In vitro studies showed that ExoCrocin and ExoCurcumin were not cytotoxic at a certain dose, and they could enter tumor cells. In vivo studies indicated that combination of the L1-E7 polypeptide with ExoCrocin or ExoCurcumin could produce a significant level of immunity directed toward Th1 response and CTL activity. These regimens showed the protective and therapeutic effects against tumor cells (the percentage of tumor-free mice: ~100%). In addition, both ExoCrocin and ExoCurcumin represented similar immunological and anti-tumor effects. Generally, the use of exosomal crocin or curcumin forms along with the L1-E7 polypeptide could significantly induce T-cell immune responses and eradicate tumor cells.Cervical cancer is the most common malignant tumor in females worldwide. Human papillomavirus (HPV) infection is associated with the occurrence of cervical cancer. Thus, developing an effective and low-cost vaccine against HPV infection, especially in developing countries is an important issue. In this study, a novel HPV L1-E7 fusion multiepitope construct designed by immunoinformatics tools was expressed in bacterial system. HEK-293T cells-derived exosomes were generated and characterized to use as a carrier for crocin and curcumin compounds. The exosomes loaded with crocin and curcumin compounds as a chemotherapeutic agent (ExoCrocin and ExoCurcumin) were used along with the L1-E7 polypeptide for evaluation of immunological and anti-tumor effects in C57BL/6 mouse model.  Cervical cancer is the fourth most common cancer in women, ranking after breast cancer, colorectal cancer and lung cancer, respectively . CervicaHPV genome contains early regions encoding regulatory proteins , late regions encoding capsid proteins (L1 and L2), and a non-coding region (or long control region)  were useCrocus sativus L.) and turmeric, respectively which have anti-tumor/ anti-metastatic/ anti-angiogenic, anti-oxidant and chemopreventive properties in vitro/ in vivo [Since ancient times, plants have been used to treat different types of lesions with promising results. More than 60% of anticancer drugs were derived from natural compounds . Bioacti in vivo –14.α mRNA, and low iNOS expression and nitric oxide production through down-regulation of NF-κB activity [in vitro [Crocin treatment showed a cardioprotective effect in doxorubicin-treated rats. The anti-inflammatory properties of crocin included the reduced levels of COX-2 and TNF-activity . Crocin in vitro . Curcumiin vitro .in vitro, and also found naturally in body fluids [The studies indicated that poor absorption, quick metabolism, and rapid systemic elimination led to the restriction of crocin and curcumin bioavailability. Thus, it is required finding effective methods of delivery , 19. Exoy fluids , 21. Sevy fluids –25. Due y fluids .in silico studies. Moreover, the HEK-293T-derived exosomes were used to deliver crocin and curcumin compounds in vitro and in vivo. Fig 1 shows the schematic model of this study as a brief.In this study, combination of immunotherapy (a polypeptide construct) with chemotherapy  was used to evaluate immune responses, and eradicate TC-1 tumors. The polypeptide construct was designed based on HPV L1 and E7 proteins using in silico predictions such as human MHC class I/ II epitope prediction (using NetMHCpan 4.0 server/ NetMHCII 2.3 server), MHC class I processing prediction (based on the immune epitope database or IEDB recommendations), conservancy analysis (using the IEDB conservancy tool), population coverage (using the IEDB population coverage tool), prediction of cytokine induction , prediction of allergenicity (using AllerTOP v2.0 and AllergenFP servers), toxicity (using ToxinPred web server), immunogenicity (using IEDB immunogenicity predictor tool), antigenicity (using ANTIGENpro and VaxiJen v2.0 servers), hemolytic potency (using hemolytic web server), cross reactivity (using peptide matching server), and peptide-MHC molecular docking . The L1 epitopes were selected from two low-risk (6 & 11) and sixteen high-risk  HPV strains, and the E7 epitopes were selected from five high-risk strains  with the highest impact on cervical cancer. Afterwards, a novel multiepitope construct using twelve selected epitopes and AAY linker was designed . For expression of the L1-E7 polypeptide in the next steps, different cutting sites were considered in its DNA construct . The potential B-cell epitopes were then determined in the full-length of multiepitope construct by Bepipred 2.0. Next, to identify the physicochemical properties and protein solubility of the designed construct, ProtParam, and Protein-Sol web servers were applied. After that, the secondary and tertiary structures were predicted by RaptorX, PSIPREDm and I-TASSER servers, respectively. Moreover, refinement of 3D structure was done by ModRefiner, and Galaxy Refine. Finally, 3D structure validation was performed using ERRAT, ProSA-web and RAMPAGE servers [In our previous study , twelve  servers .http://tools.iedb.org/mhci/), NetMHCpan4.1 (http://www.cbs.dtu.dk/services/NetMHCpan/), and SYFPEITHI (http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm) were utilized. Default threshold settings were used in prediction tools.In this study, the binding affinity of the selected epitopes to mouse MHC alleles (both class I and class II) was predicted, as well. To predict the binding affinity of CTL epitopes to mouse MHC class I alleles, three web servers such as IEDB MHC-1 prediction tool (http://tools.iedb.org/mhcii/), and SYFPEITHI (http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm) were applied. Default threshold settings were used in prediction tools.Helper T-lymphocyte (HTL) immune responses help in pathogen clearance. Herein, to predict the binding affinity of HTL epitopes to mouse MHC class II alleles, IEDB MHC-II prediction tool  was used to evaluate the formation of peptide-mouse MHC class I complex. The PDB files of mouse MHC alleles were extracted from RCSB (https://www.rcsb.org).The binding of the selected epitopes to human MHC was previously investigated . Herein,https://www.rcsb.org). Then, ClusPro 2.0 web server  have critical roles in the stimulation of host antimicrobial defenses . Moleculbu.edu/; ) was utiBamHI/NotI cloning sites of pGEX-6p2 expression vector. The Glutathione S-transferase (GST)-L1-E7 expression was performed in E. coli strains BL21 and Rosetta under different conditions including IPTG concentrations (rang of 0.1–1 mM), temperatures (30°C and 37°C), OD600 (rang of 0.5–1) and times . After confirming protein expression by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the GST-L1-E7 protein was purified by Ni-NTA beads (Qiagen) under denaturing conditions using constant concentration of urea and pH gradient (from 8 to 4.5). Elution fractions containing GST-L1-E7 protein were pooled, dialyzed in phosphate-buffered saline 1X (PBS 1X) for removing urea and other salts, and confirmed by SDS-PAGE and western blot analysis using anti-His tag antibody . Then, PreScission protease was utilized to remove GST-tag from the purified protein. Finally, the purified protein was dialyzed, and its concentration was measured by Bradford protein assay kit and NanoDrop spectrophotometer. On the other hand, the LAL assay (QCL-1000) was done to determine the contamination with bacterial endotoxin which was less than 0.4 EU/mg.The L1-E7 multiepitope DNA construct was synthesized in pUC57 cloning vector, and subsequently was subcloned in the 2 flasks containing RPMI 1640 medium supplemented with 10% exosome-depleted bovine serum  and 1% Pen-Strep antibiotic (Sigma). The cells were incubated at 37°C in 5% CO2 for 48 h. Then, 24 h after cell passage (the cell confluency was 70–80%), the culture medium was slowly collected. Finally, HEK-293T-derived exosomes were isolated using ExoQuick-TC kit (System Biosciences). According to ExoQuick-TC protocol, the collected medium was centrifuged at 3000 g for 15 min to remove cells and debris. In the next step, 1 ml of ExoQuick-TC buffer was mixed with 5 ml of culture medium, and incubated at 4°C for 24 h. The mixture was centrifuged at 3000 g for 10 min to precipitate exosomes. The Bradford test was performed to measure protein levels at this stage. After that, the pellet was resuspended in various buffers (based on kit), and finally, the solution containing pure exosome was obtained. The BCA protein assay kit (Thermo Fisher Scientific) was used to measure protein content at this stage.HEK-293T cells were cultured in 25 cm2O2) was used to detect the bands.Scanning electron microscope (FEI Nova Nano SEM 450), and western blotting were used to detect the presence of the isolated exosomes, and confirm their nature, respectively. For western blotting, 60 μl of the isolated exosomes were solubilized in 30 μl of RIPA buffer (DNAbiotech), and protein concentration was quantified by Bradford assay. HEK-293T cells (negative control) and exosome proteins  were separated using 10% SDS polyacrylamide gel. Protein bands were transferred onto nitrocellulose membrane (Bio-Rad). After incubating in BSA blocking buffer  for 2 h, the samples were incubated with anti-CD63 antibody  and also anti-CD9 antibody  for 24 h in 4°C. Then, the membrane was incubated with the mouse total anti-IgG HRP-conjugated secondary antibody (1:10000) for 1 h at room temperature. Finally, 3, 3′-Diaminobenzidine solution (DAB/ HThe crocin and curcumin solutions were prepared by dissolving crocin powder in PBS and curcumin powder (Sigma) in ethanol for the next uses. For achieving high amounts of crocin, it was extracted by adsorption chromatography using aluminum oxide 90-active according to our previous studies , 31.Two methods were investigated for loading crocin and curcumin into exosomes including sonication and freeze-thaw cycles. The crocin solution in PBS 1X (2 mM) and the curcumin solution in ethanol/PBS (2 mM) were mixed with exosomes in the fixed proportion (1: 4) , 33. The2 atmosphere. After 24 h, the cells were studied using a fluorescence microscope .Fluorescence imaging was performed to detect the entry of ExoCrocin and ExoCurcumin into TC-1 cell lines. Briefly, the cancerous TC-1 cells (5% RPMI) were seeded in 12-well plate (10000 cells/ well), and exposed to crocin, ExoCrocin, curcumin and ExoCurcumin. The plate was incubated for 24 h at 37°C in humidified incubator with 5% CO2 for 3h and next, the insoluble formazan (purple) was dissolved in dimethyl sulfoxide (DMSO). Finally, absorption  was measured at 570 nm by an ELISA reader. The non-treated cells were used as a negative control. The cell viability was calculated from the following equation:The viability of the non-cancerous HEK-293T was studied after seeding 10000 cells/ well in a 96-well plate, and then incubating with fresh RPMI medium containing the empty exosome (200μg), crocin (2 mM), ExoCrocin (2 mM crocin in 200μg exosome), curcumin (2 mM), ExoCurcumin (2 mM curcumin in 200μg exosome), and the L1-E7 polypeptide (5 μg) for 48 h. After incubation, 3--2, 5-diphenyl tetrazolium bromide  was added to each well. The cells were incubated at 37°C in a humidified 5% COv/v, G1), the L1-E7 polypeptide (5 μg) + ExoCrocin , ExoCrocin , the L1-E7 polypeptide (5 μg) + ExoCurcumin , ExoCurcumin . Groups 4–6 were injected with PBS, Montanide and the empty exosome as control groups, respectively (Table 1). The L1-E7 polypeptide was injected subcutaneously (s.c.), and the empty exosome, ExoCurcumin and ExoCrocin were administered Intraperitoneally (i.p.) in mice.Inbred C57BL/6 female mice, 5–7 week old, were obtained from the breeding stocks maintained at Pasteur Institute of Iran under specific pathogen-free conditions. All procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals which were evaluated by Islamic Azad University-Science and Research Branch . Eight groups of female C57BL/6 mice (n = 8 per group) were injected on days 0, 14 and 28 with the L1-E7 polypeptide (5 μg) + Montanide 720 adjuvant . Then, vaccinated mice were subcutaneously challenged in the right flank with 0.1 × 106 TC-1 tumor cells three weeks after the last immunization. Tumor growth and survival rates were monitored by palpation twice a week for 65 days. Tumor volume was calculated using the formula: V = (a2b)/2. Mice were euthanized when tumor diameter exceeded >5% of body weight.For Three weeks after the last immunization, sera were collected from whole blood samples of each group. The levels of total IgG, IgG1, IgG2a and IgG2b were measured in the sera with goat anti-mouse immunoglobulin antibodies  by indirect ELISA. The coated antigen was the recombinant L1-E7 polypeptide. 