ABSTRACT
The incidence rate of intrahepatic cholangiocarcinoma (ICC), which has a poor prognosis, is rapidly increasing. To investigate the intratumor heterogeneity of ICC, we analyzed single-cell RNA sequencing data from the primary tumor and adjacent normal tissues of 14 treatment-naïve patients. We identified ten major cell types, along with 45 subclusters of cells. Notably, we identified a fibroblast cluster, Fibroblast_LUM+, which was preferably enriched in tumor tissues and actively interacted with cholangiocytes. LGALS1 was verified as a marker gene of Fibroblast_LUM+, contributing to the malignant phenotype of ICC. The higher amount of LGALS1 + fibroblasts were associated with poorer overall survival in ICC patients. LGALS1 + fibroblasts activated the proliferation and migration of tumor cells by upregulating the expression levels of CCR2, ADAM15, and ß-integrin. Silencing LGALS1 in cancer-associated fibroblasts (CAFs) suppressed CAF-augmented tumor cell migration and invasion in vitro as well as tumor formation in vivo, suggesting that blockade of LGALS1 serves as a potential therapeutic approach for ICC. Taken together, our single-cell analysis provides insight into the interaction between malignant cells and specific subtypes of fibroblasts. Our work will further the understanding of the intratumor heterogeneity of ICC and provide novel strategies for the treatment of ICC by targeting fibroblasts in the tumor microenvironment.
ABSTRACT
Intrinsic boundaries formed by grain stacks of randomly oriented perovskite crystallites seriously restrict charge transport in the resultant photovoltaic devices, whereas direct passivation of these defects remains unexplored, and it is desirable to modulate perovskite growth with uniform orientation. Herein, we report a simple but effective method to regulate perovskite crystallization by employing a volatile and polymerizable monomer of hydroxyethyl methacrylate (HEMA), which can simultaneously interact with both FA+ and Pb2+ via hydrogen and coordination bonding, respectively, to seed perovskite crystallization with accelerated nucleation and retarded crystal growth. Upon thermal annealing, the gradual volatilization and partial self-condensation of the HEMA drive the perovskite growth perpendicularly to the substrate, leading to largely suppressed defect states, improved crystallinity, and a reduced Young's modulus of the perovskite film. As a result, champion efficiencies exceeding 24 and 22% with improved operational and mechanical stability of the optimized perovskite solar cells based on rigid and flexible substrates were finally achieved.
ABSTRACT
A typical magnetometer-based measurement-while-drilling (MWD) system determines the azimuth of the bottom hole assembly during the drilling process by employing triaxial accelerometers and magnetometers. The geomagnetic azimuth solution is susceptible to magnetic interference, especially strong magnetic interference and so a rotary norm constraint filtering (RNCF) method for azimuth estimation, designed to support a gyroscope-aided magnetometer-based MWD system, is proposed. First, a new magnetic dynamical system, one whose output is observed by the magnetometers triad, is designed based on the Coriolis equation of the desired geomagnetic vector. Second, given that the norm of the non-interfered geomagnetic vector can be approximated as a constant during a short-term drilling process, a norm constraint procedure is introduced to the Kalman filter. This is achieved by the normalization of the geomagnetic part of the state vector of the dynamical system and is undertaken in order to obtain a precise geomagnetic component. Simulation and actual drilling experiments show that the proposed RNCF method can effectively improve the azimuth measurement precision with 98.5% over the typical geomagnetic solution and 37.1% over the KF in a RMSE sense when being strong magnetic interference environment.
