chemokine receptor was expressed only inside, but barely on, their source cells. Moreover, exosomal CX3CR1 was capable of binding soluble CX3CL1. Therefore, our study suggests that CX3CR1 is a novel and ligand-competent exosome receptor.  •CX3CR1 is highly expressed by exosomes.•Expression of CX3CR1 is restricted within, but not on, the cells.•Exosomal CX3CR1 is capable of binding soluble CX3CL1.•CX3CL1 binding of exosomes may deprive their source cells of the chance to bind this chemokine. Among t0 nm–5 μmApart from their functionality vis-à-vis integrins, chemokine receptors on exosomes have been shown to play a role in tissue-specific homing or chemotaxis in both cells and exosomes. The CXCR4 and CCR2 on bone-marrow stromal cell-derived exosomes exert a promoting effect on the migration of multiple myeloma cells . In addiWhen cognate chemokines are bound, the chemokine receptors present on the plasma membrane are generally internalized in early endosomes and are then trafficked to MVBs . These uThe chemokine receptor CX3CR1 mediates cell adhesion and trafficking through its interaction with chemokine CX3CL1  . CX3CR1 Here, we examined the exosomal expression of CX3CR1 by using many cell types, revealing its high expression by all the exosomes used, and further showing the exosomal binding to CX3CL1. Based on the compelling results obtained in this study, CX3CR1 represents a ligand-competent chemokine receptor highly expressed by exosomes.22.1in vitro culturing and activating primary T cells as previously described [2 supply. T cells activated for 48 h were moved onto antibody-uncoated dishes and further incubated for 72 h in the same culture media supplemented with interleukin-2 (1 ng/ml) . As mentioned above, to eliminate extracellular vesicles (EVs) including exosomes in FBS, FBS was centrifuged at 76,000 g for 18 h at 4 °C using polypropylene centrifuge tubes , a swing bucket rotor  and an L60 Ultracentrifuge (Beckman Coulter) and the supernatant solution was filtered through 0.22-μm filter units . This filtrated FBS was considered to be exosome-depleted and was used in this study for exosome isolation from the cells.All the study has been done with mouse cell lines and primary cells, unless otherwise specified. The spleen cells isolated were used for escribed . In brieSeveral mouse tumor cell lines including TK1, CT26.WT, EL4, LTPA, and B16F10 cells were obtained from ATCC . RAW264.7 (mouse macrophages) and THP-1 (human monocytes) were also from ATCC. MLO-Y4 (osteocyte) and MLO-A5 (osteoblast) cells were purchased from Kerafast . All cells were cultured, according to the manufacturers’ instructions, for 48 h in RPMI1640, DMEM , or MEMα (Thermo Fisher Scientific) media supplemented with 10% EV-depleted FBS and penicillin/streptomycin.2.2C57BL/6J mice (8–11 weeks old) were purchased from CLEA Japan  and maintained at the Experimental Animal Facility of Mie University. Experimental animal protocols were approved by the Ethics Review Committee for Animal Experimentation of Mie University . Spleens, bone marrows, and lungs were separated from the mice and used to isolate cells by using a mechanical dissociation with Falcon 40-μm cell strainers . The media collected in the cell isolation procedure were used to isolate exosomes (see section 2.3g for 10 min at 4 °C to remove cells. The supernatant was spun at 2000 g for 20 min at 4 °C to eliminate apoptotic bodies. The supernatant was centrifuged in an L60 Ultracentrifuge (Beckman Coulter) at 24,000 g for 20 min at 4 °C. The supernatant was then subjected to a second centrifugation at 110,000 g for 2 h at 4 °C. The pelleted exosomes were subsequently suspended in phosphate-buffered saline (PBS) buffer . In turn, this exosome solution was passed through a 0.22-μm filter unit and spun at 110,000 g for 2 h at 4 °C. The pellet (exosomes) was suspended in PBS buffer. The concentration was measured with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The particle size was characterized by using a dynamic light scattering (DLS) device .Exosomes were isolated as previously described with minor changes ,27,28. B2.4The exosomes