3, 3′, 5, 5′-tetramethylbenzidine (TMB) was used as substrate.6 cells/ml) were seeded in 48-well plates exposed to the recombinant L1-E7 polypeptide (5 μg). Also, RPMI 5% and concanavalin A  were considered as negative and positive controls, respectively. The supernatants were harvested to assess the secretion of IFN-γ and IL-4 by a sandwich-based ELISA system  as stated by the manufacturer’s instructions. All data were reported as mean ± SD for each set of samples. The lymphocyte proliferation was assessed by MTT and indicated as stimulation index (SI).To determine the secretion of IFN-γ and IL-4 cytokines, three mice from each group were chosen three weeks after the last injection. Mice were sacrificed following ketamine/xylazine anesthesia and the spleens were removed under sterile conditions. The red blood cell-depleted pooled splenocytes (2 × 104 cells/ well) and incubated with the L1-E7 polypeptide (~ 30 μg/ ml) for 24 h. Then, the prepared splenocytes (Effector cells: E) were added to the target cells at an E: T ratio of 100:1, and incubated for 6 h. Finally, the supernatants were harvested to measure the Granzyme B concentration by ELISA (eBioscience kit) according to the manufacturer’s instruction.The P815 target cells (T) were seeded into 96-well plates . Finally, tumor growth was detected two times a week for 65 days. The L1-E7 polypeptide was injected subcutaneously (s.c.), and ExoCurcumin and ExoCrocin were administered intratumorally (i.t.) in mice.For therapeutic tests of the established TC-1 tumors, three groups of four female C57BL/6 mice were considered. Briefly, four mice in each group were subcutaneously injected with 0.1 × 10t-test. Survival rate  was evaluated using the log-rank (Mantel-Cox) test. The value of p < 0.05 was statistically considered significant. The results were determined from two-independent experiments.Statistical analyses were performed by Prism software  to determine the differences between the control and test groups using one-way ANOVA and student’s 458DLDQFPLGRKFLLQ471 (L1), 327NQLFVTVVDTTRSTN341 (L1), 45AEPDRAHYNIVTF57 (HPV16 E7), 2HGPKATVQDIVLHL15 (HPV18 E7), 46KPDTSNYNIVTF51 (HPV31 E7), 40RPDGQAQPATADYYI54 (HPV33 E7), 85RTLQQLFLSFV95 (HPV45 E7), 7TLHEYMLDLQPETTD21 (HPV16 E7), 83LRAFQQLFLNTLSFV97 (HPV18 E7), 6PTLQDYVLDLQPEAT20 (HPV31 E7), 8LKEYVLDLYPEPTDL22 (HPV33 E7), and 87LQQLFLSTLSFVCPW101 (HPV45 E7) epitopes, respectively as shown in S1 Table. The AAY linker was located between epitopes to improve the proteasomal processing of the construct.The L1-E7 multiepitope construct was composed of the ).Herein, these selected CTL and HTL epitopes bound to human MHC alleles were studied to bind mouse MHC alleles using various web servers .Finally, the L1-E7 multiepitope construct along with an amino-terminal 6xHis-tag was synthesized as a synthetic DNA construct in pUC57 vector . Furthermore, the recombinant GST-L1-E7 protein was detectable using anti-His antibody in western blotting . The protein purification was performed by affinity chromatography under denaturing method. On the other hand, SDS-PAGE analysis indicated that the L1-E7 polypeptide (~24 kDa) was successfully separated from GST-tag (~27 kDa) after incubation with PreScission protease. The concentration of L1-E7 polypeptide was obtained approximately 2.73 mg/ml.The GST-L1-E7 protein was expressed in S4A Fig). The exosomes’ shape and membrane integrity were confirmed using a transmission electron microscopy . The exosome identification was confirmed by anti-CD63 and anti-CD9 antibodies as the clear bands of ~ 45–100 kDa in western blotting. Two methods for crocin and curcumin incorporation into exosomes were used including sonication and freeze-thaw. The concentrations of crocin and curcumin loaded in exosomes by absorbance assay showed that freeze-thaw method is better than sonication under the used conditions (S5 Table). ExoCrocin and ExoCurcumin detected by TEM showed that after crocin loading in exosome, the exosomes became larger (about 150–250 nm) but curcumin-loaded exosomes had smaller sizes in comparison with ExoCrocin . Also, the membrane integrity of ExoCrocin and ExoCurcumin was proved as shown in Fig 3B1 & 3B2.HEK-293T cell-derived exosomes were prepared and quantified by Bradford assay based on ExoQuick protocol. Approximately 2.8–3.5 mg protein equivalent of exosomes per column was obtained in purification step indicating sufficient amount of exosomes. The results of scanning electron microscopy (SEM) showed that the size of the exosomes was between 30 and 100 nm  were utilized in detecting the compounds under experimental conditions. Our fluorescence microscopy data showed that both ExoCrocin and ExoCurcumin (as detected in green color) have the ability to enter TC-1 cells. The content of crocin and curcumin was increased with exosome transmission within cells .The spectral properties of crocin and curcumin . The viability percentages for crocin, curcumin, ExoCrocin and ExoCurcumin were 78–81%, 76–79%, 90–92% and 91–94%, respectively. As observed, the exosomal forms of crocin and curcumin had no significant cytotoxic effects on HEK-293T cells at a certain concentration. The untreated cells had a viability percentage about 98–99%.Fig 5). There was no significant difference in the level of IgG1 among L1-E7 polypeptide (G1), L1-E7 polypeptide + Exocrocin (G2), and L1-E7 polypeptide + ExoCurcumin (G7) groups (p > 0.05). The groups immunized with L1-E7 polypeptide + ExoCrocin, and L1-E7 polypeptide + ExoCurcumin (G2 and G7) showed significantly higher levels of total IgG, IgG2a and IgG2b in comparison with L1-E7 polypeptide . Immunization with L1-E7 polypeptide + ExoCurcumin (G7) caused higher IgG2a antibody response than those with L1-E7 polypeptide + ExoCrocin . There is no antibody response against the recombinant polypeptide in control groups .The levels of L1-E7 polypeptide, L1-E7 polypeptide + ExoCrocin, ExoCrocin, L1-E7 polypeptide + ExoCurcumin and ExoCurcumin-specific total IgG, IgG1, IgG2a and IgG2b antibodies were measured in mice sera using ELISA . The groups immunized with L1-E7 polypeptide + ExoCrocin (G2) and L1-E7 polypeptide + ExoCurcumin (G7) showed significantly higher levels of IFN-γ in comparison with ExoCrocin (G3) and ExoCurcumin groups . The levels of IFN-γ in groups injected with L1-E7 polypeptide + ExoCrocin (G2) and L1-E7 polypeptide + ExoCurcumin (G7) was higher than group injected with L1-E7 polypeptide . No significant difference in IFN-γ secretion was observed between L1-E7 polypeptide + ExoCrocin (G2) and L1-E7 polypeptide + ExoCurcumin (G7) groups (p > 0.05). The group immunized with L1-E7 polypeptide + ExoCrocin (G2) showed higher level of IL-4 than L1-E7 polypeptide (G1) and Exocrocin . However, the level of IL-4 secretion was significantly lower than IFN-γ in groups immunized with L1-E7 polypeptide (G1), L1-E7 polypeptide + ExoCrocin (G2), L1-E7 polypeptide + ExoCurcumin (G7). The ratio of IFN-γ to IL-4 was increased about 10–11 folds in groups immunized with L1-E7 polypeptide (G1), L1-E7 polypeptide + ExoCrocin (G2) and L1-E7 polypeptide + ExoCurcumin (G7) indicating direction of immune responses toward Th1 response. Moreover, ExoCrocin and ExoCurcumin could also induce cytokine secretion as compared to control groups . The high rate of splenocyte proliferation was observed in groups receiving L1-E7 polypeptide + ExoCrocin (G2) and L1-E7 polypeptide + ExoCurcumin . The group injected with L1-E7 polypeptide (G1) showed higher level of lymphoproliferation in comparison with ExoCrocin (G3) and ExoCurcumin (G8) groups (p < 0.01). Our results indicated that the combination of exosomes carrying crocin or curcumin and L1-E7 polypeptide led to better Th-cell proliferative responses .The levels of IFN-γ and IL-4 cytokines were evaluated by ELISA determining Th1 and Th2 immune responses in mice, respectively. The groups injected with L1-E7 polypeptide, L1-E7 polypeptide + ExoCrocin, ExoCrocin, L1-E7 polypeptide + ExoCurcumin, and ExoCurcumin significantly increased the secretion of IFN-γ and IL-4 compared to the control groups . The groups immunized with L1-E7 polypeptide + ExoCrocin (G2) and L1-E7 polypeptide + ExoCurcumin (G7) produced significantly higher concentrations of Granzyme B than group immunized with L1-E7 polypeptide . The level of Granzyme B in group receiving L1-E7 polypeptide + ExoCrocin (G2) was similar to that in group receiving L1-E7 polypeptide + ExoCurcumin (G7) (p > 0.05).The secretion of Granzyme B in groups immunized with L1-E7 polypeptide (G1), L1-E7 polypeptide + ExoCrocin (G2) and L1-E7 polypeptide + ExoCurcumin (G7) was significantly higher than other groups (p < 0.001). Indeed, these groups indicated 100% survival 65 days post-inoculation. The groups immunized with L1-E7 polypeptide (G1), ExoCrocin (G3) and ExoCurcumin (G8) showed significant reduction in tumor growth and 80% of them survived . Our results demonstrated that the control groups had tumor growth at 7–14 days. Generally, the simultaneous use of the recombinant L1-E7 polypeptide and exosomes harboring crocin or curcumin (G2 and G7) could protect 100% mice from tumor growth.The prophylactic effects of different regimens were evaluated on tumor growth and survival rate. All groups were monitored after inoculation with TC-1 tumor cells for 65 days. The groups immunized with L1-E7 polypeptide + ExoCrocin (G2), and L1-E7 polypeptide + ExoCurcumin (G7) showed no tumor growth compared to other groups (3) were treated by the L1-E7 polypeptide + ExoCrocin (G1) and L1-E7 polypeptide + ExoCurcumin (G2). Both groups were free of tumor growth as compared to control group .Mice with the established tumors  was proved. Moreover, the lowest energy levels were observed for interaction of the L1-E7 multiepitope construct with TLRs indicating the highest binding affinity of the construct. Then, the L1-E7 multiepitope peptide construct was reversely translated to the multiepitope DNA construct. The DNA construct was expressed in bacterial system. The purified L1-E7 polypeptide was generated (~ 24 kDa), and its concentration was determined to use in immunological assay. The results of mice immunization indicated that the L1-E7 polypeptide stimulated B- and T-cell immune responses as compared to control groups. Our previous in silico study [in vivo study showed that the L1-E7 polypeptide induced both IFN-γ and IL-4 cytokines in mice. Moreover, the level of IFN-γ secretion was significantly higher than IL-4 secretion.Despite effective prophylactic vaccines and screening modalities for cervical dysplasia/cancer, it continues to be the fourth most common cancer in women worldwide with over 500,000 new cases every year . Thus, dimmunity . In thisimmunity , the immco study  showed tco study . Herein,et al. predicted the immunogenic epitopes of L1 and L2 proteins in five types of HPV  [NSGGYGGNPGQDNRVNVGMD, GTQCSNTSVQNGDCPPLELI (HPV-11), LDDTENASAYAANAGVDNRE, STSETTYKNTNFKEYLRHGE (HPV-16), ASTQSPVPGQYDATKFKQYS, NSSILEDWNFGVPPPPTTSL (HPV-18), HPYYSIPKSDNPKKIVVPKV, KGSPCSNNAITPGDCPPLEL (HPV-31), ASTQNPVPNTYDPTKFKHYS and NSSILENWNFGVPPPPTTSL (HPV-45) [S1 Fig). Namvar et al. designed DNA constructs based on the highly conserved and potentially immunogenic L1 and L2 epitopes in 13 types of high-risk HPVs . They reported that the combination of L1 and L2 DNA constructs without adjuvant or carrier induced effective immune responses in vivo [et al. selected the peptide sequences YVARTNIYYHAGTSRLLAVGHPYFPIK , DR4, DR11 (DRB1*1101), DR1 (DRB1*0101), DR4 (DRB1*0401), DR51 (DRB5*0101)) and YGDSLFFYLRREQMFVRH ) using immunoinformatics tools to design an L1-based peptide vaccine [et al. predicted QAEPDRAHY and AEPQRHTML peptides as antigenic E7 peptides in HPV types 16 and 18, respectively [et al. identified the immunogenic CD8+ T-cell epitopes in HPV 16, 18, 31 and 45 using a two-step method consisting of molecular docking and sequence-based approaches [RAHYNIVTF, STHVDIRTL, LEDLLMGTL, LQPETTDLY & TLHEYMLDL epitopes for HPV16 E7, SSADDLRAF, FQQLFLNTL, QLFLNTLSF, TLQDIVLHL & LRAFQQLFL epitopes for HPV18 E7, TSNYNIVTF, TLQDYVLDL, GQAEPDTSNY, QAEPDTSNY & QPEATDLHCY epitopes for HPV31 E7, TLQEIVLHL, NELDPVDLL, LQQLFLSTL, QLFLSTLSF & SSAEDLRTL epitopes for HPV45 E7 [S1 Fig).Many studies focused on design of the peptide-based HPV vaccines using bioinformatics tools. Hosseini  and 45) . For L1 (HPV-45) . We studR4 DRB1*01, DR51  led to significant reduction of tumor growth after treatment. ExoGEM could enhance survival rate in tumor-challenged mice in a dose-dependent manner [et al. investigated the effects of various concentrations of curcumin  on the proliferation and morphology of SiHa cells . The percentage of cell proliferation was considerably decreased at concentration of 50 μM in a dose-dependent manner [et al. showed that intraperitoneal injection of curcumin loaded in exosome  led to a five- to ten-fold higher curcumin accumulated in peripheral blood than that of curcumin alone. At 12 hours after intraperitoneal injection, curcumin in the plasma still remained at a high level in mice injected with exosomal curcumin [Exosomes could increase the therapeutic index of drugs. Hadla and coworkers showed that doxorubicin loaded in exosomes was safer and more effective than free doxorubicin. Indeed, higher doses of the drug can be delivered by the exosome . Li et at manner . On the t manner . The stut manner –51. Sun Curcumin reduces the side effects of chemotherapy or radiotherapy resulting in improving patients’ quality of life. The findings from several studies suggest that curcumin can prevent the formation and spread of tumors or reduce their size by exerting anti-angiogenic effects, inducing apoptosis and interfering with the cell proliferation cycle. Curcumin inhibits angiogenesis in some tumors by suppressing angiogenic cytokines such as IL-6, IL-23, and IL-1β .In vitro cytotoxic assay showed that saffron extract inhibited the growth of tumor cells, whereas, non-tumor cells were less sensitive or even insensitive to the extract. Escribano et al. indicated that crocin induced apoptosis in HeLa cells . Moreover, human fibroblasts were less sensitive to saffron extract than HeLa cells, suggesting a selective cytotoxicity on tumor cells [et al. investigated the anticancer activity of crocin against HeLa cells. Cell viability was reduced 97.1, 96.4, 85.5, 78.4 and 70.2% at 25, 50, 75, 100 and 125 mg/L of crocin, respectively. These data suggested that crocin is a potential antitumor agent against cervical carcinoma [The cytotoxic effects of saffron and its components were studied in human tumor cells. or cells . In 2018arcinoma . In anotarcinoma . Our stuet al. indicated that crocin significantly promoted T cell proliferation and the secretion of IL-2 and IL-4 in a dose-dependent manner [et al. also showed the role of Hsp70-positive exosomes in the activation of macrophages by assessment of the TNF-α secretion which was about 260-fold higher than the recombinant Hsp70 [In our study, the L1-E7 polypeptide construct along with exosomes loaded with crocin or curcumin (ExoCrocin or ExoCurcumin) was injected in mice. The health of mice after injection and the lack of inflammation represented that the tested doses of L1-E7 polypeptide, ExoCurcumin and ExoCrocin had no side effects in mice, as shown in MTT assay. Our finding indicated that combination of ExoCurcumin or ExoCrocin with L1-E7 polypeptide (G2 & G7) could significantly enhance the levels of total IgG, IgG2a, IgG2b, IFN-γ, Granzyme B and lymphocyte proliferation as compared to L1-E7 polypeptide (G1), ExoCrocin (G3), ExoCurcumin (G8) and control groups (G4-G6). Moreover, the levels of IgG1 and IL-4 were increased in groups immunized with L1-E7 polypeptide (G1), L1-E7 polypeptide/ ExoCrocin (G2), and L1-E7 polypeptide/ ExoCurcumin (G7) as compared to ExoCrocin (G3), ExoCurcumin (G8) and control groups (G4-G6). However, the ratios of IFN-γ: IL-4, and IgG2a: IgG1 in groups immunized with L1-E7 polypeptide/ ExoCrocin (G2), L1-E7 polypeptide/ ExoCurcumin (G7) and then L1-E7 polypeptide (G1) were significantly higher than other groups indicating induction of immune responses toward Th1 responses. Indeed, the combination of ExoCurcumin or ExoCrocin with a polypeptide improved cellular immune responses toward Th1 response, and CTL activity. In addition, groups injected with L1-E7 polypeptide combined with ExoCrocin (G2) or ExoCurcumin (G7) elicited the same immune responses indicating similar effects of ExoCrocin and ExoCurcumin at a certain non-toxic concentration. Protective studies showed that L1-E7 polypeptide + ExoCrocin (G2), and L1-E7 polypeptide + ExoCurcumin (G7) could confer complete protection against TC-1 tumor-challenged mice  as compared to L1-E7 polypeptide (G1), ExoCrocin (G3) and ExoCurcumin (G8) . The results indicated that injection of exosomes loaded with crocin or curcumin along with L1-E7 polypeptide has a significant therapeutic effect on tumor eradication, as well. On the other hand, ExoCrocin and ExoCurcumin induced total IgG, IgG1, IgG2a, IgG2b, IFN-γ and IL-4 as compared to control groups indicating the possible immunological response of exosomal compounds, because the empty exosomes extracted from HEK-293T cells induced no considerable immune responses. For instance, in 2018, Zhang t manner . It was t manner . Vega etnt Hsp70 .in vitro and in vivo experiments. For example, in 2020, Hong et al. indicated that dendritic cell (DC)-derived engineered antigenic exosomes  had the potency to induce antigen-specific CD8+ T cell activation in vitro and in vivo [There are various reports about the efficiency of dendritic cells/ tumor cells-derived exosomes  in vivo . Howeverin vivo. It was interesting that ExoCurcumin or ExoCrocin showed similar immunological and antitumor effects at a certain non-toxic dose. However, this study is the first report for construction of a novel fusion L1-E7 polypeptide, preparation of Exosomal crocin and curcumin, immunological assay of the L1-E7 multiepitope construct with and without Exosomal crocin and curcumin, and their protective and therapeutic effects in tumor mice. However, the study of cellular uptake mechanisms of the exosomal crocin and curcumin forms require further researches in the next studies.In conclusion, our data showed that combination of ExoCurcumin or ExoCrocin with L1-E7 polypeptide (G2 & G7) could significantly enhance the levels of total IgG, IgG2a, IgG2b, IFN-γ, Granzyme B and lymphocyte proliferation indicating induction of Th1 response and CTL activity. The protective and antitumor effects of these regimens confirmed the induced cellular immune responses S1 FigMolecular docking between the CTL epitopes and mouse MHC class I alleles: The successful docking between L1 epitope (DLDQFPLGRKFLLQ) and H-2-Db with interaction score of 263.0 (A); between E7 epitope (HGPKATVQDIVLHL) and H-2-Ld with interaction score of 352.0 (B); between E7 (AEPDRAHYNIVTF) and H-2-Kb with interaction score of 226.0 (C); between E7 epitope (RPDGQAQPATADYYI) and H-2-Kd with interaction score of 267.0 (D); The mouse MHC alleles were shown as golden ribbon representation, and the CTL epitopes were shown as colored ball & stick representation.(TIF)Click here for additional data file.S2 FigA) Interaction of the multiepitope construct with TLR-2 (A), TLR-3 (B), TLR-4 (C), TLR-5 (D), TLR-8 (E), and TLR-9 (F). The multiepitope construct was shown as colored ribbon representation, and TLRs were indicated as golden ribbon presentation.(TIF)Click here for additional data file.S3 FigA) SDS-PAGE analysis: lane 1: Molecular weight , lane 2: Before induction, lane 3: 3 h after induction, and lane 4: Purified protein; B) Identification of the recombinant GST-L1-E7 protein by western blot analysis using anti-His antibody: lane 1: Molecular weight, lane 2: Before induction, lane 3: After induction, and lane 4: Purified protein.(TIF)Click here for additional data file.S4 FigA) Scanning electron microscopy ; B) Transmission electron microscopy (TEM). The vesicle shape and membrane integrity of exosomes were proved in these images.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


The sudden rise of the worldwide severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in early 2020 has called into drastic action measures to perform instant detection and reduce the rate of spread. Common clinical and nonclinical diagnostic testing methods have been partially effective in satisfying the increasing demand for fast detection point-of-care (POC) methods to slow down further spread. However, accurate point-of-risk diagnosis of this emerging viral infection is paramount as the need for simultaneous standard operating procedures and symptom management of SARS-CoV-2 will be the norm for years to come. A sensitive, cost-effective biosensor with mass production capability is crucial until a universal vaccination becomes available. Optical biosensors can provide a noninvasive, extremely sensitive rapid detection platform with sensitivity down to ∼67 fg/ml (1 fM) concentration in a few minutes. These biosensors can be manufactured on a mass scale (millions) to detect the COVID-19 viral load in nasal, saliva, urine, and serological samples, even if the infected person is asymptotic. Methods investigated here are the most advanced available platforms for biosensing optical devices that have resulted from the integration of state-of-the-art designs and materials. These approaches include, but are not limited to, integrated optical devices, plasmonic resonance, and emerging nanomaterial biosensors. The lab-on-chip platforms examined here are suitable not only for SARS-CoV-2 spike protein detection but also for other contagious virions such as influenza and Middle East respiratory syndrome (MERS). I.A.The emergence of novel respiratory tract infections in the 21st century has been a growing concern that has turned into a major cause of concern, hospitalization, and death. The growing alert turned into a full-scale, worldwide deadly pandemic in 2020 with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) following predecessor respiratory pandemics, including SARS (severe acute respiratory syndrome) in 2003, H1N1 influenza (swine flu) in 2009, and MERS (Middle East respiratory syndrome) in 2012. The coronavirus disease 2019 (COVID-19) pandemic and its rapid