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string | predicted_class
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|---|---|---|
Mice were sacrificed, tissue dissected, and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Tissues were then paraffin‐embedded, and sections were used for immunohistochemical staining 4‐HNE (1:200; Abcam) using an immunohistochemistry Select HRP/DAB kit (EMD Millipore). The images were acquired using an All‐in‐One Fluorescence Microscope BZ‐X700 (Keyence). Skeletal muscle was examined for pathology in transverse and longitudinal planes by H&E staining.
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study
| 99.94 |
Spinal cord was carefully flushed using saline, and lumbar spinal cord was dissected for further analysis. Each gastrocnemius muscle from each animal was dissected and gently stretched for 10 s before being immersed in fixing solution. Both tissues were fixed in 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer, pH = 7.4. The fixed material was sectioned at the Stanford Electron Microscopy Facility. Sections were taken between 75 and 80 nm, picked up on formvar/carbon‐coated 75 mesh Ni grids and stained for 20 s in 1:1 saturated uracetate (≈7.7%) in acetone followed by staining in 0.2% lead citrate for 3–4 min for contrast. For each spinal cord, sample two tissue blocks (volume of 5 mm3) were cut to obtain an average of 20 grids. Each grid included at least five cells, which were analyzed along non‐serial sections. Motor neurons were selected based on classic morphological features (multipolar cells with dispersed nuclear chromatin and prominent nucleoli). Mitochondrial samples were observed in a JEOL 1230 transmission electron microscope at 80 kV, and photographs were taken using a Gatan Multiscan 791 digital camera.
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study
| 100.0 |
Muscle architecture was examined based on intersarcomeric area, defined by the space intermingled between two sarcomers as well as density of mitochondria in the muscle, defined by the number of mitochondria per surface unit as described earlier. Mitochondria were defined as altered according to criteria being validated by previous morphological studies (i) significantly decreased electron density of the matrix (dilution, vacuolization, cavitation); (ii) fragmented and ballooned cristae (intracristal swelling); (iii) partial or complete separation of the outer and inner membranes; (iv) mitochondrial swelling. Quantitative analysis of mitochondrial damage was performed independently by two investigators, who reviewed each enlarged electron microscopy image for the presence of structurally abnormal mitochondria.
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study
| 100.0 |
Data are expressed as means ± SD. Statistical analysis was assessed by ANOVA. Significance in cell culture experiments was analyzed with the Tukey's post hoc test. The standard Mantel–Cox log‐rank test was used to assess survival. Significance of changes in neurological symptoms and tissue samples was analyzed with the Fisher's LSD post hoc test. All analyses were conducted with GraphPad Prism software. In animal studies, we used n = 7–14 mice/group for behavioral tests and n = 5 mice/group for biochemical analysis from the same litter; the five mice included in the analysis were selected at random. For the cell culture studies, we performed at least three independent experiments, in duplicates. An observer who was blind to the experimental groups conducted all the animal studies. From the age‐matched mice, one of eight TAT‐treated and two of sixteen P110‐treated mice were excluded from the study due to death during the surgery to implant the second pump. The data from these three mice were not included in any of the behavioral analysis.
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study
| 100.0 |
AUJ conducted all experiments and wrote and revised the manuscript. DM‐R helped design and supervise the studies and revised the manuscript. NLS and MS performed the pump implantation and animal behavior study. ADC performed the PCA analysis on the behavioral data. HV performed the pathological assessment of tissues.
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other
| 99.94 |
Patents on P110 and its utility in HD, ALS and other neurodegenerative diseases have been filed by DM‐R and AUJ, and P110 was recently licensed to Mitoconix Bioscience, a company that DM‐R founded and serves on its board, that develops new treatment for Huntington's disease. However, none of the work in her laboratory was carried out in collaboration with or with financial support from the company. AUJ and MS both advised the company, as part of technology transfer to the company, on their work related to Huntington's disease. The other authors declare that they have no conflict of interest.
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other
| 99.94 |
Amyotrophic lateral sclerosis (ALS), which clinically manifests by progressive muscle atrophy and paralysis, is a fatal neurodegenerative disease with patients commonly dying of respiratory failure or pneumonia 3–5 years from initial diagnosis. Currently, the glutamate release inhibitor, riluzole, and recently approved free radical scavenger edaravone are the only medications approved by the FDA for ALS. However, there remains a strong need for new disease‐modifying treatment strategies. Several recent studies suggested possible defects in mitochondrial dynamics in models of ALS, regardless of the causative mutation. However, whether Drp1 hyperactivation and its specific interaction with Fis1 play a role in the pathogenesis of ALS and neurodegeneration of motor neurons and whether its inhibition can reduce ALS pathology are unknown.
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review
| 99.8 |
Our laboratory developed a peptide inhibitor, P110, that blocks the increased Drp1 association with mitochondria by selectively inhibiting Drp1/Fis1 interaction under pathological conditions. Mitochondrial dysfunction was evident in fibroblasts of ALS patients carrying pathogenic mutations in SOD1 (I113T), in FUS1 (fused in sarcoma; R521G), or in TDP43 (TAR DNA‐binding protein 43; G289S) genes and was associated with an increased Drp1 association with the mitochondria. Further, expression of SOD1 G93A in motor neurons affected a range of signaling pathways. Correcting mitochondrial dysfunction by inhibition of pathological fission induced by Drp1/Fis1 interaction using P110 had a beneficial effect. Finally, therapeutic administration of P110 suppressed muscle atrophy and mitochondrial structural defects and enhanced motor activity and life span in SOD1‐G93A ALS mouse model.
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study
| 100.0 |
This study establishes critical role for Drp1 hyperactivation and its interaction with a single mitochondrial outer membrane adaptor, Fis1, as a driving force behind mitochondrial dysfunction in ALS. Specific Drp1/Fis1 inhibitors, such as P110, that help restore mitochondrial dynamics without affecting the basal mitochondrial fission, may provide novel disease‐modifying treatment approach. Further, this therapeutic approach might be useful in other types of muscular dystrophy with dysfunctional or aberrant mitochondrial dynamics.
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study
| 99.94 |
Extranodal nasal-type natural killer/T-cell lymphoma (NKTCL) is one of the Epstein-Barr virus (EBV)-related hematological malignancies, which mainly develops in the nasal cavity but can also occur in extranasal sites, either as a primary extranasal or disseminated disease (Harabuchi et al., 1996; Chen et al., 2015). NKTCL is more common in Asia than in Western countries (Au et al., 2009). Although most of the cases of NKTCL are diagnosed in the early stage of the disease, the long-term survival rate of patients is ∼46%-60% (Suzuki et al., 2010). The one-year survival rate of patients with advanced-stage disease is only 50%, despite improvements in treatment (Jaccard et al., 2011; Yamaguchi et al., 2011). The tumor cells of NKTCL derived from NK cells and, rarely, T cells are linked to EBV infection (Huang et al., 2013). However, the biological characteristics of NKTCL are not yet completely clear.
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review
| 99.9 |
Gelsolin (GSN), a Ca2+-regulated actin filament severing and capping protein, is a widespread, polyfunctional regulator of cell structure and metabolism (Li et al., 2012). GSN is a widely expressed actin regulator, and has been reported to be a multifunctional regulator of physiological and pathological cellular processes, and regulates cell migration, cell morphology, proliferation and apoptosis (Sun et al., 1999; Li et al., 2012). Previous research demonstrated that GSN was prevalently expressed in a variety of cells (Tanaka et al., 2006). A previous study revealed that the levels of GSN are decreased in various cancers, including breast, urinary bladder, colon, kidney, ovary, prostate, gastric and urinary system cancer (Tanaka et al., 2006). A study presented by Zhou et al. (2015) showed that upregulated GSN inhibits apoptosis, whereas downregulated GSN promotes apoptosis, which could be associated with the regulation of GSN in the apoptosis-associated pathways and the apoptosis factors caspase 3 and bcl-2. In addition, a study showed that GSN was observed in vitro to suppress the proliferation and invasion of 786-0 renal cell carcinoma cells (Zhu et al., 2015). A previous study found that GSN in colorectal tumor cell regulates cell invasion through its modulation of the urokinase (uPA)/urokinase receptor (uPAR) cascade, with possible vital roles in colorectal tumor dissemination to metastatic sites (Zhuo et al., 2012).
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review
| 99.5 |
GSN displayed high expression in the secondary diffuse large B-cell lymphoma (DLBCL) compared with de novo DLBCL (Ludvigsen et al., 2015). However, a recent study revealed that the level of GSN is downregulated in serums of advanced NKTCL patients (Zhou et al., 2016). Although the roles of GSN have been explored, whether the GSN can modulate cell proliferation, apoptosis and invasion in NK/T-cell lymphoma cells is currently unknown. Further investigations are required concerning the role of GSN in NK/T-cell lymphoma progression to determine whether decreased or increased GSN levels in NK/T-cell lymphoma have a direct relationship with tumorigenesis.
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study
| 99.9 |
It is well known that the PI3K/Akt/mTOR pathway is important and has been successfully targeted in many cancers, including many lymphomas (Westin, 2014). GSN-PI3K-Akt signaling could be involved in regulating the EMT transcription factors (Westin, 2014). GSN has been shown to physically associate with PI3K (Chellaiah et al., 2000) and promote its activity (Singh et al., 1996). An earlier study showed that inhibition of PI3K repressed GSN protein expression and decreased migration and invasion of hepatocarcinoma cells, which suggested that GSN is involved in the PI3K-Akt pathway (Wu et al., 2013).
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study
| 99.9 |
Here, we investigated the effects of GSN on the proliferation, apoptosis and invasion of NK/T-cell lymphoma cells in vitro, and further explored whether GSN exerts its biological function through the PI3K-Akt pathway. Our findings might contribute to the current understanding of the biological functions of GSN in NK/T-cell lymphoma.
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study
| 100.0 |
After the lentivirus-containing Lenti-Con (lentivirus-carrying vectors) and Lenti-GSN (recombinant lentivirus-carrying GSN cDNA) vectors were transfected into natural killer (YTS) cells, green fluorescence was obvious in the infected YTS cells, as observed under a fluorescence microscope, and the result indicated a successful transfection (Fig. 1A). Flow cytometry analysis showed that the transfection ratio in cells was 70-80% (Fig. 1B). Real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis and western blot analysis demonstrated that the mRNA and protein levels of GSN were both significantly increased in the YTS cells transfected with the pCDH-CMV-MCS-EF1-copGFP-GSN vector (YTS-GSN cells), when compared with the YTS cells transfected with the pCDH-CMV-MCS-EF1-copGFP vector (YTS-Con cells) (Fig. 1C,D). Fig. 1.Transfection ratio of the Lenti-virus-containing Lenti-GSN vector and GSN overexpression in YTS cells. (A) Green fluorescence was observed in the transfected YTS cells under a fluorescence microscope (×200 magnification) at 48 h, which indicated a successful transfection. (B) Flow cytometric analysis demonstrated that the transfection ratio in cells was 70-80% at 48 h. (C) qRT-PCR analysis exhibited that GSN mRNA expression was higher in YTS-GSN cells than in YTS-Con cells at 48 h. (D) Western blot analysis showed that the level of GSN protein was higher in YTS-GSN cells than in YTS-Con cells at 48 h. GSN, gelsolin; YTS cells, nontransfected cells; YTS-Con cells, YTS cells transfected with the pCDH-CMV-MCS-EF1-copGFP vector; YTS-GSN cells, YTS cells transfected with the pCDH-CMV-MCS-EF1-copGFP-GSN vector. **P<0.01, ***P<0.001.