were conjugated to 4-μm latex beads for efficient detection and then stained with fluorescently labeled monoclonal antibodies as previously shown . In brie2.5Antibodies to CD9 (HI9a), CD63 (NVG-2), CD63 (H5C6), CD81 (Eat-2), CCR9 (9B1), CXCR4 (L276F12), and CX3CR1 (SA011F11) were purchased from BioLegend . Isotype controls including Rat IgG2a, Rat IgG2b, Armenian Hamster IgG, Mouse IgG2a, and Mouse IgG1 were also from BioLegend. The antibody to CD9 (KMC8) was obtained from BD Biosciences. The antibodies to CCR7 (4B12), CCR10 (248918) were acquired from R&D Systems. The cells or microbead-conjugated exosomes were stained with the fluorescently labeled antibodies, washed twice with PBS containing 2% FBS and 2 mM ethylenediaminetetraacetic acid (EDTA) , and analyzed by using BD Accuri C6 flow cytometer and software (BD Biosciences). For this method of microbead conjugation of exosomes, only positive events can be detected and fluorescently quantified in the flow cytometry. In some experiments, total (intracellular plus surface) staining experiment was done by using FIX and PERM Kit (Thermo Fisher Scientific) according to manufacturer's instructions.2.6The exosomes conjugated to the microbeads were incubated with recombinant mouse fractalkine-His (R&D Systems) at 37 °C for 1 h, washed once with PBS containing 2% FBS and 2 mM EDTA, and labeled with phycoerythrin (PE)-anti-His Tag antibody (Biolegend). After wash, fluorescence intensity for fractalkine binding was analyzed by using BD Accuri C6 flow cytometer and software.33.1To characterize the exosomes, we examined expression of tetraspanin markers . All the exosomes isolated from different types of mouse cells used in this study were positive for indicated tetraspanins, although they exhibited the expression levels different depending on source cells A–D. We tWe sought to examine expression pattern of selected chemokine receptors on T-cell exosomes that were isolated from activated primary T cells . We disc3.2We next inquired whether CX3CR1 levels are intracellularly sustained, by using two T-cell lines (TK1 and EL4). Expressions of CCR9, CXCR4, and CX3CR1 were poor on the surface of TK1 and EL4 cells, except for CXCR4, which was moderately expressed by EL4 cells A. In theSome of macrophages have been known to express CX3CR1 ,31. UsinCX3CR1 was superior to other chemokine receptors in its exosomal acquisition, suggesting the possibility that the intracellular compartment of this chemokine receptor might largely be restricted to the membrane of MVBs . In this3.3To determine whether CX3CR1 expression by exosomes is limited to those isolated from the aforementioned cell types or is otherwise widespread, we used various cell lines to isolate their exosomes separately and examined their expression levels of CX3CR1, CCR9, and CXCR4. The exosomes from several cancer cells including B16F10 , CT26.WT , and LTPA  were chosen to test their chemokine-receptor expression levels. These cancer-cell exosomes were found to express typical tetraspanins with some variations depending on their parent cell types B. We fouNext, we isolated the exosomes from spleen, bone marrow (BM), and lung cells of mice and then confirmed for their CD9 and CD63 expressions D, in ordIn order to rule out a possibility that this marked CX3CR1 expression of the exosomes may be manifested due to any remaining EVs in their depleted FBS supplemented to culture media, we next decided to test this. PBS (1 ml), PBS (1 ml) plus EL4 EV sample (5 μg), EV-depleted FBS (1 ml), and FBS (1 ml) were first coupled to 10 μl latex beads as done with the same methods as exosomes. Then, all samples stained equally for CX3CR1 as well as CD9 and CD63 were analyzed using flow cytometry. On note, all three samples except for EL4 EV-containing conjugates did not show any positive signals for the expression of CD9, CD63, and CX3CR1 . TherefoFurthermore, because expression of CX3CR1 was remarkable on all the exosomes tested in current study, it may be open to question that the anti-CX3CR1 antibody is too blunt to discriminate the exosomes positive from negative for mouse CX3CR1. We