A method based on direct protein precipitation-ultra performance liquid chromatography was established for the determination of trace 5-fluorouracil in serum. The samples were pretreated with 6% perchloric acid as the precipitant and 5-chlorouracil as the internal standard, and the supernatant separated after vortex mixing and high speed centrifugation was used as the test sample. The separation was carried out with an Acquity HSS T3 column and the mobile phase consisting of water and acetonitrile, and the flow rate was 0. 2 mL/min; the injection volume was 5 μL, and the column temperature was 20 °C. The key parameters of this method such as specificity, accuracy, precision and linearity, were validated. Compared with other methods, this method can complete the analysis with significantly reduced time and cost. The established method is useful for therapeutic drug monitoring of 5-fluorouracil plasma concentration and it has been successfully applied in clinical cases.

Objective To establish a real-time fluorescence quantitative PCR method for detection of the different expression level of FPGS in methotrexate enantiomer-resistant A549 cell lines,and observe FPGS mRNA expression in patients with leukemia.Methods A real-time fluorescence quantitative PCR method for detection FPGS mRNA was established using SYBR Green Ⅰ as fluorescence and β-actin as reference.The method was evaluated by Ct,correlation coefficient,slope,repeatability curve,melting curve and amplification efficiency curve.The expression levels of FPGS gene in methotrexate enantiomer-resistant A549 cell lines and methotrexate resistant leukemia cells in bone marrow were detected by the method.Results The standard curves had a high linear relationship between cycle threshold and template concentration.The correlation coefficients of FPGS and β-actin were 0.996 8 and 0.998 7,and the slopes were -3.595 and -3.740,respectively.The inter-coefficient of variation was from 1.27% to 2.95%.The intra-coefficient of variation was 3.82%.The method was characterized with specific melting curve and similar amplification efficiency(slope was 0.021 7).The relative contents of FPGS mRNA were(3.51 ±0.66),(0.16 ±0.01) and(1.00 ±0.31) in L-(+)-MTX/A549 cells(L),D-(-)-MTX/A549 cells(D)and A549 parent cells,and there was statistically difference among the three groups(F = 64.45 ,P＜ 0.01)Statistical difference was observed between L and D(q =9.29,P＜0.01).After treated with MTX,the expression level of FPGS mRNA was(0.35 ± 0.04) in methotrexate resistant leukemia patients,compared with(1.00 ± 0.44) before treatment.Statistical difference was observed(t = 8.83 ,P＜ 0.01).Conclusions The real-time fluorescence quantitative PCR is suitable for the quantification of FPGS.The expression levels of FPGS in methotrexate resistant leukemia cells in bone marrow and drug resistant cells are different.Two enantiomer forms of methotrexate may play different roles in drug resistance mechanisms.

Objective: To explore the differences of temperament characteristics between children with autism spectrum disorder (ASD) and normal children, and to provide evidence for early detection of ASD children and the development of personalized treatment plans. Methods: In this case-control study, we enrolled 129 ASD children and 129 normal children aged 3-7 years. The Behavioral Style Questionnaire (BSQ) scale was used to assess the temperament. Results: ASD children got higher scores in terms of "activity level", "withdrawal", "adaptability", "emotional nature", "persistence", and "response threshold" temperament dimensionality scores(P<0.05), and lower scores in terms of "rhythmical", "response intensity" temperament dimensionality scores than normal children(P<0.05). However, there was no significant difference in "attention dispersiveness" between ASD group and control group(P>0.05). Among the children in the ASD and control group, the proportion of each temperament type was "easy to raise temperament type" (41.8% vs 62.8%), "partially easy to raise temperament type" (31.8% vs 27.9%), "partially difficult to raise temperament type" (17.1% vs 6.2%), “slow-up-towarm temperament type" (7.7% vs 2.3%) and "difficult to raise temperament type" (1.6% vs 0.8%).Statistical analysis showed that the rate of "easy to raise temperament type" was lower than that in normal children(P<0.05), while the rates of "partially easy to raise temperament type", "partially difficult to raise temperament type", "difficult to raise temperament type", and "slow-up-to-warm temperament type" in ASD children were higher(P<0.05). Conclusion There was significant difference in temperament characteristics between ASD children and normal children. The evaluation of temperament type contributes to early detection of ASD children and provides a reference for their behavioral correction.