growth rate have driven an unprecedented worldwide demand for emergency measures to mitigate its fast rate of spread.B.SARS-CoV is an enveloped, single-stranded RNA virus that exists in humans and animals and is mainly transmitted through aerosols and nearby interpersonal contact.C.The most well-known and commonly used procedure for identifying pathogens like SARS-CoV-2 relies on clinical real-time RT-PCR (reverse transcription polymerase chain reaction).POC biosensors can provide the next best alternative reliable solution to clinical diagnosis with a much faster real-time detection without compromising sensitivity and accuracy.Optical biosensing works under different physical transduction principles, such as interferometers, resonators, and plasmonics,D.A biosensor functioning mechanism, in general, consists of a sample or target analyte that binds to the bioreceptor, after which the transducer helps in converting the biorecognition data into a measurable quantity . The difThese labels require sophisticated reagent selection and modification that in turn can come with the drawback of perturbing the assay and, in some cases, making final detection a challenging task. On top of that, labeling chemistry can be expensive and time consuming. Thus, there have been many intriguing efforts in biosensing systems involving unlabeled or unmodified biomolecules (label-free biosensing)In the case of an optical biosensing platform, the sensing transduction signals are usually based on miniscule changes in refractive index, which itself results from the attachment of biomolecules to the immobilized bioreceptors. The biosensor final sensitivity and specificity are also strongly dependent on the immobilized molecules and the accessibility of target analytes to them. In a label-free biosensor, a highly sensitive biorecognition layer on the transducer surface is therefore of vital importance.All in all, for a highly sensitive optical biosensor suitable for detecting SARS-CoV-2, labeled or label free, the limit of detection is the determining factor to have a reliable POC substituting the lab-based detection methods. In Secs. II.Due to their immunity to electromagnetic interference (EMI), compactness, and high selectivity, optical biosensors have attracted special attention as a biosensing system.Effective mode index change for a perturbed waveguide can be calculated through the variation method:Sensitivity (S) and LOD are the two main criteria for evaluating the performance of the optical biosensor, which in turn depends on the strength of the interaction between the substance and light in a solution or on the surface.A.Integrated interferometer photonics is one of the most practical architectures for sensing applications. It is based on splitting the input beam into two arms through a Y junction: One arm is completely retained as the reference arm, and the other arm contains the target. The interaction of electromagnetic waves on the sensing arm will cause a phase difference with respect to the reference arm, and recombination of the two beams in the output will cause constructive or destructive interference see .Output intensity π/refractive index units (RIU) are obtained through increasing light–analyte interaction in the slot waveguide-sensing region thanks to the low refractive index subwavelength-sized slot regions. Liu et al.2 = 0.979177). The asymmetry of the MZI array is used to detect microRNA (miRNA) in human urine samples by measurement of light phase change due to attachment of complementary DNA capture probe and the target miRNA. Utilizing slot waveguide as the sensing arm plus the elimination of the tedious incubation process make it an integrated, accurate, and sensitive POC optical biosensor. Chalyan et al.4 rad/RIU, leading to a LOD <5 × 10−7 RIU enabled through surface functionalization of the biosensor , respectively. At the same time, to keep the SWG working in the subwavelength regime (Λ ≪ λ / 2neff), Λ is safely set to be 230 nm, and f is simply set to be 0.5 with no optimization. Furthermore, to make the SWG waveguide work as a single or few-mode waveguide, the width of the gratings is set to <2 μm. All in all, the ranges of w, A1, and A2 are set to be , , and , respectively. The FOM is defined to achieve the lowest bending loss, with a bend radius of 5 μm. Based on the optimization measures taken, we finally achieved a bending loss as low as 0.0279 dB/cm with the optimized  = . By adjusting the coupling gap between the insertion SWG waveguide and the designed SWGR, the Q can be as high as ∼50 000 (the resonance at 1557.6 nm) with a broad FSR of 25 nm, as shown in μm-radius SWGR [not shown in 1, A2) = , at the same waveguide–ring cross-coupling coefficients. Needless to say, as the quality factor of the ring becomes higher, the fabrication tends to be more challenging. Bulk RI sensitivity [shown in the inset of −5 RIU. Note that iDL performances can be further improved by exploiting a larger radius ring or by further achieving the critical coupling condition given the predictable higher Q while making a trade-off between the performance and the sensor size or the resonance peak extinction ratio.To develop an accurate estimate of a COVID-19 biosensing functioning mechanism, a simulation model first needs to be designed. Here, in a proposed simulation model, COVID-19 is approximated to be a solid sphere core containing RNA covered with a membrane protein with radii of ectively .50 Thus,bindingn) depends on the number of bonded virus layers, which is a function of the virus concentration and binding processing time, and is dominated by the concentration in real sensing process with a given binding time. Thus, the SWGR sensing performance can be evaluated by calculating the bindingn response of the device, with bindingn ranging from 1.35 (no binding) to 1.5 (full binding). Simulation results in To evaluate the specific sensing ability of the proposed device for COVID-19, surface sensing performance is analyzed by considering the device immersed in buffer solution bonded by several surface layers (generated in the sensing preparation process), including the ∼2–3-nm surface oxide layer, ∼10-nm functionalization layer with bonded antibody (protein layer), and virus particles layer, in the detection process. In simulations, the layers generated during the preparation process are further simplified to be a uniform layer (RI = 1.45) with a thickness of 15 nm, and the bonded virus layer is simplified as a uniform layer with a thickness of 125 nm (the maximum diameter of the COVID-19 virus) . It is nV.Ever-increasing advances and developments in the preparation and utilization of nanostructured materials have facilitated the extraction of an unprecedented range of properties from nanomaterials. Biosensing and amplification of targeted samples at extremely low concentrations for detection are among the most intriguing properties.Among the 2DM family, graphene possesses a special electron band structure showing high carrier mobility and zero bandgap characteristics, exceptional high-energy transfer efficiency, and large surface area.et al. demonstrated a functionalized graphene oxide wrapped around SiO2 that possessed superior RNA sensitivity and a limit of detection up to 1 fM, with the potential to show even higher sensitivity values.π–π stacking on its surface, make graphene a very promising candidate for development of any optical 2DM–based biosensorAs a result, graphene and its derivatives, like graphene oxide (GO), have been used in various biomedical areas, including but not limited to DNA sequencing, tissue engineering, stem cell research, and biosensing.Optical biosensors based on 2DMs and, in particular, graphene can generally be divided into three main groups for sensing: fluorescence, SPR, and SERS . Here, wA.π–π interaction between DNA probe and graphene surface.et al.et al.et al.The working mechanism of fluorescence biosensors is based on fluorescence quenching, fluorescence enhancement, or fluorescence resonance energy transfer (FRET).B.π–π stacking. The effect of adding a graphene layer to the gold surface in a prism-based SPR has been shown by Wu et al.γ (where γ > 1), where γ is the adsorption efficiency enhancement factor. It has been shown that graphene increases the sensitivity to refractive index change by 25% for L = 10 and, therefore, S increases five times in the case where graphene adsorbs four times more biomolecules (γ = 4).An SPR and LSPR biosensing platform conjugated with graphene has been developed to enhance the optical biosensor's sensitivity through amplifying detectable signal compared to conventional plasmonic materials.C.et al.2/Si surface due to electron transfer and energy transfer between the graphene and R6G dye molecules [A Raman spectroscopy working mechanism is based on measuring inelastic scattering of light, which provides a footprint regarding the molecular or material structure. However, the Raman scattering from organic molecules is weak and needs to be amplified through applicable methods to be used as a biosensing mechanism. The enhancement of Raman signal can be accomplished by adding metal nanostructures due to the SPR effect. The graphene can be used as a very effective SERS active substrate as it can extinguish photoluminescence of fluorescent dyes and eliminate fluorescence background. Xie olecules .et al. showed a highly sensitive plasmonic/magnetic hybrid nanomaterial graphene-based platform possessing an attractive combination for virus detection: plasmonic and magnetic effects. They used a magnetic field for separation of target virus from impurities along with the plasmonic substrate for fluoroimmunosensing to detect influenza virus (H1N1) with LOD of 7.27 fg/ml.et al. as a base for a facile fluorometric system for detection of influenza viral genes.2GO) has been developed to function through the surface plasmon resonance principle. The high binding affinity of targeted dengue virus envelope (DENV) E-proteins to the surface resulted in a high detection sensitivity of 0.08 pM. The low absorption of graphene and small photocurrent are a driving force to use graphene quantum dots (GQDs), which show a significant photoluminescence that can be improved by functionalization and doping of graphene.As mentioned, a wide range of graphene-based optical biosensors based on fluorescence, SERS, and SPR has been developed in recent years, among which few possess sufficient sensitivity and specificity to be utilized for SARS-CoV-2 detection .174–179 VI.Emerging pandemic and epidemic diseases, like COVID-19, bring out a high demand for advancement and research in medical detection and treatment methods. The optical biosensors provide a fast detection (<1 min) of such a virus at very low concentrations (∼1 fM). However, they need to be designed and functionalized to be the most absorptive to the target analyte. The ideal label-free biosensor is cost-effective, disposable or reusable, compact, and semiautomatic. Although most efforts in biosensors have been focused on protein biomarkers, other targets, such as small molecules and nucleic acids, are crucial in expanding the application of biosensors, including optical ones. A common challenge for optical biosensors is to reach the capability of performing the measurement in real complex samples, avoiding, or limiting, the sample preparation phase. Developing a label-free biosensor is aligned with that purpose because the need for onsite detection techniques is increasing as the world after the COVID-19 pandemic will never be anything like before.A.A., C.W., and K.M.Y. contributed equally to this work.