thus tested this by using the exosomes isolated from THP-1 cells (human monocytes) as a negative control, despite CX3CR1 expression on human exosomes remains obscure and should be carefully determined in a separate experimental setting in near future. THP-1 exosomes positive for human CD9 and CD63 were negative for CX3CR1 , indicat3.4To examine the function of exosomal CX3CR1, we explored its binding to fractalkine (CX3CL1), a ligand unique to CX3CR1 . We chosBased on the results shown in this study, two possibilities regarding the regulatory function of CX3CR1-expressing exosomes can be raised. First, those exosomes expressing CX3CR1 are thought to bind both membrane-bound and soluble CX3CL1 produced by counterpart cells before do source cells. The exosomes isolated from the two selected cell types proved to be capable of binding recombinant CX3CL1 A, thus sOurs is the first study to report exosomal expression of chemokine receptor CX3CR1. In particular, CX3CR1 was prevalently and highly expressed by all of the exosomes isolated from the different cell types tested in this work. The various source cells contained high level of CX3CR1 in their intracellular compartments, presumably on the membrane of MVBs, which are the exosomal reservoirs inside cells. Moreover, those exosomes were capable of binding fractalkine. Our study concludes that CX3CR1 is a novel and ligand-competent molecule of exosomes. In future investigations, it would be worthwhile to elucidate: i) the underlying mechanisms by which CX3CR1 expression is maintained at such high levels on all of the exosomes; and ii) the exosomal impact on any functional changes undergone by the cells or tissues that express membrane-bound CX3CL1.Eun Jeong Park: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, review & editing, Visualization, Funding acquisition. Phyoe Kyawe Myint: Investigation, Methodology. Michael G. Appiah: Investigation, Methodology. Patsorn Worawattananutai: Investigation, Methodology. Janjira Inprasit: Investigation, Methodology. Onmanee Prajuabjinda: Investigation, Methodology. Zay Yar Soe: Investigation, Methodology. Arong Gaowa: Investigation, Methodology. Eiji Kawamoto: Investigation, Methodology. Motomu Shimaoka: Conceptualization, Data curation, Supervision, Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that might have influenced the work reported in this paper.


Results showed that carboxyl functionalization could increase the amount and activity of immobilized GOx, thereby improving the electrode properties.This research revealed the effect of carboxyl-functionalization on the mesoporous carbon (MC)-fixed glucose oxidase (GOx) for promoting the properties of bioelectrodes. It showed that the oxidation time, temperature and concentration, can significantly affect MC carboxylation. The condition of 2 M ammonium persulfate, 50 °C and 24 h was applied in the study for the successful addition of carboxyl groups to MC, analyzed by FTIR. The nitrogen adsorption isotherms, and X-ray diffraction analysis showed that the carboxylation process slightly changed the physical properties of MC and that the specific surface area and pore size were all well-maintained in MC-COOH. Electrochemical characteristics analysis showed that Nafion/GOx/MC-COOH presented better electrocatalytic activity with greater peak current intensity (1.13-fold of oxidation peak current and 4.98-fold of reduction peak current) compared to Nafion/GOx/MC. Anodic charge-transfer coefficients (α) of GOx/MC-COOH increased to 0.77, implying the favored anodic reaction. Furthermore, the GOx immobilization and enzyme activity in MC-COOH increased 140.72% and 252.74%, leading to the enhanced electroactive GOx surface coverage of Nafion/GOx/MC-COOH electrode (22.92% higher, 1.29 × 10 Fuel cells, which convert chemical fuels into electricity, have been considered as a renewable power source for electronic equipment. Due to the minimal risk of false-positive responses, enzymes are the most promising bio-receptors used in bio-sensors . Unlike Carbon materials are generally used in electroanalytical investigations due to the low