Objective: To develop an approach for simultaneous detection of multi-mycotoxins in fresh fruits, so as to provide technical supports for mycotoxins surveillance in fresh fruits. Methods: Fresh fruits were collected from markets and homogenized. Then, 2 g of fresh fruits were added with 10 mL of 0.1% formic acid ( 99∶1, v/v ) in acetonitrile and wortexed for 10 min. Following extraction with 1 g of sodium chloride and 4 g of anhydrous sodium sulfate, samples were centrifuged and 5 mL of the supernatant was cleaned up with 25 mg C18. Following centrifugation, the supernatant was dried under nitrogen. The residue was dissolved in 300 μL of methanol-acetonitrile mixture solution ( 1∶1, v/v ), and mixed evenly in 700 μL of the distilled water. Samples were then eluted in gradient series of 0.1% formic acid and 5 mmol ammonium formate and methanol-acetonitrile mixture solution ( 1∶1, v/v ). The 15 mycotoxins were determined using liquid chromatography-tandem mass spectrometry ( LC-MS/MS ) with electrospray ion source (ESI+/ESI-) under multiple reaction monitoring. In addition, a matrix-matched standard curve was employed for quantitative analysis. Results: There was a good linear relationship for 15 mycotoxins at concentrations of 0.25 to 10 ng/mL ( R2>0.992 ), the LC-MS/MS method showed the detection limits of 0.1-1.0 μg/kg, the spiked recovery rates of 71.68%-117.50%, and the relative standard deviations ( RSDs ) of 0.01%-13.60%. The detection rate of mycotoxins was 27.09% in 203 fresh fruits sold in markets. Conclusions The optimized LC-MS/MS method can be used for simultaneous determination of multi-mycotoxins in fresh fruits.

Objective : To develop an analytical method of ibotenic acid (IBA) and muscimol (MUS) in wild mushroom by dansyl chloride (DNSCl) derivatization-liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to provide technical support for etiological identification of mushroom poisoning events. Methods : The sample was extracted with hydrochloric acid solution, derived by bimolecular DNSCl, diluted and inorganic salts precipitated with acetonitrile. The extract was separated by a waters XBridgeTM BEH C18 column and measured by LC-MS/MS. Results : The limits of detection for IBA and MUS in wild mushroom were 0.15 mg/kg and 0.1 mg/kg, respectively. Good linear relationship was obtained for IBA and MUS at the range of 0.5-250 mg/kg with the correlation coefficient of 0.997 and 0.999, respectively. The average recoveries at three spiking levels were 84.5%-102.0% with relative standard deviations (RSDs, n=6) of 4.7%-8.6% for IBA. The average recoveries were 88.6%-95.4% with RSDs (n=6) of 4.9%-7.5% for MUS. Conclusion The optimized sample extraction and bimolecular DNSCl derivatization conditions can achieve rapid and accurate analysis of IBA and MUS in wild mushroom poisoning sample.

Objective To test the protective immunity in mice induced by recombinant Schistosoma japonicum Sj14FABP through several adjuvant formulations. Methods The recombinant Schistosoma japonicum Sj14FABP was prepared by expression in E. coli as a GST fusion protein (rSj14/GST) and used to vaccinate outbred Kunming mice by using complete Freund's adjuvant (FCA)/incomplete Freund's adjuvant (FIA), Bacillus Calmette-Guerin (BCG) and the immunostimulating complex (ISCOM) as adjuvant respectively. Results The purified recombinant protein rSj14/GST was immunogenic in mice, and 34.3% and 36.0% worm reduction rates were obtained in outbred Kunming mice immunized intradermally with BCG adjuvant and immunized subcutaneously with ISCOM adjuvant respectively, compared with non-vaccinated control group. However, intramuscularly vaccination with rSj14/GST in FCA/FIA was not protective, although the high level of IgG antibody was induced. Conclusion Both BCG and ISCOM are suitable adjuvants for rSj14/GST.