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study
| 100.0 |
Transfection ratio of the Lenti-virus-containing Lenti-GSN vector and GSN overexpression in YTS cells. (A) Green fluorescence was observed in the transfected YTS cells under a fluorescence microscope (×200 magnification) at 48 h, which indicated a successful transfection. (B) Flow cytometric analysis demonstrated that the transfection ratio in cells was 70-80% at 48 h. (C) qRT-PCR analysis exhibited that GSN mRNA expression was higher in YTS-GSN cells than in YTS-Con cells at 48 h. (D) Western blot analysis showed that the level of GSN protein was higher in YTS-GSN cells than in YTS-Con cells at 48 h. GSN, gelsolin; YTS cells, nontransfected cells; YTS-Con cells, YTS cells transfected with the pCDH-CMV-MCS-EF1-copGFP vector; YTS-GSN cells, YTS cells transfected with the pCDH-CMV-MCS-EF1-copGFP-GSN vector. **P<0.01, ***P<0.001.
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study
| 100.0 |
To explore the effects of GSN on YTS cell proliferation and colony formation, CCK-8 and colony formation assays were performed. Our results from CCK-8 assay revealed that cell proliferation of the YTS-GSN cells was significantly suppressed, compared with that of YTS-Con cells (Fig. 2A). In addition, the results of colony formation assay demonstrated that GSN resulted in a decrease in the clonogenic survival of YTS-GSN cells, compared with YTS-Con cells (Fig. 2B). These results suggested that GSN had inhibitory effects on YTS cell proliferation. Fig. 2.GSN overexpression inhibits proliferation, colony formation and invasion of YTS cells and promotes apoptosis. (A) CCK-8 assays revealed that GSN overexpression inhibited YTS cell proliferation. (B) Representative photomicrographs of colony formation assay of YTS cells transfected with lenti-Con and lenti-GSN plasmids for 12 days are shown. Statistical analysis of colony formation assay showed that GSN overexpression caused a decrease in the clonogenic survival of YTS cells compared with YTS-Con cells. (C) Representative photomicrographs of flow cytometric analysis are shown. Statistical analysis of flow cytometric analysis showed that GSN overexpression significantly increased the apoptosis rate in YTS cells at 48 h. The apoptosis rate was the sum of the late apoptosis in the first quadrant and the early apoptosis in the fourth quadrant. (D) Representative photomicrographs of transwell invasion assay in cells at 48 h. Statistical analysis showed that GSN obviously suppressed YTS cell invasion. **P<0.01.
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study
| 100.0 |
GSN overexpression inhibits proliferation, colony formation and invasion of YTS cells and promotes apoptosis. (A) CCK-8 assays revealed that GSN overexpression inhibited YTS cell proliferation. (B) Representative photomicrographs of colony formation assay of YTS cells transfected with lenti-Con and lenti-GSN plasmids for 12 days are shown. Statistical analysis of colony formation assay showed that GSN overexpression caused a decrease in the clonogenic survival of YTS cells compared with YTS-Con cells. (C) Representative photomicrographs of flow cytometric analysis are shown. Statistical analysis of flow cytometric analysis showed that GSN overexpression significantly increased the apoptosis rate in YTS cells at 48 h. The apoptosis rate was the sum of the late apoptosis in the first quadrant and the early apoptosis in the fourth quadrant. (D) Representative photomicrographs of transwell invasion assay in cells at 48 h. Statistical analysis showed that GSN obviously suppressed YTS cell invasion. **P<0.01.
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study
| 100.0 |
Next, we detected the effects of GSN on apoptosis and YTS cell invasion. Flow cytometry analysis revealed that GSN overexpression caused a significant increase in apoptotic YTS cells (Fig. 2C). Transwell assay showed that invasion of YTS-GSN cells was significantly inhibited, compared with YTS-Con cells (Fig. 2D).
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study
| 100.0 |
To further confirm the potential mechanism of the effects of GSN on YTS cells, western blot analysis was performed to detect the components of the PI3K/Akt pathway. As shown in Fig. 3, Akt expression in the three experimental groups was not significantly different. Moreover, phosphorylation of Akt is characteristic of PI3K activation. The levels of PI3K and p-Akt in YTS-GSN cells were both significantly decreased, compared with levels in YTS-Con cells. The results revealed that upregulation of GSN can inhibit the PI3K/Akt pathway. Fig. 3.GSN overexpression inhibits the PI3K/Akt pathway in YTS cells. (A,B) Western blot analysis revealed that AKT expression in the three experimental groups was not significantly different. The levels of PI3K and p-AKT in YTS-GSN cells were significantly reduced at 48 h, compared with those in YTS-Con cells. **P<0.01.
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study
| 100.0 |
GSN overexpression inhibits the PI3K/Akt pathway in YTS cells. (A,B) Western blot analysis revealed that AKT expression in the three experimental groups was not significantly different. The levels of PI3K and p-AKT in YTS-GSN cells were significantly reduced at 48 h, compared with those in YTS-Con cells. **P<0.01.
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study
| 100.0 |
To confirm whether blocking the PI3K/Akt pathway inhibits cell proliferation and invasiveness and promotes apoptosis of YTS cells, LY-294002, a specific inhibitor of PI3K, which can significantly inhibit the protein expression of p-Akt and PI3K, but not Akt, was used to treat cells (Fig. 4A,B). As expected, CCK-8 and colony formation assays showed that blocking the PI3K-Akt pathway caused increased cell proliferation and colony formation of YTS cells (Fig. 4C,D). Flow cytometry analysis and transwell invasion assay exhibited that blocking the PI3K/Akt pathway resulted in increased apoptosis and diminished cell invasion ability in YTS cells (Fig. 4E,F). Fig. 4.Blocking the PI3K/AKT pathway inhibits YTS cell proliferation and invasion and promotes apoptosis. (A,B) Western blot analysis revealed that LY294002 treatment (5 μM and 20 μM) significantly suppressed p-AKT and PI3K in a dose-dependent manner, while AKT expression in the three experimental groups was not significantly different. (C) CCK-8 assay showed that LY294002 treatment had an inhibitory effect on cell proliferation of YTS cells in a dose-dependent manner at 48 h. (D) Colony formation assay showed that LY294002 treatment caused a decrease in the clonogenic survival of YTS cells in a dose-dependent manner. (E) Flow cytometric analysis exhibited that LY294002 treatment had a promotive effect on proliferation of YTS cells in a dose-dependent manner at 48 h. (F) Transwell invasion assay showed that LY294002 treatment had an inhibitory effect on the cell invasion ability of YTS-GSN cells in a dose-dependent manner at 48 h. *P<0.05,**P<0.01.
|
study
| 100.0 |
Blocking the PI3K/AKT pathway inhibits YTS cell proliferation and invasion and promotes apoptosis. (A,B) Western blot analysis revealed that LY294002 treatment (5 μM and 20 μM) significantly suppressed p-AKT and PI3K in a dose-dependent manner, while AKT expression in the three experimental groups was not significantly different. (C) CCK-8 assay showed that LY294002 treatment had an inhibitory effect on cell proliferation of YTS cells in a dose-dependent manner at 48 h. (D) Colony formation assay showed that LY294002 treatment caused a decrease in the clonogenic survival of YTS cells in a dose-dependent manner. (E) Flow cytometric analysis exhibited that LY294002 treatment had a promotive effect on proliferation of YTS cells in a dose-dependent manner at 48 h. (F) Transwell invasion assay showed that LY294002 treatment had an inhibitory effect on the cell invasion ability of YTS-GSN cells in a dose-dependent manner at 48 h. *P<0.05,**P<0.01.
|
study
| 100.0 |
NKTCL is a common kind of malignant lymphoma, and usually develops in the nasal cavity but can also occur in extranasal sites, either as a primary extranasal or disseminated disease (Takeuchi et al., 2014). A recent study demonstrated that the level of GSN is significantly decreased in serums of advanced NKTCL patients (Zhou et al., 2016). However, the potential effects of GSN on NK/T-cell lymphoma cells and molecular mechanisms remain unclear.
|
study
| 99.9 |
GSN is a protein that is broadly expressed intracellularly, including in the cytoplasm and mitochondria, and exists in both intracellular and an extracellular forms (Wen et al., 1996). Previous studies revealed that the expression of GSN is decreased in many cancers, including NKTCL (Tanaka et al., 2006; Zhou et al., 2016). Deng et al. (2015) showed that the upregulation of GSN promotes cell growth and motility and speculate, which is involved in the progression of human oral cancers. Nevertheless, a study has revealed that overexpression of GSN reduces the proliferation and invasion of colon carcinoma cells (Li et al., 2016). Our study results indicate that GSN overexpression significantly suppressed cell proliferation and invasion in YTS cells. A previous study showed that GSN suppresses apoptosis by negatively regulating the expression of apoptosis-associated genes in hepatocarcinoma cells (Zhou et al., 2015). Our results further showed that overexpression of GSN significantly increased apoptosis in YTS cells. Abedini et al. (2014) revealed that GSN plays roles as both an effector and inhibitor of apoptosis, which underlines its association with a wide variety of cancer types. According to the above results, GSN has different effects on cell proliferation, apoptosis and invasion in different cancers, which may be caused by GSN activating or inactivating different signaling pathways in varying cancers.
|
review
| 54.25 |
The PI3K/Akt pathway plays a vital role in cell survival by suppressing apoptosis and promoting cell proliferation (Vivanco and Sawyers, 2002). Akt, an essential serine/threonine kinase, is a crucial component of the PI3K signaling pathway, and its activation has been involved in the genesis or progression of many human malignancies (Blume-Jensen and Hunter, 2001; Vivanco and Sawyers, 2002). Previous studies showed that AKT1 and AKT2, the target genes of PI3K, are overexpressed in breast, gastric and ovarian cancers (Staal, 1987; Bellacosa et al., 1995). Many studies demonstrated that the constitutively active PI3K or Akt is oncogenic in cell systems and animal tumor models (Chang et al., 2003; Liu et al., 2015). Several studies have shown that Akt/PKB is involved in immune activation, cell proliferation, apoptosis and cell survival through activating the transcription of a variety of genes (Fowles et al., 2015; Warfel and Kraft, 2015). Our study revealed that significant upregulation of GSN inhibited the PI3K/Akt pathway in YTS cells. A previous study revealed that the cytoskeletal protein GSN was a vital determinant of cell invasion and scattering by inhibiting E-cadherin expression through the HGF-PI3K-Akt signaling pathway in gastric cancer (Huang et al., 2016). In addition, it has been reported that constitutive PI3K/Akt activation promotes the progress of prostate cancer from an organ-confined disease to a highly invasive and even possibly metastatic disease. Due to its role as a vital regulator of cell survival, Akt has been considered as a crucial factor in tumorigenesis (Nowinski et al., 2015). Consistent with that, in our study, blocking the PI3K/Akt pathway inhibited cell proliferation and invasion of YTS cells, while promoting apoptosis.
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study
| 99.94 |
We speculate that GSN overexpression inhibits cell proliferation and invasion and promotes apoptosis of YTS cells, at least partially through suppressing the PI3K/Akt signaling pathway, which is closely related to NKTCL and might have an antitumor effect. However, to our knowledge, relevant reports on the association between GSN and NKTCL are relatively few. Therefore, the specific pathogenesis requires further investigation.