ABSTRACT
OBJECTIVE: Elucidating complex ecosystems and molecular features of gallbladder cancer (GBC) and benign gallbladder diseases is pivotal to proactive cancer prevention and optimal therapeutic intervention. DESIGN: We performed single-cell transcriptome analysis on 230 737 cells from 15 GBCs, 4 cholecystitis samples, 3 gallbladder polyps, 5 gallbladder adenomas and 16 adjacent normal tissues. Findings were validated through large-scale histological assays, digital spatial profiler multiplexed immunofluorescence (GeoMx), etc. Further molecular mechanism was demonstrated with in vitro and in vivo studies. RESULTS: The cell atlas unveiled an altered immune landscape across different pathological states of gallbladder diseases. GBC featured a more suppressive immune microenvironment with distinct T-cell proliferation patterns and macrophage attributions in different GBC subtypes. Notably, mutual exclusivity between stromal and immune cells was identified and remarkable stromal ecosystem (SC) heterogeneity during GBC progression was unveiled. Specifically, SC1 demonstrated active interaction between Fibro-iCAF and Endo-Tip cells, correlating with poor prognosis. Moreover, epithelium genetic variations within adenocarcinoma (AC) indicated an evolutionary similarity between adenoma and AC. Importantly, our study identified elevated olfactomedin 4 (OLFM4) in epithelial cells as a central player in GBC progression. OLFM4 was related to T-cell malfunction and tumour-associated macrophage infiltration, leading to a worse prognosis in GBC. Further investigations revealed that OLFM4 upregulated programmed death-ligand 1 (PD-L1) expression through the MAPK-AP1 axis, facilitating tumour cell immune evasion. CONCLUSION: These findings offer a valuable resource for understanding the pathogenesis of gallbladder diseases and indicate OLFM4 as a potential biomarker and therapeutic target for GBC.
Subject(s)
Gallbladder Neoplasms , Single-Cell Analysis , Tumor Microenvironment , Gallbladder Neoplasms/pathology , Gallbladder Neoplasms/immunology , Gallbladder Neoplasms/genetics , Gallbladder Neoplasms/metabolism , Humans , Tumor Microenvironment/immunology , Adenoma/pathology , Adenoma/genetics , Adenoma/immunology , Adenoma/metabolism , Adenocarcinoma/pathology , Adenocarcinoma/genetics , Adenocarcinoma/immunology , Male , Macrophages/immunology , Macrophages/metabolism , B7-H1 Antigen/metabolism , B7-H1 Antigen/genetics , Cholecystitis/pathology , Cholecystitis/metabolism , Gene Expression Profiling/methods , Polyps/pathology , Polyps/genetics , Polyps/immunology , Granulocyte Colony-Stimulating FactorABSTRACT
Anguillid herpesvirus 1 (AngHV), the causative agent of "mucus sloughing and hemorrhagic septicemia disease", causes serious infectious diseases in farmed eel. Among the effective prevention and control strategies, vaccination is one of the most effective approaches. However, no vaccine for AngHV is available. Our study developed a formalin-inactivated AngHV vaccine and evaluated its performance in American eels. Initially, AngHV-FJ, a strain of AngHV, was inactivated completely by 0.1 % formaldehyde, mixed with adjuvant Montanide ISA 763 A VG (763A). Then, vaccines containing different amount of antigen (3 × 106 PFU, 3 × 105 PFU, 3 × 104 PFU, 3 × 103 PFU) were immunized in each American eels. The results showed that the 3 × 105 PFU/fish was the proper dose. The inactivated AngHV vaccine was proven safe for American eels by back intramuscular injection. The results of twice immunization showed that antibody production peaked in the 8th week after the first immunization, and the antibody titer was 1:64,000. Furthermore, the immunized fishes challenged with AngHV (105 PFU/ml immersion) showed a significantly lower incidence rate (33.33 %) than the control group (95.65 %). The survival of the fish in the vaccine group (94.44 %) was significantly higher than the control group (60.87 %). The relative survival rate of the vaccinated group was 85.80 %. Also, vaccine group tissue collected at 7th d post-challenge showed reduced tissue damage and a lower virus load than the control group. The expression of cytokines of IL-1ß, IFN-α, IFN-γ, Mx1, RIG-1, and IRF-3, were significantly lower in the vaccine group than the control group at the 7th and 14th d post-challenge. Overall, the formalin-inactivated AngHV vaccine was safe and had immune protective effects against AngHV infection.
