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Zhuofeng Yu, Pinjing He, Liming Shao, Hua Zhang, Fan Lü
Since municipal solid waste (MSW) landfill harbours miscellaneous wastes, pollutants and microorganisms, it gradually becomes a huge potential reservoir for breeding antibiotic resistance genes (ARGs). The objective of this study was to determine the prevalence and diversity of ARGs associated with various mobile genetic elements (MGEs) in MSW landfill leachates. The relationship of ARGs with leachate characteristics was also studied to explore the influence of landfill age. Seven sulfonamides (sulfapyridine, sulfadiazine, sulfathiazole, sulfamethoxazole, sulfamerazine, sulfamethazine and sulfaquinoxaline), three encoded ARGs (sul-I, sul-II and sul-III) and four types of MGEs (plasmids, transposons, integrons and insertion sequences) were quantified in leachates with landfill ages ranging from 3 months-6 years...
October 18, 2016: Water Research
Alice Nomura, Kaustav Majumder, Bhuwan Giri, Patricia Dauer, Vikas Dudeja, Sabita Roy, Sulagna Banerjee, Ashok K Saluja
NF-κB has an essential role in the initiation and progression of pancreatic cancer and specifically mediates the induction of epithelial-mesenchymal transition and invasiveness. In this study, we demonstrate the importance of activated NF-κB signaling in EMT induction, lymphovascular metastasis, and neural invasion. Modulation of NF-κB activity was accomplished through the specific NF-κB inhibitor (BAY 11-7085), triptolide, and Minnelide treatment, as well as overexpression of IKBα repressor and IKK activator plasmids...
October 24, 2016: Laboratory Investigation; a Journal of Technical Methods and Pathology
Jaroslaw Smieja, Marzena Dolbniak
This paper deals with development of signaling pathways models and using plasmid-based experiments to support parameter estimation. We show that if cells transfected with plasmids are used in experiments, the models should include additional components that describe explicitly effects induced by plasmids. Otherwise, when the model is used to analyze responses of wild type, i.e. non-transfected cells, it may not capture their dynamics properly or even lead to false conclusions. In order to illustrate this, an original mathematical model of miRNA-mediated control of gene expression in the NFκB pathway is presented...
December 1, 2016: Mathematical Biosciences and Engineering: MBE
Nadine Czekalski, Stefanie Imminger, Elisabeth Salhi, Marjan Veljkovic, Karolin Kleffel, David Drissner, Frederik Hammes, Helmut Bürgmann, Urs von Gunten
Ozone, a strong oxidant and disinfectant, seems ideal to cope with future challenges of water treatment, such as micropollutants, multiresistant bacteria (MRB) and even intracellular antibiotic resistance genes (ARG), but information on the latter is scarce. In ozonation experiments we simultaneously determined kinetics and dose-dependent inactivation of Escherichia coli and its plasmid-encoded sulfonamide resistance gene sul1 in different water matrixes. Effects in E. coli were compared to an autochthonous wastewater community...
October 24, 2016: Environmental Science & Technology
Nadja Förster, Inga Mewis, Hansruedi Glatt, Michael Haack, Regina Brigelius-Flohé, Monika Schreiner, Christian Ulrichs
Leaves of Moringa oleifera are used by tribes as biological cancer medicine. Scientific investigations with M. oleifera conducted so far have almost exclusively used total plant extracts. Studies on the activity of single compounds are missing. Therefore, the biological effects of the two main aromatic multi-glycosylated glucosinolates of M. oleifera were investigated in the present study. The cytotoxic effects of M. oleifera glucosinolates were identified for HepG2 cells (NRU assay), for V79-MZ cells (HPRT assay, SCE assay), and for two Salmonella typhimurium strains (Ames test)...
