Objective To establish a purification,enrichment and test method of tetrodotoxin in blood.Methods Through the investigation of various hydrophilic chromatographic columns,the comparison of extraction effects of different types of solid phase extraction columns and the interference analysis of mixed peaks on qualitative ion pairs,the matrix influence of tetrodotoxin was reduced,and the detection sensitivity and qualitative accuracy were improved.Results Tetrodotoxin is highly polar and easily inhibited by the matrix,while conventional precipitation protein method has low sensitivity and isomer double peaks,and the C18 column is not reserved.After comprehensive comparative analysis,the weak cation exchange column PWC column is finally used for purification and enrichment.Complete elution was achieved using 0.5 mL 10%formic acid and 50%acetonitrile aqueous solution.Seperation was performed on an Atlantis HILIC column,with qualitative ion pairs set at m/z,320.10>162.15 and 284.15.The detection limit of the method was 0.061 ng/mL.Conclusion The established PWC solid-phase extraction-LC/MS detection method demonstrates significant purification efficacy,minimal matrix influence,unobstructed chromatographic peaks,markedly improved detection sensitivity.This approach is operationally simple,and applicable to forensic casework.

Objective To establish a purification,enrichment and test method of tetrodotoxin in blood.Methods Through the investigation of various hydrophilic chromatographic columns,the comparison of extraction effects of different types of solid phase extraction columns and the interference analysis of mixed peaks on qualitative ion pairs,the matrix influence of tetrodotoxin was reduced,and the detection sensitivity and qualitative accuracy were improved.Results Tetrodotoxin is highly polar and easily inhibited by the matrix,while conventional precipitation protein method has low sensitivity and isomer double peaks,and the C18 column is not reserved.After comprehensive comparative analysis,the weak cation exchange column PWC column is finally used for purification and enrichment.Complete elution was achieved using 0.5 mL 10%formic acid and 50%acetonitrile aqueous solution.Seperation was performed on an Atlantis HILIC column,with qualitative ion pairs set at m/z,320.10>162.15 and 284.15.The detection limit of the method was 0.061 ng/mL.Conclusion The established PWC solid-phase extraction-LC/MS detection method demonstrates significant purification efficacy,minimal matrix influence,unobstructed chromatographic peaks,markedly improved detection sensitivity.This approach is operationally simple,and applicable to forensic casework.

ObjectiveThe bioinformatics method was used to screen ferroptosis differential genes （FRGs） closely related to ulcerative colitis （UC）， and animal experiments were conducted to verify whether the mechanism of Xiezhuo Jiedu recipe in treating UC is related to the regulation of ferroptosis. MethodThe differentially expressed genes （DEGs） of colonic mucosa tissue of UC patients were obtained from the GEO database， and the intersection of the genes with ferroptosis genes was used to obtain FRGs. The core FRGs were obtained by cluster analysis， minimum absolute contraction and selection operator （LASSO） regression， and receiver operating characteristic curve （ROC） curve analysis. In animal experiments， the UC mouse model was prepared by making the mouse freely drink 2.5% dextran sodium sulfate （DSS）. Xiezhuo Jiedu recipe and mesalazine were given by gavage for seven days， and the inflammatory infiltration of colonic mucosa was observed by hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of E3 ubiquitin ligase （FBXW7）， zinc finger protein （ZFP36）， solute carrier family 7 member 11 （SLC7A11）， and Toll-like receptor 4 （TLR4） in colon tissue. The protein expression levels of FBXW7， ZFP36， SLC7A11， and TLR4 in colon tissue were detected by Western blot. ResultDataset GSE87466 was screened from the GEO database， and its intersections with the ferroptosis gene were analyzed to obtain 21 FRGs. After cluster analysis， LASSO regression， and ROC analysis， core FRGs （FBXW7， ZFP36， SLC7A11， and TLR4） were obtained. Immunoinfiltration analysis showed significant differences in the expression of initial B cells， M1 macrophages， plasma cells， and M2 macrophages in the colonic mucosa tissue of UC mice， and there was a significant correlation between core FRGs and these immune cells. Further animal experiments showed that the colonic mucosa tissue of mice in the model group was disorganized and infiltrated by a large number of inflammatory cells. The inflammation of the colonic mucosa tissue of mice in each group was relieved to varying degrees after treatment with Xiezhuo Jiedu recipe and mesalazine， while the colonic mucosa tissue of mice in the high-dose group of Xiezhuo Jiedu recipe showed almost no inflammatory changes. Compared with the normal group， the protein and mRNA expressions of FBXW7， ZFP36， SLC7A11， and TLR4 in the model group were significantly increased， and the expression of core FRGs in colonic mucosa tissue of mice in all groups was significantly down-regulated after treatment with Xiezhuo Jiedu recipe and mesalazine. ConclusionFBXW7， ZFP36， SLC7A11， and TLR4 are ferroptosis genes closely related to the pathogenesis of UC， and Xiezhuo Jiedu recipe can significantly alleviate colonic mucosa inflammation in mice by down-regulating core ferroptosis genes.