Subject(s)
Anguilla , Fish Diseases , Animals , Vaccines, Inactivated , Formaldehyde/pharmacology , ImmunityABSTRACT
Cirrhosis is a progressive and diffuse liver disease characterized by liver tissue fibrosis and impaired liver function. This condition is brought about by several factors, including chronic hepatitis, hepatic steatosis, alcohol abuse, and other immunological injuries. The pathogenesis of liver cirrhosis is a complex process that involves the interaction of various immune cells and cytokines, which work together to create the hepatic homeostasis imbalance in the liver. Some studies have indicated that alterations in the immune microenvironment of liver cirrhosis are closely linked to the development and prognosis of the disease. The noteworthy function of mesenchymal stem cells and their paracrine secretion lies in their ability to promote the production of cytokines, which in turn enhance the self-repairing capabilities of tissues. The objective of this review is to provide a summary of the alterations in liver homeostasis and to discuss intercellular communication within the organ. Recent research on MSCs is yielding a blueprint for cell typing and biomarker immunoregulation. Hopefully, as MSCs researches continue to progress, novel therapeutic approaches will emerge to address cirrhosis.
Subject(s)
Liver Diseases , Mesenchymal Stem Cells , Humans , Liver Cirrhosis/pathology , Liver Diseases/pathology , Mesenchymal Stem Cells/pathology , Fibrosis , CytokinesABSTRACT
Alpha-fetoprotein (AFP)-secreting hepatocellular carcinoma (HCC), which accounts for ~75% of HCCs, is more aggressive with a worse prognosis than those without AFP production. The mechanism through which the interaction between tumors and the microenvironment leads to distinct phenotypes is not yet clear. Therefore, our study aims to identify the characteristic features and potential treatment targets of AFP-negative HCC (ANHC) and AFP-positive HCC (APHC). We utilized single-cell RNA sequencing to analyze 6 ANHC, 6 APHC, and 4 adjacent normal tissues. Integrated multi-omics analysis together with survival analysis were also performed. Further validation was conducted via cytometry time-of-flight on 30 HCCs and multiplex immunohistochemistry on additional 59 HCCs. Our data showed that the genes related to antigen processing and interferon-γ response were abundant in tumor cells of APHC. Meanwhile, APHC was associated with multifaceted immune distortion, including exhaustion of diverse T cell subpopulations, and the accumulation of tumor-associated macrophages (TAMs). Notably, TAM-SPP1+ was highly enriched in APHC, as was its receptor CD44 on T cells and tumor cells. Targeting the Spp1-Cd44 axis restored T cell function in vitro and significantly reduced tumor burden when treated with either anti-Spp1 or anti-Cd44 antibody alone or in combination with anti-Pd-1 antibody in the mouse model. Furthermore, elevated IL6 and TGF-ß1 signaling contributed to the enrichment of TAM-SPP1+ in APHC. In conclusion, this study uncovered a highly suppressive microenvironment in APHC and highlighted the role of TAM-SPP1+ in regulating the immune microenvironment, thereby revealing the SPP1-CD44 axis as a promising target for achieving a more favorable immune response in APHC treatment.
