The hapten of Flumequine(FLU) with four carbon atoms spacer arm(FLUABA) was synthesized and coupled to bovine serum albumin(BSA) as immunogen using activated ester method. Balb/c mice were immunized by the artificial immunogen and the splenocytes of immunized mice were fused with Sp2/0 cells to obtain the monoclonal antibody(McAbs). A hybridoma cell line(DB6-E7) secreting anti-flumequine McAbs was obtained by limited dilution method and screened by indirect enzyme-linked immunosorbent assay(ELISA) using heterogenous coating antigen. The results showed that the subtype of the McAb was IgG_1, and the affinity was 8.19×10~8 L/mol. The haptens of FLU, FLUABA and FLUACA, with different space arm, were separately linked to ovalbumin(OVA) for heterologous or homologous coating antigen. The results of indirect ELISA and indirect competitive ELISA(icELISA) indicated that the heterologous coating antigen could improve the sensitivity of ELISA significantly. By heterologous coating antigen(FLU-OVA), the icELISA showed an IC_(50) value of 26.33 μg/L, LOD of 4.0 μg/L, and the workable range of 8-114 μg/L (IC_(20)-IC_(80)). Cross-reactivity studies showed that the McAbs were quiet specific for FLU, no cross-reactivity(<0.1%) was detected between the obtained McAbs and the quinolones compounds or other structural similarity compounds. The developed icELISA for FLU can satisfy the detection criteria of flumequine in animal food-products.

Objective To study the in vitro and in vivo effects of pulsed electromagnetic fields (PEMFs) on osteoclast and osteoblast precursor cells.MethodsTo observe the in vitro effect of PEMFs,femur bone marrow cells of 8 week old female SD rats were collected.According to different treating doses,rats were divided into four treatment groups and one control group.After the treatment,the clones of granuloeyte/maerophage colony forming unit(CFU-GM) and fibroblast colony forming units(CFU-F) were measured respectively.To observe the combined in vitro and in vivo effect of PEMFs,8 week old female SD rats were randomly divided into three groups: 2-70 group,ovariectomization (OVX) group and SHAM group.Rats in the 2-70 group and OVX group were bilateral ovariectomized,while rats in the SHAM group were sham-ovariectomized.12 weeks after ovariectomization,the 2-70 group was exposed to PEMFs while the other groups were left untreated.Then,femur bone marrow cells of the rats were collected.According to the way whether the groups were treated with PEMFs,the cells were divided into six groups: 2-70 with/without treatment,OVX with/without treatment,SHAM with/without treatment.After the treatments,the clones of CFU-GM and CFU-F were measured respectively.Resultsin vitro effect of PEMFs: Compared with the control group,the CFU-GMin the treated groups reduced while the CFU-F increased.PEMFs effect in vitro and in vivo: The CFU-F in treated groupsincreased,whileno.significantdifferencesofCFU-GMwerefoundamongthegroups.Conclusion PEMFs has inhibitory effect on osteoelast precursor cells and enhances the proliferation of osteoblast precursor cells when simply applied in vitro.When PEMFs was applied in combined manner of in vitro and in vivo,it shows that PEMFs enhance the proliferation of osteoblast precursor cells but has no inhibitory effects on osteoelast precursor cells.

To detect furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone ( AMOZ ) in fish sample, an Eu3+ labeling time-resolved fluoroimmunoassay ( TRFIA ) was developed. The effects of experimental conditions including AMOZA-OVA concentration, dilution of antibody, and reaction time on the sensitivity of TRFIA were explored. The results showed that the optimized assay conditions were as follows:the AMOZA-OVA concentration was 0. 25 μg/mL; the antibody was diluted 5í104 folds, and the competitive reaction time was 50 min. Under optimal conditions, the method showed a detection limit of 0. 01 ng/mL, an IC50 of 0. 26 ng/mL and a linear range (IC20-IC80) of 0. 025-2. 83 ng/mL. The recoveries of AMOZ in fish at three spiked levels ranged from 78 . 0% to 86 . 0%, and the relative standard deviations were less than 15%. Good correlation between the ic-TRFIA and high performance liquid chromatography-tandem mass spectrometry was obtained for spiked food samples. The proposed ic-TRFIA method was suited for the determination of AMOZ residue in food samples.