The humidity sensor is an essential sensing node in medical diagnosis and industrial processing control. To date, most of the reported relative humidity sensors have a long response time of several seconds or even hundreds of seconds, which would limit their real application for certain critical areas with fast-varying signals. In this paper, we propose a flexible and low-cost humidity sensor using vertically aligned carbon nanotubes (VACNTs) as electrodes, a PDMS-Parylene C double layer as the flexible substrate, and graphene oxide as the sensing material. The humidity sensor has an ultrafast response of ~20 ms, which is more than two orders faster than most of the previously reported flexible humidity sensors. Moreover, the sensor has a high sensitivity (16.7 pF/% RH), low hysteresis (<0.44%), high repeatability (2.7%), good long-term stability, and outstanding flexibility. Benefiting from these advantages, especially the fast response, the device has been demonstrated in precise human respiration monitoring , noncontact electrical safety warning for bare hand and wet gloves, and noncontact pipe leakage detection. In addition, the facile fabrication of the flexible platform with the PDMS-Parylene C double layer can be easily integrated with multisensing functions such as pH sensing, ammonium ion sensing, and temperature sensing, all of which are useful for more pattern recognition of human activity. Based on different sensing mechanisms and readout signals, humidity sensors can be categorized into several types, including resistance9, capacitance10, optical type11, surface acoustic wave5, impedance12, and field-effect transistor13. In addition, the sensing materials are also diverse, such as metal oxides15, carbon nanotubes16, graphene17, graphene oxide18, hydrogels19, and polyimides20. With the development of flexible and soft electronics, in recent years, flexible humidity sensors have become an essential component for wearable applications32. For example, Zhu33, Duan28, and Alrammouz34 reported a cost-saving, environmentally friendly paper-based flexible humidity sensor and demonstrated its applications for breathing, noncontact sensing, skin humidity detection, etc. Komazaki35, Ma36, and Rauf37 incorporated the sensing materials of PDMS-CaCl2, polyimide, and metal-organic frameworks with flexible textile substrates for humidity sensing. Zheng25 fabricated a transparent and flexible humidity sensor using biocompatible, natural biomaterial silk fibroin films as the substrate. In addition to the above paper and silk fibroin substrates, other flexible materials are also being explored as substrates for flexible humidity sensors, including polyimide27, PDMS38, cellulose/KOH composites39, polyethylene terephthalate (PET)40, and polycarbonate (PC)15. In addition, to be incorporated with flexible substrates, metals41, graphene42, ITO43, and graphite44 have been investigated as sensing electrodes for humidity sensors. Most of the electrodes are directly deposited or printed on the surface of the flexible substrate, and the adhesion performance is not robust and easily peels off at the crease area. Furthermore, since electrode materials are normally inorganic rigid materials, they tend to be damaged during bending or other deformations. The common solution is to put the electrode layer in the neutral plane to reduce the stress issue46. However, it cannot always be realized given the device structure, especially when two layers of electrodes are needed. Moreover, when targeting wearable electronics applications, flexibility should only be the prerequisite, other merits  are still vital. However, most of the current works focus on enhancing the sensitivity and changing different materials for better flexibility34. Very few works have put effort into improving the response time. Kano24 and He47 proposed two types of flexible humidity sensors with fast response/recovery times of approximately several tens of milliseconds using sensing materials of silicon-nanocrystal films and tunable graphene polymer heterogeneous nanosensing junctions, respectively. For one of the most important applications of humidity sensors, respiration monitoring, the breathing rate of coughing, asthma, and choking is much higher than the normal respiration frequency (~0.33 Hz). The humidity sensor with a slow response would not catch the sudden change of breathing signal48. In addition, although some RH sensors with a slow response could still sense fast-breathing behavior with their transient response, the information they offer is usually only the respiration strength. For example, since the humidity level is nearly the same for different breathing conditions , sensors with larger response times can sense changes in respiration, but the sensor signal variation amplitudes between fast breathing, normal breathing, and deep breathing would be quite different49. In addition, due to the accumulation effect, for the fast-breathing signal, the overall change in the record data has a distinct drift29. Moreover, for some fast-breathing conditions, the monitoring signal will have some sharp corners because the sensor cannot be efficient enough to precisely follow the breathing signals and catch clear breathing patterns36. They cannot truly provide the whole waveform of the breath, so they cannot precisely indicate the real respiration condition29.Humidity sensing is essential in broad applications, including process control in industry, medical facilities, environmental monitoring of greenhouses, cleanrooms, and heating, ventilation, air conditioning (HVAC) systemsHerein, for the first time, we present a flexible relative humidity sensor integrating a PDMS-Parylene C double-layer thin film as the flexible substrate, vertically aligned carbon nanotubes as the electrodes, and graphene oxide as the sensing material. VACNTs are stuck into flexible substrates and have ultra-robust performance under deformation and folding. The sensor achieves an ultrafast response, high sensitivity, good repeatability, and good long-term stability as a whole application package. In addition, a high-performance relative humidity sensor is demonstrated in real applications for human respiration monitoring, pipe leakage detection, and noncontact electric safety warning, showing its potential as a flexible or curved humidity-sensing node in wearable electronics or industry monitoring.The detailed fabrication process is shown in Fig. Polydimethylsiloxane (PDMS) and Parylene C were used to fabricate the double-layer flexible substrate, which was used to peel off the VACNT electrodes from the silicon wafer. The PDMS and Parylene C dimers were purchased from Dowsil and Galentis, respectively. To reduce the PDMS adhesion to the silicon substrate and ensure the integrity of the PDMS-Parylene C film during the peeling-off process from the Si wafer, 100 μl trichloro  silane (PFOCTS) (Merck & Co.) was evaporated and coated onto the surface of the wafer using the following procedure. The wafers and opened PFOCTS container were put into the same vacuum chamber, which was pumped for 5 min to reach base vacuum pressure and kept sealed. It took 3 h for PEOCTS to evaporate from the container and coat onto the wafer surface. After the surface treatment, a degassed PDMS precursor mixer (monomer/curing agent: 10/1) was spun on the surface of the wafer to cover the VACNTs using a spin coater with a rotation speed of 500 rotations/min for 40 s  as a sensing material was used in the devices  and G-band (1596 cm−1)  Fig. , indicat−1) Fig. , while C−1) Fig. . The XPS2O < 5 ppm) was divided into two channels and controlled with two mass flow meters. One channel worked as the source of dry air and another was wetted through the deionized water as a moisture source. A reference relative humidity sensor  was mounted into the testing chamber aside from our device. Arduino Yun board and RS LCR meter were used to record the reference RH value and capacitance of the flexible sensor, respectively. Figure y axis scaled as a logarithm. Figure 42. Besides, the absorbed water molecules are beneficial in strengthening the polarization effect and increasing the dielectric constant, leading to an increase of sensor capacitance with rising RH21. The high sensitivity of 16.7 pF/% RH was achieved at the excitation frequency of 1 kHz referring to the relative humidity change from 10% (4.84 pF) to 90% (1340.2 pF). Flexible sensors with different film thicknesses were achieved by three types of GO concentrations in the diluted GO solutions . Figure HRH was calculated by the following equationCD_RH and CA_RH are the capacitance values tested at certain RH values in the desorption and absorption process, respectively, and Sc is the sensitivity of the device. A low hysteresis of the flexible humidity sensor was achieved, which was smaller than 0.44%. The larger hysteresis mainly occurred in the high humidity regime, which may be caused by the residual water molecules in the bottom part of the graphene oxide5. The repeatability   are shown in Supplementary Fig. SAs a flexible sensor, the performance under static deformation was tested by bending both nonsensing and sensing areas under a circumstance with an RH of 85%. Figure 2, and the results show that the largest capacitance change of the sensor for exhaling breath with up to 4% CO2 is only 1.8%. Finally, a comparison table of different humidity sensors is summarized  of the breathing signal due to their slow response time. Based on the ultrafast response of the proposed flexible humidity sensor, it is practical to employ this sensor for monitoring respiration precisely, especially since it can capture comprehensive information for abnormal conditions, such as choking, apnea, and asthma. The flexible sensor was mounted inside of the hollow cylinder  with a double side adhesive Fig. . The res54 Fig. .Fig. 5Hu42. The peak capacitances for different spacer heights are 1.22 nF (5 mm), 1.01 nF (10 mm), and 0.96 nF (15 mm). Moreover, the pipe movement time was also tested with different moving periods, including 9 s, 15 s, and 20 s, at the same space height of 5 mm. As the moving speed increased, the peak values decreased from 1.22 nF (20 s) to 1.12 nF (15 s) and then to 1.06 nF (9 s), as shown in Fig. Pipe leakage tests are important for fluid pipelines, especially for water pipelines in the tunnels of subways and other difficult inspection areas. Water leakage can change the humidity, which can be monitored by the humidity sensor, and a warning signal can be sent to the control center to ensure safety and prevent property damage. We provide a simple testing method to demonstrate this pipe leakage detection application shown in Fig. Electric safety is not only an important aspect in the industry but also a critical issue in family and public areas. Current electric safety detection technology is based on signals such as leakage current and current overload generated after the event to identify the accident. We propose a novel method to monitor dangerous situations where people approach electric adapters. The flexible sensor can be easily attached to the surface of the electric adapter shown in Fig. In this work, we successfully demonstrate a flexible humidity sensor using vertically aligned carbon nanotubes as robust conductive electrodes, GO as the sensing material, and a PDMS-Parylene C double layer as the flexible substrate. Its fabrication process is a novel generic method for the development of flexible sensors, which can be transferred to realizing other types of flexible sensing functions using the same platform. The device is characterized by demonstrating an ultrafast response (20.8 ms)/recovery (19.9 ms), high sensitivity (16.7 pF/% RH), low hysteresis (<0.44%), and high repeatability (2.7%). The ultrafast-response characteristic makes it feasible for the accurate sensing of human respiration, including abnormal behaviors such as choking, apnea, and asthma. Future work will focus on the deep analysis of the recorded data for medical information extraction, including breathing rate and breathing mean ventilation. Moreover, the device is also capable of leakage detection in water pipelines and electric safety monitoring, which are promising applications for personnel working in laboratories, mines, petrochemical industries, and other vulnerable industries.Supplemental Material


