The contents of cholesterol in a number of vegetable oils were determined by saponification-gas chromatography ( FID) . There was quite a large possibility that cholesterol peak was seriously interfered by saponification-gas chromatography ( FID) , so it was difficult to set a cholesterol content value to differentiate vegetable oil from waste oil. Solid phase extraction ( SPE) sample pretreatment was chosen and the process conditions were optimized. The optimal conditions were as follows:0. 25 g of oil samples, 20 mL of 0. 6%ethyl ether-hexane ( V/V) as eluent ( get rid of fatty acid glycerides) and 20 mL of 15% ethyl ether-hexane ( V/V) as eluent ( obtain cholesterol) . An obvious cholesterol-content-difference between vegetable oils and waste oils was found by SPE-GC. The detected cholesterol contents in 84 vegetable oil samples were all less than 50 μg/g and the contents of 11 waste oil samples among the 13 waste oils were greater than 50 μg/g. Therefore, cholesterol content in vegetable oil of more than 50 μg/g could be determined as suspected waste oil, not vice versa by SPE-GC. The correlation coefficient R2 was 0. 9999 in 0-760 mg/L concentration range, and the detection limit was 6. 0 μg/g. Relative standard deviations at two concentration levels of 17. 7 and 695 mg/L were 1. 6% and 1. 5% respectively. The recovery was 103%.

Objective To investigate the effects of Plasmodium vivax merozoite surface protein 1(PvMSP1)on differentia-tion,maturation and function of dendritic cells(DC)and the mechanisms of PvMSP1 on the activation of DC via toll like receptors (TLR). Methods DCs were incubated with different doses of PvMSP1(1.0,10.0,100.0μg/ml)in vitro. The changes of CD83, CD86,and HLA-DR on DC were detected by flow cytometry(FCM);the expressions of cytokine IL-10 and IL-12 of DC were mea-sured by ELISA;the expressions of TLR4 and TLR9 mRNA of DC were measured by RT-PCR;the proliferation induction to autol-ogous lymphocytes of DC was measured by MTT. Meanwhile,the untreated DC and LPS inducing DC were as the negative control and positive control,respectively. All the data were analyzed statistically. Results Compared with the untreated DC,the propor-tions of CD83,CD86 and HLA-DR on DC induced by LPS and PvMSP1 increased significantly(all P<0.05);the expressions of IL-10 and IL-12 of DC induced by LPS increased significantly(P<0.01),and those induced by PvMSP1 also increased signifi-cantly(all P<0.05). In the LPS inducing group,the TLR4 mRNA production increased(P<0.05)and the TLR9 mRNA produc-tion had no significantly changes(P>0.05). In the PvMSP1-treated group,the DC TLR4 mRNA production increased(P<0.01) and the TLR9 mRNA production had no significantly changes(P>0.05);DC stimulated the proliferation of autologous lympho-cytes. Conclusion PvMSP1 enhances DC differentiation and maturation,and the mature DC induced by PvMSP1 has the ability of antigen presenting. The route for PvMSP1 inducing DC maturation might be TLR4 pathway rather than TLR9 pathway.

Objective To construct a eukaryotic expression system of IFN-λ,examine the expression of IFN-λand evaluate its bio-functions including anti-proliferation and anti-viral activity.Methods The genes of human IFN-λ1 /2 (hIFN-λ1 /2)were cloned from the mRNA of poly I∶C treated HuH-7 cells.The PCR product was examined with DNA sequencing.The genes of IFN-λ1 /2 were sub-cloned into pcDNA3 vector.The correct insertion of the gene IFN-λ1 /2 was identified with enzyme digestion.The constructed pcDNA3-IFN-λ1 /2 plasmids were transfected into COS-7 cells and IFN-λ1 /2 protein was checked in the supernatant and lysis of transfected cells using Western blotting analysis.The human esophageal carcinoma YES5 and T.Tn cells were treated with the IFN-λ1 /2 from the transfected cells and the proliferation of carcinoma cells were measured with CCK-8 kit.In the treated carcinoma cells,the apoptosis and antivirus related molecules such as caspase-3,ISG15 and MxA was analyzed with Western blotting or Quantitative real time PCR.Results The sequence of hIFN-λ1 /2 fragment matched that of the gene bank and the gene of the cytokines was inserted into pcDNA3 vector correctly.With Western blotting analysis,IFN-λ1 /2 protein was detected in the pcDNA3-IFN-λ1 /2 transfected COS-7 cells.The IFN-λ1 /2 from the transfected COS-7 cells inhibited the growth of YES5 and T.Tn cells, activated apoptosis related caspase-3,and up-regulated the anti-virus gene expression of ISG15 and MxA.Conclusion COS-7 cells can express IFN-λ1 /2 after transfection with pcDNA3-IFN-λ1 /2,suggesting that eukaryotic expression system of IFN-λis established.IFN-λ1 /2 from the system can perform bio-functions,such as proliferation inhibition,apoptosis induction and anti-viral gene up-regulation,which indicates that the system can contribute to further investigations of IFN-λbio-activity and its clinical application.