October 24, 2016: Food & Function
Jian Hu, Qingmei Wang, Li Huang, Yuanqing Zeng, Zheng Hu, Dixian Luo
Objective To prepare rabbit anti-aldo-keto reductase family 1 member B10 (AKR1B10) polyclonal antibody and identify its specificity. Methods AKR1B10 cDNA was amplified by reverse transcription PCR (RT-PCR) and inserted into prokaryotic expression vector pET-15b to form recombinant plasmid pET-15b-AKR1B10. The recombinant plasmid pET-15b-AKR1B10 was transformed into E.coli DH5α. Isopropylthio-β-D-galactoside (IPTG) was used to induce the expression of the recombinant protein His-tagged AKR1B10 in E.coli DH5α...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Jia Guo, Ming Jiang, Lingjing Jin, Yanxin Yin, Hui Sun, Lihua Yu, Yuting Mao, Jianmin Fang
Objective To construct lentiviral vectors for the expression of monovalent antibody against human c-mesenchymal epithelial transition factor (c-Met) using anti-c-Met chimeric antibody ch3E1D7 plasmid, and test the affinity and neutralizing ability of the purified monovalent antibody in transfected HEK293T cells. Methods The anti-c-Met monovalent antibody was designed, namely mono3E1D7. Three different lentiviral expression vectors of the monovalent antibody were then constructed using genetic engineering technology...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Guanghong Fu, Dan Gong, Jia Wan
Objective To investigate the effect of E5 protein of human papillomavirus type 16 (HPV16 E5) on keratinocyte growth factor-triggered autophagy. Methods Firstly, pCI-neo-E5 recombinant plasmid was constructed. After that, the recombinant plasmid harboring pCI-neo-E5 was transfected into HaCaT cells using Lipofectamine(TM)2000 reagent, and the mRNA transcriptional level of 16 E5 was evaluated by quantitative real-time PCR (qRT-PCR). Then, the expression of LC3BIIwas detected by Western blotting and the localization of LC3B was observed by immunofluorescence in pCI-neo-E5-HA-transfected HaCaT cells triggered with keratinocyte growth factor...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Wenjing Yang, Xianguo Fu, Juan Liao, Xiaoyan Guo, Fenghua Lan
Objective To construct a eukaryotic expression vector of human fragile X mental retardation 1 (FMR1) gene and establish stably transfected HeLa cells. Methods The full-length FMR1 cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pEGFP-N2 using restriction enzyme. The recombinant plasmid pEGFP-N2-FMR1, after identified by restriction digestion and DNA sequencing, was transfected into HeLa cells by lipofectamine 2000. The stably transfected cell line was obtained by screening with G418...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Xin Chen, Zheng Jiang
Objective To observe the effect of aplysia Ras homolog I (ARHI) gene on human colon cancer cell (SW480) apoptosis. Methods The pcDNA3.1-FLAG-ARHI plasmid was adopted to transfect the SW480 cell line, and G418 screening to obtain stably ARHI-transfected SW480 cells. Reverse transcription PCR and Western blotting were used to detect mRNA and protein expression of ARHI, respectively. The flow cytometry was applied to detect the effect of ARHI overexpression on colon cancer cell apoptosis. The levels of protein kinase B (Akt), phosphorylated Akt (p-Akt), p53 and Bcl-2 were determined by Western blotting...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Jinlan Nie, Quanfang Huang, Shimei Tan, Xing Lin
Objective To investigate the effect of Raf kinase inhibitor protein (RKIP) over-expression on the proliferation of LX-2 human hepatic stellate cells. Methods Recombinant plasmid pcDNA3.1-RKIP was transfected into LX-2 cells. G418 was used to screen and culture stably infected cells. MTT assay and colony formation assay were used to examine the effect of RKIP over-expression on cell proliferation and colony formation, respectively. Western blotting was performed to assess the expressions of RKIP, α-smooth muscle actin (α-SMA), type 1 collagen (Col1) and matrix metalloproteinase 1(MMP-1) and MMP-2 as well as extracellular signal-regulated kinases/mitogen-activated protein kinase (ERK/MAPK) signaling pathway-related proteins...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Junxiong Cheng, Dan Guo, Xi Chen, Ting Wang, Jiayi Huang
Objective To construct a recombinant adenovirus expressing siRNA targeting human sclerostin (SOST) gene, and test the function of MG-63 cells while co-cultured with MDA-MB-231 cells infected by Ad-siSOST. MethodsAccording to the RNA sequence of SOST gene, two pairs of primers which contained 3 siRNA sequences were designed, and a pB2B plasmid was taken as template to amplify 2 DNA sequences. Both of the 2 DNA sequences were ligated to pAdTrace-OK by Gibson DNA Assembly way. After homologous recombination between recombinant shuttle plasmid and adenovirus vector plasmid, the adenovirus was packaged in HEK-293 cells...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Fang Chen, Weifeng Zhang, Junli Zhao, Peiyan Yang, Rui Ma, Haibin Xia
Objective To prepare Rev-erbβ knockout HEK293 cells using clustered regularly interspaced short palindromic repeats/Cas 9 nuclease (CRISPR/Cas9) gene editing technology. Methods The knock-in or knockout of Rev-erbβ gene could be realized by single-guide RNA (sgRNA)-mediated Cas9 cutting of target DNA, and followed by DNA homologous recombination or non-homologous end joining-mediated DNA repair. Firstly, four sgRNAs were designed for Rev-erbβ gene. The sgRNA1 and sgRNA2 with the higher activity were respectively used to construct pCMV-hCas9-U6-Rev-erbβ sgRNA1 and pCMV-hCas9-U6-Rev-erbβ sgRNA2...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Nathan J Alves, Kendrick B Turner, Kyle A DiVito, Michael A Daniele, Scott A Walper
To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin...