ABSTRACT
Eels are important aquaculture species for which an increasing number of reference genes are being identified and applied. In this study, five housekeeping genes [RPL7 (ribosomal protein L7), 18 S (18 S ribosomal RNA), EF1A (elongation factor 1α), ACTB (ß-actin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)] were chosen to evaluate their reliability as reference genes for quantitative real-time PCR (qPCR) for the study of Anguilla anguilla. The expression of the selected genes in different eel tissues was determined using qPCR at different growth stages or upon challenge by Anguillid herpesvirus (AngHV), and the expression levels of these genes were then compared and evaluated using the geNorm and NormFinder algorithms. Then, RefFinder was used to comprehensively rank the examined housekeeping genes. Interestingly, the expression of the evaluated housekeeping genes exhibited tissue-dependent and treatment-dependent variations. In different growth periods A. anguilla tissues, the most stable genes were the following: ACTB in mucus; 18 S in skin and kidney; RPL7 in muscle, gill, intestine and brain; EF1A in heart and liver; and GAPDH in spleen. In contrast, in AngHV-challenged A. anguilla tissues, the most stable genes were the following: 18 S in mucus; RPL7 in skin, gill, heart, spleen, kidney and intestine; EF1A in muscle and liver; and ACTB in brain. Further comparison analysis indicated that the expression of RPL7 and EF1A was stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV. Nonetheless, the expression level of GAPDH in eel tissues was lower, and it was unstable in several tissues. These results indicated that the selection of reference genes for qPCR analysis in A. anguilla should be made in accordance with experimental parameters, and both RPL7 and EF1A could be used as reference genes for qPCR study of A. anguilla at different growth stages or upon challenge by AngHV. The reference genes identified in this study could improve the accuracy of qPCR data and facilitate further studies aimed at understanding the biology of eels.
Subject(s)
Anguilla , Genes, Essential , Animals , Real-Time Polymerase Chain Reaction/methods , Genes, Essential/genetics , Anguilla/genetics , Reproducibility of Results , Gene Expression ProfilingABSTRACT
Anguillid herpesvirus 1 (AngHV) is an important viral pathogen affecting eel. This study was designed to investigate the potential molecular mechanisms and immune response elicited at the protein levels in the skin mucus of AngHV-infected Anguilla anguilla. Tandem mass tag (TMT)-labelling proteomics with the liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for performing quantitative identification of the proteins. In addition, the quantitative protein amount was detected by parallel reaction monitoring (PRM) analysis. A total of 3486 proteins were identified, of which 2935 were quantified. When a protein fold change was greater than 1.3 or less than 0.76, it indicated a differentially expressed protein (DEP). Overall, 187 up-regulated proteins and 126 down-regulated proteins were detected, and most of the DEPs were enriched in the CAMs pathway, intestinal immune pathway, herpes simplex virus 1 infection pathway, phagosome pathway and p53 signaling pathway. The results of the DEPs detected by PRM were highly consistent with the results of the TMT-labelled quantitative proteomic analysis. The findings of this study provide an important research basis for further understanding the pathogenesis of AngHV.
Subject(s)
Anguilla , Fish Diseases , Animals , Chromatography, Liquid , Herpesviridae , Mucus , Proteomics , Tandem Mass Spectrometry , Tumor Suppressor Protein p53ABSTRACT
Schizosaccharomyces japonicus have been found as dominant yeast species coexisting with Saccharomyces cerevisiae during late stages of spontaneous fermentation of local grapes in Guizhou, China. Therefore, this study further investigated the impacts of the two indigenous yeast species on typicality of crystal grape (Niagara) wine. Five indigenous and one commercial S. cerevisiae strains were firstly selected based on their genotypes and fermentation traits in synthetic medium. All the six S. cerevisiae exhibited high tolerance to glucose, temperature, and SO2. The two killer active strains FBKL2.996 and FBKL2.9126 showed relative higher tolerance capacity of ethanol, pH and osmotic pressure than the other four S. cerevisiae strains. Further pure fermentation of six strains using crystal grape must exhibited different contents of volatile compounds, with commercial strain CECA producing the highest levels of acetate esters (phenethyl acetate), ethyl esters (ethyl caprylate, ethyl hexanoate), n-caprylic acid isobutyl ester, and terpenes (linalool) whereas being ranked the last in the sensory analysis. The co-inoculation of indigenous S. japonicus with each of the six S. cerevisiae strains increased the acetate esters (mainly isoamyl acetate) by 2-3 times in crystal grape wine. The indigenous S. cerevisiae FBKL2.9128 showed the most obvious variation of volatile compounds between pure and mixed fermentation, exhibiting the significant increase of isoamyl acetate, ethyl decanoate, ethyl caprylate, ethyl hexanoate, methyl decanoate, and dodecanal when co-inoculated with S. japonicus. Sugar addition in immature crystal grape juice increased the ethanol, glycerol, and some volatile compounds such as ethyl butyrate, but decreased the volatile compounds with floral and animal odors. The aroma sensory analysis confirmed the decrease of varietal aroma in wines with sugar addition when comparing with wines made from immature crystal grape. The results of this study provide basic information on the impact of indigenous S. cerevisaie and S. japonicus, and sugar addition on typicality of crystal grape wine, which would help to improve the wine flavor made from crystal grape in southwest China.