OBJECTIVE To identify the role of mixed lineage kinase domain like protein(MLKL)in cerebral small vessel disease(CSVD)and explore the underlying mechanism.METHODS Transient bilateral common carotid artery occlusion(tBCCAO)was used to establish a mouse model of CSVD.Immunofluorescence staining and Western blotting were used to observe the expres-sions of RIPK3/MLKL signaling molecules in brain tissues at 7,14 and 28 d after tBCCAO.Open field test,rotarod test,Y-maze and novel object recognition test were used to observe the effect of MLKL knockout on cognitive func-tion after tBCCAO.Blood-brain barrier(BBB)disruption was observed by sodium fluorescein permeability test and the expressions of tight junction proteins.Immunoflu-orescence staining and Western blotting were used to detect the expression of microglia marker Iba-1,astro-cyte marker GFAP,and NLRP3/Caspase-1 signaling mol-ecules in the hippocampus of CSVD mice.ELISA was used to detect the level of inflammatory factors(TNF-α,IL-1β,IL-18)in hippocampus.RESULTS The expres-sions of RIPK3/MLKL signaling molecules increased in cortex and hippocampus after tBCCAO,especially on day 14.The expression of pMLKL mainly increased in neurons,glia cells and endothelial cells in CSVD mice.MLKL knockout improved the cognitive functions such as motor learning,spatial learning and working memory,and object recognition ability in CSVD mice.MLKL knock-out alleviated the leakage of sodium fluorescein and attenuated the down-regulation of tight junction proteins at 1 d and 14 d after tBCCAO.At 14 d after tBCCAO,MLKL knock out inhibited the activations of microglia and astrocytes,attenuated the expressions of NLRP3/cas-pase-1 molecules,and decreased the levels of inflamma-tory factors in the hippocampus of mice.CONCLUSION Genetic inhibition of MLKL exerts protective effects against cognitive impairment by ameliorating BBB dam-age and neuroinflammation in a mouse cerebral small vessel disease model.