The gene fragment encoding the egg-shell precursor protein of Schistosoma japonicum was amplified with RT-PCR by using PCR primer designed according to the 423 bp cDNA fragment of the Philippine strain of S.japonicum, the corresponding mDNA fragment of Chinese strain as template and then the 5' and 3' ends of this gene cDNA were amplified with 5' RACE and 3' RACE by using a series of primers designed according to the result of sequencing. Result of sequence analysis showed that this fragment, named as Sj423, contained a complete open reading frame (ORF) of gene encoding the egg-shell precursor protein of S.japonicum.(Chinese strain). As demonstrated by sequencing analysis. No intron could be detected in this gene fragment. This gene was subsequently expressed in E.coli after cloning into the expression vector pET28c(+). The molecular mass of the expressed product of this gene was 20.9 kDa as revealed by SDS-PAGE analysis, and Western blot analysis showed that the recombinant protein expressed could react well with the rabbit antiserum against the worm antigen of S.japonicum;indicating the good antigenicity of this expressed product.

Objective To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum.Methods Total RNA was extracted from adult worms of S.japonicum by Trizol reagent and mRNA was isolated from the total RNA.The ds cDNA was synthesized by reverse transcription using random primer.Directional EcoRⅠ/HindⅢ linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoRⅠand HindⅢ,which resulted in ds cDNA with EcoRⅠand HindⅢ adhering ends.The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector.After packaging in vitro,the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library.Plaque assay and PCR were used to evaluate the library.Seven known objective genes of S.japonicum were screened by PCR to detect the representation of the library.Result Primary library capacity was 4.98?106 pfu,and the titer of amplified library was 3.85?1011 pfu/mL.The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%,in which 95.6% inserted cDNA fragments were longer than 300 bp in length.All the seven known objective genes of S.japonicum were amplified from the library.Conclusion The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

Objective:To investigate the effect of iodine nutrition status and sorts of indexes on homocysteine (HCY) in the second trimester of pregnancy under conditions with normal thyroid function.Methods:481 second-trimester pregnant (13th to 27th week of pregnancy) women with normal thyroid function were selected for the study. Urinary iodine concentration (UIC), thyroid auto-antibody, HCY and biochemical indexes were measured. The HCY levels were compared among subjects with different iodine nutritional status, and factors related to HCY level were analyzed.Results:Patients were stratified into iodine deficiency, iodine adequate, iodine more than adequate and iodine excess groups and the proportion were 57.0% ( n=274), 29.7% ( n=143), 10.8% ( n=52) and 2.5% ( n=12), respectively. The overall median UIC was 134.1 μg/L. There was significant difference in HCY levels between iodine excess group and iodine adequate group(1.83 μmol/L vs. 2.46 μmol/L, P=0.036). Spearman correlation analysis showed that HCY was negatively correlated with iodine excess, free triiodothyronine (FT3), thyroglobulin antibody, and fasting blood glucose( r=-0.101, P=0.026; r=-0.099, P=0.03; r=-0.192, P<0.01; r=-0.099, P=0.03), and was positively correlated with TPOAb ( r=0.177, P<0.01). Multiple linear regression analysis showed that HCY was independently negatively correlated with iodine excess (β=-1.505, P=0.043) and FT3 (β=-0.661, P=0.008). Conclusion:Up to 57% women in the second trimester of pregnancy are with iodine deficiency and normal thyroid function. Moderate iodine excess and elevated FT3 levels are beneficial to the decrease of HCY levels and thus reduce the risk of vascular complications in pregnant women.

Objective To construct a T7 phage display cDNA library from the lung of Microtus fortis for further screening the schistosomiasis-resistence-related genes of Microtus fortis. Methods mRNA was isolated from total RNA extracted from the lungs of Microtus fortis by TRIzol reagent, and was used to synthesize double strain cDNA by the reverse transcription. Then the double strain cDNA was given with EcoRⅠ and Hind Ⅲ adhering ends by ligation with the directional EcoRⅠ/Hind Ⅲ linkers and digestion with EcoRⅠ and Hind Ⅲ. The double strain cDNA fragments longer than 300 bp in length were fractionated by the Mini Column, and ligated into the T7 Select 10-3b vector with EcoRⅠ and Hind Ⅲ adhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library. Results The library constructed here contained 1.5?106 clones and the titer of the amplied library was 1.1?1012 pfu/ml. The PCR identification results of 100 clones picked at random showed that 91% clones were recombinant and 90% of recombinant clones contained cDNA fragments longer than 300 bp in length. Conclusion A T7 phage display cDNA library from the lung of Microtus fortis is successfully constructed.

