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Pathogenicity and Virulence
Published in Julius P. Kreier, Infection, Resistance, and Immunity, 2022
Plasmids carrying genes for antibiotic resistance known as R factors were first observed in the genus Shigella in 1959. These plasmids are readily transferable by conjugation to bacterial cells of the same species as well as to cells of different species and genera and can confer resistance to many antibiotics.
Methods of Evaluation in Orthopaedic Animal Research
Published in Yuehuei H. An, Richard J. Friedman, Animal Models in Orthopaedic Research, 2020
The basic terminology given here is adapted from the reviews by Shore and Kaplan.251,252 A gene is a unit of heredity, consisting of a segment of chromosomal DNA that is required for production of a functional protein or RNA. The gene contains both coding and regulatory regions. A transgene is a foreign gene which has been spliced into an animals original genomic DNA. mRNA is a type of RNA that contains protein coding information. Nucleotide sequence refers to the order of nucleotides in a given segment of DNA or RNA. Translocation is the transfer of a portion of DNA from one chromosome to another. A probe is a DNA or RNA molecule that is labeled, or tagged, and can then be used to locate a complementary DNA or RNA strand through hybridization. Vectors are DNA molecules that are used as carrier molecules for cloned DNA sequences. They contain information which allows recombinant molecules to be replicated in host bacterial cells. A plasmid is a small circular double-stranded DNA molecule which is found in bacteria and replicates independently of the host chromosome. They are commonly used as vectors in molecular cloning. A recombinant DNA molecule is a DNA molecule containing segments of DNA from different origins, such as a piece of human DNA that has been joined to a plasmid DNA. A clone is a term used to describe identical segmental DNA molecules produced by recombinant DNA technique. Molecular cloning is a process by which a specific segment of DNA is isolated and then numerous identical copies, or clones, of that segment of DNA are generated.
Can we accelerate the osteoporotic bone fracture healing response?
Published in Peter V. Giannoudis, Thomas A. Einhorn, Surgical and Medical Treatment of Osteoporosis, 2020
Martijn van Griensven, Elizabeth Rosado Balmayor
As the typical plasmid gene delivery is not really feasible for a clinical setting, an innovative method was explored for clinical feasibility concerning gene therapy. In this case, the gene is introduced as messenger RNA to the cell, thereby preventing the need for the gene to enter the nucleus. The messenger RNA molecule is able to immediately be translated to protein once present in the cytoplasm. Messenger RNA molecules are, however, labile and immunogenic (61). Messenger RNA molecules may activate the immune system via toll-like receptors. In order to overcome this instability, a poly(A) tail of 120 nucleotides was added (62). Further stability and a decrease of immunogenicity were achieved by modifying uridine and cytidine nucleotides with thio- and methyl-groups, respectively (63).
High-sugar, high-fat, and high-protein diets promote antibiotic resistance gene spreading in the mouse intestinal microbiota
Published in Gut Microbes, 2022
Rong Tan, Min Jin, Yifan Shao, Jing Yin, Haibei Li, Tianjiao Chen, Danyang Shi, Shuqing Zhou, Junwen Li, Dong Yang
One study found that horizontal gene transfer (HGT) is common in the human intestinal microbiota and occurs by various mechanisms, the most typical of which are transduction and conjugation transfer.15 Through conjugation transfer, many species in the same habitat can obtain ARGs, which are usually transferred across species boundaries.16 The high density of bacteria in the intestine can facilitate the transfer of plasmids between the same species or between different species.17 The high diversity of ARGs in human bacteria may lead to antibiotic resistance of human pathogens in the future. Once ARGs enter human pathogens, they may be transmitted to symbiotic bacteria through pathogens, then further spread among symbiotic bacteria, causing greater harm.18 In 2017, the World Health Organization published the first list of “priority pathogens” possessing antibiotic resistance.19 The list consists of 11 species, including Staphylococcus aureus, Helicobacter pylori, and Salmonella typhi. Eight of these can participate in DNA uptake through natural ability, and the three Enterobacteriaceae on the list potentially have this ability.20 Conjugation-mediated plasmid transfer may be the main reason to promote the emergence of highly pathogenic or antibiotic resistant pathogens.21 The main reason why they are considered represent a great threat to human health is that they can not only cause serious diseases, but are also resistant to currently effective antibiotics.
Suitability of transiently expressed antibodies for clinical studies: product quality consistency at different production scales
Published in mAbs, 2022
Sara Rodriguez-Conde, Sophie Inman, Viv Lindo, Leanne Amery, Alison Tang, Uche Okorji-Obike, Wenjuan Du, Berend-Jan Bosch, Paul J. Wichgers Schreur, Jeroen Kortekaas, Isabel Sola, Luis Enjuanes, Laura Kerry, Katharina Mahal, Martyn Hulley, Olalekan Daramola
Traditionally, transient gene expression (TGE) has been the technology used for production of therapeutic glycoproteins at early drug development stages because it allows for rapid production of high-quality material.1 This technology involves introducing plasmid DNA, which encodes the protein of interest, into mammalian cells. The cells then express the recombinant protein over a limited period of time, typically up to 14–21 days. Several methods are used to transfer plasmid DNA into mammalian cells for TGE. Some of the most common chemical agents used for transfection are calcium phosphate, polyethyleneimines (PEIs) and cationic lipids. In particular, PEIs are frequently used due to the high transfection efficiency and relatively low cost compared to lipid-based reagents. An alternative to chemical-based transfection is the use of electroporation methods, such as the MaxCyte® STXTM flow electroporation system. Using this approach, Steger et al. were able to produce 3.5 g of antibody from less than 3 L of culture.2 Although currently this technology has been tested only at shake flask culture scale, it has the potential to be scaled up to several liters in bioreactors.
Layer-by-Layer technique as a versatile tool for gene delivery applications
Published in Expert Opinion on Drug Delivery, 2021
Dmitrii S. Linnik, Yana V. Tarakanchikova, Mikhail V. Zyuzin, Kirill V. Lepik, Joeri L. Aerts, Gleb Sukhorukov, Alexander S. Timin
Plasmid DNA (pDNA) is one of the most commonly used forms of nucleic acid for gene therapy. This circular double-stranded molecule is a preferred choice of gene vectors because it is less susceptible to nucleases and experiences less physical impact during delivery compared to linear DNA and RNA molecules, thus generally obviating the necessity to add nuclease inhibitors into the LbL system. Plasmids have the capacity to carry many genes that can be expressed simultaneously or under different conditions, if various promoters and other regulatory elements are used. The cloning capacity of plasmids, the simplicity of manipulation by various cloning approaches, and their ease of production on a large scale also make them a genetic carrier of choice for delivery. However, there are also several disadvantages when using plasmids as genetic material for delivery. Expression of genes encoded on plasmids requires nuclear localization of the delivered nucleic acid. Moreover, unmethylated double-stranded DNA (dsDNA) can be recognized as a potentially dangerous agent by receptors of the innate immune system, including Toll-like receptor (TLR) 9, stimulator of interferon genes (STING), and NLR family pyrin domain containing 3 (NLRP3) inflammasome. Thus, a pretreatment of cells with specific inhibitors of these pathways, or co-delivery of these inhibitors together with plasmid DNA, could result in increased transfection efficiency and higher expression levels [67].