Objective To explore the expression of mocro RNA(miR)-142-5p and suppressor of cytokine signaling 1(SOCS1)mRNA in peripheral blood mononuclear cells(PBMC)of mice and clinical patients with ankylosing spondylitis(AS)and their impact on immune function.Methods The mRNA levels of miR-142-5p and SOCS1 in PBMC of 30 patients with AS(Patient group)and 30 healthy controls(Health group)treated in the First People's Hospital of Shangqiu from January 2022 to March 2023 were measured by quantitative real time PCR(qRT-PCR).AS mice models were induced by bovine proteoglycan combined with complete Freund's adjuvant,and these mice were divided into control group,model group,NC group and antagomir group.Normal saline was injected into tail vein in control group and model group,and NC-antagomir and miR-142-5p-antagomir were injected into tail vein in NC group and antagomir group,respectively.After 2 weeks of treatment,the arthritis symptom scores of mice in each group were evaluated.The morphology of ankle joint was evaluated by hematoxylin eosin(HE)staining.The levels of Th1 cytokine interferon-γ(IFN-γ),Th2 cytokine interleukin-4(IL-4),Th17 cytokine interleukin-17(IL-17)and Treg cytokine forkhead box protein P3(FOXP3)in PBMC of mice were detected by ELISA method.The mRNA and protein expression of miR-142-5p,SOCS1,IFN-γ,IL-4,IL-17 and FOXP3 in PBMC and ankle joints were detected by qRT-PCR and Western blot.Results Compared with Health group,the level of miR-142-5p in PBMC of patient group was increased(1.00±0.21 vs 3.03±0.99,t=10.997,P＜0.001),while the level of SOCS1 mRNA was decreased(1.00±0.18 vs 0.41±0.09,t=15.956,P＜0.001).Compared with control group,miR-142-5p level(1.00±0.04 vs 4.00±0.52)and the mRNA and protein levels of IFN-γ and IL-17 in ankle joint tissue of model group were increased,while the mRNA and protein levels of SOCS1,IL-4 and FOXP3 were decreased,with significant differences(t=23.356,31.420,48.056,47.224,38.035,29.007,54.183,28.123,55.155,26.758,45.346,all P＜0.05).The arthritis symptom score was increased(7.83±0.94 vs 0.00±0.00,t=22.212,P＜0.05),and the ankle joint structure was damaged.Serum IFN-γ,IL-17 levels,IFN-γ/IL-4 ratio(0.81±0.08 vs 2.08±0.33)and IL-17/FOXP3 ratio(0.41±0.03 vs 1.27±0.10)were increased,and the differences were statistically significant(t=15.382,35.779,8.934,35.130,all P＜0.05).Compared with NC group,miR-142-5p level(3.89±0.33 vs 1.47±0.10),the mRNA and protein levels of IFN-γ and IL-17 in ankle joint tissue of antagomir group were decreased,while the mRNA and protein levels of SOCS1,IL-4 and FOXP3 were increased,and the differences were statistically significant(t=18.846,22.969,43.454,32.617,23.259,20.881,41.832,11.994,32.977,15.190,35.834,all P＜0.05).The arthritis symptom score was decreased(7.42±1.24 vs 2.75±0.75,t=13.233,P＜0.05),and the shape of the ankle joint of the rats was improved.Serum IFN-γ,IL-17 levels,IFN-γ/IL-4 ratio(1.22±0.11 vs 1.91±0.19)and IL-17/FOXP3 ratio(0.69±0.05 vs 1.23±0.12)were decreased,and the differences were statistically significant(t=8.688,22.972,3.785,22.007,all P＜0.05).Conclusion MiR-142-5p was highly expressed in AS.Down-regulation of miR-142-5p using antagonists may reduce Th1/Th2 ratio and Th17/Treg ratio through up-regulation of SOCS1,there by improving the immune balance of AS mice and inhibiting the progression of AS.

Objective To compare the two kinds of fixation methods of A-V fistula needle in the patients with maintaining hemodialysis (MHD).Methods Ninety MHD patients were divided into the control group and the study group,and the control group received the traditional fixation,and the study group received the bridge fixation.The incidence rates of minimal,minor,major and total bleeding caused by pulling out needles were compared between two groups.Results No differences were found in the incidence rates of minimal,minor,major and total bleeding caused by pulling out needles between two groups (P =0.116,0.488,0.238,respectively),but the number of total bleeding in the study group was significantly lower than that of the control group (P =0.014).Condusions The bridge fixation of the A-V fistula needle in MHD patients can reduce the incidence of bleeding causes by pulling out needles.

Seven novel linear polyketides,talaketides A-G(1-7),were isolated from the rice media cultures of the mangrove sed-iment-derived fungus Talaromyces sp.SCSIO 41027.Among these,talaketides A-E(1-5)represented unprecedented unsaturated lin-ear polyketides with an epoxy ring structure.The structures,including absolute configurations of these compounds,were elucidated through detailed analyses of nuclear magnetic resonance(NMR)and high-resolution mass spectrometry(HR-MS)data,as well as elec-tronic custom distributors(ECD)calculations.In the cytotoxicity screening against prostate cancer cell lines,talaketide E(5)demon-strated a dose-dependent inhibitory effect on prostate cancer PC-3 cell lines,with an IC50 value of 14.44 μmol·L-1.Moreover,com-pound 5 significantly inhibited the cloning formation of PC-3 cell lines and arrested the cell cycle in S-phase,ultimately inducing ap-optosis.These findings indicate that compound 5 may serve as a promising lead compound for the development of a potential treat-ment for prostate cancer.

