The culture of suspended Vero cells is facing difficulties such as low cell viability and long doubling time. To investigate the main reasons for the slow growth and low viability of suspended Vero cells, this study conducted transcriptomic analysis of suspended Vero cells (Vero-XF) and adherent Vero cells (Vero-AD) to screen the differentially expressed genes (DEGs) affecting the growth of suspended cells. In addition, epidermal growth factor (EGF) was supplemented to the culture system to improve the growth of Vero-XF. The results showed that compared with the Vero-AD group, the Vero-XF group had 7 376 significant DEGs. Kyoto encyclopedia of genes and genomes enrichment analysis revealed that the DEGs were mainly enriched in the autophagy and mitophagy pathways. Eleven DEGs were selected and verified by quantitative real-time PCR, which showed up-regulated expression of ATG9B, WIPI2, LAMP2, OPTN, Rab7a, and DEPTOR and down-regulated expression of ATG4D, being consistent with the results of transcriptomic analysis. In addition, the Vero-XF group showed significantly up-regulated expression of ATG101, ATG2A, and STX17 and insignificant change in the expression of NBR1, compared with the Vero-AD group. The protein levels of LC3 and P62 in Vero-XF and Vero-AD were determined by Western blotting, which showed up-regulated expression of LC3Ⅱ/Ⅰ and down-regulated expression of P62 in Vero-XF, indicating a higher level of autophagy. Finally, the exogenous supplementation of EGF at 10, 20, and 30 μg/L in the culture system reduced the autophagy level of Vero-XF by 22.35%, 48.15%, and 71.29%, increased the specific growth rate by 15.48%, 33.33%, and 57.14%, and decreased the apoptosis rate by 2.84%, 15.46%, and 16.23%, respectively. The results of this study preliminarily reveal that the activation of autophagy is one of the reasons for the slow growth of Vero-XF, which provides reference for the subsequent culture of suspended Vero cells.
Animals ; Vero Cells ; Autophagy/genetics* ; Chlorocebus aethiops ; Epidermal Growth Factor/pharmacology* ; Gene Expression Profiling ; Transcriptome ; Cell Survival

Gut bacterial nitroreductases play an important role in reduction of various nitroaromatic compounds to the corresponding N-nitroso compounds, hydroxylamines or aromatic amines, most of which are carcinogenic and mutagenic agents. Inhibition of gut nitroreductases has been recognized as an attractive approach for reducing mutagen metabolites in the colon, so as to prevent colon diseases. In this study, the inhibitory effects of 55 herbal medicines against Escherichia coli(E. coli) nitroreductase (EcNfsA) were examined. Compared with other herbal extracts, Syzygium aromaticum extract showed superior inhibitory potency toward EcNfsA mediated nitrofurazone reduction. Then, the inhibitory effects of 22 major constituents in Syzygium aromaticum against EcNfsA were evaluted. Compared with other tested natural compounds, ellagic acid, corilagin, betulinic acid, oleanic acid, ursolic acid, urolithin M5 and isorhamnetin were found with strong to moderate inhibitory effect against EcNfsA, with IC₅₀ values ranging from 0.67 to 28.98 mol·L^-1. Furthermore, the inhibition kinetic analysis and docking simulation demonstrated that ellagic acid and betulinic acid potently inhibited EcNfsA (K_i < 2 μmol·L ^-1) in a competitively inhibitory manner, which created strong interactions with the catalytic triad of EcNfsA. In summary, our findings provide new scientific basis for explaining the anti-mutagenic activity of Syzygium aromaticum, where some newly identified EcNfsA inhibitors can be used for developing novel agents to reduce the toxicity induced by bacterial nitroreductase.
Ellagic Acid/pharmacology* ; Escherichia coli ; Kinetics ; Nitroreductases/pharmacology* ; Plant Extracts/pharmacology* ; Syzygium

Small proteins specifically refer to proteins consisting of less than 100 amino acids translated from small open reading frames (sORFs), which were usually missed in previous genome annotation. The significance of small proteins has been revealed in current years, along with the discovery of their diverse functions. However, systematic annotation of small proteins is still insufficient. SmProt was specially developed to provide valuable information on small proteins for scientific community. Here we present the update of SmProt, which emphasizes reliability of translated sORFs, genetic variants in translated sORFs, disease-specific sORF translation events or sequences, and remarkably increased data volume. More components such as non-ATG translation initiation, function, and new sources are also included. SmProt incorporated 638,958 unique small proteins curated from 3,165,229 primary records, which were computationally predicted from 419 ribosome profiling (Ribo-seq) datasets or collected from literature and other sources from 370 cell lines or tissues in 8 species (Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Danio rerio, Saccharomyces cere-visiae, Caenorhabditis elegans, and Escherichia coli). In addition, small protein families identified from human micro-biomes were also collected. All datasets in SmProt are free to access, and available for browse, search, and bulk downloads at http://bigdata.ibp.ac.cn/SmProt/.