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study
| 100.0 |
The natural killer (NK) cell line YTS was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS, Takara Biotechnology Co., Ltd., Dalian, China), 1% nonessential amino acids (NEAA, Invitrogen), 1% sodium pyruvate (Sigma-Aldrich), 10 mM HEPES (PAA, Invitrogen), 2 mM L-glutamine (Biochrom, Berlin, Germany), and 1% penicillin-streptomycin (100 μg/ml; Invitrogen Life Technologies, Beijing, China) and 5% CO2 at 37°C.
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other
| 99.9 |
The human embryonic kidney (HEK) 293T cell line was purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The 293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Hyclone, Logan, UT) supplemented with 10% FBS, 10 mM HEPES, 1% penicillin-streptomycin and 5% CO2 at 37°C.
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other
| 99.9 |
A specific primer was designed using Primer Premier 5.0 software (Shanghai Shenggong Biology Engineering Technology Service, Shanghai, China) according to the nucleotide sequences of the human GSN gene, as reported in Genebank (www.ncbi.nlm.nih.gov/genbank/; reference sequence: NM_000177). The primer sequence for GSN was as follows: DCE-GSN-F: 5′-ATTCTAGAGCTAGCGAATTCATGGCTCCGCACCGCCCCG-3′; and DCE-GSN-R: 5′-CCTTCGCGGCCGCGGATCCTCAGGCAGCCAGCTCAGCC-3′. The coding DNA sequence region of the GSN gene was amplified in a thermal cycler (Gene Amp PCR system 2400, Perkin-Elmer, Foster City, CA, USA) according to the manufacturer's instructions. The target DNA gene fragment was subcloned into the DCE lentiviral vector to construct a GSN overexpression lentiviral vector (lenti-GSN).
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study
| 100.0 |
293T cells were cultured in 10-cm cell culture dishes (3×106 cells/dish). The lentiviral vector packaging system was made as follows: a solution of 500 μl was first prepared consisting of 12 μg plasmid pCMV-Δ8.2, 10 μg pCMV-VSV-G, 22 μg transfer expression plasmid lenti-GSN, and 125 μl 2 mM CaCl2 in deionized distilled water. CaCl2/DNA was then added dropwise while vortexing to a volume of 2×HEPES-buffered saline (HBS) to a total of 1 ml, and was added to the cells at a density of 80%. GFP expression was observed by fluorescent microscopy after 24 h. The supernatant was harvested by centrifugation at 3000 rpm for 5 min at 4°C after 48 h and the ratio of positive cells was measured by using FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The high-concentration lentiviral concentrate was used to infect the YTS cells.
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study
| 100.0 |
YTS cells were seeded in 24-well plates (4×104 cells/well). The viral supernatant with Lenti-Con (lentivirus-carrying vectors) and Lenti-GSN (recombinant lentivirus-carrying GSN cDNA) were added into the cells at a density of 70%-80%, respectively. After 72 h, the transfection ratio was determined under a fluorescence microscope and was measured by flow cytometry. The cells with a transfection ratio of >70% served as the target cells and were identified by qRT-PCR analysis.
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study
| 99.94 |
Total RNA was isolated from cultured cells using TRIZOL reagent (Invitrogen). 2 μg total RNA was then reverse-transcribed using the Transcriptor First Strand cDNA synthesis Kit (Roche, Mannheim, Germany) with random hexamers. GSN mRNA was detected using Fast SYBR green PCR master mix (PE Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol, and the primer sequence for GSN and GAPDH was as follows: GSN-QF: 5′-GCT GAG GTT GCC GCT GGT G-3′, and GSN-QR: 5′-TGT GTT GGT TGC ATT TCC TTT TTG-3′; GAPDH-F: 5′-TGG TAT CGT GGA AGG ACT CAT GAC-3′, and GAPDH-R: 5′-ATG CCA GTG AGC TTC CCG TTC AGC-3′. Relative mRNA expression of GSN was calculated with the comparative threshold cycle (Ct) (2−ΔΔCt) method.
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study
| 99.94 |
CCK-8 assay was performed to detect the growth of YTS, Lenti-Con-transfected YTS, as well as Lenti-GSN-transfected YTS cells. Cells were seeded in 96-well plates at a density of 1×104 cells/well and incubated for 24, 48, 72 or 96 h in a humidified incubator. Subsequently, 10 μl CCK-8 solution (7Sea PharmTech, Shanghai, China) was added to the wells at the indicated times. After incubation for 3-4 h, absorbance was detected using the using a multilabel counter (Enspire Multimode Plate Reader, PerkinElmer) at 450 nm.
|
study
| 100.0 |
For colony formation assays, all six-well culture plates containing the bottom and soft layers were used. The cells were plated in soft agarose as follows: cells were harvested from monolayer culture, washed and resuspended at 4×104 cells/ml in fully supplemented RPMI 1640 culture medium and molten 1.5% agarose (to a final concentration of 0.3%) on Day 0, then 0.5 ml of the cellular suspension was applied to the base layer (1×104 cells/well) and allowed to set at 4°C for 6 min. Duplicate soft agarose cultures were established to assess colony formation. Cultures were placed in an incubator at 37°C, 5% CO2 and 100% relative humidity for 12 days. The number of colonies containing ≥50 cells was counted using a light microscope.
|
study
| 100.0 |
Cell apoptosis was detected using the Annexin V-phycoerythrin (PE)/7-amino-actinomycin D (7-AAD) Apoptosis Detection Kit (Nanjing KeyGen Biotech, Nanjing, China) according to the manufacturer's instructions. Cells were seed in six-well plates at 5×105 per well. The cells were harvested and washed twice in PBS. Then, 1×106 cells were resuspended in 500 μl binding buffer. The suspension was stained with 1 μl Annexin V-PE in the dark for 10 min at room temperature, and 5 μl 7-AAD was added to the suspension and maintained for 10 min at room temperature in the dark. Cell apoptosis was analyzed using a BD FACSAria II cell sorter (BD Biosciences, Franklin Lakes, NJ, USA).
|
study
| 99.94 |
For invasion assays, transwell filters (Corning Incorporated, Corning, NY, USA) were coated with Matrigel (BD Biosciences) for 24 h, and 2×105 cells were seeded into the upper compartment of the chambers with 100 μl serum-free RPMI-1640 medium. The lower chamber of the transwell was filled with culture media containing 10% FBS as a chemo-attractant. After 48 h incubation, noninvaded cells on the top of the transwell were scraped off with a cotton swab. Cells successfully translocated were fixed with 10% formalin and counted under a light microscope.
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study
| 99.94 |
Total protein was extracted from cells using lysis buffer (Roche Diagnostics, Basel, Switzerland). Protein samples (30 μg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) ultrafiltration membrane (Sigma-Aldrich) for 2 h at 4°C. The membranes were blocked with 5% nonfat milk for 1 h at room temperature. The membranes were washed three times for 5 min each with 15 ml TBS Tween 20 (TBST; Cell Signaling Technology). The membranes were incubated with primary antibodies overnight at 4°C. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated antibody for 2 h at 37°C. Antigen-antibody complexes were visualized by enhanced chemiluminescence (ECL) blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK) and GAPDH served as the internal reference. The primary antibodies used in this study are as follows: GSN, PI3K, Akt, p-Akt and GAPDH antibody (1:1000; Cell Signaling Technology). An HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody (Santa Cruz Biotechnology).
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study
| 99.94 |
Malformations of the external ear are known to cause cosmetic1,2 and psychological disturbances3 with numerous etiologies ranging from burns, cancer, trauma and congenital defects. Physical aberrations of the ear have been reported as a significant factor in limiting social integration4 and plastic surgeons have used various operative techniques to correct them. Ear reconstruction, however, has been historically very challenging5,6 owing to the need to reconstruct the complex nature of the ear’s shape. Currently, surgeons use costal cartilage grafts to recreate the ear’s framework. However, this approach causes donor site morbidity7 as well as requiring extensive surgical expertise8–10. In addition, the histological make up of elastic cartilage differs to costal cartilage11 and this influences the mechanical properties with the auricular elastic cartilage possessing a higher degree of flexibility12. This is due to its extracellular matrix (ECM) possessing a high density of elastic fibres. Hyaline cartilage however has a rich amorphous gelatinous matrix with a high density of collagen type 2 fibres as well as chondroitin sulphate11. This discrepancy in mechanical properties does not make costal cartilage grafts ideal for ear reconstruction.
|
review
| 99.9 |
Since the advent of material science and biomaterials, other options have been experimented with. MedPor® ear scaffolds composed of porous polyethylene have been applied clinically, however, they have reported a high incidence of extrusion post implantation13. The use of different synthetic scaffolds for auricular repair has demonstrated some difficulties in mimicking the native ECM as well as cytotoxicity and degeneration obstacles13. An alternative option is the use of decellularized scaffolds, as they mimic the ECM. A native ECM offers a more biocompatible approach for auricular repair and decellularization of ear cartilage has so far successfully offered a scaffold, which is capable of inducing cartilage formation14. However, the understanding into decellularization protocols for human auricular cartilage is limited. In the study by Utomo et al.15, the number of cycles required to achieve optimum decellularization of ear cartilage is not specified, this makes it difficult to replicate by other research groups. Also their study design does not compare different decellularization protocols which limits the evaluation of their proposed method. Gong et al.16 have briefly referred to a protocol however they do not provide intricate detail of the experimental steps. In addition, their decellularized cartilage was of a porcine source.
|
review
| 99.9 |
The aim of this study was to identify a protocol that could optimize the decellularization of human auricular cartilage. The technique was based on the success of previous methods applied in the case of tracheal17,18 and ear decellularization15. Our methodology consisted of applying three different decellularization protocols to human ear cartilage consisting of both physical and chemical methods. Physical methods of decellularization such as freeze thaw can help disrupt cell membranes19 as well as induce apoptosis of chondrocytes20. However the effect on degradation of the extracellular matrix structure has been proven to be insignificant. Szarko et al. reported no differences in glycosaminoglycan and collagen content after freeze thaw21 as well as reporting no change in the mean complex stiffness level. The process however initiates decellularization by inducing necrosis of cells whilst maintaining the three dimensional matrix structure. This reduces the duration of exposure to chemical methods of decellularization19. Chemical methods whilst effective can impair the mechanical properties of the matrix as Elder et al. noted decreased matrix strength when the exposure time of cartilage to SDS was increased from 2 hours to 8 hours22. Therefore a combination with physical decellularization techniques can help reduce the exposure time of cartilage to chemical methods. Freeze thaw action has been reported to accelerate the decellularization process in various tissues with lower nuclear content detected when combined with chemical techniques23. This study is the first to compare multiple decellularization protocols for human auricular cartilage and in doing so has identified a pathway for accelerating the process whilst preserving the structure of the native matrix.
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study
| 99.94 |
Procurement of auricular elastic cartilage was conducted by careful dissection of cadaveric human ears removing the skin and underlying perichondrium. This was subsequently followed by harvesting 5 mm × 5 mm sections from the elastic ear cartilage. Specimens were then split up into assessment for GAG (glycosaminoglycans), DNA, collagen and for histological analysis with H&E, DAPI, Masson’s Trichrome, Scanning Electron Microscopy (SEM) as well as assessing for mechanical properties (n = 3). For native tissue a total of 21 samples were used. Figure 1 provides a summary of the steps involved and details of analysis time points as well as assessment criteria.Figure 1Summary of steps for cartilage harvest.