Background Long-term exposure to free silica particles will lead to fibrosis of lung tissue, and abnormal expression of microRNA (miRNA) may affect the occurrence and process of fibrosis. Objective To observed possible intervention effect of miR-204-3p overexpression adenovirus on silicosis fibrosis induced by silica dust using a silicosis rat model via non-exposed intratracheal instillation. Methods Forty SD rats were randomly divided into four groups: control group, silicosis model group, miRNA-NC group, and miR-204-3p intervention group. Under ether anesthesia, rats in the silicosis model group, miRNA-NC group, and miR-204-3p intervention group were injected with 1 mL (50 mg·mL⁻¹) of free silica dust suspension into the trachea, while the control group was injected with the same volume of normal saline. After 30 d of dust exposure, the miR-204-3p intervention group was injected with rno-mir-204 adenovirus vector to overexpress miR-204-3p, and the miRNA-NC group was given empty virus vector. After 30 d of normal feeding, the animals were sacrificed by chloral hydrate anesthesia, and the lung tissue was taken for subsequent experiments. The relative expression level of miR-204-3p in lung tissue of rats in each group was detected by real-time fluorescence quantitative PCR (RT-qPCR). HE staining, Masson staining, and Sirius red staining were used for pathological observation. Immunohistochemistry was used to detect the expression of Fibronectin and Collagen I in lung tissue of rats in each group. RT-qPCR was used to detect the relative gene expression levels of fibrosis markers Fibronectin, Vimentin, Collagen I, and Collagen III in lung tissue of rats in each group. Western blot was used to detect the protein expression levels of fibrosis markers Fibronectin, Vimentin, Collagen I, and Collagen III in lung tissue of rats in each group. Results The anatomical features of lung tissue in the control group were pink lung tissue with soft texture and smooth surface, while those in the silicosis model were grayish white tissue with hard texture and scars and grayish white silicon nodules on the surface. Compared with the silicosis model group, the color of lung tissue in the miR-204-3p intervention group became ruddy, the surface was smooth, and the texture became soft. The staining results showed that the alveolar wall of the control group was thin, there were a small number of capillaries in the alveoli, and the alveolar structure was clear and complete. In the silicosis model group, the alveolar wall became thicker, the pulmonary septum was partially broken, the alveolar structure was defective, and a large amount of collagen fibers were deposited. The alveolar structure of the miR-204-3p intervention group was relatively clear and there was a small amount of collagen fiber deposition. RT-qPCR results showed that compared with the control group, the relative expression levels of miR-204-3p in lung tissue of the silicosis model group and the miRNA-NC group were decreased (P<0.05), and the relative expression level of miR-204-3p in lung tissue of the miR-204-3p intervention group was increased (P＜0.05). The results of immunohistochemistry showed that compared with the control group, the expression levels of Fibronectin and Collagen I in lung tissue of the silicosis model group were increased (P＜0.05). Compared with the silicosis model group, the relative expression levels of Fibronectin and Collagen I in lung tissue of the rats in the miR-204-3p intervention group were significantly decreased (P<0.05). The results of RT-qPCR and Western blot showed that compared with the control group, the relative protein and gene expression levels of fibrosis factors Fibronectin, Vimentin, Collagen I, and Collagen III in lung tissue of the silicosis model group increased (P＜0.05). Compared with the silicosis model group, the relative gene and protein expression levels of fibrosis factors Fibronectin, Vimentin, Collagen I, and Collagen III in lung tissue of rats in the miR-204-3p intervention group were decreased (P＜0.05). Conclusion Silica dust can cause lung fibrosis in rats, and overexpression of miR-204-3P in vivo can reduce silicosis fibrosis in rats caused by silica dust.

Objective : To investigate the effect of chromosome instability(CIN) associated gene polypeptide N-acetylgalactosaminyltransferase 7(GALNT7) on proliferation and apoptosis of HCT116 colon cancer cells. Methods : The HCT116 cell line withGALNT7knockdown was constructed by lentiviral infection. The correlation betweenGALNT7and CIN was verified by chromosome spread assay. The effect ofGALNT7on cell proliferation was detected by live cell counting, and the effect ofGALNT7on cell cycle distribution was detected by flow cytometry and Western blot. Caspase-3 activity and Western blot assays were used to detect the effect ofGALNT7on apoptosis. Results : HCT116 cells showed a slower proliferation rate upon knocking down ofGALNT7, and exhibited a more scattered karyotype distribution and a phenotype of increased degree of CIN. Inhibition ofGALNT7in HCT116 cells resulted in cell cycle arrest, upregulation of P21 and downregulation of CDK6 protein levels, as well as increased levels of Caspase-3 activity, cleaved PARP1 and PUMA protein expression, and decreased levels of BCL-2 protein expression. Conclusion TheGALNT7gene may promote proliferation and inhibit apoptosis of HCT116 colon cancer cells through the suppression of CIN generation.