background current, relatively wide potential window, and chemical inertness. Carbon materials are also appropriate for various types of analysis  in both 2O2 and NADH [Nano-carbon materials such as graphene ,13,14 caand NADH ,24. It mand NADH . Howeverand NADH . The infand NADH .To further activate the surface of MC and overcome the deficiencies mentioned above, a number of methods that apply strong oxidation agents  to generate some oxygenated functionalities have been explored. It is known that functional nanomaterials can cause a synergistic effect among biocompatibility, conductivity, and catalytic activity to speed up signal transduction, leading to highly sensitive biosensing . For exaTo explore the carboxylic functionalization conditions for MC, oxidation temperature, oxidant concentration, and oxidation time were studied. The functional groups, skeleton structure, pore volume and specific surface area of MC after carboxyl functionalization (MC-COOH) were investigated by Fourier transform infrared (FTIR) spectrometer, wide-angle X-ray diffraction (XRD) diffractometer, and nitrogen adsorption specific surface area analyzer (BET). The influence of-COOH group on the amount and enzyme activity of immobilized GOx was analyzed. Finally, the electrode was prepared using 5% Nafion solution, and the electrochemical properties were analyzed by cyclic voltammetry (CV).3 g−1, surface area of 150–250 m2 g−1, 699640, Sigma-Aldrich Corp. St. Louis, MO, USA) was oxidized using an alike process to that reported in the literature [4)2S2O8, APS) with different concentrations ranging from 0 to 2 M in 2 M sulfuric acid (H2SO4). In a conventional oxidation experiment, 300 mg of MC was added to 20 mL of the APS solution. To utilize DF-101S heat-collecting magnetic stirrer , the mixture was stirred at 30 rpm for diverse periods of time up to a maximum of 96 h and at various temperatures ranging from 30 to 80 °C. All the oxidized samples were filtered, washed a few times with distilled water until the washing water was without sulfates, and dried overnight in vacuum drying oven .MC  at room temperature in a centrifuge tube. The consequent intermixture of GOx solution and the supports was then incubated at 10 °C with agitating at 220 rpm  for 6 h to attain adsorption equilibrium [The immobilization experiment was conducted by suspending 20 mg of MC-COOH and MC in 1 mL 0.05 M pH 7.0 PBS buffer, which contains 10 mg mlilibrium . After iThe amount of GOx immobilized on MC and MC-COOH particles was computed by ascertaining the original and terminal concentration of GOx in the liquid solution through ultraviolet spectrophotometry at 450 nm . The glucose oxidase activity of GOx/MC-COOH and GOx/MC was analyzed by utilizing a glucose oxidase assay Kit . In each set of experiments, three biological replicates were performed.The glucose bioelectrode was made through pipetting 20 mg of the dispersions of GOx/MC and GOx/MC-COOH in a pH 7.0 PBS buffer solution with 0.5% Nafion onto carbon cloth as an anode . After d−1 region were acquired on a Nicolet 6700 (USA) spectrometer using the usual KBr pellet technique. XRD analyses were performed on an Empyrean diffractometer  using copper anode radiation over the range of 10°–80°. Nitrogen sorption isotherms were measured with an ASPS 2460  analyzer at 77.4 K. In front of measurement, the powders were degassed at 80 °C under vacuum for more than 6 h.FTIR spectra of the samples in the 400–4000 cm−1 scan in the potential range from −1.0 to 1.0 V (v.s. Ag/AgCl) in air-saturated 0.1 M pH 7.0 PBS supplemented with 10 mM glucose at room temperature.By using an Autolab PGSTAT 302 N (Switzerland) potentiostat, all electrochemical processes were analyzed. CV was performed in a three-electrode system, which contains a platinum piece counter electrode, an Ag/AgCl electrode, and a working electrode. CV scans were performed at 20–200 mV·sTo investigate the influence of carboxylation conditions on GOx immobilization on MC-COOH, various reaction conditions, including different oxidation temperatures, APS concentrations, and reaction times, were investigated, shown as As shown in The amounts of immobilized GOx in MC-COOH with various APS concentrations were presented in Moreover, Above all, the results showed that the oxidation temperature, APS concentration, and reaction time could significantly influence MC carboxylation and consequently improve GOx immobilization. From the above experiments, it was found that the adsorption capacity of GOx increased the most  under 2 M APS, 50 °C and 24 h carboxylation conditions. Thus, the MC oxidized by this condition was selected in the subsequent experiments to prepare electrodes.