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study
| 100.0 |
The protocol started with an initial dry 12 hour freeze thaw of the specimens followed by thawing at room temperature. This was followed by another 12 hour wet freeze thaw cycle in phosphate buffer solution (PBS, Sigma-Aldrich, USA) at −20 °C. The addition of a wet freeze thaw cycle in PBS allowed for cellular remnants to be washed away from the scaffold after thawing at room temperature. In addition it exerted greater mechanical pressure on the cartilage in physically decellularizing it. The cartilage was subsequently washed in deionized water overnight under agitation at room temperature. The next step involved transferring the tissue specimens to 4% sodium deoxycholate solution (Sigma-Aldrich, USA) under agitation at room temperature for four hours. This was subsequently followed by a wash in PBS solution for 30 minutes under agitation at room temperature. Next, the tissue specimens were submersed in 2% deoxyribonuclease/DNase (Sigma-Aldrich, USA) for 3 hours. This was followed by an overnight wash in deionized water with agitation at room temperature. The process was repeated for subsequent cycles however the initial freeze thaw steps were only performed once at the beginning of the protocol (Fig. 2).Figure 2Summary of steps for protocol A, B and C.
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study
| 100.0 |
Protocol B was an adaptation of protocol A and involved the same steps however specimens were submersed in 0.25% trypsin(Sigma-Aldrich, USA) for three hours after being subject to treatment with 2% DNAse (Sigma-Aldrich, USA). This was followed by an overnight wash in deionized water as before. Trypsin was used only for 7 cycles so as to avoid damaging the ECM.
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other
| 99.7 |
Following fixation in 4% paraformaldehyde (PFA), the specimens were paraffin embedded. They were then subjected to dehyrdration and rehydration cycles. Specimens were subsequently stained in Hematoxylin for 10 minutes. They were then briefly washed in running tap water for 3 seconds after which they were differentiated in acid alcohol for 6 seconds. This was a 1% concentration solution consisting of hydrochloric acid and 70% ethanol. Specimen were run through tap water until becoming blue in colour. Staining was conducted in 1% eosin for 5 minutes. Slides were analysed with the use of light microscopy (EVOS, XL Core) (n = 3).
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other
| 61.47 |
Following fixation in 4% PFA, the specimens were paraffin embedded and were then subjected to dehydration and rehydration cycles. Nuclei were subsequently stained with celestine blue-haemalum sequence and rinsed in distilled water. Subsequent staining was conducted with Hematoxylin for 5 minutes. Staining was then conducted in ponceau-acid fuchsin solution for 5 minutes. Specimens were later differentiated in 1% aqueous phosphotunstic acid for 10 minutes. Counterstain in light green was performed. Specimens were finally dehydrated and mounted on to slides and examined with light microscopy (EVOS, XL, Core) (n = 3).
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other
| 89.7 |
Preparation of slides for 4,6-diamidino-2-phenylindole (DAPI) staining involved incubation of the slides at 60 °C to remove wax in which they were initially fixated within. This was for 30 minutes. They were then submersed three times with stock solution of xylene for 5 minutes each time. After this step, they were taken through absolute IMS for 5 minutes, followed by 90% IMS for 5 minutes and then 70% IMS. Finally, one drop of DAPI stain was applied. Three slides were prepared with DAPI for each time point from the different protocols and assessed under fluorescent microscopy (Invitrogen FL Cell Imaging System) (n = 3).
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study
| 99.9 |
DNA measurement was conducted with a DNeasy quantification kit supplied by Qiagen and performed according to manufacturer conditions. A proteinase K–Buffer ATL working solution was prepared which contained 20 μl proteinase K stock solution and 180 μl Buffer ATL per sample. Mixing by vortex and centrifugation at 3000 rpm for 10 minutes was performed and the samples were then incubated overnight at 56 °C to ensure lysis of the tissue. Following this, 410 μl of premixed Buffer AL–ethanol was added to each sample. After vigorous vortex for 15 seconds, samples were then centrifuged again at 3000 rpm for 10 minutes to allow homogenous distribution of the lysate. The supernatant was then removed from each sample and centrifuged again at 6000 rpm for ten minutes. Then 500 μl of buffer AW1 was added to each sample and re-centrifuged for 5 minutes at 6000 rpm. Next, 500 μl of the Buffer AW2 was added to each one of the samples. These were centrifuged for 15 minutes at 6000 rpm. A nanodrop spectrophotometer (Thermo Scientific NanoDrop Lite Spectrophotometer) was used to measure the DNA concentration at a 260 nm wavelength (n = 3).
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study
| 99.94 |
In brief, the tissue samples being placed within eppendorf tubes after which the volume was made up to 100 μl with distilled water. Then 1 ml of Blyscan dye reagent was added to each specimen. Glycosaminoglycan standards were then prepared by employing aliquots with the following concentration of GAG: 1.0, 2.0, 3.0, 4.0 & 5.0 μg. The specimens were subsequently placed on a mechanical agitator at room temperature for 30 minutes to allow binding of the blyscan dye to the sulphated glycosaminoglycans. The next step involved centrifuging the specimens at 10000 rpm for 10 minutes. The specimens were then inverted and drained removing the unbound dye solution. The next step involved releasing of the bound dye, which, was achieved by adding 1 ml of dissociation reagent to each one of the cartilage samples. The specimens were then mixed by way of a vortex. The contents were transferred to a well plate which was clearly labelled. This was inserted within a photospectrometer to calculate absorption ratios of the samples. The absorption ratios for the standards were plotted on a graph and these were used to calculate the content of GAG (n = 3).
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study
| 100.0 |
Collagen content was measured using the Quickzyme Biosciences (UK) kit and performed according to manufacturer conditions. In brief the cartilage specimens were inserted within eppendorf micro-centrifugation tubes. To each sample 6 M HCl was then added and made up to a volume of 100 μl. The specimens were then incubated at a temperature of 95 °C for 20 hours in an oven to commence hydrolysis. They were then allowed to cool at room temperature. Next the samples were centrifuged for 10 minutes at 13000 rpm. The supernatant was then transferred from each tube to fresh ones whilst taking care not to pipette the black particles which represented fat degradation products. These can interfere with the absorption of light. 100 μl of water was added to each one of the samples so as to dilute the hydrolysate. Then 35 μl of the new mixture was used for analysis with a spectrophotometer (Thermo Scientific Fluoroskan Ascent FL Microplate Fluorometer and Luminometer). It contained a Quartz-halogen pump, filters as well as a photomultiplier tube. It had an emission wavelength range of 360 nm to 670 nm. The fluorometric sensitivity was 2 fmol fluorescein/well in a black 96 well plate (n = 3).
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study
| 99.94 |
Following fixation in 4% PFA for 48 hours the specimens were washed in deionized water for 10 mins. The specimens were then dehydrated in ascending ethanol washes (50%, 70, 90%, 100%) for 10 mins each. Following gold coating, the specimens were placed on carbon coated alumminin stubs before being imaged (FEI Quanta 200 F, n = 3).
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other
| 99.6 |
Cartilage specimens were tested using indentation compression using a Mach-1 material testing machine (Biomomentum, Canada). Each specimen was loaded to 300 g at 1 mm/sec via a 1 kg load cell. The Young’s modulus was reported for native tissue as well as the end points for protocol A at day 35, protocol B and C at day 14 with N = 3 at each time point. In addition, the percentage weight reduction in comparison to native for the different protocols was recorded.
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study
| 100.0 |
All statistical analysis was conducted with the aid of SPPSS version 24. One way ANOVA as well as the standard T-test were applied for comparing the mean content of DNA, GAG and collagen across our different protocols including their separate time points. Further analysis was conducted through post-HOC bonferoni assessment.
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study
| 100.0 |
This was performed with DAPI as well as H&E which are shown in Figs 2 and 3 respectively. Collagen was also assessed with masons trichrome. Cell quantification was conducted using imageJ software with intact nuclei in each image being counted using the cell counter plugin for the DAPI and H&E slides.Figure 3DAPI (4′,6′-diamidino-2-phenylindole) analysis of cartilage specimens assessing for decellularization with quantification of nuclei count per slide (n = 3) for different protocols using imageJ. Error bars for standard deviation.
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study
| 100.0 |
Assessing the nuclear count with DAPI showed that both protocol B at day 14 and protocol A at day 35 had a significantly lower cell number in comparison to native (P-value < 0.05). These results have been confirmed with H&E staining (Fig. 4) which too has shown a significant reduction in nuclear count for both protocol B at day 14 and protocol A at day 35 (P-value < 0.05) relative to native samples.Figure 4H&E stained cartilage sections demonstrating decellularization of native tissue with quantification of nuclei count per slide (n = 3) for different protocols using imageJ. Error bars for standard deviation.
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study
| 100.0 |
Purple stained nuclei evident within native cartilage of H&E stained specimens (Fig. 4). Progressive loss of nuclei with increased exposure to detergent/enzymatic decellularization in protocol A with no nuclei visible after 35 days. Protocol B demonstrates that after 14 days of decellularization using trypsin for the first 7 cycles, adequate decellularization can be achieved. Figure 4 displays absence of nuclei after 14 days. In the absence of trypsin as in protocol A, 35 days were required to achieve a similar histological picture of decellularization.
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study
| 100.0 |
Protocol C with EDTA demonstrates that purple stained nuclei are still evident after 14 days of decellularization. The trypsin protocol however has shown to be quicker in removing cells with fewer cellular remnants visible after 14 days. This has been confirmed with cell quantification with protocol B at day 14 having a 0.70 fold lower mean cell count compared to protocol C at the same time point.
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study
| 99.94 |
The collagen content was found to either remain relatively the same as native or to increase in concentration for all protocols (Fig. 5).Figure 5Mean collagen concentration for native samples and the different protocols with error bars for standard deviation. ANOVA analysis (P value: 0.076) indicating no significant difference between the respective study groups.
|
study
| 100.0 |
For protocol A at day 35 there was a 0.22 fold reduction in collagen after decellularization compared to native which was not significant (P-value: 0.23). The same was the case for protocol C at day 14 where there was a comparable reduction of only 0.2 fold (P-value: 0.23). In protocol B at day 14 there was an increase in collagen content compared to native of 20% from 333 μg/ml to 400 μg/ml however this was insignificant (P-value: 0.37). There was no change in collagen content between day 7 and 14 of protocol B with mean values of 400 μg/ml at both time points. For protocol C the average collagen content decreased from 300 μg/ml at day 7 to 267 μg/ml at day 14 which marked a 0.11 fold reduction however this was not considered significant. Comparison of the collagen content for native cartilage, day 35 of protocol A, day 14 of protocol B and C showed no significant difference at these time points (P-value: 0.076). This has been reflected by histology below where there was a similar density of collagen stained with masons trichrome amongst the different protocols (Fig. 6).Figure 6Masson’s trichrome stain of elastic cartilage. Same density distribution of collagen fibres between lacunae amongst different protocol groups. (A) Native. (B) Protocol A, day 28. (C) Protocol B, day 7. (D) Protocol B, day 14. (E) Protocol C, day 7. (F) Protocol C, day 14.
