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OBJECTIVE@#Antimicrobial resistance (AMR) has become a global concern and is especially severe in China. To effectively and reliably provide AMR data, we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology, and screened multiple AMR genes in broiler fecal samples.@*METHODS@#A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection. A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.@*RESULTS@#The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction. The sensitivity rate, specificity rate, positive predictive value, negative predictive value and correct indices were 99.30%, 98.08%, 95.31%, 99.79%, and 0.9755, respectively. Utilizing this assay, we demonstrate that AMR genes are widely spread, with positive detection rates ranging from 0 to 97.07% in 273 broiler fecal samples. blaCTX-M, blaTEM, mcr-1, fexA, cfr, optrA, and intI1 showed over 80% prevalence. The dissemination of AMR genes was distinct between the two farms.@*CONCLUSION@#We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples. The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.
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Objective Previous studies suggested that overerpression of Slit2 results in abnormal Alzheimer's disease-like behavior and cognition impairment in mice. The aim of this study is to investigate the relationship between overerpression of Slit2 and accumulation and clearance of amyloid-β in aging mice by comparing the differential expression of genes for accumulation and clearance of amyloid-β in aging Tg-Slit2 and Tg-2576 mice. Methods 14-month old male C57BL/6, Tg-Slit2 and Tg-2576 mice were used to detect the expression of Aβ1 - 40 and Aβ1 - 42 in brain by immunohistochemistry. Further, the total RNA in the brain of these mice were extracted, identified and inversely transcripted to cDNA, then the cDNA was detected by PCR array. The expression of genes in the brain of Tg-Slit2 and Tg-2576 mice were analyzed. Results Comparing with the Tg-2576 mice in the same age, accumulation of Aβ was not found in the brain of Tg-Slit2 and C57BL/6 mice. The result from PCR array analysis showed that comparing with the same aged C57BL/6 mice, there were 16 up-regulated genes and 8 down-regulated genes in the brain of Tg-Slit2 mice and 14 up-regulated genes and 17 down-regulated genes in the brain of Tg-Slit2 mice. The expression of amyloid beta precursor protein (APP) in the brain of the three group mice was not changed. The expression of presenilin 2 ( Psen2) related with Aβ production was significantly up-regulated in the Tg-2576 mice. In addition, the expression of low density lipoprotein receptor-related protein ( LRP) 6 and 9 were markedly decreased in the Tg-2576 mice. Notably, these genes were not changed in the brain of the aging Tg-Slit2 mice. Conclusions The accumulation of Aβ in the brain are not found in 14-month Tg-Slit2 mice, In addition, different from Tg-2576 mice, the significant changes of expression of Aβ-related genes is not found in the brain of Tg-Slit2 mice.
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Objective To develop a PCR-array method for detecting common purulent meningitis pathogens including Streptococcus pneumoniae,Escherichia coli,Haemophilus influenzae type B,Neisseria meningitidis,S.agalactiae and Listeria monocytogenes in children.Methods The amplification efficiency,limit of detection (LOD) and cross-reactivity were validated with individually real-time PCR using genomic DNA of the six pathogenic bacteria.The sensitivity and specificity of the PCR-array method were evaluated using artificial cerebrospinal fluid(CSF),and the consistency between the PCR-array method and the golden method of CSF culture was evaluated using clinical samples.Results The primers and probes of the pathogens in PCR-array had high specificity,and there was no cross reaction between them.The LOD of the PCR-array method was 10 cfu/ml and very sensitive.The sensitivity and specificity of the PCR-array method could reach 95％ in the evaluation of artificial CSF,and had a good consistency with the clinical gold standard method.Conclusion The PCR-array method with high sensitivity and specificity can simultaneously detect six common pathogens in children with purulent meningitis at 2.5 h,which could provide reference for the diagnosis of purulent meningitis.