Objective:To evaluate the effectiveness and safety of ultrasound-guided percutaneous polidocanol sclerotherapy of the thyroglossal duct cysts in children.Methods:A retrospective analysis of 26 children who were treated with ultrasound-guided percutaneous puncture sclerosis for thyroglossal duct cysts in Beijing Children′s Hospital, Capital Medical University from October 2019 to April 2022, the size of the cyst before treatment was recorded, and the cyst volume in accordance with V(ml) =1/6 πabc cyst volume was calculated. The follow-up time was the 1st, 3rd, 6th, and 12th months after sclerotherapy. And the cyst volume and volume reduction ratio at different time points was calculated. According to changes in the cystic volume, the treatment was considered effective if the cyst volume reduction rate was≥50%. Cosmetic grading scores (CGS) were performed pre-treatment and at the last follow-up after sclerotherapy using the WHO grading system. Complications were recorded during the follow-up period.Results:The cyst volume before treatment was 2.67 (3.78)ml, and the cyst volume at the 1st, 3rd, 6th and 12th month after treatment were 0.66(1.83), 0.45(0.87), 0.40(0.70), 0.38 (0.63)ml, respectively, there were significant differences between pre-treatment and each time point after-treatment (all P<0.001); the volume reduction rate at the last follow-up was (81.48±14.57)%. The reduction rate of cyst volume was more than 50% at the last follow-up in 22 children, the treatment efficiency was 84.62% (22/26). The cosmetic grading scores at the last follow-up after sclerotherapy was 1.74(1.50), and it decreased significantly compared with the pre-treatment score 3.85(0)( P<0.001). There was no skin pigmentation, pain in the injection area, local urticaria and blistering after sclerotherapy, no adverse reactions such as cyst bleeding and drunkenness-like reaction, and no serious complications occurred. There were 2 cases of cysts that presented infection with fever, and no serious complications occurred. Conclusions:Ultrasound-guided percutaneous sclerotherapy is a safe and effective minimally invasive treatment for thyroglossal duct cysts in children.

Objective:To study the distribution and antibiotics resistance of the main pathogens of neonatal purulent meningitis in different regions of China.Methods:A retrospective descriptive clinical epidemiological study was conducted in children with neonatal purulent meningitis which admitted to 18 tertiary hospitals in different regions of China between January 2015 to December 2019. The test results of blood and cerebrospinal fluid, and drug sensitivity test results of the main pathogens were collected. The distributions of pathogenic bacteria in children with neonatal purulent meningitis in preterm and term infants, early and late onset infants, in Zhejiang Province and other regions outside Zhejiang Province, and in Wenzhou region and other regions of Zhejiang Province were analyzed. The chi-square test was used for statistical analysis.Results:A total of 210 neonatal purulent meningitis cases were collected. The common pathogens were Escherichia coli ( E. coli)(41.4%(87/210)) and Streptococcus agalactiae ( S. agalactiae)(27.1%(57/210)). The proportion of Gram-negative bacteria in preterm infants (77.6%(45/58)) with neonatal purulent meningitis was higher than that in term infants (47.4%(72/152)), and the difference was statistically significant ( χ2=15.54, P=0.001). There were no significant differences in the constituent ratios of E. coli (36.5%(31/85) vs 44.8%(56/125)) and S. agalactiae (24.7%(21/85) vs 28.8%(36/125)) between early onset and late onset cases (both P>0.05). The most common pathogen was E. coli in different regions, with 46.7%(64/137) in Zhejiang Province and 31.5%(23/73) in other regions outside Zhejiang Province. In Zhejiang Province, S. agalactiae was detected in 49 out of 137 cases (35.8%), which was significantly higher than other regions outside Zhejiang Province (11.0%(8/73)). The proportions of Klebsiella pneumoniae, and coagulase-negative Staphylococcus in other regions outside Zhejiang Province (17.8%(13/73) and 16.4%(12/73)) were both higher than those in Zhejiang Province (2.9%(4/137) and 5.1%(7/137)). The differences were all statistically significant ( χ2=14.82, 12.26 and 7.43, respectively, all P<0.05). The proportion of Gram-positive bacteria in Wenzhou City (60.8%(31/51)) was higher than that in other regions in Zhejiang Province (38.4%(33/86)), and the difference was statistically significant ( χ2=6.46, P=0.011). E. coli was sensitive to meropenem (0/45), and 74.4%(32/43) of them were resistant to ampicillin. E. coli had different degrees of resistance to other common cephalosporins, among which, cefotaxime had the highest resistance rate of 41.8%(23/55), followed by ceftriaxone (32.4%(23/71)). S. agalactiae was sensitive to penicillin, vancomycin and linezolid. Conclusions:The composition ratios of pathogenic bacteria of neonatal purulent meningitis are different in different regions of China. The most common pathogen is E. coli, which is sensitive to meropenem, while it has different degrees of resistance to other common cephalosporins, especially to cefotaxime.