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study
| 100.0 |
Masson’s trichrome stain of elastic cartilage. Same density distribution of collagen fibres between lacunae amongst different protocol groups. (A) Native. (B) Protocol A, day 28. (C) Protocol B, day 7. (D) Protocol B, day 14. (E) Protocol C, day 7. (F) Protocol C, day 14.
|
study
| 98.75 |
The results for collagen content follows the effect of normalization15 with depletion of GAG allowing for collagen to redistribute within the structure of ECM, therefore appearing to measure at an almost similar concentration with no statistical difference to native. This is due to a decrease in the wet weight of the cartilage specimens. The collagen level measures in association to a reduced wet weight post decellularization therefore appearing at a higher concentration. The wet weight decreased consistently for all protocols at their end points. The percentage decrease relative to native tissue is demonstrated in Fig. 7.Figure 7Mean percentage reduction in wet weight compared to native (N = 3). Error bars for SD.
|
study
| 100.0 |
The results depict that GAG content was reduced the most when samples were tested on day 35 of protocol A as it was undetectable (Table 1). Protocol B at day 14 experienced a 0.88 fold reduction and protocol C at day 14 underwent a comparable decrease of 0.94 fold compared to naïve (Table 1). Assessment of protocol A at day 28 as well as protocol B and C at days 14 showed a significant difference at these time points (P-value < 0.05, one away ANOVA). Post-HOC bonferoni assessment of protocol B (day 14) and protocol C (day 14) was insignificant (P-value: 0.072) but protocol B (day 14) showed a significantly higher GAG level compared to protocol A, day 28 (P-value: 0.022). Assessment of protocol A (day 28) and day 14 of protocol C also demonstrated a significantly higher GAG level (P-value < 0.05) in the latter group. All time points underwent a reduction in GAG content when compared to native (P-values < 0.05).Table 1Mean concentration of GAG for different time points.ProtocolMean GAG concentration (n = 3)Native3.3 μgProtocol A day 210.1 μgProtocol A day 280.1 μgProtocol A day 35<0.1 (undetectable)Protocol B day 70.5 μgProtocol B day 140.4 μgProtocol C day 70.8 μgProtocol C day140.2 μg
|
study
| 100.0 |
Normalising the GAG content to percentage reduction in wet weight shows that protocol A at day 35 underwent the greatest depletion in GAG level which corresponded with the highest reduction in the wet weight fraction (Fig. 7). This effect was also seen in protocol B at day 14 which demonstrated the second highest reduction in GAG and wet weight fraction.
|
study
| 100.0 |
Protocol A at day 35 demonstrated a reduction in the DNA content when compared to native (P-value: 0.0026). This was also the case at day 28 (P-value < 0.05). Day 21 however did not produce a significant reduction in the content of DNA compared to native (P-value: 0.074).
|
study
| 100.0 |
There was a significant difference between day 35 of protocol A as well as day 14 of both protocol B and C (P-value < 0.05). Significant differences also existed between protocol A at day 35 and protocol C at day 14 (P-value < 0.05) as well as between protocol B, day 14 and protocol C, day 14 (P values: <0.05). However there was believed to be no difference between protocol A day 35 as well as protocol B day 14 (P-value: 0.23). Results for DNA are demonstrated in Fig. 8.Figure 8Mean DNA content for different protocols (N = 3). Error bars for SD.
|
study
| 100.0 |
Normalising DNA content to the percentage reduction in the wet weight fraction there was a consistent decrease at the end points of the three protocols (Fig. 7) correlating with a decrease in DNA content. Protocol A at day 35 underwent the highest reduction (0.79 fold) in DNA content compared to native and equally showed the greatest percentage reduction in wet weight fraction. The same trend was observed in in the case of protocol B and C at 14 days with the former showing a greater decrease in DNA content and reduction of the wet weight fraction.
|
study
| 100.0 |
SEM was used to assess the cartilage structure. Results of SEM (Fig. 9) showed obliteration of the matrix structure in protocol A at day 35 in comparison to native. Protocol B and C however showed preservation with a similar morphology to that of native.Figure 9Scanning electron microscopy of cartilage specimens (high magnification). (A) Native. (B) Protocol A, day 35. (C) Protocol B, day 14. (D) Protocol C, day 14.
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study
| 100.0 |
The Young’s modulus decreased significantly post decellularization in comparison to native samples for protocol A at day 35 (P < 0.05). For protocol B at day 14 however there was no significant difference on comparison to native (P value: 0.1270). This was also the case for protocol C at day 14 (Fig. 10).Figure 10Mean Young’s modulus (MPa) for native samples, protocol A, B and C (N = 3). Error bars for standard deviation.
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study
| 100.0 |
Literature on decellularization of human auricular cartilage is currently in-sufficient. Utomo et al.15 have provided a protocol however it didn’t specify the number of decellularization cycles required to produce an optimized scaffold. Similarly in the study by Gong et al.16, they have not clarified details of experimental steps. In addition the cartilage is of a porcine source only. This study is the first to compare multiple protocols for decellularization of human auricular cartilage with the detergent/enzymatic method proposed in protocol B having shown to have been the most effective. The steps for protocols A to C were chosen based on the success of methods from previous study groups. The process of freeze thaw has shown to accelerate decellularization23 when applied to different tissues and this can minimize the duration of exposure to chemical forms. Adding a wet freeze thaw cycle in PBS allowed for cellular remnants to be washed away from the scaffold after thawing at room temperature as well as exerting greater mechanical pressure on the cartilage in physically decellularizing it. Detergent/enzymatic methods can damage the structure of the ECM if prolonged22 and so physical techniques can shorten the exposure time. The use of EDTA in protocol C was adapted from Utomo et al.15 who had used it successfully in their decelullarization process. In addition the use of DNAse as well as SDC amongst the different protocols has been reported to effectively decellularize cartilage in the literature17. Trypsin was used in protocol B as it has shown to accelerate decellularization in the case of hyaline cartilage of the trachea as reported by Gomez et al.18.
|
study
| 99.1 |
Data in this study has shown that trypsin can certainly be effective. Histological analysis with DAPI stain has demonstrated that cells were successfully removed by day 14 with a significant reduction in comparison to native. Findings were supported by the H&E results which demonstrated a similar pattern to the DAPI staining. This was statistically proven with DNA showing a significant reduction after 14 cycles to native (P < 0.05). Staining with Masson’s Trichrome showed that collagen was still present within the extra cellular matrix which is important since it’s vital to maintain the biomechanical properties as well as acting as a biochemical cue when it comes to directing cell behaviour19. In addition the mean Young’s Modulus was maintained post decellularization at the end of protocol B compared to native.
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study
| 100.0 |
SEM showed that the three dimensional hierrarchial arrangement of the matrix was maintained at day 14 for protocol B. Protocol A was not ideal since it not only necessitated 35 days to decellularize the cartilage but in doing so it obliterated the ECM’s three dimensional (3D) structure. Surface characterization of cartilage post decellularization with SEM has also been reported by Utomo15 as well as Gomez18 and is an effective method of ECM analysis to asses for gross changes in the scaffold’s structure.
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study
| 100.0 |
Glycosaminoglycans constitute an important part of the ECM structure for providing biochemical cues to guide cell behaviour24. Numerous studies have been conducted where their presence has been beneficial for directing differentiation of cells. Murphy et al.24 have emphasized the importance of GAG in increasing differentiation of mesenchymal stem cells towards a chondrogenic lineage. The role of intrinsic ECM properties including GAG content influencing cell behaviour has been demonstrated by Reilly et al. also25. Whilst it’s important to retain GAG for influencing stem cell activity, a reduction in content may also have a benefit as it reduces the density of the ECM and increases pore size26. This would allow for greater cellular infiltration if recellularization is attempted. Therefore the ideal scaffold for cartilage should have a balance of reduced GAG whilst not completely depleting its level so that cell differentiation can be guided. This has certainly been reflected by the scaffold produced in the trypsin protocol within this study where GAG has been reduced but not completely lost. Utomo et al.15 have reported an almost similar outcome for decellularization of ear cartilage with a 75.3% reduction compared to an 88% for the trypsin protocol at day 14 in this study.
|
study
| 99.94 |
Whilst the depletion of DNA to a significant amount was desirable for the decellularization pathway in protocol A, a 35 day cycle however lead to significant morphological changes with denaturation of the 3D ECM structure on SEM when compared with native. Protocol C with EDTA did not produce the same level of reduction in DNA as did protocol B with trypsin after 14 cycles of chemical decellularization. Protocol B however, was able to reduce it in a short time whilst preserving the three dimensional structure of the cartilage.
|
study
| 100.0 |
Results demonstrated that the average collagen content either increased or stayed the same. This phenomenon of normalization has previously been reported within the literature15. It occurs as a result of a decrease in the GAG content after decellularization. This induces a compensatory redistribution of the collagen content and an increase to the wet weight contribution of cartilage. Similar results have been obtained in other studies15. Collagen is vital for provision of biomechanical strength to the scaffold as well as guiding chondrogenic differentiation27. Therefore it is important that it is retained within the native ECM. Assessment with Masson’s Trichrome showed that collagen was retained post decellularization and this is comparable to the study by Utomo15 in which immunohistochemical staining has also shown the retention of collagen fibres.
|
study
| 100.0 |
This study has so far optimized the decellularization process for human auricular cartilage. This technique can be incorporated further with biofabrication strategies in developing hybrid scaffolds for auricular reconstruction. Biofabrication through the use of 3D printing has been able to recreate the complex shape of human ears28 and enabled targeted cell seeding as well as growth factor delivery29. It has allowed for the use of hydrogels also termed “bio-inks” to be coated on to three dimensional ear scaffolds providing an aqueous environment in which chondrogenic differentiation of stem cells can occur12. Bio-inks can also be constituted by soluble forms of decellularized extra cellular matrices which have shown to be more biocompatible options30. By optimizing the production of a decellularized ECM, this can be combined with bioprinting strategies to improve the overall process of auricular regeneration.
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study
| 99.94 |
Human ear cartilage can be successfully decellularized in the production of cartilage scaffolds. Using trypsin, the decellularization process produces scaffolds within a 14 day period that have optimal structural and biomechanical properties which mimic the native ECM. The use of decellularized scaffolds for elastic cartilage regeneration looks promising for auricular reconstruction.
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study
| 99.94 |
In the past few years, paper-based inkjet printing biosensor and bioanalytical tools have been extensively used for the rapid detection of biomolecule interactions. Many research activities increasingly concentrate on microfluidic devices fabricated with glass and polymer surfaces, which have attracted great attention because of their potential miniature form and automation. Specifically, studies have validated these paper-based microfluidic biosensors, i.e., surface-modified miniaturized microfluidic devices, as a novel analytical tool for sequential analytical measurements. It includes chemiluminescent methods1, surface-Raman spectroscopy2, electrochemical3, and FRET-based fluorescent detection methods4–6. On the other hand, the design and fabrication of these microfluidic devices would be complex, highly expensive, and time- consuming. Hence, a growing need exists for a cost-effective and similar method for the detection of biomolecules. Nitrocellulose (NC) membranes7,8, filter paper9, parchment paper9, chromatographic paper8, or glass fiber paper-like6 substrate have been utilized as the paper-based material, because they possess high protein/enzyme binding capability that makes it available for bio-molecular immobilization. These porous membranes or paper operates based on capillary action to transport and react with liquid samples. So far, paper-based inkjet printing microfluidic sensors have been employed for sandwich ELISAs7, α-amylase detector for disease diagnosis10, detection of acetylcholinesterase (AChE) inhibitors11, and micro-colorimetric biochemical (glucose/glucose oxidase, DNA/hydrogen peroxidase and biotin/streptavidin) detection method12. Similarly, automated paper-based inkjet printing sandwich ELISA was fabricated on a piece of nitrocellulose membrane to analyze human chronic gonadotropin (hCG). However, this method also includes multiple steps, and crucial printing patterns are required to obtain quantitative outcomes7. Different paper-based microfluidic devices have reported FRET-based fluorescent assay for the direct detection of protein, nucleic acid, and upconversion phosphors (UCPs) suitable for molecular diagnosis4,6. Recently, portable paper-based sensor bis (dithiocarbamato) copper (II) complex functionalized carbon nanodots (CDs) for the detection of mercuric ion (Hg2+) were developed by printing CuDTC2-CD solution on cellulose acetate paper using a commercial inkjet printer5. Although various inkjet-printing paper-based ELISA platforms with colorimetric detection for drug screening, and molecular diagnosis and enzyme inhibitory analysis have been successfully developed in the recent years, a paper-based inkjet-printing technique is still not widely applied for FRET detection. Paper-based assays are commonly utilized for detecting biologically small molecules and macromolecules because of their effective accessibility and fewer false-positive results13–16. A variety of applications based on paper and inkjet printing-based diagnosis have been reported for molecular diagnosis17–23, RNA detection and analysis for Ebola virus diagnosis13, C-reactive protein (CRP) monitoring24, multiplexed point-of-care diagnostic devices to detection of nucleic acids, malaria and dengue14–16,25,26.