−1, 1638.64 cm−1, and 1523.94 cm−1 (−1 belonged to the stretching frequency of C=O in carboxylic group (O=C−OH) [−1 could be attributed to the symmetric COO- oscillation and/or aromatic ring stretching coupled with greatly conjugated keto groups [−1 was ascribed to the -OH oscillation [−1 were observed in both MC-COOH and MC, attributed to C-O-C asymmetric oscillations in ether groups of oxidized mesoporous carbon, which were observed in both MC-COOH and MC [According to the FTIR analysis results, three bands centered at approximately 3451.50 cm.94 cm−1 a, A were(O=C−OH) . The bano groups . The broillation ,40. The H and MC ,42. ThesIn the wide-angle XRD patterns b, the MC4 hysteresis loops and an obvious capillary condensation step [4 hysteresis loops, often did not close before approaching the saturation pressure, were also structured by slit-shaped pores, reflecting the characteristics of activated carbon materials [0 values between 0.6 and 0.8, bespeaking that MC-COOH had an abundant uniform mesoporous structure [Nitrogen adsorption/desorption isotherms, and the corresponding pore size distribution curves of the MC and MC-COOH samples were presented in ion step ,48. Variion step . Type H4aterials . The hystructure ,51.2 adsorption isotherm at 77 K, as shown in In addition, the pore size distribution curve based on the adsorption data explicitly identified a limited pore size distribution within 4–12 nm b and shoCV scans of Nafion/MC, Nafion/MC-COOH, Nafion/GOx/MC and Nafion/GOx/MC-COOH were obtained, as shown in As is well known, glucose is the substrate of GOx, whose presence will result in an oxidation reaction and increase the oxidative peak current on electrode surface. Previous studies presented that the redox peak current signal shows the increase due to the electron transfer that occurs between the active sites of glucose oxidase and electrodes . Thus, tThere were two cathodic peaks observed in the CV curve of the bio-electrode constructed with Nafion/GOx/MC-COOH. In the presence of oxygen, the reduced enzyme is oxidized very quickly at the surface of the electrode ,56,58:G2 reduction by GOx, the maximum cathodic peak at −0.430 V was speculated to be the peak of reducing oxygen. The similar phenomenon were also observed in other bio-electrodes constructed with GOx as the catalyst. Two peaks appeared in the CV curves of GOD-graphene/PANI/AuNPs modified GCE in an N2-saturated PBS [2 saturated PBS [2 saturated 0.1 M PBS [The catalytic regeneration of the enzyme in its oxidized form causes the loss of reversibility and the increase in size of the reduction peak ,59,60. Iated PBS . The graated PBS . It was ated PBS . Moreoveated PBS . In Figu.1 M PBS . However.1 M PBS ,58,59,646H12O6 with different scan rates. We found that peak potential shifted toward more positive while in reverse toward more negative as the scan rate increased. This phenomenon was typical for irreversible and quasi-reversible systems [1,B1) showed that the electron transfer was a surface-controlled electrochemical process [P in the voltammetric curve , the Epc and Epa linearized in accordance with the logarithm of the scan rates (v), which slopes were −2.3RT/anF and 2.3RT/(1−a)nF, respectively . Hence, the anodic charge-transfer coefficients (α) of Nafion/GOx/MC and Nafion/GOx/MC-COOH were calculated to be 0.67 and 0.77. The α increased as the amount of fixed GOx increased, starting with α = 0.67 (GOx concentration was 1.22 mg GOx/20 mg MC) up to approximately α = 0.77 (GOx concentration was 2.81 mg GOx/20 mg MC) [To further study the properties