OBJECTIVE: To assess the value of chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) for the prenatal diagnosis of chromosomal mosaicisms. METHODS: A total of 775 pregnant women who had visited the Prenatal Diagnosis Center of Yancheng Maternal and Child Health Care Hospital from January 2018 to December 2020 were selected as study subjects. Chromosome karyotyping analysis and CMA were carried out for all women, and FISH was used to validate the suspected mosaicism cases. RESULTS: Among the 775 amniotic fluid samples, karyotyping has identified 13 mosaicism cases, which yielded a detection rate of 1.55%. Respectively, there were 4, 3, 4 and 2 cases for sex chromosome number mosaicisms, abnormal sex chromosome structure mosaicisms, abnormal autosomal number mosaicisms and abnormal autosomal structure mosaicisms. CMA has only detected only 6 of the 13 cases. Among 3 cases verified by FISH, 2 cases were consistent with the karyotyping and CMA results, and clearly showed low proportion mosaicism, and 1 case was consistent with the result of karyotyping but with a normal result by CMA. Eight pregnant women had chosen to terminate the pregnancy (5 with sex chromosome mosaicisms and 3 with autosomal mosaicisms). CONCLUSION For fetuses suspected for chromosomal mosaicisms, CMA, FISH and G-banding karyotyping should be combined to determine the type and proportion of mosaicisms more precisely in order to provide more information for genetic counseling.
Female ; Pregnancy ; Humans ; Mosaicism ; In Situ Hybridization, Fluorescence ; Chromosome Disorders/genetics* ; Prenatal Diagnosis/methods* ; Chromosome Aberrations ; Sex Chromosome Aberrations ; Microarray Analysis/methods* ; Chromosomes

OBJECTIVETo investigate the polymorphisms of the coding gene and the human T cell epitopes of antigen GlnA1, Mpt70, LppX, GroES and LpqH on Mycobacterium tuberculosis complex (MTBC) strains in thirteen provinces of China.

METHODSA total of 173 clinical MTBC isolates from thirteen provinces were selected to test the gene sequences of the five antigens, using PCR and DNA sequencing methods. Sequences were compared and sliced by BioEdit, and the variations of the human and nonhuman T cell epitopes were analyzed. The rates on synonymous mutation (dS), non-synonymous mutation (dN) and dN/dS values were calculated by Mega 6.0 software.

RESULTSAmong the 173 strains, there were two non-synonymous mutations in the non-epitope region of glnA1, one non-synonymous mutations in epitope domain of mpt70, one non-synonymous mutation and one synonymous mutation in the epitope domain of lpqH; while groES showed no mutation. lppX had five non-synonymous mutations and one synonymous mutation in the epitope domain. Nine strains presented higher polymorphism at the same gene locus of position 152 in lppX. And seven of the fifteen epitopes contained in lppX were altered and the dN/dS value of this gene was 0.19.

CONCLUSIONSData from the human T cell epitope domains of MTBC antigens Mpt70, LppX and LpqH contained epitope diversity, indicated that these antigens may have involved in diversifying the selection to evade the host immunity. GlnA1 had the polymorphism in epitope domain, which might have little influence on the immuno-response. While GroES seemed relatively conservative, it could play an important role on identification, diagnosis and the development of potential Mycobacterium tuberculosis vaccine.

Antigens, Bacterial ; genetics ; Bacterial Proteins ; genetics ; China ; Epitopes, T-Lymphocyte ; genetics ; Humans ; Mycobacterium tuberculosis ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Sequence Analysis, DNA