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review
| 99.9 |
In the present study, for the first time, fluorescence resonance energy transfer (FRET) determination with cyclic AMP (cAMP)-specific phosphodiesterase 4B (PDE4B) inhibitory assay using an inkjet-printing technique is proposed. FRET signal measures the interaction between two molecules labeled with two different fluorophores (i.e., the donor and the acceptor), by the transfer of energy from the excited donor to the acceptor. Various methods are available in the extent literature to quantify and plot the FRET signal27, but the measurement that they offer involves numerous practical difficulties, including calculation error, difficult interpretation, and high sensitivity. We propose here a quantitative method by using a non-fabricated parchment paper surface to measure the FRET system with controlled amounts (nanoliter volume) of donor and acceptor fluorophores using a conventional inkjet printer equipped with four cartridges. The reaction sample solutions, including cAMP, PDE4B, roliparm or roflumilast, Eu- anti cAMP, and ULight cAMP are sequentially printed on parchment paper through a layer-by-layer process. This paper demonstrates successful completion between Eu chelate- labeled cAMP tracer (donor) and ULight- anti-cAMP dye (acceptor) on parchment paper. After printing, Eu chelate- labeled cAMP tracer is excited, and the energy emitted by Eu chelate was transferred by FRET to ULight molecule on paper, detected at 665 nm using a fluorescent microscope. In the absence of free cAMP maximum, the FRET signal was achieved on paper, while a decrease in the FRET signal was recorded when free cAMP produced by PDE4B inhibitors compete with Eu-cAMP, binding with ULight-mAb. Parchment papers are found to be a unique substrate to measure fluorescent energy transfer, due to their insignificant self-absorption, which facilitates efficient interaction of reaction components. This new parchment paper-based enzyme-inhibitor interaction/FRET assay offers several major advantages, including low reagent consumption, cost-effectiveness, no need of a fabrication process, stability, and biodegradability. In contrast to standard FRET measurements, the inkjet printing-based FRET determination precisely evaluates the inhibitory mole 50 (IM50) with an effective number of mole of PDE4B inhibitor printed per spot. IM50 is the amount of inhibitor used to react with different concentrations of substrate printed on paper. Hence, the methodology reported here constitutes an innovative approach towards the quantitative determination of FRET signals generated on paper.
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study
| 99.94 |
PDE4B is a member of the phosphodiesterase family of proteins, which plays a critical role in regulating intracellular levels of cyclic AMP (cAMP)28. cAMP-specific 3′,5′-cyclic phosphodiesterase 4B is a therapeutic target for the treatment of several inflammatory disorders29. Here, we present the development of a simple, cost-effective, less time-consuming, and highly specific PDE4B assay on a non-fabricated parchment paper surface using inkjet-printing technology. Mainly, this assay is based on the competition between the europium (Eu) chelate-labeled cAMP tracer and sample cAMP for binding sites on cAMP-specific monoclonal antibodies (mAb) labeled with the ULight dye on parchment paper. PDE4B enzyme and their respective inhibitors (roliparm and roflumilast)30,31 were utilized to study FRET-based enzyme-inhibitor interactions on paper. In connection with this, Fig. 1 represents the scheme of the present study. When ULight-monoclonal antibody is bound to the Eu-labeled cAMP tracer, a light pulse at 320/340 nm excites the Eu chelate molecule of the cAMP tracer. The energy emitted by the excited Eu chelate is transferred by FRET to ULight molecules on the antibodies, which in turn emit fluorescence at 665 nm. Residual energy from the Eu chelate will produce fluorescence at 615 nm. After printing the reaction components on parchment paper, fluorescence energy transfer between Eu-cAMP and ULight-anti-cAMP provides a specific binding signal in the absence of free cAMP (Fig. 1A), while energy transfer does not occur in the presence of free cAMP (Fig. 1B). FRET between Eu-cAMP and ULight-anti-cAMP provides a particular signal, that is utilized here as an optical method to quantify cAMP-specific PDE4B and their respective inhibitor interactions on paper. In the presence of PDE4B, the cAMP is degraded into AMP, which is not recognized by the ULight-mAb. This leads to an increase in FRET signal, which is proportional to the concentration of degraded cAMP (Fig. 1C). Similarly, in the presence of PDE4B inhibitor, cAMP remains intact and competes with the Eu-cAMP tracer for binding to the Ulight-mAb. This results in a decrease in FRET signal, which is proportional to increased inhibitor concentration (Fig. 1D).Figure 1Schematic diagram. The schematic diagram shows that the reaction components were printed on parchment paper, and the fluorescence energy transfer between Eu-cAMP and ULight-anti-cAMP was determined under four different conditions, including: (A) absence of free cAMP; (B) presence of free cAMP; (C) presence of cAMP and PDE4B enzyme; and (D) presence of cAMP, PDE4B enzyme, and PDE4B inhibitor.
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study
| 100.0 |
Schematic diagram. The schematic diagram shows that the reaction components were printed on parchment paper, and the fluorescence energy transfer between Eu-cAMP and ULight-anti-cAMP was determined under four different conditions, including: (A) absence of free cAMP; (B) presence of free cAMP; (C) presence of cAMP and PDE4B enzyme; and (D) presence of cAMP, PDE4B enzyme, and PDE4B inhibitor.
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other
| 96.56 |
Firstly, optimal cAMP activity was measured to evaluate the cAMP-specific PDE4B enzyme inhibitory assay. The spot size 0.3 cm with an area of 0.071 cm2/spot was used for printing. The cAMP (Y cartridge), Eu-cAMP tracer (M cartridge), ULight anti-cAMP mAb (C-cartridge), and Buffer (K cartridge) were sequentially printed on parchment paper based on a layer-by-layer process using a conventional inkjet printer (Fig. 2A). Two different sets of experiments (W cAMP and W/O cAMP) were performed to verify cAMP activity in the presence of Eu-cAMP tracer and ULight anti-cAMP mAb. The reaction components were printed on parchment paper in reference to Fig. 2A. Hence, two different spot areas were printed, W and W/O cAMP. Immediately, Eu-cAMP tracer and ULight anti-cAMP mAb were printed on the cAMP pre-printed spot surface and incubated for 30 min (Fig. 2B,C). Competitive fluorescent energy transfer occurred in the presence and absence of cAMP measured at the 665-emission range using a fluorescent microscope, and the FRET images were analyzed with MetaMorph software. Figure 2B(1 and 2) and 2C(1 and 2) represent the monochrome and fluorescent dye color image of the FRET signal acquired with W and W/O cAMP, respectively. Three different regions (R1, R2, and R3) were selected to confirm the uniform FRET signal distributed on parchment paper, and their representative individual intensity profiles and percentage intensity profiles are shown in Fig. 2B(3 and 4) and 2C(3 and 4), respectively. Individual and average FRET signals corresponding to R1, R2, and R3 that acquired W and W/O cAMP are graphically represented as Fig. 2D.Figure 2Optimization of cAMP activity using the inkjet-printing method. (A) Represents printing order. (B) FRET signals were acquired with cAMP, and their respective images were illustrated as: 1. monochrome image; 2. fluorescent dye color image; 3. intensity profile of three different regions; and 4. percentage intensity profile of three regions. (C) FRET signals were acquired without cAMP, and their respective images were shown as: 1. monochrome image; 2. fluorescent dye color image; 3. intensity profile of three different regions; and 4. percentage intensity profile of three regions. (D) Fluorescence signal generated on a single spot was measured. The graph illustrates the individual fluorescence signal produced with and without cAMP by three different regions/spot areas.
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study
| 100.0 |
Optimization of cAMP activity using the inkjet-printing method. (A) Represents printing order. (B) FRET signals were acquired with cAMP, and their respective images were illustrated as: 1. monochrome image; 2. fluorescent dye color image; 3. intensity profile of three different regions; and 4. percentage intensity profile of three regions. (C) FRET signals were acquired without cAMP, and their respective images were shown as: 1. monochrome image; 2. fluorescent dye color image; 3. intensity profile of three different regions; and 4. percentage intensity profile of three regions. (D) Fluorescence signal generated on a single spot was measured. The graph illustrates the individual fluorescence signal produced with and without cAMP by three different regions/spot areas.
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study
| 100.0 |
Although significant FRET signals were acquired with W and W/O cAMP, it is important to assess the amount of cAMP involved in the reaction. In our previous work, we developed a novel method using an inkjet printer to evaluate the number of mole printed on the surface based on volume printed per spot and their respective solution density32. This approach enhanced the application of the inkjet-printing technique to study enzyme inhibitory assay. The described method32 was applied in the present study to evaluate the number of mole of cAMP printed on paper. Initially, the reaction sample solution densities were evaluated using a pycnometer and expressed in g/mL (Supplementary Table S1). The printed volume was evaluated using solution densities, and cartridge weight loss before and after printing. Thus, the printed volume was calculated using the formula: printed volume = (cartridge weight loss/solution density) * area. Supplementary Table S2 shows that 27.8 nL of cAMP was printed/spot area to react with 30 nL of Eu-cAMP tracer and 30 nL of ULight anti-cAMP mAb, which were printed on the same spot area. The effective mole printed on the surface was estimated using estimated printed volume and initial solution stock concentration using a standard equation: number of mole = molarity * volume. It was determined that 2.79 × 10−16 mole of cAMP was printed per spot area.