ObjectiveThe differential expression of microRNAs （miRNAs） between the active stage and the remission stage of ulcerative colitis （UC） was analyzed by bioinformatics method， and the regulatory relationship was constructed by screening the differentially expressed genes （DEGs）. The mechanism of Xizhuo Jiedu recipe in the treatment of UC was speculated and verified by animal experiments. MethodThe miRNAs data set of colonic mucosa tissue of UC patients was obtained from the gene expression database （GEO）， and the most differentially expressed miRNAs were screened by GEO2R， Excel， and other tools as research objects. TargetScan， miRTarbase， miRDB， STRING， TRRUST， and Matescape databases were used to screen key DEGs， predict downstream transcription factors （TFs）， gene ontology （GO）， and conduct Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. The key signaling pathways were selected for animal experiments. In animal experiments， the UC mouse model was prepared by making the mouse freely drink 2.5% dextran sodium sulfate （DSS）. Xiezhu Jiedu recipe and mesalazine were given by gavage for seven days， and the inflammatory infiltration of colonic mucosa was observed by hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of miR-155-5p in colon tissue. Immunohistochemistry and Western blot were used to detect the protein expression levels of cytokine signal transduction inhibitor （SOCS1）， phosphorylated transcriptional signal transductor and activator 3 （p-STAT3）， phosphorylated Janus kinase 2 （p-JAK2）， and retinoic acid-associated orphan receptor-γt （ROR-γt）. The expression levels of transforming growth factor-β （TGF-β）， interleukin-17 （IL-17）， interleukin-6 （IL-6）， and interleukin-10 （IL-10） in serum were detected by enzyme linked immunosorbent assay （ELISA）. ResultThe GSE48957 dataset was screened from the GEO database， and miR-155-5p was selected as the research object from the samples in the active and remission stages. 131 DEGs were screened. The GO/KEGG enrichment analysis was closely related to biological processes such as positive regulation of miRNA transcription and protein phosphorylation， as well as signaling pathways such as stem cell signaling pathway， IL-17 signaling pathway， and helper T cell 17 （Th17） cell differentiation. The Matescape database was used to screen out 10 key DEGs， among which SOCS1 was one of the key DEGs of miR-155-5p. Further screening of the TFS of key DEGs revealed that STAT3 was one of the main TFs of SOCS1. The results of animal experiments showed that Xiezhu Jiedu Recipe could effectively down-regulate the mRNA expression of miR-155-5p and protein expression of p-STAT3， p-JAK2， and ROR-γt in colon tissue of UC mice and the expression of IL-17 and IL-6 in serum of UC mice， up-regulate the protein expression of SOCS1 and the expression of TGF-β and IL-10， increase the level of anti-inflammatory factors， and reduce inflammatory cell infiltration. ConclusionIt is speculated that Xizhuo Jiedu recipe may interfere with SOCS1 by regulating the expression of miR-155-5p in UC mice， inhibit the phosphorylation of STAT3， inhibit the differentiation of CD4⁺ T cells into Th17 cells， reduce the levels of pro-inflammatory factors （IL-17 and IL-6）， and increase the levels of anti-inflammatory factors （TGF-β and IL-10）. As a result， the inflammation of colon mucosa in UC mice was alleviated.

Objective:The aim of the current study is to understand the sleep disorders in children with autism and related factors.Methods:A case-control study method was conducted in this study which included 198 children autism who met the criteria of the Diagnostic and Statistical Manual of Mental Disorders,Fourth Edition (DSM-Ⅳ) and 233 cases of gender-and age-matched normal children.Their general demographic and sleep data were collected and the children with autism were assessed with Autism Behavior Checklist,Child Behavior Checklist and Autism Spectrum Screening Questionnaire.Correlation analysis and regression analysis were performed to compare sleep quality,sleep problems and the related factors of the two groups.Results:The scores of difficulty falling asleep,sleep restlessness,taking hold or shaking,restless sleep,interrupted sleep,nightmares,sleepwalking,night terrors,sleep snoring,nocturnal enuresis,sleep apnea,mouth breathing,sweating and sleep scores were higher in the autism group compared to the normal control group before falling asleep (P ＜ 0.05),while the proportion of daytime sleep time in the autism group was lower than the normal control group (P ＜0.05).With sleep status as dependent variable,linear regression analysis was conducted with children's birth weight,walking month age,speaking month age,father age,and autism spectrum as independent variables.The multiple linear regression analysis showed the scores of,sensory behaviors,body and object use behaviors,and self-help behaviors in Autism Behavior Checklist for autistic children were positively associated with their sleep (β =0.21,0.21,0.29,P ＜ 0.05).However,paternal age at birth in the autism group had negative associatin with the sleep of autistic children (β =-0.16,P ＜0.05).In the autism group,their total scores of Autism Behavior Checklist and the scores of language and social behavior in Autism Behavior Checklist were not associated with their sleep status score (P ＞ 0.05).Conclusion:It suggests that children with autism may have more sleep problems,and their sleep problems are related to autism symptoms including the scores of,sensory behaviors,body and object use behaviors,and self-help behaviors in Autism Behavior Checklist for autistic and their paternal age at birth.