Pancreatic lipase (PL), a crucial enzyme in the digestive system of mammals, has been proven as a therapeutic target to prevent and treat obesity. The purpose of this study is to evaluate and characterize the PL inhibition activities of the major constituents from Fructus Psoraleae (FP), one of the most frequently used Chinese herbs with lipid-lowering activity. To this end, a total of eleven major constituents isolated from Fructus Psoraleae have been obtained and their inhibition potentials against PL have been assayed by a fluorescence-based assay. Among all tested compounds, isobavachalcone, bavachalcone and corylifol A displayed strong inhibition on PL (IC < 10 μmol·L). Inhibition kinetic analyses demonstrated that isobavachalcone, bavachalcone and corylifol A acted as mixed inhibitors against PL-mediated 4-methylumbelliferyl oleate (4-MUO) hydrolysis, with the K values of 1.61, 3.77 and 10.16 μmol·L, respectively. Furthermore, docking simulations indicated that two chalcones (isobavachalcone and bavachalcone) could interact with the key residues located in the catalytic cavity of PL via hydrogen binding and hydrophobic interactions. Collectively, these finding provided solid evidence to support that Fructus Psoraleae contained bioactive compounds with lipid-lowering effects via targeting PL, and also suggested that the chalcones in Fructus Psoraleae could be used as ideal leading compounds to develop novel PL inhibitors.

Aberrant regulation of miRNA genes contributes to pathogenesis of a wide range of human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a RNA/DNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs regulated by TDP-43 using RNA-Seq coupled with an siRNA-mediated knockdown approach. TDP-43 knockdown affected the expression of a number of miRNAs. In addition, TDP-43 down-regulation led to alterations in the patterns of different isoforms of miRNAs (isomiRs) and miRNA arm selection, suggesting a previously unknown role of TDP-43 in miRNA processing. A number of TDP-43 associated miRNAs, and their candidate target genes, are associated with human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p. In contrast, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Reduced expression of miR-500a-3p is associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Cancer-associated genes LIF and PAPPA are possible targets of miR-500a-3p. Our work suggests that TDP-43-regulated miRNAs may play multifaceted roles in the pathogenesis of cancer.
Animals ; Cells, Cultured ; DNA-Binding Proteins ; metabolism ; Electrophoretic Mobility Shift Assay ; Humans ; Immunoprecipitation ; Mice ; MicroRNAs ; genetics ; metabolism ; Neoplasms ; genetics ; metabolism

Objective To investigate the application of intraoperative neuromonitoring (IONM) during thyroidectomy for external branch of superior laryngeal nerve(EBSLN).Methods From Jan.2017 to Jun.2017,138 patients undergoing thyroidectomy were randomly divided into monitor group (n=69) and the control group (n=69).The monitor group were used IONM for EBSLN,while the control group were used conventional area protection.Results The overall incidence of EBSLN injury was 1.4％(1/69) in the monitor group,and the overall incidence of EBSLN injury was 11.6％(8/69) in the control group.There was statistical significance between the two groups.Conclusion The application of IONM in thyroidectomy can exactly identify EBSLN,and reduce the possibility of EBSLN injury remarkably.

BACKGROUND:In the clinical treatment of diabetic retinopathy, the therapeutic effects of different filers on the retina and optic nerve after the removal of the vitreous are lack of comprehensive and detailed reports. OBJECTIVE:To observe the changes of retina and optic nerve in patients with diabetic retinopathy after filing cyclic polydimethylsiloxane. METHODS: Totaly 147 patients with diabetic retinopathy undergoing vitreous removal were enroled, including 77 males and 70 females, aged 35to70 years. The vitreous from 74 patients with diabetic retinopathy was filed with cyclic polydimethylsiloxane (test group). The cyclic polydimethylsiloxane was taken out at 3 months post-operation. The vitreous from another 73 patients with diabetic retinopathy was filed with the whole fluorine propane (control group). The changes of retina, optic nerve, intraocular pressure, and the occurrence of vitreous re-hemorrhage and cataract after operation were observed in these two groups. RESULTS AND CONCLUSION: After 7 days of folow-up, the intraocular pressure of patients in the test group was higher than that in the control group (P < 0.05). After 6 months of folow-up, there was no significant difference in the intraocular pressure between these two groups. After 6 months of folow-up, cyclic polydimethylsiloxane was successfuly removed in the test group, retinal blood vessels became thinning with the presence of white sheath, and the number of optic nerves was higher than the control group (P < 0.05). The incidence of vitreous re-hemorrhage and cataract in the test was higher than the control group (P < 0.05). These results demonstrate that after the removal of the vitreous, cyclic polydimethylsiloxane filing can lead to optic atrophy in patients with diabetic retinopathy.

