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https://www.readbyqxmd.com/read/28212413/scarless-deletion-of-up-to-seven-methyl-accepting-chemotaxis-genes-with-an-optimized-method-highlights-key-function-of-chem-in-salmonella-typhimurium
#1
Stefanie Hoffmann, Christiane Schmidt, Steffi Walter, Jennifer K Bender, Roman G Gerlach
Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus...
2017: PloS One
https://www.readbyqxmd.com/read/28211929/calcium-phosphate-nanoparticles-prepared-from-infusion-fluids-for-stem-cell-transfection-process-optimization-and-cytotoxicity-analysis
#2
Quazi T H Shubhra, Ayako Oyane, Hiroko Araki, Maki Nakamura, Hideo Tsurushima
This is the first study to report the use of infusion fluids for particle-mediated gene delivery with DNA-immobilized calcium phosphate (CaP) nanoparticles (NPs). In conventional CaP systems, CaP NPs are fabricated in labile supersaturated CaP solutions which are prepared from chemical reagents. In the present study, we fabricated CaP NPs via coprecipitation in labile supersaturated CaP solutions that were prepared from infusion fluids (even the water used was of injectable quality) instead of chemical reagents and demonstrated their gene delivery capabilities for the hard to transfect pluripotent stem cell (C3H10T1/2) along with the easy to transfect CHO-K1 cell...
February 17, 2017: Biomaterials Science
https://www.readbyqxmd.com/read/28211920/evaluation-of-acridine-orange-derivatives-as-dna-targeted-radiopharmaceuticals-for-auger-therapy-influence-of-the-radionuclide-and-distance-to-dna
#3
Edgar Pereira, Letícia do Quental, Elisa Palma, Maria Cristina Oliveira, Filipa Mendes, Paula Raposinho, Isabel Correia, João Lavrado, Salvatore Di Maria, Ana Belchior, Pedro Vaz, Isabel Santos, António Paulo
A new family of (99m)Tc(I)- tricarbonyl complexes and (125)I-heteroaromatic compounds bearing an acridine orange (AO) DNA targeting unit was evaluated for Auger therapy. Characterization of the DNA interaction, performed with the non-radioactive Re and (127)I congeners, confirmed that all compounds act as DNA intercalators. Both classes of compounds induce double strand breaks (DSB) in plasmid DNA but the extent of DNA damage is strongly dependent on the linker between the Auger emitter ((99m)Tc or (125)I) and the AO moiety...
February 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28211908/the-n-terminal-domain-of-schmallenberg-virus-envelope-protein-gc-is-highly-immunogenic-and-can-provide-protection-from-infection
#4
Kerstin Wernike, Andrea Aebischer, Gleyder Roman-Sosa, Martin Beer
Schmallenberg virus (SBV) is transmitted by insect vectors, and therefore vaccination is one of the most important tools of disease control. In our study, novel subunit vaccines on the basis of an amino-terminal domain of SBV Gc of 234 amino acids ("Gc Amino") first were tested and selected using a lethal small animal challenge model and then the best performing formulations also were tested in cattle. We could show that neither E. coli expressed nor the reduced form of "Gc Amino" protected from SBV infection...
February 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28211892/high-throughput-in-situ-cell-electroporation-microsystem-for-parallel-delivery-of-single-guide-rnas-into-mammalian-cells
#5
Shengtai Bian, Yicen Zhou, Yawei Hu, Jing Cheng, Xiaofang Chen, Youchun Xu, Peng Liu
Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells...
February 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28210928/bacterial-resistance-to-arsenic-protects-against-protist-killing
#6
Xiuli Hao, Xuanji Li, Chandan Pal, Jon Hobman, D G Joakim Larsson, Quaiser Saquib, Hend A Alwathnani, Barry P Rosen, Yong-Guan Zhu, Christopher Rensing
Protists kill their bacterial prey using toxic metals such as copper. Here we hypothesize that the metalloid arsenic has a similar role. To test this hypothesis, we examined intracellular survival of Escherichia coli (E. coli) in the amoeba Dictyostelium discoideum (D. discoideum). Deletion of the E. coli ars operon led to significantly lower intracellular survival compared to wild type E. coli. This suggests that protists use arsenic to poison bacterial cells in the phagosome, similar to their use of copper...
February 16, 2017: Biometals: An International Journal on the Role of Metal Ions in Biology, Biochemistry, and Medicine
https://www.readbyqxmd.com/read/28210900/false-responses-of-renilla-luciferase-reporter-control-to-nuclear-receptor-tr4
#7
Dongyun Zhang, Sam S Atlasi, Krishna K Patel, Zihao Zhuang, Anthony P Heaney
Renilla luciferase reporter is a widely used internal control in dual luciferase reporter assay system, where its transcription is driven by a constitutively active promoter. However, the authenticity of the Renilla luciferase response in some experimental settings has recently been questioned. Testicular receptor 4 (TR4, also known as NR2C2) belongs to the subfamily 2 of nuclear receptors. TR4 binds to a direct repeat regulatory element in the promoter of a variety of target genes and plays a key role in tumorigenesis, lipoprotein regulation, and central nervous system development...