This method was validated at the lower limit of detection (LOD) and the lower limit of quantitation (LLOQ) of 5 ng/mL and 10 ng/mL, with linearity of 10–5000 ng/mL (r2 > 0.99) and 10–2500 ng/mL (r2 > 0.99), in rat plasma and tongue homogenates, respectively. The intra- and inter-day precision (CV%) and accuracy (RE%) were within 15% for LLOQ, low-, medium- and high-quality control samples. The mean extraction recoveries were around 50% and 80% for plasma and tongue homogenates, respectively. This assay was successfully applied to pharmacokinetics study following intravenous administration of OXP, as well as tongue tissue distribution after 1 h and 4 h of a novel oral mucosal patch application.Oxaliplatin (OXP), a third-generation platinum-based chemotherapy drug, was often indirectly analyzed via total platinum by an ICP-MS because it was difficult to directly quantify using an LC-MS/MS method, due to its instability, bad column separability and severe MS signal inhibition. Here, we developed and validated a specific, sensitive and reproducible LC-MS/MS method for the quantification of OXP itself in rat plasma and tongue tissue on a SCIEX 4000 QTRAP However, those products do not make a significant contribution to the cytotoxicity of OXP even though they are known to be more cytotoxic than OXP in cellular assays + exhibited m/z values of 397, 398, 399 and 401 +-2HCOOH). For the IS, m/z 131, a stable conjugated π bond system [CH=CH-N=N-C6H5]+, is formed after the loss of a molecule of CH3COCH3. Those daughter ions were stable to be used in quantification. Since stable isotope-labeled OXP was not available on the market, antipyrine was chosen as an IS based on its appropriate retention time and no interference in this method. The product ion mass spectra for OXP is shown in  and 401 . The str® F5 column , Acquity HSS-T3  and ACE Excel 2 Super C18 column , and Phenomenex Lux 5u Cellulose-1 column . However, all of them showed serious signal inhibition for plasma samples, suggesting that the OXP signal might be interfered with by endogenous substances. With a longer Phenomenex Lux 5u Cellulose-1 column, the simple isocratic elution could separate OXP and IS more efficiently than the other columns, where retention times of OXP and IS were 3.48 min and 4.54 min, respectively. Notably, 1.25 mM of ammonia formate was needed for the mobile phase to obtain consistent signal, indicating that the pH of mobile phase could affect the quality of the LC-MS/MS analyzing method. Different pH of mobile phase has been investigated, it being observed that adding 0.05% and 0.1% of formic acid to mobile phase under same condition could lead to double peak at high concentration, and the neutral mobile phase without adding acidic or alkaline solution could lead to unstable sensitivity of OXP. The final UHPLC method was optimized by selecting and testing different types of column, mobile phase compositions and flow rates to obtain better peak shapes, less carryover and higher sensitivity. As shown in OXP and internal standard (IS) were separated on a Phenomenex Lux 5u Cellulose-1 column . Several other short stationary phase columns were tested, including the KinetexAs shown in 2) were at least 0.99  for all calibration curves. The accuracy was within 85–115% for plasma and tongue homogenate calibration standards at all concentration levels. LOD of the assay was 5 ng/mL and LLOQ was 10 ng/mL for both plasma and tongue homogenates, where LOD had at least 3:1 and LLOQ had at least 5:1 signal-to-noise ratio , MQC , HQC  in rat plasma and tongue homogenates with five replicates . The intA dilution integrity study was performed to check if sample dilution changed signals compared to the predicting concentration. All of them were within ±15% of nominal concentration, indicating that the dilution was reliable for quantification of samples with higher OXP concentration than ULOQ.The extraction recovery and matrix effect from different biological matrices are shown in OXP stability in rat plasma and tongue homogenates was tested for short-term stability, freeze-thaw stability, auto-sampler stability, and long-term stability at four QC levels with triplicates . Bench-t0→90min) was 200.4 ± 35.6 min∙μg/mL. The apparent volume of distribution (Vd) was 867.0 ± 75.3 mL/kg, the apparent clearance (CL) was 127.8 ± 25.2 mL/min/kg, and total body mean residence time (MRT) and terminal elimination half-life (T1/2) were 6.9 ± 0.7 and 16.2 ± 2.5 min, respectively (No noticeable signs of discomfort were observed in rats after IV administration of OXP 25 mg/kg BW) in 5% glucose solution. The mean plasma concentration versus time profile is shown in  mg/kg BWectively . The finThe OXP concentration in plasma after patch administration was lower than LOD. We determined the concentration of OXP in tongue homogenates at 1 h and 4 h following patch application, the mean ± SD of tongue tissue concentrations were 2620 ± 432 ng/g and 2834 ± 664 ng/g, respectively. The percentage values of OXP in patch residue and tongue tissues at 1 h and 4 h after supra-lingual application are presented in Several drug dosage formulations have been reported for intraoral application, including the implantable tablet, mucoadhesive patch, film, microsphere, ointment, cream, and hydrogel ,33,34,35OXP, antipyrine , and ammonia formate were purchased from Sigma Aldrich . Acetonitrile and high-performance liquid chromatography (HPLC)-grade water were obtained from VWR Chemicals BDH . Blank rat plasma used for the method’s development and validation was purchased from Innovative Research .Adult male Sprague-Dawley rats were used in all animal studies and were purchased from Envigo RMS  and tissues were from the non-treated rats. The animal experiments were approved (Protocol #9080) by the Institutional Animal Care and Use Committee (IACUC) at Texas Southern University (TSU) and were conducted according to the National Institute of Health “Guide for the Care and Use of Laboratory Animals, 8th Edition”.® MS/MS system . System control and data analysis were performed using the Analyst® software 1.6.2 .The UHPLC-MS/MS system includes a Shimadzu Nexera X2 UHPLC system  and a 4000 QTRAPv/v) acetonitrile in water containing 1.25 mM ammonia formate. The injection volume of each sample was 10 μL. The total run time was 5.5 min. The flow rate was 0.8 mL/min.OXP separation was carried out on a Phenomenex Lux 5u Cellulose-1 column  with an isocratic elution of 50% , 39 and 28 V for collision energy (CE), and 13 and 9 V for collision cell exit potential (CXP), respectively.MRM data were collected by the MS using a Turbo V™ source with ESI positive mode to detect the specific precursor-to-product ion transitions, Stock solutions of OXP and IS were dissolved in water at a concentration of 1 mg/mL and stored at 4 °C until use. Standard samples were prepared by spiking OXP into blank rat plasma and rat tongue homogenates, respectively, at concentrations ranging 10–5000 ng/mL and 10–2500 ng/mL for rat plasma and tongue homogenates, respectively. Briefly, a series of working solutions of OXP were prepared by diluting stock solution in water to the appropriate concentrations  for standards  in plasma and tissue homogenates. Working solutions were then spiked into blank rat plasma and blank tissue homogenates with 10 times dilution to make calibration standards. The final concentrations of standard samples were 10, 50, 250, 500, 1000, 2500, 5000 ng/mL in plasma. The final concentrations of calibration standards for tongue homogenates were 10, 50, 250, 500, 1000, 2500 ng/mL in tissue homogenates. Calibration standards were freshly prepared daily.The working solutions for QC samples were independently prepared by diluting OXP standard solutions with water to give the concentrations of 100, 250, 20,000, and 40,000 ng/mL, which were 10 times as that of nominal concentrations: 10 (LLOQ), 25 (LQC), 2000 (MQC) and 4000 ng/mL (HQC) for rat plasma. For tongue homogenates, the working solution concentrations of QC samples were 100, 250, 8000, and 20,000 ng/mL, and the corresponding nominal concentrations were 10 (LLOQ), 25 (LQC), 800 (MQC) and 2000 ng/mL (HQC).w/v) using the Biospec Tissue TearorTM  to make tongue homogenates. Rat plasma or tongue homogenates were extracted by a protein precipitation method described in [w/v) ammonium formate. After vortexing for 15 s, the extracted samples were centrifuged at 14,000 rpm for 20 min at 4 °C. Finally, 10 μL of supernatant was injected into the LC-MS/MS system. Sample preparation was performed on top of ice pads.Each weighted tongue tissue sample was homogenized in a clean scintillation vial with water . Carryover in the blank sample following the ULOQ should not be greater than 20% of the LLOQ for OXP and 5% for the IS.Intra-day accuracy and precision were conducted at four QC levels of OXP concentrations in plasma and tongue homogenates on a single assay by five replicates. Inter-day accuracy and precision were determined by five replicates of four QC levels on three consecutive days. Accuracy was determined by the RE%. Precision was determined by the CV%. Calculated results of inter-day and intra-day precision and accuracy should be ≤15%, except for the LLOQ, where the absolute value of calculated concentrations should be ≤20%.The dilution integrity of rat plasma performed to determine the accuracy of extended linearity beyond the ULOQ (5000 ng/mL). The dilution factors for dilutions of QC were 2, 5 and 10. Diluted quality control  samples were further diluted five and 10 times using plasma to measure the accuracy and precision of the dilution. Another DQC (10 μg/mL) plasma sample was further diluted two and five times to measure the accuracy and precision of the dilution.In this study, matrix effects were evaluated by comparing the peak area of the post-extracted blank rat plasma or tongue homogenates to the peak area of neat solution at four QC levels  in five replicates. The calculated equation of the matrix effect is as follows:The extraction recovery was evaluated by comparing the peak area of the pre-extracted blank rat plasma or tongue QC sets to the peak area of the post-extracted QC sets. The calculated equation of extraction recovery is as follows:The stability of OXP in rat plasma or tongue homogenates was examined by short-terms, freeze-thaw, post-prepared auto-sampler, and long-term stability studies. All stability experiments were conducted at four QC levels  with five replicates. The short-term bench-top stability was tested by placing prepared QC samples at 4 °C for 6 h. Auto-sampler stability was performed by placing QC samples inside of autosampler of LC-MS/MS for 6 h, the auto-sampler temperature was set to 15 °C. Freeze-thaw stability was conducted by three cycles of freezing at −80 °C and thawing at 4 °C. Long-term stability was evaluated by placing the QC samples in −80 °C. OXP concentrations in all stability samples were compared to that of freshly made QC samples.2O, on a supporting material. The concentration of OXP in each patch was 1.1 μg/cm2.The polymer patches were fabricated by Cellink Bio X using a high-OXP-concentration solution containing 30 mg of OXP, 0.05 g of PAA, 0.05 g of 3,4-dihydroxyphenylalanine (DOPA), 0.1 g of CMC, and 5 mL of Hn = 3) and 4 h (n = 3), respectively, at a dose of 150 μg/kg BW under anesthesia. Serial blood samples (about 100 μL each) were collected at 1, 2, 3 and 4 h from the start of the patch application to the end of patch application and stored in heparinized tubes. Blood samples were centrifuged at 3000 rpm for 20 min to separate the plasma samples, then stored at −80 °C until analysis. Tongues were removed immediately after euthanasia, rinsed with deionized water three times, sliced into small pieces, and then homogenized in water (1:6 w/v). Blank tongues were prepared separately using drug-free rats. The PK parameters for each rat were estimated using the Phoenix WinNonlin software 8.2 . Non-compartmental analysis was used to determine the PK parameters of OXP after IV administration [The validated method was applied to investigate the plasma profiles of OXP after IV administration of a 25 mg/kg BW OXP in 5% glucose solution, and tongue distribution of OXP after a supralingual administration of 150 μg OXP patch/kg BW in rats. A group of three adult male SD rats was used for the PK studies. Serial blood samples (approximately 150 μL for each) were collected in heparinized tubes at various time points up to 24 h after dosing. Plasma was separated immediately by centrifugation of the blood samples at 3000 rpm for 20 min and kept at −80 °C until analysis. For the tongue tissue distribution and blood diffusion studies, six adult male SD rats were used for the study. A mucoadhesive patch formulation containing OXP was applied onto the dorsal tongue surface of a rat for 1 h (stration .In this study, we developed a rapid, sensitive, and reliable UHPLC-MS/MS method for measuring OXP in rat plasma and tongue homogenates. This method was properly validated over the concentration ranges of 10–2500 ng/mL and 10–5000 ng/mL for tongue homogenates and plasma, respectively. We have successfully applied this method to the PK study after IV administration of OXP and showed that the plasma concentration was quantifiable until 1.5 h after administration. Topical delivery of OXP to rat tongue tissue was also determined and showed similar tongue concentrations of OXP at 1 h and 4 h after patch application, indicating a burst penetration of OXP into tongue tissue before 1 h and a long-term sustained release until 4 h of the application.