ObjectiveTo investigate the value of serum cell cycle protein-dependent kinase 1 （CDK1） and aurora kinase A （AURKA） in the diagnosis of patients with hepatitis B virus-related hepatocellular carcinoma （HBV-HCC）. MethodsA total of 50 HBV-HCC patients， 50 patients with hepatitis B virus-related liver cirrhosis （HBV-LC）， and 50 chronic hepatitis B （CHB） patients who were hospitalized in Department of Gastroenterology， Gansu Provincial Hospital， from June 2022 to December 2023 were enrolled， and 50 healthy individuals， matched for age and sex， who received physical examination at Physical Examination Center during the same period of time were enrolled as control group. Related data were recorded for all patients， including age， sex， complications， and the results of routine blood test， liver function， and coagulation for the first time after admission. ELISA was used to measure the serum levels of CDK1 and AURKA. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups； the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups and the least significant difference Bonferroni test was used for further comparison between two groups； the chi-square test or the Fisher’s exact test was used for comparison of categorical data between groups. The Spearman correlation analysis was used to investigate the correlation between CDK1 and AURKA， and the receiver operating characteristic （ROC） curve and the area under the ROC curve （AUC） were used to investigate the value of CDK1 and AURKA in the diagnosis of HBV-HCC. ResultsThere were significant differences in liver function parameters between the HBV-HCC patients and the control group （all P<0.05）； there were significant differences between the CHB group and the HBV-HCC group in albumin， Glb， direct bilirubin， aspartate aminotransferase （AST）， gamma-glutamyl transpeptidase （GGT）， and alkaline phosphatase （all P<0.05）； there were significant differences between the HBV-LC group and the HBV-HCC group in Glb， AST， and GGT （all P<0.05）. The HBV-HCC group had significantly higher serum levels of CDK1 and AURKA than the HBV-LC group， the CHB group， and the control group （all P<0.05）. There was a significant positive correlation between CDK1 and AURKA in the overall study population and the HBV-HCC patients （r=0.526 6 and 0.815 2， P<0.001）. With the control group as reference， CDK1 had an AUC of 0.832 3 in the diagnosis of HBV-HCC， with a sensitivity of 92.86% and a specificity of 75%， and AURKA had an AUC of 0.886 6 in the diagnosis of HCC， with a sensitivity of 95.80% and a specificity of 74%. With the CHB group as reference， CDK1 had an AUC of 0.833 3 in the diagnosis of HBV-HCC， with a sensitivity of 93.75% and a specificity of 75%， and AURKA had an AUC of 0.972 7 in the diagnosis of HBV-HCC， with a sensitivity of 95.83% and a specificity of 91.67%. With the HBV-LC group as reference， CDK1 had an AUC of 0.608 5 in the diagnosis of HBV-HCC， with a sensitivity of 66.67% and a specificity of 54.17%， and AURKA had an AUC of 0.762 2 in the diagnosis of HBV-HCC， with a sensitivity of 95.83% and a specificity of 47.92%. ConclusionThe serum levels of CDK1 and AURKA increase with the progression of hepatitis B-associated chronic liver disease， and significant increases in serum CDK1 and AURKA have a certain value in the diagnosis of HBV-HCC.