February 16, 2017: Molecular and Cellular Biochemistry
https://www.readbyqxmd.com/read/28210248/coexistence-of-blaoxa-48-and-truncated-blandm-1-on-different-plasmids-in-a-klebsiella-pneumoniae-isolate-in-china
#8
Lianyan Xie, Yi Dou, Kaixin Zhou, Yue Chen, Lizhong Han, Xiaokui Guo, Jingyong Sun
Objectives: To describe the genetic environment, transferability, and antibiotic susceptibility of one clinical Klebsiella pneumoniae isolate harboring both blaOXA-48 and blaNDM-1 on different plasmids from a Chinese hospital. Methods: The isolate was subjected to antimicrobial susceptibility testing and multilocus sequence typing using Etest and PCR. The plasmids harboring blaOXA-48 and blaNDM-1 were analyzed through conjugation experiments, S1-nuclease pulsed-field gel electrophoresis, and hybridization with specific probes...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28210241/outer-membrane-proteins-form-specific-patterns-in-antibiotic-resistant-edwardsiella-tarda
#9
Bo Peng, Chao Wang, Hui Li, Yu-Bin Su, Jin-Zhou Ye, Man-Jun Yang, Ming Jiang, Xuan-Xian Peng
Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28209967/optimization-of-the-production-of-knock-in-alleles-by-crispr-cas9-microinjection-into-the-mouse-zygote
#10
Aurélien Raveux, Sandrine Vandormael-Pournin, Michel Cohen-Tannoudji
Microinjection of the CRISPR/Cas9 system in zygotes is an efficient and comparatively fast method to generate genetically modified mice. So far, only few knock-in mice have been generated using this approach, and because no systematic study has been performed, parameters controlling the efficacy of CRISPR/Cas9-mediated targeted insertion are not fully established. Here, we evaluated the effect of several parameters on knock-in efficiency changing only one variable at a time. We found that knock-in efficiency was dependent on injected Cas9 mRNA and single-guide RNA concentrations and that cytoplasmic injection resulted in more genotypic complexity compared to pronuclear injection...
February 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28209827/first-complete-genome-sequence-of-marinilactibacilluspiezotolerans-strain-15r-a-marine-lactobacillus-isolated-from-coal-bearing-sediment-2-0-kilometers-below-the-seafloor-determined-by-pacbio-single-molecule-real-time-technology
#11
Yuli Wei, Junwei Cao, Jiasong Fang, Chiaki Kato, Weicheng Cui
Marinilactibacillus piezotolerans strain 15R is a facultatively anaerobic heterotrophic lactobacillus isolated from deep marine subsurface sediment nearly 2 km below the seafloor in the northwestern Pacific. We report here the first whole-genome sequence of strain 15R. The identified genome sequence has 2,767,908 bp, 35.4% G+C content, and predicted 2,552 candidate protein-coding sequences, with no identified plasmids.
February 16, 2017: Genome Announcements
https://www.readbyqxmd.com/read/28209826/complete-genome-sequence-of-the-polychlorinated-biphenyl-degrading-bacterium-pseudomonas-putida-kf715-nbrc-110667-isolated-from-biphenyl-contaminated-soil
#12
Hikaru Suenaga, Atsushi Yamazoe, Akira Hosoyama, Nobutada Kimura, Jun Hirose, Takahito Watanabe, Hidehiko Fujihara, Taiki Futagami, Masatoshi Goto, Kensuke Furukawa
Pseudomonas putida KF715 (NBRC 110667) utilizes biphenyl as a sole source of carbon and degrades polychlorinated biphenyls (PCBs). Here, we report a complete genome sequence of the KF715 strain, which comprises a circular chromosome and four plasmids. Biphenyl catabolic genes were located on the largest plasmid, pKF715A.
February 16, 2017: Genome Announcements
https://www.readbyqxmd.com/read/28209599/role-of-shp2-protein-tyrosine-phosphatase-in-sert-inhibition-by-enteropathogenic-e-coli-epec
#13
Megha Singhal, Christopher Manzella, Vinay Soni, Waddah A Alrefai, Seema Saksena, Gail A Hecht, Pradeep K Dudeja, Ravinder K Gill
Enteropathogenic E. coli (EPEC), one of the diarrheagenic E. coli pathotypes, is among the most important food-borne pathogens infecting children worldwide. Inhibition of serotonin transporter (SERT), that regulates extracellular availability of serotonin (5-HT), has been previously implicated in EPEC-associated diarrhea. EPEC was shown to inhibit SERT via activation of protein tyrosine phosphatases (PTPase), albeit the specific PTPase involved is not known. Current studies aimed to identify EPEC activated PTPase and its role in SERT inhibition...