of Nafion/GOx/MC A and Naf systems , indicat systems . The goo systems   . This fi−1, which was 252.74% higher than the GOx/MC activity. The GOx immobilized on MC-COOH had a unit enzyme activity of 406.60 U mg−1, which was 1.65 times greater than the unit enzyme activity of free GOx. The GOx immobilized on MC-COOH exhibited maximal activity. The total charge (Q) of the electrode was calculated from the integration of the CV peaks for estimating the electrical activity of the reductase or oxidase. The surface coverages (Γ) of electroactive GOx in Nafion/GOx/MC and Nafion/GOx/MC-COOH were 1.05 × 10−8 mol cm−2 and 1.29 × 10−8 mol cm−2, respectively. The value was calculated according to Γ = Q/nFA, where Q is the charge obtained from the integrals of the anodic peak, n is the number of electrons transferred, F is the Faraday constant, and A is the electrode area (2.25 cm2) [−12 mol cm−2) [Additionally, the activity of the GOx immobilized on MC and MC-COOH was investigated . The act.25 cm2) . This vaol cm−2) , which sThe results showed that the oxidation temperature and time for MC functionalization could significantly influence the amount of enzyme immobilized on MC-COOH. When the oxidative temperature increased from 30 to 60 °C and the oxidative time increased from 6 h to 48 h, the ability of the mesoporous carbon to immobilize GOx increased. This enhanced enzyme adsorption might be due to the remarkable binding affinity of the oxygen-containing groups (mainly COOH groups) on the surface for the functionalized MC . When th2 groups on the GOx surface, giving rise to the amide bond (CO-NH) formation [Compared to those of the enzyme immobilized on MC, the amount and activity of the enzyme immobilized on MC-COOH increased by 130.47% and 252.74%, respectively. The reason may be that the carboxylic groups endow MC-COOH with hydrophilicity and ionic character, thus facilitating the interactions of immobilization. The basal plane itself was electrochemically inert with no such sites ideally contained. GOx was fixed onto the MC-COOH electrode by covalent coupling to construct the enzymatic glucose electrode. Incubation of GOx solution with the MC-COOH electrode led to sufficient contact between the COOH groups of MC and NHormation . Besides−1, which was 1.65 times greater than that of GOx immobilized on MC. Thus, the enzyme is not denatured or inactivated, and the glucose oxidation by GOx might be the main reason for producing the anodic peak. The scan rate dependence of the peak currents of the Nafion/GOx/MC and Nafion/GOx/MC-COOH electrodes is plotted in 1, B1. The linear dependence of the peak currents on the scan rate indicates that the redox reaction observed is due to GOx immobilized on the electrode [The -COOH groups on the carboxylated mesoporous carbon had no effect on the GOx activity or structure, even though these groups could not only physically adsorb GOx but also covalently couple with GOx, thereby increasing the amount of immobilized enzyme and the GOx surface coverage of the enzyme electrode. The results of cyclic voltammetry of Nafion/MC and Nafion/MC-COOH, as shown in lectrode . Thus, ilectrode .−9 mol cm−2) [−10 mol cm−2) and MWCNT/PyBA-GOxEDC (1.15 × 10−9 mol cm−2) [Additionally, the GOx surface coverage of the Nafion/GOx/MC-COOH electrode was calculated to be 22.92%, which was higher than that of the Nafion/GOx/MC electrode. Moreover, this surface coverage was more extensive than that of GOx coated on the surface of a carbon-ceramic electrode (CCE) (1.8 × 10ol cm−2) , MWCNT/Pol cm−2) . The widol cm−2) .The work of the authors and other references have been compared and shown in the 2 (up to 4.98 times), rising anodic charge-transfer coefficients (α) (from 0.67 to 0.77), compared to Nafion/GOx/MC. Results showed that carboxylation is a promising strategy for improving the bio-electrode performance through favoring the active catalyst surface and current intensity.Electrodes constructed by the immobilization of GOx on MC-COOH exhibited good electrochemical performance. Carboxylated MC was prepared using APS as the oxidant. During this process, oxygen surface groups could be generated, with the pore volume and specific surface area well-maintained. According to single-factor experiments, the conditions of 50 °C, a 2 M APS concentration and 24 h were chosen for the carboxyl addition. Electrochemical characteristics analysis showed that Nafion/GOx/MC-COOH presented better electrocatalytic activity with enhancing peak current intensity (up to 1.13-fold), increasing reduction peak current of O