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The Wnt signaling pathway and its key component β-catenin have critical roles in the development of diseases such as tumors in mammals. However, little has been reported about involvement of the Wnt/β-catenin signaling pathway in canine mammary tumors (CMTs). The present study detected expression of 30 Wnt signaling pathway-related genes in CMTs; the results are potentially useful for molecular-based diagnosis of CMTs and the development of new targeted therapies. Significant upregulations of dickkopf-1 protein, secreted frizzled-related sequence protein 1 (SFRP1), frizzled 3, β-catenin, and lymphoid enhancer-binding factor 1 (LEF1) were detected in highly malignant CMTs compared to levels in normal mammary gland tissues; moreover, highly significant upregulation of WNT5A was observed in low malignancy CMTs. Downregulation was only detected for SFRP4 in malignant CMT samples. The subcellular location of β-catenin and cyclin D1 in 100 CMT samples was investigated via immunohistochemical analysis, and significantly increased expressions of β-catenin in cytoplasm and cyclin D1 in nuclei were revealed. Western blotting analysis revealed that the expression of β-catenin and LEF1 increased in in the majority of CMT samples. Taken together, the results provide important evidence of the activation status of the Wnt pathway in CMTs and valuable clues to identifying biomarkers for molecular-based diagnosis of CMT.
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beta Catenin , Biomarkers , Blotting, Western , Cyclin D1 , Cytoplasm , Diagnosis , Down-Regulation , Immunochemistry , Lymphoid Enhancer-Binding Factor 1 , Mammals , Mammary Glands, Human , Polymerase Chain Reaction , Up-Regulation , Wnt Signaling PathwayABSTRACT
Objective:To treat the chronic non-atrophic gastritis patients induced by Helicobacter pylori with Qingweizhitong Weiwan combined with standard triple therapy,and to detect the differential expression of related immflammation genes with PCR array,and to clarify its mechanism.Methods: Ten patients with chronic non-atrophic gastritis complicated with Helicobacter pylori infection were used as treatment group and 10 health people were used as health control group. The patients in treatment group were treated with Qingweizhitong Weiwan combined with standard triple therapy for 14 d. The blood samples of the subjects in treatment group and health control group were collected before and after treatment,and QIAGEN human antibacterial response PCR array was performed to test the total RNA inperipheral blood and to analyze the differential expressions of 84 inflammation-related genes.Results:The differential expressions of 20 inflammation-related genes were found.Compared with health control group,the expression levels of 20 genes in treatment group before treatment were up-regulated (Fold-change>2);after treatment,the expression levels of 20 genes were down-regulated,and 11 of them were similar to the level in health control group (Fold-change< 2).More specifically,part of 20 genes was related to NLRP3 inflammasome.Compared with health control group,the gene expression levels of CASP1,IL1B,NLRP3,and PYCARD in treatment group before treatment were up-regulated (P <0.05).Compared with before treatment,the expression levels of CASP1,IL1B,NLRP3,and PYCARD in treatment group after treatment were down-regulated (P <0.05).Conclusion:The mechanism of Qingweizhitong Weiwan combined with standard triple therapy in the treatment of chronic non-atrophic gastritis patients induced by Helicobacter pylori may be related to inhibiting the expressions of NLRP3 inflammasome-related genes and interfering the antimicrobial innate immune response.
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Background and purpose:Bladder cancer is the most common urological tumor, and its pathogen-esis is still not fully understood. The study was aimed to observe the expressions of key genes in many tumor-associated signaling pathways in normal bladder tissue and bladder carcinoma, and to provide further evidence for the subsequent study of bladder cancer recurrence and metastasis.Methods:Twenty-seven cases of bladder cancer specimens were col-lected, and normal bladder tissues and bladder cancer tissues were distinguished by frozen section. Then, the expressions of 84 genes of cancer-related signaling pathways in bladder cancer tissues and normal bladder tissues were screened by Cancer Pathway Finder PCR Array produced by QIAGEN company.Results:Compared with the normal bladder tissues, the bladder carcinoma tissues had 8 up-regulated genes and 19 down-regulated genes. In this study, the impact of epithe-lial-mesenchymal transition (EMT) signaling pathway was selected as a research direction in which theGSC,KRT14,DSP were up-regulated,SNAI2,SNAI3 were down-regulated. ThereforeGSC,KRT14,DSP,SNAI2 andSNAI3 were chosen as target genes, and verified by qRT-PCR in many examples. The result showed that the expressions ofGSC gene in bladder cancer tissues were up-regulated, but with no statistical significance;KRT14,DSP expressions in bladder cancer were higher than those in normal bladder tissues (P<0.05);SNAI2,SNAI3 expressions in bladder cancer were lower than those in normal bladder tissues (P<0.05), andSNAI3 showed the most obvious expression differences.Conclusion:KRT14,DSP andSNAI3 may play an important role in bladder cancer’s occurrence, development and metastasis.