February 16, 2017: American Journal of Physiology. Gastrointestinal and Liver Physiology
https://www.readbyqxmd.com/read/28209206/rethinking-the-role-of-alpha-toxin-in-clostridium-perfringens-associated-enteric-diseases-a-review-on-bovine-necro-haemorrhagic-enteritis
#14
REVIEW
Evy Goossens, Bonnie R Valgaeren, Bart Pardon, Freddy Haesebrouck, Richard Ducatelle, Piet R Deprez, Filip Van Immerseel
Bovine necro-haemorrhagic enteritis is an economically important disease caused by Clostridium perfringens type A strains. The disease mainly affects calves under intensive rearing conditions and is characterized by sudden death associated with small intestinal haemorrhage, necrosis and mucosal neutrophil infiltration. The common assumption that, when causing intestinal disease, C. perfringens relies upon specific, plasmid-encoded toxins, was recently challenged by the finding that alpha toxin, which is produced by all C...
February 16, 2017: Veterinary Research
https://www.readbyqxmd.com/read/28208635/advances-in-non-viral-dna-vectors-for-gene-therapy
#15
Cinnamon L Hardee, Lirio Milenka Arévalo-Soliz, Benjamin D Hornstein, Lynn Zechiedrich
Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic...
February 10, 2017: Genes
https://www.readbyqxmd.com/read/28208239/narcissus-yellow-stripe-virus-and-narcissus-mosaic-virus-detection-in-narcissus-via-multiplex-taqman-based-reverse-transcription-pcr-assay
#16
Jing Jin, Jianguo Shen, Wei Cai, Xie Guohui, Furong Liao, Fangluan Gao, Junfeng Ma, Xihong Chen, Zujian Wu
AIMS: Development of a multiplex TaqMan RT-qPCR assay to simultaneously detect Narcissus yellow stripe virus (NYSV) and Narcissus mosaic virus (NMV), frequently causing mixed narcissus infection. Feasibility verification was confirmed in natural samples. METHODS AND RESULTS: Primers and probes were designed based on the conserved CP gene regions of NYSV or NMV and their suitability for singleplex and multiplex TaqMan RT-qPCR assays as well as for conventional RT-PCR...
February 16, 2017: Journal of Applied Microbiology
https://www.readbyqxmd.com/read/28208218/antibiotic-resistance-in-escherichia-coli-in-husbandry-animals-the-african-perspective
#17
REVIEW
Carla Andrea Alonso, Myriam Zarazaga, Rym Ben Sallem, Ahlem Jouini, Karim Ben Slama, Carmen Torres
In the last years different surveillances have been published in Africa, especially in Northern countries, regarding antimicrobial resistance among husbandry animals. Information is still scarce, but the available data shows a worrying picture. Although the highest resistance rates have been described against tetracycline, penicillins and sulphonamides, prevalence of plasmid mediated quinolone resistance genes and extended-spectrum β-lactamase (ESBL) are being increasingly reported. Among ESBLs, the CTX-M-1 group was dominant in most African surveys, being CTX-M-15 the main variant both in animals and humans, except in Tunisia where CTX-M-1 was more frequently detected among E...
February 16, 2017: Letters in Applied Microbiology
https://www.readbyqxmd.com/read/28207825/discovery-of-a-new-family-of-relaxases-in-firmicutes-bacteria
#18
Gayetri Ramachandran, Andrés Miguel-Arribas, David Abia, Praveen K Singh, Isidro Crespo, César Gago-Córdoba, Jian An Hao, Juan Roman Luque-Ortega, Carlos Alfonso, Ling J Wu, D Roeland Boer, Wilfried J J Meijer
Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes...
February 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28207166/a-set-of-isomeric-episomal-plasmids-for-systematic-examination-of-mitotic-stability-in-saccharomyces-cerevisiae
#19
Ruben Hohnholz, Kim Julia Pohlmann, Tilman Achstetter
Yeast episomal shuttle vectors (YEp type) are commonly used in fundamental research and biotechnology whenever elevated product levels are desired. Their instability, however, poses an impediment not only in industrial scale fermentation. In order to analyze instability which might be linked to plasmid structure, a series of YEp type plasmids identical in size has been assembled, differing only in the overall arrangement of the fragments used. Performance of the eight plasmid isoforms was studied with respect to mitotic stability...
February 16, 2017: Yeast
https://www.readbyqxmd.com/read/28207002/detecting-natural-adaptation-of-the-streptococcus-thermophilus-crispr-cas-systems-in-research-and-classroom-settings
#20
Alexander P Hynes, Marie-Laurence Lemay, Luc Trudel, Hélène Deveau, Michel Frenette, Denise M Tremblay, Sylvain Moineau
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRISPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization...
March 2017: Nature Protocols
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