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study
| 100.0 |
Inkjet bioprinting was performed and PDE4B enzyme reaction curve was obtained at three different concentrations (stock concentrations: high −10 µM, medium −5 µM, and low −0.5 µM) using 2.79 × 10−16 mole of cAMP as a substrate on parchment paper. cAMP, PDE4B, Eu-cAMP tracer, and ULight anti-cAMP mAb were sequentially printed on parchment paper based on a layer-by-layer process using a conventional inkjet printer. Figure 3 illustrates the optimization condition under which the FRET signal correlates linearly with time. As shown in Fig. 3A,B, the signal increased significantly as a function of time up to 50 min with a higher concentration of PDE4B; whereas, the signal reached a plateau after only 30 min when medium and low concentration of enzyme were used. Figure 3A represents the FRET images acquired using a fluorescent microscope, and Fig. 3B corresponds to their respective fluorescent intensity determined using Metamorph software. Figure 3C presents the intensity profile of fluorescent signals produced on parchment paper, which confirms the linear correlation between the fluorescent signal and time point. The intensity profile image clearly supports that the FRET signal increased linearly as a function of time up to 50 min with a higher concentration of PDE4B. Similarly, non-linear signals were generated on paper in the presence of medium and low concentration of PDE4B (Fig. 3C). As previously mentioned, the volume printed and number of mole of PDE4B printed per spot area were evaluated. The number of mole of PDE4B printed per spot area corresponds to 2.81 × 10−13 mole, 1.40 10−13 mole and 1.40 10−14 mole, which was ejected from the cartridges filled with high –10 µM, medium −5 µM, and low −0.5 µM concentration of PDE4B, respectively (supplementary Table S2). In Fig. 3D, the graphical plot presents the FRET signal generated in response to the enzymatic reaction on paper. The optimized condition here verifies that 2.81 × 10−13 mole of PDE4B printed/spot degrading 2.79 × 10−16 mole of cAMP leads to an increase in FRET signal, while a significantly lesser FRET signal was generated in the presence of 1.40 10−13 mole and 1.40 10−14 mole of PDE4B. In order to develop a paper-based sensitive assay and ensure cAMP activity using inkjet-printing technique, 2.81 × 10−13 mole of PDE4B and reaction time of approximately 50 min were selected for subsequent experiments.Figure 3Optimization of PDE4B enzyme activity using the inkjet-printing method. (A) Fluorescence signal generated on parchment paper; (B) graph represents the average fluorescence intensity of the individual images (3A); (C) illustrates the intensity profile of the individual images (3A). Figure data A, B, and C were acquired as a function of dose and time point; and (D) the X-axis represents the number of mole of PDE4B printed per spot, the primary Y-axis represents the FRET signals produced in the presence of cAMP and PDE4B, and the secondary Y-axis represents the ejection volume of PDE4B.
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| 100.0 |
Optimization of PDE4B enzyme activity using the inkjet-printing method. (A) Fluorescence signal generated on parchment paper; (B) graph represents the average fluorescence intensity of the individual images (3A); (C) illustrates the intensity profile of the individual images (3A). Figure data A, B, and C were acquired as a function of dose and time point; and (D) the X-axis represents the number of mole of PDE4B printed per spot, the primary Y-axis represents the FRET signals produced in the presence of cAMP and PDE4B, and the secondary Y-axis represents the ejection volume of PDE4B.
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study
| 100.0 |
On the basis of the sensitivity and specificity studies, the practical application of the inkjet printing-based system for determining PDE4B inhibitory activity was investigated. For this purpose, the printing-output setting was fixed as 100, to print cAMP, PDE4B, Eu-cAMP tracer, ULight anti-cAMP mAb, and buffer. In addition, the printing-output setting (C value) was altered by a two-order difference per spot (from 0 to 88) to vary the amount of roliparm or roflumilat to be printed per spot area. Figure 4A(1)–A(4) and Fig. 5A(1)–A(4) show the response order of printing cAMP, PDE4B, and roliparm/roflumilast. Finally, the buffer was printed to avoid drying. Reaction samples printed-paper was incubated for 30 min inside of an airtight container. Immediately, Eu-cAMP tracer and ULight anti-cAMP mAb (Fig. 4A(6) and (7) and Fig. 5A(6) and (7)) were printed from different cartridges on the same spot area, and incubated for 50 min. FRET signals were detected at 665 nm using a fluorescent microscope.Figure 4Inkjet-printing based determination of PDE4B inhibition by roliparm. (A) Represents printing order; (B) fluorescence signal generated on parchment paper as a function of C value; (C) graph represents the average fluorescence intensity of the individual images (4A) as a function of C value; and (D) the X-axis represents the number of mole of roliparm printed per spot, the primary Y-axis represents the FRET signals produced in the presence of cAMP, PDE4B and roliparm, and the secondary Y-axis represents the ejection volume of roliparm. Error bars represent the standard deviation of three independent measurements.Figure 5Inkjet-printing based determination of PDE4B inhibition by roflumilast. (A) Represents printing order; (B) fluorescence signal generated on parchment paper as a function of C value; (C) graph represents the average fluorescence intensity of the individual images (4A) as a function of C value; and (D) the X-axis represents the number of mole of roliparm printed per spot, the primary Y-axis represents the FRET signals produced in the presence of cAMP, PDE4B and roflumilast, and the secondary Y-axis represents the ejection volume of roflumilast. Error bars represent the standard deviation of three independent measurements.
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study
| 100.0 |
Inkjet-printing based determination of PDE4B inhibition by roliparm. (A) Represents printing order; (B) fluorescence signal generated on parchment paper as a function of C value; (C) graph represents the average fluorescence intensity of the individual images (4A) as a function of C value; and (D) the X-axis represents the number of mole of roliparm printed per spot, the primary Y-axis represents the FRET signals produced in the presence of cAMP, PDE4B and roliparm, and the secondary Y-axis represents the ejection volume of roliparm. Error bars represent the standard deviation of three independent measurements.
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study
| 100.0 |
Inkjet-printing based determination of PDE4B inhibition by roflumilast. (A) Represents printing order; (B) fluorescence signal generated on parchment paper as a function of C value; (C) graph represents the average fluorescence intensity of the individual images (4A) as a function of C value; and (D) the X-axis represents the number of mole of roliparm printed per spot, the primary Y-axis represents the FRET signals produced in the presence of cAMP, PDE4B and roflumilast, and the secondary Y-axis represents the ejection volume of roflumilast. Error bars represent the standard deviation of three independent measurements.
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study
| 100.0 |
The fluorescent signals were generated as an effect of enzyme inhibitors. Roliparm (Fig. 4B)/roflumilast (Fig. 5B) spotted per area shows that the decrease in FRET signals is directly related to the increase in PDE4B inhibitors spotted on same spot surface area. Individual acquired images clearly verify the variance for roliparm (Fig. 4B)/roflumilast (Fig. 5B) printed per spot with respect to the printing-output settings (C value). In order to classify FRET signals and their corresponding C values; fluorescent images were categorized as S1 (2, 12, 22, 32, 42, 52, 62, 72, 82), S2 (4, 14, 24, 34, 44, 54, 64, 72, 84), S3 (6, 16, 26, 36, 46, 56, 66, 76, 86), and S4 (8, 18, 28, 38, 48, 58, 68, 78, 88). Figure 4C and Fig. 5C represent the fluorescent signal generated on paper, where the X-axis represents the C value and the Y-axis represents the FRET signal. The graph clearly shows a linear reduction in the FRET signal, confirming the PDE4B enzyme inhibitory activity on paper. As previously mentioned, to perform the PDE4B inhibitory assay, 2.79 × 10−16 mole of cAMP and 2.81 × 10−13 mole of PDE4B were printed on parchment paper. It was found that 0.29 × 10−13 mole to 2.72 × 10−13 mole of roliparm and 0.04 × 10−13 mole to 2.5 × 10−13 mole of roflumilast were printed on the surface. Reproducibility of FRET signal on the parchment paper was confirmed by measuring the FRET signals of 3 different sets using three different inkjet-printed parchment papers with same parameters. A graph was plotted to corroborate the volume of roliparm/roflumilast, mole printed per spot area, and their corresponding FRET signals generated on paper. As shown in Fig. 4D and Fig. 5D, the X-axis represents mole of roliparm/roflumilast printed per spot area, and the primary Y-axis represents the volume of roliparm/roflumilast printed per spot area and the secondary Y-axis represents the resultant FRET signals produced on parchment paper. Error bars in the plot represent the standard deviation of three independent measurements. The inhibitory mole 50 (IM50) of roliparm and roflumilast, which is required to reduce the PDE4B activity by half, was determined as 2.46 × 10−13 mole and 1.86 × 10−13 mole, respectively (supplementary Table S3).
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study
| 100.0 |
cAMP-specific 3′,5′-cyclic phosphodiesterase 4B (PDE4B) was found to constitute an excellent therapeutic target. Inhibition of PDE4B suppresses inflammation33. Roliparm and roflumilast were reported as potential PDE4B inhibitors for the treatment of inflammation34–36. Although different methods, including, ELISA, gene cloning and radioisotope/alumina acid methods, are available to screen PDE4B inhibitors, these methods are still highly time-consuming. For example, cloning usually takes more than two weeks to establish PDE4B gene-expressing vectors. Consequently, even more time is required for compound screening. Likewise, to perform drug screening using radioisotope/alumina acid methods, handling a liquid system and preparation of alumina plates is inconvenient37–40. In comparison, the inkjet printing-based screening approach offers a user-friendly method and is much less time-consuming. The fundamental strategy in this work utilizes non-fabricated parchment paper as a substrate to sense PDE4B inhibitor activity, and in turn, this sensing event produces FRET signals. We present an alternative system, which will substantially enhance the ability to screen future FRET signal-based assay for novel analyses. Our study involves paper-based inkjet bioprinting for FRET signal determination via evaluating the competition between Eu-labelled cAMP tracer and cAMP for binding sites on cAMP- specific mAb labeled with ULight. After printing the reaction components on parchment paper, antibodies bound to the Eu-labelled cAMP. When Eu-chelate molecules were excited at 320/340 nm, the energy is transferred by FRET to ULight molecules, which in turn emitted fluorescence at 665 nm. cAMP-specific PDE4B inhibition results in the production of FRET-based antibody interaction, which displayed a uniquely characteristic low-background signal on parchment paper. Therefore, FRET signals determined at a lower optical background enables us to analyze threshold values sensitively. The experimental results clearly confirm the compatibility and applicability of this inkjet-printing system for quantitative FRET determination. Previous studies have reported an inkjet printing-based multi-enzyme printing system (ADH–DP), to print enzyme and substrate on a piece of parchment paper for certain applications, such as biosensing9. Our previous study proposed a novel concept IM50 for the quantitative determination of inhibitor efficacy using a conventional inkjet printer containing four cartridges32. In addition, earlier reports have verified that gradient color codes preset on a computer can be utilized to quantitatively print enzymes, proteins, and inhibitors on paper. This basic background allowed us to predict the number of mole of component used to react with enzymes/inhibitors. It was also found that a controlled spot size and pattern design played critical roles in creating a paper-based enzymatic assay9,32.
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study
| 100.0 |
Earlier, conventional methods-based cAMP-dependent PDE4B assay was found to be highly sensitive for drug screening, and the optimal working range of cAMP required to inhibit the activity of PDE4B enzyme was determined to be 0.5 µM/1 µM. Under this experimental condition, the IC50 value to inhibit PDE4B activity by roliparm and roflumilast was determined as 132 nM and 0.8 nM, respectively41,42. In the present study, a commercially available inkjet printer equipped with four cartridges was used to achieve an efficient outcome of PDE4B inhibitor activity against PDE4B enzyme in a dose-dependent manner. Gradient color codes were utilized to print gradient concentration of inhibitors per spot area. To obtain the highest sensitivity and reproducibility, the number of moles of cAMP and PDE4B were optimized using the inkjet-printing method. Our results confirm that Eu-cAMP recognizes ULight anti-cAMP in the absence of cAMP, which was confirmed by the increased FRET signal. However, cAMP remains intact and competes with the Eu-cAMP tracer for binding to the Ulight-mAb in the presence of cAMP, and thus decreases in FRET signals were recorded. Since ejection volume to be printed per spot can be controlled by adjusting the printing-output settings, the number of mole printed per spot can be evaluated. It was determined that 2.79 × 10−16 mole of cAMP successfully competes with 30 nL of Eu-cAMP tracer and ULight anti-cAMP mAb printed per spot. This optimal condition for producing the highest fluorescent intensity was selected. Similarly, three different concentrations of PDE4B were utilized to screen PDE4B activity using the inkjet printer. Here, 2.81 × 10−13 mole of PDE4B was utilized to interact with 2.79 × 10−16 mole of cAMP, where Eu-cAMP was accessible to react with ULight anti-cAMP. The maximum fluorescent signal on parchment paper was achieved in 50 min reaction time. The fluorescent signal was directly proportional to the FRET signal detected in the absence of cAMP. Our results demonstrate that FRET signals were detected on parchment paper at a lower femtomole of cAMP and picomole of PDE4B, which is proven to be more sensitive and reliable than the conventional method.