Due to the low molecular weight and simple structure, the production of specific antibodies against acrylamide is unavailable. In this study, a novel hapten was synthesized through the derivatization of acrylamide and 4-mercaptophenylacetic acid. The hapten was then coupled to carrier protein and used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to the acrylamide derivative ( hapten) was obtained. The antibody was labeled with horseradish peroxidase ( HRP) and used to develop a direct competitive enzyme-linked immunosorbent assay ( dc-ELISA) . The dc-ELISA was used to determine the content of acrylamide derivative, and then transferred to the content of acrylamide. The assay showed an IC50 value of 45. 49 μg/L, a limit of detection of 3. 0 μg/L and the linear range of 9. 2-195 μg/L for acrylamide. The recovery of acrylamide from spiked food sample was determined ranging from 83 . 6% to 112 . 7%. Good correlations between the results of dc-ELISA and standard HPLC-MS/MS were obtained. The proposed dc-ELISA is suitable for the determination of acrylamide in food samples.

4-Amino dimethyl phthalate as the hapten was coupled to carrier protein and then used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to dimethyl phthalate ( DMP) was thus obtained, and on the basis of this, an indirect competitive chemiluminescent enzyme-linked immunoassay ( icCLEIA ) was developed. The experimental parameters of icCLEIA were optimized as follows: the concentration of coating antigen was 50 μg/L, the primary antibody concentration was 92. 5 μg/L, the secondary antibody concentration was 1μg/mL, distilled water (pH 6. 0) was used as diluent solution and the competitive reaction time was 40 min. Under the optimal conditions, the icCLEIA exhibited a linear working range from 0. 74μg/L to 30. 32μg/L with the limit of detection of 0. 29μg/L. The cross-reactivity of thirteen structural analogues was lower than 5%. The recovery of DMP from spiked liquor and soy sauce samples ranged from 80 . 2% to 116 . 0% and the average RSD was less than 3 . 6%. The detection results of the spiked liquor and soy sauce samples were consistent with those by standard gas chromatography-mass spectrometry method. The developed icCLEIA method exhibited a practical potential for detecting DMP residue in food samples.

Objective To investigate the correlation of miR-181a and miR-181b with fucosyltransferase FUT1, the functional mechanism was elucidated in a colorectal cancer ( CRC).Methods It collected 32 pairs of tissue samples , 18 males and 14 females in the first affiliated hospital of Dalian Medicinal University, from March 2014 to January 2016.The expression of miR-181a and miR-181b was detected by RT-PCR in CRC tissues , adjacent tissues , serum of colorectal cancer patients and healthy people, and CRC cell lines SW620 and SW480 with differently metastatic ability.The relationship of FUT1 and miR-181a, miR-181b expression were verificated by Pearson's correlation curve.FUT1 was identified the target of miR-181a and miR-181b by Network prediction softwares ( TargetScan Human 7.1, microRNA.org and Starbase v2.0) and luciferase assay.The effects of miR-181a and miR-181b expression on the proliferation, migration, invasion and angiogenesis of SW 480 and SW620 cells were further detected by CCK8, wound healing, transwell and tube foramtion assays.T-test was used for comparison between two independent samples , and one-way anova was used for comparison between multiple samples . Pearson correlation coefficient was used for correlation analysis .Results The levels of miR-181a and 181b in CRC tissues were much lower than in tumor-adjacent tissues (3.12 ±1.88 vs 6.44 ±2.32, t=11.74;3.16 ± 1.77 vs 5.52 ±2.45, t=3.24 ;P<0.05).The levels of miR-181a and 181b in serum of colorectal cancer patients were much lower than in healthy people (1.32 ±0.25,2.57 ±0.48,t=10.26;0.91 ±0.14,1.63 ± 0.29,t=5.19;P<0.05 ) .The levels of miR-181a and miR-181b in SW620, SW480 CRC cells were detected to be much lower than in normal colorectal epithelial cells [(0.65 ±0.10, 0.50 ±0.09) vs 1.0;(0.60 ±0.12,0.42 ±0.03)vs 1.0;t=3.08, P<0.05].FUT1 was highly expressed in CRC tissues and SW620 (t=5.23, P<0.05).Based on the network prediction softwares and luciferase assays , FUT1 was the common target of miR-181a and miR-181b.Over expression of miR-181a or miR-181b inhibited FUT1 level and attenuated the capacity of cell migration , invasion and proliferation in SW 620.Down-regulation of miRNAs in SW480 increased FUT1 expression and promoted the capability of cell migration , invasion and proliferation.Downregulation of the two miRNAs attenuated the capability of cell invasion in SW 480, which was blocked by the reductive FUT1.Conclusion MiR-181a and miR-181b mediated the progression of CRC cells by targeting FUT1.