Objective To investigate the effects of IL-1 β on proliferation and migration of gallbladder cancer cells.Methods The secretion of IL-1 β in tissues of gallbladder cancer, chronic cholecystitis and normal gallbladder as well as in supernatant of gallbladder cancer cell lines (GBC-SD, SGC996) and HIBEpic cells was determined by enzyme-linked immunosorbent assay (ELISA) method.The levels of IL-1 β mRNA in GBC-SD, SGC996 and HIBEpic cells were measured by RT-PCR assay.The effects of exogenous IL-1 β on the proliferation of GBC-SD and SGC996 cells in vitro and in vivo were evaluated using WST-1 assay and xenograft tumor model, respectively.The effects of exogenous IL-1 β on the migration of GBC-SD and SGC996 cells in vitro were measured by Tranwell assay.The levels of Twist protein in GBC-SD and SGC996 ceils were examined by western blot assay after treatment with exogenous IL-1 β.In addition, the proliferation and migration of GBC-SD and SGC996 cells after gene silencing of Twist by Twist-siRNA were also evaluated.Results The level of IL-1β protein in normal gallbladder was low (66.4 ± 35.0)pg/ml,while it was significantly increased in gallbladder cancer and chronic cholecystitis [(616.4 ± 95.7) pg/ml and (422.3 ± 48.9) pg/ml, P ＜ 0.05].The levels of IL-1 βin GBC-SD and SGC996 cell culture medium [(587.4 ± 99.8) pg/ml and (657.2 ± 76.6) pg/ml] were much higher than those in the HIBEpic cells [(38.4± 12.1)pg/ml, P ＜ 0.05].Exogenous IL-1β promoted the proliferation of GBC-SD and SGC996 cells both in vitro and in vivo as well as migration in vitro (P ＜ 0.05).The level of Twist protein was significantly increased after treatment with exogenous IL-1 β.In addition, gene silencing of Twist blocked IL-1 β-induced proliferation and migration of GBC-SD and SGC996 cells.Conclusion IL-1 β promoted proliferation and migration of gallbladder cancer cells via Twist activation.

Objective To establish the quality standard of Radix Toddaliae Asiaticae. Methods Thin layer chromatography ( TLC) and high performance liquid chromatography ( HPLC) were used to identify and determine chloride nitidine and toddalolactone in Radix Toddaliae Asiaticae. The moisture and total ash contents were detected according to the methods recorded in appendix of Chinese Pharmacopeia (2010 edition) . Results Toddalolactone and chloride nitidine were detectable by TLC, the spots were clear and the dissociation was good. The established HPLC method was simple and accurate. The linear ranges of toddalolactone and chloride nitidine in Radix Toddaliae Asiaticae were 2.84~42.6 μg/mL and 25.6~385 μg/mL, and their recovery rates were 99.2 % ( RSD=1.12%) and 100 % ( RSD=0.71%) , respectively. The content of moisture was in the range of 75.8~98.9 mg/g and that of total ash was in the range of 12.4~33.6 mg/g. Conclusion The developed method is specific and accurate, and can provide useful reference for establishing quality standard of Radix Toddaliae Asiaticae.

Objective To explore the value of plasma C-peptide levels in early prediction of type 2 diabetes mellitus with peripheral sensory neuropathy.Methods The vibration perception threshold,pain,temperature sensation,touch-pressure sensation,ankle reflex was detected in 500 eases of type 2 diabetes mellitus,and the patients were divided into 4 groups according to peripheral sensory nerve test results:normal group (159 cases),mildly abnormal group (120 cases),moderately abnormal group (121 cases) and severely abnormal group (100 cases).Fasting and 2-hour postprandial C-peptide levels were determined and analysed with peripheral sensory nerve changes.The receiver-operating characteristic (ROC) curve was used to find the best critical point for diagnosis of diabetic peripheral sensory neuropathy.Results The fasting C-peptide among 4 groups had no significant difference (F =1.632,P ＞0.05).Two-hour postprandial C-peptide from normal group to mildly abnormal group and then moderately abnormal group gradually increased [(1.110 ± 0.526),(1.324 ± 0.490),(1.573 ± 0.716) μ g/L],while 2-hour postprandial C-peptide in severely abnormal group was significantly decreased and lower than that in normal group,and there were significant differences (P＜ 0.05).The max Youden Index was 0.366 when 2-hour postprandial C-peptide was 1.173 μ g/L.Conclusions The fasting C-peptide might be not related to early diabetic peripheral sensory neuropathy,but 2-hour postprandial C-peptide might be closely related to early diabetic peripheral sensory neuropathy.It is helpful to detect the early diabetic peripheral sensory neuropathy if we can take a dynamical observation of 2-hour postprandial C-peptide.