Buckypaper consisting of a carbon nanotube (CNT) sheet has a great potential for sensing and structural applications due to the exceptional piezoresistive and mechanical properties of CNTs. In this work, buckypaper was impregnated with the epoxy resin to improve the fragility and handling capability. The mechanical properties of the buckypaper/epoxy composite were determined by the tensile and nanoindentation tests. A thermogravimetric analyzer (TGA) was used to evaluate the thermal stability. Strain and temperature sensing performances of the buckypaper/epoxy composite based on the piezoresistive effect were investigated using a meter source. Experimental results indicated that the elastic modulus and ultimate strength of the buckypaper/epoxy composite were increased by 82% and 194%, respectively, in comparison with the pristine buckypaper, while the strain and temperature sensitivities were decreased by 33% and 0.2%, respectively. A significant increase of the tensile strength accompanied with a moderate decrease of the strain sensitivity demonstrates that the overall performance of buckypaper/epoxy composite is better than that of pristine buckypaper. Carbon nanotubes (CNTs) discovered by Iijima  with excBuckypaper, a paper-like material, consists of CNTs formed by van der Walls forces . BuckypaBuckypaper is a promising material for a variety of applications. However, the free-standing buckypaper suffers a major drawback in terms of mechanical properties, which limit its commercial applications. Efforts have been devoted to develop buckypaper composite with high elastic modulus and tensile strength. Che et al.  proposedIn this work, multi-walled carbon nanotubes (MWCNTs) purchased from Conjutek Co., New Taipei City, Taiwan were used as received. The diameter and length of MWCNTs are in the range of 10–20 nm and 100–200 μm, respectively, while the purity is greater than 98.5%. An amount of 50 mg of MWCNTs were dispersed in an aqueous solution of 250 mL. In addition, a surfactant Triton X-100 of 5 g was added into the solution to separate the MWCNTs bundles into individual MWCNTs. The suspension was dispersed using a sonicator . The sonication process was operated at pulse mode with 10 s on and 20 s off for 2 h. Once the sonication process was completed, the suspension was filtered through a PVDF (Polyvinylidene Fluoride) microporous membrane with pore size 0.45 μm and diameter 47 mm. The experimental setup of the vacuum filtration is shown in The pristine buckypaper was impregnated with epoxy using a vacuum infiltration technique. The low viscosity epoxy Mungo 4200A part A and curing agent 4200B part B provided by Uchess Co.  were used. The weight ratio between the epoxy and curing agent was 2:1 as recommended by the manufacturer. The curing agent was added into the liquid epoxy, and slowly stirred for 10 min. Then, the mixture was degassed in a vacuum chamber at room temperature for 15 min to remove trapped air induced by the stirring. Consequently, epoxy resin was poured onto the buckypaper and placed in a vacuum chamber. A vacuum pressure of 0.1 atm was applied to infiltrate the buckypaper with the epoxy resin for 15 min. After that, the buckypaper/epoxy composite was placed between two steel plate and a force equivalent to a pressure of 20 KPa was applied to the system for 4 h. The buckypaper/epoxy composite was then cured in situ at room temperature for 24 h. A typical free standing and flexible buckypaper/epoxy composite is shown in The surface morphology and tensile fracture of the pristine buckypaper and buckypaper/epoxy composite were observed using a field emission scanning electron microscopy  conducted with the accelerated