Objective To investigate the function of JAK2-STAT3 signaling pathway in pancreatic injury and systematic inflammatory response in rats with acute necrotizing pancreatitis ( ANP) . Methods SD rats were randomly divided into the ANP group (n=48), ANP+JAK2 inhibitor Ruxolitinib group (ANP+R group, n=48), ANP+STAT3 inhibitot Stattic group (ANP+S group, n=48), ANP+Ruxolitinib+Stattic group (ANP+R+S group, n=48), and sham operation group (SO group, n=48). 5％ sodium taurocholate injection via retrograde pancreatobiliary duct was used to establish ANP model. Blood samples from abdominal aorta and pancreatic tissue were collected after 3 h, 6 h, 12 h and 18 h after modeling. Serum amylase (AMY) and tumor necrosis factor-α(TNF-α) and interleukin-4 (IL-4) were tested. JAK2 and STAT3 mRNA expression and protein expression of p-JAK2 and p-STAT3 in pancreas were examined by RT qPCR and western blot, respectively. Results AMY, TNF-α and IL-4 in plasma, and JAK2 mRNA, STAT3 mRNA, p-JAK2 protein and p-STAT3 protein at different time points in ANP group were all obviously higher than those in SO group(P<0. 05). Serum AMY, TNF-αand IL-4 in ANP+R group, ANP+S group and ANP+R+S group at different time points were lower than those in ANP group [12 h (5391 ± 1009),(6130 ± 1227),(4818 ± 992)U/L vs (8524 ± 1360) U/L;(147.25 ± 27.85),(156.25 ± 23.17),(127.87 ± 21.39) ng/L vs (187.58 ±20.09)ng/L;(45.89 ±16.95),(50.19 ±15.87),(38.87 ±14.03)ng/L vs (58.85 ±9.34)ng/L] . JAK2 mRNA and p-JAK2 protein,STAT3 mRNA and p-STAT3 protein in ANP+R group and ANP+R+S group at different time points were obviously lower than those in ANP group (12 h 0. 357 ± 0. 091 vs 0. 597 ± 0. 121,1. 115 ± 0. 203 vs 1. 217 ± 0. 213,0. 361 ± 0. 089 vs 0. 489 ± 0. 097,0. 965 ± 0. 189 vs 1. 128 ± 0. 217, 0. 362 ± 0. 092 vs 0. 597 ± 0. 121,1. 107 ± 0. 212 vs 1. 217 ± 0. 213,0. 297 ± 0. 087 vs 0. 489 ± 0. 097,0. 713 ± 0. 184 vs 1. 128 ± 0. 217). STAT3 mRNA and p-STAT3 protein in ANP+S group were obviously lower than those in ANP group(0. 319 ± 0. 107 vs 0. 489 ± 0. 097,0. 849 ± 0. 177 vs 1. 128 ± 0. 217), and the difference was statistically different (P<0.05). Conclusions The activation of JAK2-STAT3 signaling pathway in pancreas may play a key role in the pathogenesis of systematic inflammatory response in ANP.