Subject(s)
Vitis , Wine , Acetates/analysis , Esters/analysis , Ethanol/analysis , Saccharomyces cerevisiae , Schizosaccharomyces , Sugars/analysis , Vitis/chemistry , Wine/analysisABSTRACT
A novel theranostic co-prodrug SCB has been designed by combining a co-prodrug from CDDO-Me and SAHA with a biotin-coupled near-infrared (NIR) probe hemicyanine via redox-responsive linker thiolactate to enhance the tumor theranostic efficacy and reduce the toxic side effects using both active and passive targeting strategies. SCB displayed reactive oxygen species (ROS)- and glutathione (GSH)-dependent release of NIR fluorescence and two parent drugs. Furthermore, the administration of SCB caused selective illumination of the tumor tissues for >24 h, thereby guiding precise removal of a tumor from intraoperative mice. Importantly, SCB exhibited highly efficient tumor inhibition, exerted selective combination therapy through prodrug mode, and minimized the adverse effects. Finally, SCB induced mitochondrial depolarization, DNA damage, and cell apoptosis through ROS generation and downregulation of HDAC6 protein, as verified by H2AX, Bax, cleaved-PARP, and Mcl-1 proteins. Thus, we suggest that SCB can provide a new platform for both precise diagnosis-guided tumor removal and selective combination therapy with high safety.
Subject(s)
Nanoparticles , Neoplasms , Prodrugs , Animals , Cell Line, Tumor , Drug Therapy, Combination , Glutathione/metabolism , Mice , Neoplasms/diagnosis , Neoplasms/drug therapy , Oleanolic Acid/analogs & derivatives , Oxidation-Reduction , Precision Medicine , Prodrugs/metabolism , Prodrugs/pharmacology , Prodrugs/therapeutic use , Reactive Oxygen Species , Theranostic NanomedicineABSTRACT
BACKGROUND: Objective to systematically evaluate the diagnostic value of long noncoding RNA (lncRNA) in gastric cancer (GC) in the Chinese population. METHODS: PubMed, Web of Science, EMBASE, Cochrane Library, CNKI, and Wanfang Database were searched. According to the search strategy and inclusion and exclusion criteria, 2 staff members screened the relevant kinds of literature from January 2010 to December 2020 and extracted the relevant data. Revman5.3, Meta-Disc1.4, and Stata15.1 software were used to analyze the relationship between lncRNA from exosomes and the diagnosis of GC. The combined values of sensitivity (SEN), specificity (SPE), positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio (DOR) and their corresponding 95% confidence intervals (CIs) were calculated. The summary receiver operating characteristic curve was drawn and the area under the ROC curve (AUC) value was calculated. RESULTS: In 9 studies, 1314 samples were included, including 792 cases in the case group and 522 cases in the control group. The combined SEN was 0.82 (95% CI: 0.77-0.86), the combined SPE was 0.78 (95% CI: 0.72-0.83), the combined positive likelihood ratio was 3.7 (95% CI: 2.9-4.6), the negative likelihood ratio was 0.23 (95% CI: 0.18-0.29), and the DOR was 16 (95% CI: 12-23), AUC was 0.87 (95% CI: 0.84-0.90). Subgroup analysis showed that the SEN, SPE, likelihood ratio, DOR, and AUC of plasma-derived lncRNA in the diagnosis of GC were better than those of serum. CONCLUSIONS: Exosome-derived lncRNA may be a new potential biomarker for the clinical diagnosis of GC.