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study
| 100.0 |
In our previous work, we observed a gradient increase in the volume used to print per spot according to the gradient increase of the printing setting32. Here, the C cartridge was filled with PDE4B inhibitors and the C value was set as the gradient range, and thus the gradient number of moles of roliparm/roflumilast was printed on parchment paper. In contrast, a decrease in fluorescent signal was recorded as the number of mole of inhibitor increased per spot. Paper-based inkjet printing-PDE4B inhibitory assay verifies that 0.29 × 10−13 to 2.72 × 10−13 mole of roliparm/ 0.04 × 10−13 to 2.5 × 10−13 mole of roflumilast (PDE4B inhibitor) printed per spot effectively interacts with 2.81 × 10−13 mole of PDE4B enzyme and 2.79 × 10−16 mole of cAMP, which permits a linear reduction in fluorescent signal. Consequently, from the observed results, it is evident that paper-based inkjet printing-PDE4B inhibitory analysis ensures specific and rapid FRET determination on paper. Compared to conventional well plate method, inkjet printing-based FRET signal determination utilizes a lesser reaction volume up to the nL range. The number of mole of PDE4B inhibitor reacting with PDE4B enzyme significantly verified this, which was four-orders less than the standard assay method. In the presence of cAMP, PDE4B, roliparm/ roflumilast, Eu-cAMP tracer and ULight-anti-cAMP; PDE4B enzyme activity were inhibited by roliparm/roflumilast, while Eu-cAMP recognizes free cAMP. Hence, Eu-cAMP is less available to bind with ULight-anti-cAMP. Conversely, the level of energy transferred to ULight was decreased. Therefore, a significant reduction in fluorescent signal was recorded as a function of the increase in the number of mole of PDE4B inhibitor reacted on paper. The methodology reported here facilitates an innovative approach towards the determination of FRET signals generated on paper. Since this system is relatively inexpensive, readily available and involves simple operating procedures, non-professionals can effectively perform the analysis.
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study
| 100.0 |
In summary, we have demonstrated a conventional inkjet-printing system for simultaneous detection of FRET signals and mole of sample consumed in the assay. It utilizes four cartridges as a generalized platform to study enzyme inhibitory activity, whereby FRET signals are generated on parchment paper. The four-cartridge printing system permits determination of mole of enzyme and inhibitor printed per spot as low as the femtomole range. Only a single sample concentration application was required for conducting the entire experiment. Given this, this paper-based enzyme inhibitory assay platform offers the major advantages of low-cost, less sample consumption, consecutive results, and fluorescent signal analysis. We expect that the sensitivity of the experiment can be further improved, and that this approach offers great potential as a novel tool for applicability of FRET determination.
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study
| 100.0 |
Hank’s Balanced Salt Solution (HBSS) (1×) (no pheno red) (Invitrogen), HEPES Buffer Solution (1 M) pH 7.2 to 7.5 (Invitrogen), IBMX (Sigma), cAMP standard, Eu-cAMP tracer, ULightTM-anti-cAMP, cAMP detection buffer, and BSA stabilizer were purchased from PerkinElmer. A commercially available HP Officejet Pro 8100 printer was used to print enzyme/inhibitor solutions. This printer was chosen because a high quality of output resolution was certified by the manufacturer, and these cartridges can be easily refilled. Readily available non-fabricated parchment paper was utilized as a substrate in the inkjet printer for the first time. The parchment paper was purchased from Lebus Corporation. The paper is made with 100% pure cellulose fiber. The basic weight and the size is 24 Lb., and 8 ½”x11”, respectively.
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study
| 99.9 |
All of the reagents used in the experiment were freshly prepared, and the experimental procedures were carried out at room temperature under a dark condition. A stimulation buffer consisting of 1 × HBSS, 5 mM HEPES, 0.5 mM IBMX, and 0.1% BSA (pH 7.4) was freshly prepared. cAMP (10 nM) and PDE4B (0.5 µM, 5 µM, and 10 µM) were prepared using the stimulation buffer, and Eu-cAMP tracer (1/150 dilution) and ULight-anti-cAMP (1/300 dilution) were prepared using the cAMP detection buffer. Roliparm (10 µM) and roflumilast (10 µM) were prepared using DMSO. Commercial inks were completely removed, washed with deionized water and 100% ethanol, and then completely air-dried in a hot air oven. Finally, printing solutions were refilled into the respective cartridges.
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study
| 99.94 |
A commercially available HP Officejet Pro 8100 printer was set to operate at normal speed. Adobe Photoshop software was utilized to attain the RGB color code, and the Microsoft Power Point tool was used to attain the desired printing patterns. Solutions of cAMP, PDE4B, Eu-cAMP tracer, ULight anti-cAMP mAb were prepared with stimulation buffer containing 180 μL of PEG-400 and 100 μL of tert-butanol per 1 mL. The printing-output setting was fixed as 100, to print cAMP, PDE4B, Eu-cAMP tracer, ULight anti-cAMP mAb, and buffer. The printing-output setting to define ejection volume was altered by two-orders of difference per spot (from 0 to 88) to vary the amount of roliparm or roflumilast. The time interval between printing each element (cAMP, PDE4B, Eu-cAMP tracer, ULight anti-cAMP mAb, and buffer) was optimized as 10 min. Under optimized condition, printing different components layer-by-layer at the same spot created a homogeneous layer that effectively provided the mixing of assay components.
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study
| 100.0 |
To evaluate cAMP-specific PDE4B inhibitor activity, C, M, Y, and K cartridges were refilled with reaction sample solutions. To optimize cAMP activity using a conventional inkjet printer, cAMP (Y cartridge), Eu-cAMP tracer (M cartridge), and ULight anti-cAMP mAb (C cartridge) were sequentially printed on parchment paper, and incubated for 50 min at room temperature (25 °C and a relative air humidity-RH, of ∼50%) inside of an airtight container. To evaluate the optimal PDE4B enzyme concentration using a conventional inkjet printer, cAMP (Y cartridge) and PDE4B (M cartridge) were consequently printed on parchment paper, and incubated for 30 min at room temperature (25 °C and a relative air humidity-RH, of ∼50%) inside of an airtight container. Then, Eu-cAMP tracer (Y cartridge) and ULight anti-cAMP mAb (M cartridge) were sequentially printed on the same spot and then incubated at room temperature. PDE4B-cAMP interaction and their respective FRET signals were detected based on concentration and in a time-dependent manner. Similarly, inhibition of PDE4B enzyme was investigated using the inkjet-printing system. To verify this, cAMP (Y cartridge), and PDE4B (M cartridge), and roliparm/roflumilast (C cartridge) were successively printed on parchment paper, and incubated for 30 min at room temperature inside of an airtight container. Then, Eu-cAMP tracer (Y cartridge) and ULight anti-cAMP mAb (M cartridge) were serially printed on the same spot and then incubated for 50 min at room temperature. Our previous report discussed the impact of measuring solution density and volume printed per spot. Hence, the number of mole printed was evaluated using solution density, and volume printed on the surface, as described by Lee et al.18. Briefly, solution density was measured by pycnometer, and is expressed as mass per unit volume. The density was calculated using the following formula: ds = (Ws − Wb)/(Ww − Wb), where Ws is solution weight, Wb is bottle weight, and Ww is water weight (supplementary Table S1). Therefore, printed volume was evaluated using cartridge weight loss before and after printing, and solution densities (volume printed = (cartridge weight loss/solution density) *area). The effective number of mole of substance printed on the surface was estimated using printed volume and initial solution stock concentration by means of a standard equation: number of mole = molarity * volume.
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study
| 100.0 |
FRET signals generated on parchment paper were analyzed using a fluorescent microscope (OLYMPUS, IX73), and the fluorescent images were captured using a charge-coupled device (CCD) as a function of wavelength. At 320/340 nm, the Eu-cAMP tracer was excited on parchment paper, which was mounted on the sample stage. The laser beam was purified by an interference filter, and it was reflected by a dichromatic mirror and focused on the paper with a 20 × objective lens. The fluorescence emission was transferred to Ulight-anti-cAMP at 665 nm, and was collected by the same microscope objective lens, which passed through a dichromatic mirror and was directed. A charge -coupled device (CCD) camera was used to detect the transmitted fluorescent beam. Light scattering was removed by a filter, which was placed in front of the CCD camera. The background (without sample) and fluorescent (with sample) images were captured. Image analysis was performed using commercially available software, MetaMorph, Version 7.1.3.0 (Molecular Devices). The average threshold values were acquired, and their percentage fluorescence intensity profiles were evaluated using the above-mentioned software.
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study
| 99.94 |
Fibrosis is a common response to hepatic damage, which is characterized by producing and depositing extracellular matrix (ECM) . Excessive fibrosis characterizes a series of liver diseases, such as chronic hepatitis, alcoholism-induced liver damage, and hepatic autoimmune disorders . During this pathological process, hepatic stellate cells (HSCs) are the main executor of fibrogenesis. HSC activation increases the expression and secretion of collagen and other ECM components. It also stimulates hepatic microenvironment cells, such as macrophages, endothelial cells, and inflammatory cells, resulting in the promotion of fibrogenesis in an autocrine or paracrine manner [3, 4]. Therefore, understanding the molecular mechanism based on HSC activation is essential for diagnosis and treatment of hepatic fibrosis.
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review
| 98.8 |
Peroxisome proliferator-activated receptor (PPAR)γ is a fundamental nuclear receptor that regulates lipid metabolism, insulin sensitivity, and fat deposition. It plays an extremely important role in liver physiological metabolism . However, increasing researches have implicated that PPARγ is a key mediator in HSC activation and phenotypic alteration, thus maintaining HSCs in a quiescent phase [6, 7]. Recently, oxidative stress is considered to be involved in HSC activation and hepatic fibrogenesis . A large amount of publications has also reported that HSCs exposed to hypoxia could be activated through HIF1α and its downstream target genes or signaling pathways [9–11]. Based on those evidences, we hypothesized that the effect of PPARγ on HSCs is the mechanisms underlying the role of hypoxia in liver fibrogenesis. In fact, PPARγ has been found to be regulated by hypoxia in several diseases. Wang et al. reported that hypoxia decreased UCP2 via HIF-1-mediated suppression of PPARγ, leading to chemoresistance of non-small cell lung cancer . Jiang et al. found that hypoxia inhibited PKG-PPARγ axis in rat distal pulmonary arterial smooth muscle cells (PASMCs) and distal pulmonary arteries . However, it is not clear whether these cellular events are associated with the pathogenesis of hepatic fibrosis and its key functionary mechanisms. We therefore performed in vivo and in vitro experiments to test the above hypothesis.
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study
| 99.94 |
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