Objective: To compare the safety between cervical conization (CC) alone and hysterectomy for patients with adenocarcinoma in situ (AIS) of the cervix. Methods: Patients diagnosed with AIS after CC during 2007–2021 were identified by computerized databases at Women’s Hospital of Zhejiang University School of Medicine. A total of 453 AIS patients were divided into 2 groups according to uterus preservation: hysterectomy group (n=300) and CC(s) alone group (n=153). The prevalence of residual disease and disease recurrence was compared between patients treated by CC(s) alone and hysterectomy. The prevalence of residual disease in specimens from women who had a hysterectomy and repeat CC were compared between positive and negative margins of CC. The factors influencing residual disease and disease recurrence were assessed. Results: Among 310 specimens from women who had a hysterectomy or repeat CC, the prevalence of residual disease was 50.6% (45/89) for a positive margin and 2.3% (5/221) for a negative margin (p=0.000). Four patients had recurrence of vaginal intraepithelial neoplasia in those treated by hysterectomy and one had recurrence of cervical squamous intraepithelial neoplasia in those treated by CC(s) alone. The prevalence of recurrence was 0.7% (1/153) for CC(s) alone and 1.3% (4/300) for hysterectomy (p=0.431). Hysterectomy did not influence residual disease or disease recurrence. Conclusion CC is an efficacious and safe option for patients with AIS of the cervix provided the margin is negative.

Quantitative real-time RT-PCR (RT-qPCR) is being widely used in microRNA expression research. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in microRNA RT-qPCR studies. The aim of this study was to identify the most stable reference gene(s) for quantification of microRNA expression analysis in uterine cervical tissues. A microarray was performed on 6 pairs of uterine cervical tissues to identify the candidate reference genes. The stability of candidate reference genes was assessed by RT-qPCR in 23 pairs of uterine cervical tissues. The identified most stable reference genes were further validated in other cohort of 108 clinical uterine cervical samples: (HR-HPV- normal, n = 21; HR-HPV+ normal, n = 19; cervical intraepithelial neoplasia [CIN], n = 47; cancer, n = 21), and the effects of normalizers on the relative quantity of target miR-424 were assessed. In the array experiment, miR-26a, miR-23a, miR-200c, let-7a, and miR-1979 were identified as candidate reference genes for subsequent validation. MiR-23a was identified as the most reliable reference gene followed by miR-191. The use of miR-23a and miR-191 to normalize expression data enabled detection of a significant deregulation of miR-424 between normal, CIN and cancer tissue. Our results suggested that miR-23a and miR-191 are the optimal reference microRNAs that can be used for normalization in profiling studies of cervical tissues; miR-23a is a novel microRNA normalizer.
Cervical Intraepithelial Neoplasia/diagnosis/genetics/*metabolism/pathology ; Cervix Uteri/*metabolism/pathology ; Early Detection of Cancer ; Female ; Gene Expression Profiling/*standards ; Humans ; MicroRNAs/genetics/*metabolism/standards ; Microarray Analysis ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; Uterine Cervical Neoplasms/diagnosis/genetics/*metabolism/pathology

Objective To examine the dose-response relationship between gestational weight gain rate and the neonate birth weight.Methods A total of 18 868 women with singleton gestations who delivered between January 2006 and December 2013 were included in this study.Maternal and neonate details of these women were drawn from the Perinatal Monitoring System database.Gestational weight gain rate was defined as the total weight gain during the last and first prenatal care visits divided by the interval weeks.Both Multiple logistic regression analysis and restricted cubic spline methods were performed.Confounding factors included maternal age,education,pre-pregnancy body mass index (BMI),state of residence,parity,gestational weeks of prenatal care entry,and sex of the neonate.Results The adjusted odds ratio for macrosomia was associated with gestational weight gain rate in lower pre-pregnancy BMI (OR=3.15,95％CI:1.40-7.07),normal (OR=3.64,95％CI:2.84-4.66) or overweight (OR=2.37,95％CI:1.71-3.27).The odds ratios of low birth weight appeared a decrease in those women with lower pre-pregnancy BMI (OR=0.28,95％CI:0.13-0.61) while the normal weight (OR=0.37,95％CI:0.22-0.64) group with gestational weight gain,the rate showed an increase.Association of gestational weight gain rate for macrosomia was found a S-curve in those term delivery women (non-linearity test P＜0.000 1).However,L-curve was observed for low birth weight and gestational weight gain rate in term births (non-linearity test P＜0.000 1).Conclusion A S-curve was seen between gestational weight gain rate and term delivered macrosomia while L-curve was observed among term delivered low birth weight neonates.