Subject(s)
Exosomes/genetics , RNA, Long Noncoding/genetics , Stomach Neoplasms/diagnosis , China , Humans , ROC Curve , Sensitivity and Specificity , Stomach Neoplasms/geneticsABSTRACT
Recently, the culture of American eels (Anguilla rostrate) in China has been impacted by emergence of a disease with signs of haemorrhagic gill necrosis. The gills of diseased eels are covered with petecchia and they bleed when the operculum is pressed. In this study, a novel American eel adomavirus (AEAdoV) was isolated from the diseased eels using the eel ovary cell line (EO). The virus proliferated in the EO cells with a maximum TCID50 /ml of 106.29 ± 0.23 at 6 days post-infection. The virions were non-enveloped with a diameter of 75-85 nm and shown to be a DNA virus upon 5-iodo-2'-deoxyuridine (IDU) treatment. PCR assays showed that AEAdoV encodes a superfamily 3 helicases (S3H) replicase and shared high similarities with Anguilla marmorata adomavirus (MEAdoV). Although no clinical signs or mortality was observed among the eels injected with AEAdoV, the virus was reisolated from livers, kidneys and gills of injected eels at 35 days post-injection. Our results suggested that AEAdoV exhibited a latent infection in A. rostrata. The pathogenicity of the AEAdoV needs to be confirmed further.
Subject(s)
Anguilla/virology , DNA Viruses/classification , Fish Diseases/virology , Necrosis/veterinary , Animals , Aquaculture , China , DNA Viruses/isolation & purification , DNA Viruses/pathogenicity , Gills/pathology , Gills/virology , Necrosis/virology , PhylogenyABSTRACT
Anguillid herpesvirus 1 (AngHV) is one of the vital pathogenic agents found in the wild and cultured eel populations, which has brought significant losses to eel culture industry in China. In this study, AngHV ORF95 was characterized. Bioinformatics analysis showed that ORF95 putatively encodes a structural protein that is homologous to hemagglutinin-esterase (HE) protein of infectious salmon anemia virus (ISAV). Temporal transcription and expression analysis indicated that ORF95 is a viral late gene. Subcellular localization analysis revealed that ORF95 was predominantly localized in the cytoplasm. Further, western blot analysis indicated that ORF95 is a structural protein of virion envelope. These results provide a novel basis to make further efforts to clarify the function of ORF95 in the process of AngHV infection and the possibility to use ORF95 as antigen to develop AngHV subunit vaccine.
Subject(s)
Eels/virology , Hemagglutinins, Viral/genetics , Herpesviridae/genetics , Open Reading Frames/genetics , Viral Fusion Proteins/genetics , Animals , Eels/genetics , Fish Diseases/genetics , Fish Diseases/virology , Herpesviridae/isolation & purification , Herpesviridae/pathogenicity , Isavirus/genetics , Virion/genetics , Virion/pathogenicityABSTRACT
Post-polymerization modification (PPM) is one of the most powerful strategy for preparing polymers with functional groups that cannot be synthesized by direct polymerization. So far, numerous experimental efforts have been devoted to the stability issue of monomer structures during the PPM process, but little attention was paid to chemical linkages. However, for hyperbranched polymers, a minor change of linkage unit could lead to a significant influence on the overall stability and performance of polymer materials. In this work, we investigated the chemical stability of long-subchain hyperbranched polystyrenes with ester, aryl ether, and carbon-carbon bonds as branching linkages under a few most popular PPM conditions, including NaOH hydrolysis reaction, TFA-promoted hydrolysis reaction, BBr3-catalyzed methoxy-hydroxyl conversion reaction, and LiAlH4 carbonyl reduction reaction. Related results are summarized into a synthetic route map that can provide practical and intuitive guidance for preparing functional long-subchain hyperbranched polystyrenes and other type of polymers by PPM for future applications.