Objective To observe the incidence of acute radiation pneumonitis (ARP) in patients with nonsmall cell lung cancer treated by three-dimensional conformation radiotherapy or intersity-modulated radiation therapy,and investigate the predictive value of high dose volume and low dose volume of the total and ipsilateral lung on ARP.Methods 174 patients with non-small-cell lung cancer verified by pathology who received radical radiotherapy in our hospital from Aug 2013 to Nov 2015 were screened.Radioactive dose was 60 Gy-70 Gy,with 97 patients' dose of 60-64 Gy and 77 patients' dose of 65-70 Gy.Chemotherapy was done by paclitaxel + pentium first line treatment.155 patients were treated with radiotherapy alone and 19 with radiotherapy combined with chemotherapy.The V5～ V30,MLD (mean lung dose),V5ipis ～ V30ipsi,MLDipsi were calculated from the dose volume histogram system.The ARP was evaluated according to Common Toxicity Criteria 4.0 (CTC4.0).Univafiate and nultivariate analysis and receiver operating characteristic (ROC) curves were performed to assess the correlated factors of ARP.Results Of 174 patients,26 patients (14.94％) developed radioactive pneumonia at grade 2,11 patients (6.32％) at grade 3,2 patients (1.15％) at grade 4.The correlative factors included the radiochemotherapy regimen (sequential chemotherapy to radiotherapy or concurrent radiochemotherapy),V5,V10,V20,V30,MLD of the total lung and ipsilateral lung according to univariate analysis.Multivariate analysis of all the factors mentioned above showed the V5,V20ipsi and MLD were the independent predictive factors associated with ARP.ROC analysis indicated that the cut-off point of V5,V20ipsi,MLD were 62.38％,47.14％and 16.85Gy respectively.According to the V5,V20ipsi value,174 patients were divided into three groups:B group V5＜62％+V20ipsi≥47％,C group V5≥62％+V20ipsi＜47％,D group V5≥62％+V20ipsi≥47％.The chisquare test between the C group and D group has statistical significance.Conclusion V5,V20ipsi,and MLD are the independent risk factors of ARP.Compared with V5,limiting V20ipsi threshold is more likely to result in ARP.

OBJECTIVE: To observe the expression of Fas receptor and ligand in human laryngocarcinoma cell line, Hep-2 and to investigate the possible mechanism of immune escape through Fas/FasL pathway in Hep-2 cell. METHOD: The mRNA and protein expressions of Fas and FasL in Hep-2 cell were analyzed by RT-PCR and flow cytometry (FCM). Growth curve of Jurkat cell was drawer based on the results of MMT, and apoptosis of Jurkat cell were determined by FCM and Hoechst 33342 staining after coculturing with Hep-2 cell. RESULT: The expressions of Fas and FasL in Hep-2 cell line were evaluated by flow cytometry and the mean fluorescence intensity were (32.91 +/- 5.6) and (25.57 +/- 7.1) respectively. After coincubation with Hep-2 cell (1 X 10(9)/L), the apoptosis rates of Jurkat cells were (38.95 +/- 0.11) % and (13.28 +/- 0.14) %, with planting concentration at 1 x 10(8)/L and 5 x 10 (8)/L respectively. In contrast, the apoptosis rate of Jurkat cultured separately was (7.53 +/- 0.17)%. The proliferation of Jurkat cell was obviously inhibited after coculture. However, the apoptosis rate was significantly decreased after adding neutralizing antibody of FasL. CONCLUSION Laryngocarcinoma cell could induce apoptosis of T lymphocytes through Fas-FasL system, thus it provided a potential mechanism to escape from immune surveillance of host.
Apoptosis ; Cell Line ; immunology ; Cell Line, Tumor ; Fas Ligand Protein ; metabolism ; Flow Cytometry ; Humans ; T-Lymphocytes ; immunology ; Tumor Escape ; fas Receptor ; metabolism