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Objective To analyze the effect of high and low dose radiation on the expressions of Th1,Th2 and Th3 /Tr1 related-genes in mice thymocytes and investigate the possible underlying molecular mechanism.Methods ICR mice were randomly divided into low-dose group (0.075 Gy),high-dose group (2.0 Gy) and sham-control group.The mouse thymus tissue was extracted at 16 hours after irradiation and the expressions of Th1-Th2-Th3 related genes were measured by PCR array.Results Eight genes were up-regulated and five genes were down-regulated after low dose radiation (0.075 Gy);while 54 genes were up-regulated and three genes were down-regulated after high dose (2.0 Gy) radiation.These genes included Th1 cell related genes,Th2 cell related genes,Th3/Tr1 cell related genes,Th1/Th2 immune response genes and transcription factor related genes.Low dose radiation induced up-regulation of Stat4 and Socs1 of genes related to the Th1 cells,and it induced down-regulation of IL-4ra,Cebpb,Gata3 and Tgfb3 associated with Th2 and Th3 cells,which lead to Sftpd genes up-regulation of Th1 immune response eventually.The high dose radiation up-regulated all of Th1,Th2 and Th3/Tr related genes and also enhanced the expressions of Cd86,IL-18,IL-10 and Irf4 genes related to Th2 immune response,but it did not alter the gene expression of Th1 immune response.Conclusions Low-dose radiation induces Th1-type immune response,while high doses radiation triggers Th2 type immune response.
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Objective To observe gene different expression of unfolded protein response signaling pathway in human osteoblasts under the excessive fluoride ,and explore the role of endoplasmic reticulum stress in fluorosis .Methods Human osteoblasts were cultured with fluoride ,intervening for 24 h .Cell viability and apoptosis were inspected by MTS assay and flow cytometer respective‐ly .The UPR signaling pathway was examined by real time PCR array ,and protein expressions were detected by Western blot .Re‐sults T he cell survival rates w ere (100 .678 5 ± 2 .830 3 )% ,(105 .393 4 ± 2 .538 4 )% ,(106 .125 7 ± 2 .048 3 )% ,(77 .977 3 ± 2 .544 3)% (P<0 .05) ,(30 .237 7 ± 0 .632 73)% (P<0 .05) treated with sodium fluoride at the concentration 0 ,5 ,10 ,20 ,40 ,80 mg/L respectively .Apoptosis rate inspected by flow cytometer was 4 .8% in 5 mg/L group ,13 .8% in 10 mg/L group ,37 .0% in 20 mg/L group ,58 .9% in 40 mg/L group ,63 .2% in 80 mg/L group (P<0 .05) .Only 1 gene was down regulated and 14 genes were up regulated .Western blot analysis showed BIP ,ATF4 ,CHOP and IRE1 both showed their protein expression gradually up regula‐ted with fluorine dose .XBP1 expression gradually increased in NaF 5-20 mg/L ,and its expression decreased at 40 and 80 mg/L . Conclusion Sodium fluoride can cause osteoblasts endoplasmic reticulum stress pathway through PTEN and IRE1 pathway ,and at high concentrations can cause apoptosis of osteoblast .