ABSTRACT
In the present study, a new hepatic tissue-origin cell line from European eel Anguilla anguilla has been developed and characterized. This cell line designated EL has been maintained in Leibovitz L-15 supplemented with 10% fetal bovine serum over 72 months, and subcultured more than 90 times. The EL cell line consisted predominantly of fibroblast-like cells, which could survive over 100 days in vitro, and could grow at 15-32°C. The optimum temperature for growth was 27°C. The chromosome analysis revealed a modal diploid karyotype of 2n = 38. The origin of this cell line was confirmed by the 18S recombinant (r)RNA sequencing. The susceptibility test indicated significant cytopathic effects in the EL cells with regard to the Rana grylio virus and the Herpesvirus anguillae. The viral replication was confirmed by transmission electron microscopy and polymerase chain reaction analysis. Following poly (I:C) exposure, the expression levels of the immune-related molecules interferon regulatory factor-7 (irf7) and transforming growth factor-ß (TGF-ß) were downregulated in EL cells, whereas the expression levels of the rf3 and the cytochrome P450 (CYP450) were upregulated. All four genes were significantly upregulated following inflammation by lipopolysaccharide (LPS). These data suggested the application of EL cell line for viral identification, as well as for immunodiagnosis and pharmacological targeting.
Subject(s)
Anguilla , Cell Culture Techniques , Hepatocytes/cytology , Liver/cytology , Animals , Cell LineABSTRACT
A new cell line derived from the kidney of European eel, Anguilla anguilla, has been established and characterized. This cell line, designated as EK (eel kidney), has been maintained in Leibovitz's L-15 supplemented with 10% fetal bovine serum for over 24â months, and subcultured more than 60 times. This cell line consists predominantly of fibroblast-like cells, and can grow at 15-37°C under an optimum temperature of 26°C. The origin of this cell line was confirmed by polymerase chain reaction (PCR) amplification and 18s recombinant (r)RNA sequencing. The chromosome analysis of EK cells at passage 58 revealed an ananeuploid karyotype. The EK cells were successfully transfected with the Pegfp-N1 plasmid, suggesting its potential in genetic studies. The susceptibility test showed a significant cytopathic effect (CPE) in EK cells for Rana grylio virus, and the viral replication was evidenced with quantitative real-time PCR (qRT-PCR) assay. After poly (I:C) stimulation, the expression of the immune-related molecules including interferon regulatory factor-3 (irf3), interferon regulatory factor-7 (irf7) and cytochrome P450 (CYP450) were significantly upregulated in EK cells, while the expression of transforming growth factor (TGF-ß) was downregulated. These results suggested the potential of EK cell line as a model in gene engineering, virus identification and environmental toxicology.
ABSTRACT
Engineering nanoparticles of reasonable surface poly(ethylene glycol) (PEG) length is important for designing efficient drug delivery systems. Eliminating the disturbance by other nanoproperties, such as size, PEG density, etc., is crucial for systemically investigating the impact of surface PEG length on the biological behavior of nanoparticles. In the present study, nanoparticles with different surface PEG length but similar other nanoproperties were prepared by using poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) copolymers of different molecular weights and incorporating different contents of PCL3500 homopolymer. The molecular weight of PEG block in PEG-PCL was between 3400 and 8000â¯Da, the sizes of nanoparticles were around 100â¯nm, the terminal PEG density was controlled at 0.4 PEG/nm2 (or the frontal PEG density was controlled at 0.16 PEG/nm2). Using these nanoproperties well-designed nanoparticles, we demonstrated PEG length-dependent changes in the biological behaviors of nanoparticles and exhibited nonmonotonic improvements as the PEG molecular weight increased from 3400 to 8000â¯Da. Moreover, under the experimental conditions, we found nanoparticles with a surface PEG length of 13.8â¯nmâ¯(MWâ¯=â¯5000â¯Da) significantly decreased the absorption with serum protein and interaction with macrophages, which led to prolonged blood circulation time, enhanced tumor accumulation and improved antitumor efficacy. The present study will help to establish a relatively precise relationship between surface PEG length and the in vivo behavior of nanoparticles.