voltage of 10 kV. The thermal stability of the pristine buckypaper and buckypaper/epoxy composite were examined by a thermogravimetric analyzer (TGA)  under nitrogen atmosphere from room temperature to The fabricated pristine buckypaper and buckypaper/epoxy composite were cut in a rectangular strip with length 30 mm and width 10 mm. The prepared buckypaper sensor was attached on an aluminum test specimen. The aluminum specimen was subjected to a four-point bending test. The resistance changes of the pristine buckypaper and buckypaper/epoxy composite sensors in response to the applied strain of the four-point bending test were measured by a source meter . The gauge factor (GF) defined as the ratio between the relative resistance change and applied strain is used to characterize the strain sensitivity as followed.To determine the temperature sensitivity, the pristine buckypaper and buckypaper/epoxy composite sensors were placed in a thermal chamber. The temperature was increased from room temperature to 100 °C at a step of 5 °C. A K-type thermocouple was employed next to the sensor to record the temperature. The resistance changes of the pristine buckypaper and buckypaper/epoxy composite sensors were measured by a source meter (Keithley 2450). Temperature coefficient of resistance (TCR) defined as follow is used to evaluate the temperature sensitivity.The SEM images of surface morphologies of the pristine buckypaper and buckypaper/epoxy composite are shown in To evaluate the impurity and epoxy resin, TGA tests were conducted on the MWCNTs, pristine buckypaper, and buckypaper/epoxy composite using a thermogravimetric analyzer . TGA tests were operated in the nitrogen environment and carried out from room temperature to The stress vs. strain curves resulted from the tensile tests for the pristine buckypaper and buckypaper/epoxy composite are plotted in In the nanoindentation tests, the indentation depth was increased from 300 nm to 1000 nm. The piezoresistive property of the buckypaper/epoxy composite was investigated by a source meter . The buckypaper/epoxy composite with dimensions of 30 × 10The pristine buckypaper and buckypaper/epoxy composite were cut into rectangular strip with dimensions of 30 mm × 10 mm and placed in a vacuum oven. To investigate the temperature sensitivity of the pristine buckypaper and buckypaper/epoxy composite, the temperature was varied from room temperature to 100 °C. The resistance changes in response to the temperature change for both the pristine buckypaper and buckypaper/epoxy composite were recorded as shown in In this work, the pristine buckypaper was infiltrated with epoxy resin. The effect of epoxy resin on the mechanical properties and sensing performance was investigated. The cross-section morphology examined by the field emission scanning electron microscopy illustrated that the epoxy resin was successfully infiltrated through the buckypaper. MWCNTs were uniformly distributed and surrounded by the epoxy resin. The mechanical properties of the buckypaper/epoxy composite determined by the tensile tests show that the elastic modulus and tensile strength are increased by 82.6% and 194%, respectively, in comparison with the pristine buckypaper. The improvement of tensile properties can be attributed to a strong interaction between the MWCNTs and epoxy, leading to a better load transfer. The sensing capability of the buckypaper/epoxy composite based on the piezoresistive effect was investigated using a source meter. It was found that the buckypaper/epoxy composite exhibits a positive piezoresistive behavior to the applied strain and a negative piezoresistive response to the temperature change. The gauge factor used to characterize the strain sensitivity of the buckypaper/epoxy composite is decreased by 32.8% in comparison with the pristine buckypaper. However, the temperature sensitivity of the buckypaper was not affected by the infiltration of the epoxy resin. A good linearity and stability of the piezoresistive response illustrate a great feasibility of the buckypaper/epoxy composite in the applications of strain and temperature sensing.