October 20, 2016: Research in Microbiology
Ketrin Cristina da Silva, Marcos Paulo Vieira Cunha, Louise Cerdeira, Maria Gabriela Xavier de Oliveira, Mirela Caroline Vilela de Oliveira, Cleise Ribeiro Gomes, Nilton Lincopan, Terezinha Knöbl, Andrea Micke Moreno
This study reports the high-virulence phylogenetic backgrounds of CMY-2- and CTX-M-2-producing avian pathogenic Escherichia coli strains isolated from turkeys sent to slaughter and condemned by airsacculitis in Brazil. Among 300 air sac samples, seven E. coli strains produced plasmid-mediated CMY-2-type AmpC, of which three carried also the blaCTX-M-2 Extended Spectrum Beta-Lactamase encoding gene. Interestingly, the transfer of the blaCMY-2 gene was positive for three E. coli strains, being associated with the presence of IncI1 plasmids...
October 7, 2016: Diagnostic Microbiology and Infectious Disease
Yaming Gu, Yizhen Guo, Changyong Wang, Jiake Xu, Jianping Wu, Thomas Brett Kirk, Dong Ma, Wei Xue
It is a promising way to treat the multi drug resistance (MDR) of tumor cells in both of drug and gene methods. A polyamidoamne dendrimer functionalized graphene oxide (GO-PAMAM) was designed, which could load doxorubicin (DOX) and MMP-9 shRNA plasmid at the same time in order to achieve effective treatment to breast cancer. GO-PAMAM has a high loading capacity to DOX and pH-controlled DOX release. Besides, it has efficient gene transfer ability, the transfection efficiency is significantly better than PEI-25k in the presence of serum, and it can significantly inhibit the expression of MMP-9 protein in MCF-7 cells...
January 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
Bao-Zhong Zhang, Jianpiao Cai, Bin Yu, Yanhong Hua, Candy Choiyi Lau, Richard Yi-Tsun Tsun Kao, Kong-Hung Sze, Kwok-Yung Yuen, Jian-Dong Huang
BACKGROUND: Clostridium difficile-associated disease (CDAD) constitutes a great majority of hospital diarrhea cases in industrialized countries and is induced by two types of large toxin molecules: toxin A (TcdA) and toxin B (TcdB). Development of immunotherapeutic approaches, either active or passive, has seen a resurgence in recent years. Studies have described vaccine plasmids that express either TcdA and/or TcdB receptor binding domain (RBD). However, the effectiveness of one vector encoding both toxin RBDs against CDAD has not been evaluated...
October 22, 2016: BMC Infectious Diseases
Marta Marchetti, Camille Clerissi, Yasmine Yousfi, Carine Gris, Olivier Bouchez, Eduardo Rocha, Stéphane Cruveiller, Alain Jauneau, Delphine Capela, Catherine Masson-Boivin
Experimental evolution is a powerful approach to study the process of adaptation to new environments, including the colonization of eukaryotic hosts. Facultative endosymbionts, including pathogens and mutualists, face changing and spatially structured environments during the symbiotic process, which impose diverse selection pressures. Here we provide evidence that different selection regimes, involving different times spent in the plant environment, can result in either intra- or extracellular symbiotic adaptations...
October 22, 2016: Molecular Ecology
Zhen Lei, Abdul Sami Shaikh, Wenshuai Zheng, Xiao Yu, Jingui Yu, Jingxin Li
Itch, the unpleasant sensation that evokes a desire to scratch, accompanies numerous skin and nervous system disorders. However, the molecular mechanisms of itch are unclear. Acid sensing ion channel 3 (ASIC3) is a sensor of acidic and primary inflammatory pain. The whole-cell patch-clamp technique was used to determine the effect of Chloroquine (CQ)on ASICs currents in primary sensory neurons or the Chinese hamster ovary (CHO) cells transfected with rat-ASIC1a or- ASIC3. Site-directed mutagenesis of plasmid was performed...
October 22, 2016: Journal of Neurochemistry
Katsutoshi Yamasaki, Masaru Komatsu, Tamotsu Ono, Hisaaki Nishio, Noriyuki Sueyoshi, Kaneyuki Kida, Kaori Satoh, Hirofumi Toda, Isao Nishi, Masahiro Akagi, Tetsu Mizutani, Isako Nakai, Tomomi Kofuku, Tamaki Orita, Takuya Zikimoto, Tatsuya Nakamura, Yasunao Wada
Six Klebsiella pneumoniae clinical isolates resistant to various cephalosporins and cephamycins were identified in a Japanese general hospital, a tertiary care hospital, between November 2009 and April 2010. All K. pneumoniae isolates carried blaGES-4 and blaSHV-1, while 2 K. pneumoniae isolates also harbored blaCTX-M-15. The pulsed-field gel electrophoresis patterns revealed that these 6 K. pneumoniae isolates were almost identical, suggesting their clonal relatedness. Plasmid profiles and conjugation assays revealed that these blaGES-4 genes were located on similar conjugative plasmids...
October 18, 2016: Journal of Infection and Chemotherapy: Official Journal of the Japan Society of Chemotherapy
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