Subject(s)
Antineoplastic Agents/administration & dosage , Docetaxel/administration & dosage , Drug Carriers/chemistry , Lactones/chemistry , Nanoparticles/chemistry , Polyethylene Glycols/chemistry , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Docetaxel/pharmacokinetics , Docetaxel/therapeutic use , Drug Carriers/metabolism , Female , Lactones/metabolism , Mice, Inbred BALB C , Mice, Inbred ICR , Mice, Nude , Molecular Weight , Nanoparticles/metabolism , Neoplasms/drug therapy , Polyethylene Glycols/metabolismABSTRACT
Reduction-responsive polymer micelles are highly promising drug carriers with better tumor therapeutic effect, which can be achieved by controlled drug release under stimulation. Gold nanorods (AuNRs) have attracted considerable attention due to their unique optical and electronic properties when used for biomedical applications. Herein, the lipoic-acid-functionalized reduction-responsive amphiphilic copolymer poly(ε-caprolactone)-b-poly[(oligoethylene glycol) acrylate] (LA-PCL-SS-POEGA) with a disulfide group between the two blocks was prepared to modify AuNRs via Au-S bonds. The size and morphology of AuNRs@LA-PCL-SS-POEGA were measured by dynamic laser light scattering (DLS) and transmission electron microscopy (TEM) methods. The stabilities of AuNRs@LA-PCL-SS-POEGA in different types of media were studied by UV/vis spectroscopy and DLS techniques. The results show that AuNRs@LA-PCL-SS-POEGA gradually aggregate in a concentrated salt solution containing 150 mM dithiothreitol (DTT), but exhibit high stability in a non-reducing environment. Near infrared (NIR)-induced heating of AuNRs@LA-PCL-SS-POEGA was investigated in an aqueous solution under NIR laser irradiation (808 nm), revealing that AuNRs@LA-PCL-R-POEGA maintain excellent photothermal conversion efficiency after modification. When compared with non-reduction responsive AuNRs@LA-PCL-CC-POEGA, the in vitro internalization of AuNRs@LA-PCL-SS-POEGA demonstrates that the reduction-responsive polymer could enhance the cellular uptake of nanoparticles measured by inductively coupled plasma mass spectrometry (ICP-MS) and TEM.
ABSTRACT
In this work, we analyze the catalytic mechanism of P4VP-RuII(bda) polyelectrolyte-metal complex (PMC) as a water oxidation catalyst and elucidate how the unique slow diffusion and multi-charge properties of the polyelectrolyte ligand dominate the catalytic process. Four poly(4-vinyl pyridine)-Ru(bda) (P4VP-Ru) PMCs with different chain lengths and controlled Ru loading amounts were prepared and used as catalysts for catalytic water oxidation. These catalysts present excellent catalytic performance with turnover numbers (TON) from â¼1200 to â¼1700 because of the good hydration properties. Surprisingly, the combined catalysis kinetics and kinetic isotope effect (KIE) studies for P4VP-Ru PMCs confirm the single-site water nucleophilic attack (WNA) mechanism in catalysis, rather than the interaction between two metal oxide units (I2M). A combination of dynamic light scattering characterization, zeta-potential measurement and molecular dynamics simulation reveals that the slow diffusion and multi-charge properties of the polyelectrolyte ligand are responsible for the observed mechanism difference between the P4VP-Ru PMC system and small-molecule multi-nuclear system, though the two systems actually own a similar structural feature (flexible linkages between Ru centers). Our experimental and simulation results highlight the fact that though the existence of flexible linkages between Ru centers could provide large conformation entropy for the occurrence of Ru-dimerization in small-molecule and neutral polymer systems, the entropy elasticity could not overcome the electrostatic interaction energy in the PMC system. Clearly, this work unambiguously clarified why both intra-chain and inter-chain Ru-dimerization (I2M) are prohibited for the PMC system from a perspective of macromolecular chemistry and physics.