PYCR1 was upregulated in HCC compared to adjacent nontumor tissues (P < 0.05). These data were verified using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry analysis. Additionally, patients with low PYCR1 expression showed a higher overall survival rate than patients with high PYCR1 expression. Furthermore, PYCR1 overexpression was associated with the female sex, higher levels of alpha-fetoprotein, advanced clinical stages (III and IV), and a younger age (< 45 years old). Silencing of PYCR1 inhibited cell proliferation, invasive migration, epithelial-mesenchymal transition, and metastatic properties in HCC in vitro and in vivo. Using RNA sequencing and bioinformatics tools for data-dependent network analysis, we found binary relationships among PYCR1 and its interacting proteins in defined pathway modules. These findings indicated that PYCR1 played a multifunctional role in coordinating a variety of biological pathways involved in cell communication, cell proliferation and growth, cell migration, a mitogen-activated protein kinase cascade, ion binding, etc. The structural characteristics of key pathway components and PYCR1-interacting proteins were evaluated by molecular docking, and hotspot analysis showed that better affinities between PYCR1 and its interacting molecules were associated with the presence of arginine in the binding site. Finally, a candidate regulatory microRNA, miR-2355-5p, for PYCR1 mRNA was discovered in HCC. Overall, our study suggests that PYCR1 plays a vital role in HCC pathogenesis and may potentially serve as a molecular target for HCC treatment.Overexpression of pyrroline-5-carboxylate reductase 1 (PYCR1) has been associated with the development of certain cancers; however, no studies have specifically examined the role of PYCR1 in hepatocellular carcinoma (HCC). Based on The Cancer Genome Atlas expression array and meta-analysis conducted using the Gene Expression Omnibus database, we determined that  Furthermore, RNA sequencing (RNA-seq), bioinformatics analyses, and molecular docking were used to elucidate protein interactions that are associated with the function of PYCR1. The results will probably provide us a potential drug target for therapy or a biomarker for diagnosis and prognosis. These results will help us better understand the role of PYCR1 in the pathogenesis and development of HCC.In the present study, we aimed to analyze the relationship between PYCR1 silencing and HCC 2 at 37 °C.The HCC cell lines used in this study included Huh7 cells and LM3 cells. Because the PYCR1 expression of these two cells were higher than the other cells . The HCCThe following antibodies were used in immunoblotting and immunofluorescence studies: anti-β-actin , anti-PYCR1 , anti-E-cadherin , anti-β-catenin , anti-N-cadherin , and anti-vimentin .Reagents and materials used for the analysis of cell proliferation, migration, and invasion and for RNA-seq included Cell Counting Kit‑8 , Transwell plates , RNA Nano 6000 assay kit , and NEB Next® Ultra™ RNA library prep kit for Illumina® .A total of 106 tumor samples and adjacent nontumor liver tissues were obtained from patients who had been pathologically diagnosed with HCC and undergone a partial hepatectomy at The Affiliated Hospital of the Guilin Medical University. None of the patients received adjuvant chemotherapy or radiotherapy prior to surgery, and all cases were independently confirmed by two senior pathologists. The clinicopathological information for all cases was retrieved from the hospital's clinical database. This study was approved by the ethics committee of the Guilin Medical University, and informed consent was obtained from the patients.http://www.ncbi.nlm.nih.gov/geo/). Multiple probes were used for the detection of the PYCR1 gene, and the mean expression value was calculated for each probe PYCR1 mRNA in HCC tissues were extracted from the datasets using the R software, and then a meta-analysis was performed. Standardized mean differences and 95% confidence intervals were calculated for pooled values and indicated expression differences. Cochran's Q and I2 tests were used to evaluate heterogeneity. Based on the level of heterogeneity, two models were applied to the overall meta-analysis. A random-effect model was chosen when there was significant heterogeneity among all samples , and a fixed-effect model was used when there was no significant heterogeneity, as indicated by statistical analysis Seven microarray datasets  were downloaded from the Gene Expression Omnibus database (https://xena-browser.net/). Gene expression was quantified experimentally using the Illumina HiSeq 2000 RNA-seq platform. Three TCGA datasets including gene expression RNA-seq  dataset, phenotype (n=438) dataset, and miRNA expression RNA-seq  dataset were used in the study, which belongs to TCGA Liver Cancer cohort. A total of 413 samples, including 363 HCC tissues and 50 adjacent non-HCC tissues were selected for this study. Detailed patient information and PYCR1 expression levels are summarized in Table Clinical and RNA-seq data for TCGA cohorts were downloaded from the Xena Public Data Hubs (https://www.mirnet.ca), miRDB (http://www.mirdb.org), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/index.php), and TargetScan (http://www.targetscan.org/vert_72/) were used to predict the upstream regulatory miRNAs of PYCR1. Only miRNAs appearing in two or more sets of the results were chosen as candidate miRNAs.Four frequently used online tools, including miRNet  according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a SYBR Green master mix  with a StepOne sequence detection system (Applied Biosystems). The qRT-PCR cycling conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 58-60 °C for 60 s, and extension at 72 °C for 10 s. The mRNA expression level was quantified using the 2Proteins were extracted using RIPA lysis buffer  containing a protease inhibitor cocktail . Protein quantification was performed using a bicinchoninic acid protein assay kit . Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and then incubated with primary antibodies, including β-actin , PYCR1 , E-cadherin , β-catenin , N-cadherin , and vimentin , at 4 ℃ overnight. After being washed with Tris-buffered saline with Tween 20 for four times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Immunoreactive bands were visualized using the BeyoECL Plus kit (Beyotime).Formalin-fixed and paraffin-embedded 4 μm-thick tissue sections were deparaffinized, rehydrated, and heated with citric acid buffer for antigen retrieval for 15 min. The samples were then placed in 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity and treated with blocking serum for 20 min. The slides were incubated with an anti-PYCR1 primary antibody  at a 1:900 dilution at 4 ℃ overnight, followed by incubation with a biotinylated secondary antibody for 25 min. The negative control slides were incubated with PBS only. The slides were rinsed in phosphate-buffered saline , then counterstained with hematoxylin, dehydrated, and mounted in a neutral resin. The staining intensities and the proportions of positive cells in all tissues were analyzed and scored by two experienced pathologists. The results were analyzed using immunoreactivity scores based on the staining intensity and the proportion of stained cells. The staining intensity was scored as follows: 0 = unstained; 1 = weakly stained; 2 = moderately stained; and 3 = strongly stained. The proportion of stained cells was scored as follows: 0 = negative; 1 = 1-10%; 2 = 11-50%; 3 = 51-80%; and 4 > 80%. The intensity score was multiplied by the proportion of staining score to obtain an immunoreactivity score. A total score greater than 3 was considered positive expression; that of 1 to 3 was considered low expression; and 0 was considered negative expression.PYCR1 was achieved using the following three interfering RNAs (RNAis): RNAi 1, 5'-gaGGGTCTTCACCCACTCCTA-3'; RNAi 2, 5'-tgAGAAGAAGCTGTCAGCGTT-3'; and RNAi 3, 5'-caCAGTTTCTGCTCTCAGGAA-3'. The RNAis were obtained from Shanghai GeneChemCo and were inserted between the AgeI and EcoRI sites of the GV248 vector. The short hairpin RNAs (shRNA) of GV-NC-shRNA and GV-PYCR1-shRNA were constructed as control lentivirus and shPYCR1 lentivirus for further experiment. To establish stable HCC cell lines with silenced PYCR1, 1.5 μg/mL puromycin was added to the culture media 48 h after the initiation of virus infection, and the expression of the green fluorescent protein (GFP) was observed. Cells were then trypsinized and cultured in a 100 cm2 dish to allow for expansion. The silencing of PYCR1 was confirmed by qRT-PCR and immunoblotting assays. Empty vectors were used as controls.Downregulation of . Briefly, Huh7 cells (1.5 × 103) from the shNC and shPYCR1 groups were seeded into a 96-well plate. After 24, 48, 72, 96, and 120 h of treatment, 10 μL of the CCK-8 solution was added to each well, and incubation continued for 2 h. The absorbance was measured at 450 nm using a microplate reader . Each experiment was performed in triplicate.Cell proliferation was assessed using the CCK-8 solution according to the manufacturer's instructions6 cells/well) and incubated for 24 h to reach 80% confluence. A sterile 10 μL pipette tip was then used to gently and slowly scratch the cell monolayer. Images of the wound were captured in 10 random fields using a light microscope  at a 100× magnification. The gap width in the PYCR1-silenced cell groups was compared to that in the control using the Photoshop software at different time points. Each experiment was repeated three times.Huh7 and LM3 cells were seeded in a serum-free DMEM into 6-well plates . Cells (1 × 104) of Huh7 and LM3 were suspended in 200 µL of a serum-free DMEM, seeded into the upper Transwell chamber, and cultured at 37 °C for 24 h, while 600 µL of DMEM containing 10% FBS was simultaneously added to the lower chamber. The invaded cells were fixed with methanol, stained with a 2.5% crystal violet stain solution (Solarbio), and then observed and photographed using an optical microscope. Five visual fields were randomly selected for quantification in each group.The 5/mL, the cells were incubated with fluorescent antibodies at room temperature for 25 min, then pelleted, and resuspended. The samples were analyzed using a flow cytometer (BD Biosciences) and the FlowJo software.Allophycocyanin-conjugated annexin V and 7-aminoactinomycin D  were used to analyze the cell apoptosis rate. Huh7-shNC and Huh7-shPYCR1 cells were washed twice with PBS and treated with 0.25% pancreatin (without EDTA) to facilitate digestion. After adjusting the cell density to 1 × 107 cells in 100 µL of DMEM) were injected subcutaneously into the upper left flank region of the mice. Tumor diameters were measured using digital calipers every 4 days. After 4 weeks of observation, the mice were sacrificed under anesthesia, and xenograft tissues were collected. All animal procedures were performed in accordance with the protocol approved by the Guilin Medical University Experimental Animal Center.Male BALB/c nude mice (6-7 weeks old) were obtained from the Shanghai Institute of Materia Medica . Cells stably transfected with the shPYCR1 or shNC , and RNA integrity and concentration were measured using the RNA Nano 6000 assay kit for the Bioanalyzer 2100 system (Agilent Technologies). A total of 2 μg of RNA from each