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BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in response to viral infection. The aim of this study was to explore the function and mechanism of MAPK signaling pathway in enterovirus 71 (EV71) infection of human rhabdomyosarcoma (RD) cells. METHODS: Apoptosis of RD cells was observed using annexin V-FITC/PI binding assay under a fluorescence microscope. Cellular RNA was extracted and transcribed to cDNA. The expressions of 56 genes of MAPK signaling pathway in EV71-infected RD cells at 8 h and 20 h after infection were analyzed by PCR array. The levels of IL-2, IL-4, IL-10, and TNF-α in the supernatant of RD cells infected with EV71 at different time points were measured by ELISA. RESULTS: The viability of RD cells decreased obviously within 48 h after EV71 infection. Compared with the control group, EV71 infection resulted in the significantly enhanced releases of IL-2, IL-4, IL-10 and TNF-α from infected RD cells (p < 0.05). At 8 h after infection, the expressions of c-Jun, c-Fos, IFN-i, MEKK1, MLK3 and NIK genes in EV71-infected RD cells were up-regulated by 2.08-6.12-fold, whereas other 19 genes (e.g. AKT1, AKT2, E2F1, IKK and NF-κB1) exhibited down-regulation. However, at 20 h after infection, those MAPK signaling molecules including MEKK1, ASK1, MLK2, MLK3, NIK, MEK1, MEK2, MEK4, MEK7, ERK1, JNK1 and JNK2 were up-regulated. In addition, the expressions of AKT2, ELK1, c-Jun, c-Fos, NF-κB p65, PI3K and STAT1 were also increased. CONCLUSION: EV71 infection induces the differential gene expressions of MAPK signaling pathway such as ERK, JNK and PI3K/AKT in RD cells, which may be associated with the secretions of inflammatory cytokines and host cell apoptosis.
Subject(s)
Humans , Enterovirus A, Human/genetics , Mitogen-Activated Protein Kinases/genetics , Rhabdomyosarcoma/virology , Cytokines/genetics , Enzyme-Linked Immunosorbent Assay , Enterovirus A, Human/enzymology , Enterovirus A, Human/physiology , Gene Expression Regulation, Neoplastic , Mitogen-Activated Protein Kinases/physiology , Polymerase Chain Reaction , Rhabdomyosarcoma/enzymology , Rhabdomyosarcoma/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Time Factors , Tumor Cells, Cultured , Up-Regulation , Virus ReplicationABSTRACT
Objective To explore the different apoptotic gene expressions and apoptotic signaling transduction of human rhabdomyosarcoma (RD) cells infected by enterovirus 71 (EV71) in different stage.Methods The survival of EV71-infected RD cells was observed by trypan blue staining.The apoptotic morphology and rates of RD cells were surveyed and detected by Annexin V-FITC/PI staining and flow cytometry,respectively.PCR array was employed to analyze 88 apoptotic gene expressions from EV71-infected RD cells at 8 h and 20 h postinfection (p.i),respectively.Results After RD cells were infected with EV71 (MOI =5) at 8 h p.i,the viability was significantly decreased.Flow cytometry data demonstrated that the apoptotic rates of EV71-infected RD cells had increased to 18.0％ and 19.1％ at 20 h p.i in early and later stage respectively.RT-PCR array studies revealed significant variations in the expression of apoptotic genes.Among 88 genes,only the expression of IFN-β1 was upregulated 5.22 folds,whereas 47 genes including ACIN1,Akt,Apaf1,caspase and CIDEB were found to be downregulated that were lower than 2 folds at 8 h p.i.However,28 genes including FasL,CD40L,TNF,caspase-10 and caspase-3 were induced more than 2 folds after EV71 infection at 20 h.Conclusion The downregulation of apoptosis-related genes is associated with viral apoptosis-suppressing effect in RD cells in the early stage of EV71 infection.The death receptor signaling pathways including Fas/FasL and TNF/TNFR are activated to induce cell apoptosis in the late stage of EV71 infection.Moreover,host cell can also inhibit apoptosis by regulating signal pathway of CD40/CD40L,NF-κB/RelA and PI3K/Akt activation.