sample was used as an input material for the RNA sample preparation. Sequencing libraries were generated using the NEB Next® Ultra™ RNA library prep kit for Illumina® according to the manufacturer's instructions. After library analysis, RNA-seq was completed using the Illumina HiSeq X Ten sequencing platform, and paired-end sequence reads were obtained.https://www.bioinformatics.babraham.ac.uk/). Clean RNA-seq reads were aligned to the GRCh38 human genome using HISAT v2.1.0 with default settings Quality control of the raw sequencing data was performed using the FastQC tool v0.11.9  networks were established using the STRING database.https://www.rcsb.org/). The preparation of the PDB files, including the removal of extraneous water molecules, addition of hydrogen molecules, and adjustment of pH-sensitive protonation, was performed using the SYBYL-X 2.0 software. Protein-protein docking was performed using the docking program HEX 8.0.0. The KFC2 (Knowledge-based FADE and Contacts) server was used to calculate protein-protein binding interface hotspots, which are defined as small fractions of residues accounting for a large fraction of the binding affinity -1) was calculated to assess the docking profile in our study. Further details were recorded in The structures of PYCR1 and its interacting proteins were downloaded from the RCSB Protein Data Bank (PDB) database . Data are presented as the mean ± standard deviation. One-way analysis of variance was used to compare differences between multiple groups, and Student's Data were extracted from seven published and publicly available HCC datasets. Only datasets containing both HCC and their adjacent nontumor samples were used for further analysis. A meta-analysis approach was applied to these datasets as outlined in Figure PYCR1 mRNA was analyzed in 413 samples, including 363 HCC tissues and 50 adjacent nontumor tissues, from the TCGA dataset. Increased PYCR1 expression was found in the HCC tissues compared with the adjacent nontumor tissues  levels, higher clinical staging (stages III and IV), and a younger age (< 45 years old) . However, recurrence-free survival indicated that there was no difference in the prognosis between high and low PYCR1-expressing patients . Furthermore, we quantified the expression levels of proteins associated with epithelial-mesenchymal transition (EMT) and determined whether their expression was affected by downregulation of PYCR1. Our results suggested that the expression of E-cadherin and β-catenin was significantly increased in cells with downregulated PYCR1 expression, whereas that of N-cadherin and vimentin was decreased . Based on these parameters, 569 genes were found. After removing noncoding genes, 425 DEGs were identified, of which 298 were upregulated and 127 were downregulated following PYCR1 silencing  cascade, ion binding, immune stimulus, cell component, transport, and catalytic activity. PPI analysis further demonstrated that many of the proteins were associated with each other  for PYCR1 and its eight target proteins were determined to be as follows: apoptogenic protein 1 , glutamate ionotropic receptor kainite type subunit 2 , RhoGD12 , dual specificity phosphatase 9 , chorionic gonadotropin subunit alpha , retinol-binding protein 4 , nuclear receptor subfamily 1 group H member 4 , and serpin family E member 1 . The complexes generated during docking are shown in Figure total values reflect the possibility of binding, with a lower value correlating with easier and stronger binding to other proteins The structures of eight interacting proteins, which were associated with key pathway modules, such as MAPK pathways, cell migration, cell communication, and cell proliferation/growth, and identified downstream of PYCR1, were found in the PDB database. The information obtained from the PDB files and HEX docking results is shown in Table PYCR1 mRNA. Based on the TCGA RNA-seq data, only miR-2355-5p was differentially expressed between HCC tissues and adjacent nontumor tissues and was downregulated in HCC samples compared to adjacent nontumor samples , and a younger age (< 45 years old). Furthermore, PYCR1 upregulation was found to be associated with a poor OS. These results suggest that PYCR1 expression may be an effective predictive factor for OS in patients with HCC. Similar correlations between PYCR1 expression and clinical features  were also observed in our patients with HCC; however, there was no association between PYCR1 expression and AFP level or sex. This discrepancy may be due to a largely Caucasian population in the TCGA database, whereas our HCC tissues were exclusively obtained from Chinese patients. Thus, ethnic diversity may impact the effect of PYCR1 on HCC development. Additional HCC samples from patients of different ethnicities should be included in further studies.In the present study, PYCR1 induces apoptosis and cell cycle arrest PYCR1 in Huh7 and LM3 HCC cells and to determine the effect of PYCR1 on various cellular functions. The results revealed that silencing PYCR1 induced a marked inhibition of cell proliferation, migration, and invasion. Further, the results of the HCC xenograft mouse study indicated that silencing PYCR1 significantly inhibited the tumor growth.A previous study has shown that silencing of PYCR1 caused increased expression of E-cadherin and β-catenin and decreased expression of N-cadherin and vimentin. These results suggest that PYCR1 promotes HCC migration and invasiveness by inducing EMT.EMT has been shown to play an important role in tumorigenesis by inhibiting epithelial function and upregulating the expression of mesenchymal-related genes to enhance invasiveness and metastasis PYCR1 silencing. Two specific genes, namely, secreted phosphoprotein 1 (SPP1) and C-X-C motif chemokine 12 (CXCL12) were identified to be strongly associated with PYCR1. Previous studies have reported that PYCR1 can activate SPP1 to regulate cell adhesion To better understand the downstream molecular events involved in the effects of PYCR1 on HCC invasiveness and metastasis, we performed RNA-seq to obtain DEG profiles following PYCR1. Furthermore, HRG inhibits the tumor growth by facilitating clearance of dying tumor cells The results of our functional analysis showed that the identified DEGs were primarily enriched in 14 functional categories, including cell communication, MAPK cascade, and cell migration. Cell communication is crucial for morphogenesis, cell differentiation, homeostasis, growth, and cell-cell interaction PYCR1 silencing. IRS1 is a cytoplasmic substrate of the insulin receptor and insulin-like growth factor 1 receptor and plays prominent roles in human malignancies. Phosphorylation of IRS1 leads to the recruitment of downstream effectors and to the activation of the MAPK cascade, which promotes the activation of the PI3K cascade PYCR1 silencing. Silencing of PYCR1 could interfere cell proliferation through the alteration of IRS1/JNK signaling pathway which subsequently inhibited MAPK pathways.A previous study has also suggested that p38 MAPK pathways may be activated by PYCR1 and by increased expression of its downstream targets that regulate cell proliferation and metastasis PYCR1 has been associated with the inhibition of proliferation and invasiveness of breast cancer cell lines via activation of the AKT/ERK signaling pathway Other studies have reported that APOA1 may inhibit the formation of tumor vessels and induce an antitumor immune microenvironment, thereby preventing tumor progression PYCR1-silenced cells. In particular, glycosylation, cell differentiation, homeostasis, cell adhesion, neuron/nervous system, cell component, and immune stimulus categories were identified. Taken together these results suggest that downregulation of PYCR1 induces the activation of these functional pathways, which may subsequently influence the development of HCC.Many other functional categories were found to be associated with the DEGs identified in total value  compared with those of two other complexes (APOA1-PYCR1 and GRIK2-PYCR1). Further, complexes of PYCR1 with proteins that are most strongly related to cell migration and cell communication exhibited the lowest Etotal values, namely, SERPINE1-PYCR1  and NR1H4-PYCR1 . Previous research has suggested that lower Etotal values are associated with easier and stronger binding between a ligand and a receptor The results of our molecular docking analysis suggest that PYCR1 binds to its interacting proteins via binding hotspots. In the MAPK cascade, the DUSP9-PYCR1 complex exhibited the lowest EAnalysis of hotspots revealed that several amino acid residues in DUSP9 and SERPINE1 exclusively contributed to PYCR1 binding, whereas no hotspots were identified in other protein-protein complexes involved in the MAPK cascade, cell migration, and cell-cell communication. Moreover, several hotspots in the CGA-PYCR1 complex were found to be involved in cell proliferation and growth. These results suggest that the CGA-PYCR1 complex is multifunctional. Interestingly, all of the identified PYCR1 hotspots included Arg, which may imply that Arg is vital for the binding between PYCR1 and its interacting proteins. It has been reported that the depletion of Arg may suppress the proliferation of HCC cells because reduced Arg levels would interfere with intracellular glutamine metabolism and inhibit the synthesis of proteins and thymidine in silico prediction revealed that miR-2355-5p might be an upstream regulator for PYCR1 mRNA. Importantly, when PYCR1 levels were upregulated, miR-2355-5p was downregulated in HCC tissues. These results indicated that miR-2355-5p could serve as a negative regulator of PYCR1 expression, and low miR-2355-5p expression could promote the accumulation of PYCR1 in HCC tissues. Furthermore, miR-2355-5p has been reported to be a master regulator of target genes in other diseases, including chondrosarcoma and intervertebral disc degeneration Various studies have demonstrated that noncoding RNAs participate in HCC development PYCR1 expression was silenced, the proliferation, migration, invasion, metastasis, and EMT, as well as the tumorigenic capacity, were significantly inhibited in HCC cells. The mechanisms responsible for these effects involved many proteins, which were associated with many functional categories and signaling pathways. Analysis of protein-protein interactions indicated that specific hotspots might facilitate the formation of HCC-specific PYCR1 complexes and promote the development of HCC. We therefore propose that PYCR1 may be an effective novel target for the development of diagnostic or therapeutic options for HCC.In summary, our results suggest a role of PYCR1 in inducing the development of HCC. PYCR1 was found to be upregulated in HCC tissues compared to paired adjacent nontumor tissues. Using a TCGA dataset, we found that patients with low PYCR1 expression exhibited a higher OS rate than in patients with high PYCR1 expression. Meanwhile, higher PYCR1 expression was observed in females than in males and correlated with elevated AFP levels, higher clinical staging (stages III and IV), and a younger age (< 45 years old). In Chinese patients, two features, namely, higher clinical staging (stages III and IV) and a younger age (<45 years old), were related to higher PYCR1 expression. When Supplementary figures and tables.Click here for additional data file.