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https://www.readbyqxmd.com/read/29790644/crispr-cas9-assisted-multiplex-genome-editing-technique-in-escherichia-coli
#1
Xu Feng, Dongdong Zhao, Xueli Zhang, Xiang Ding, Changhao Bi
Genome editing for site-specific chromosome modification is one of the most significant techniques in biological research. While conventional techniques usually deal with one genomic locus at a time, multiple genomic targets are often required to be modified to develop microbial cell factories. Thus, it is necessary to develop techniques for simultaneous editing of multiple loci. In this work, we developed a CRISPR/Cas9 assisted multiplex genome editing (CMGE) technique in Escherichia coli. With this editing method, all functional parts were assembled into replicable plasmids, and stringent inducible expression systems were used to control Cas9 gene expression, which was to decouple transformation from editing process to increase editing efficiency...
May 23, 2018: Biotechnology Journal
https://www.readbyqxmd.com/read/29789390/the-assembly-of-lipid-droplets-and-their-roles-in-challenged-cells
#2
REVIEW
W Mike Henne, Michael L Reese, Joel M Goodman
Cytoplasmic lipid droplets are important organelles in nearly every eukaryotic and some prokaryotic cells. Storing and providing energy is their main function, but they do not work in isolation. They respond to stimuli initiated either on the cell surface or in the cytoplasm as conditions change. Cellular stresses such as starvation and invasion are internal insults that evoke changes in droplet metabolism and dynamics. This review will first outline lipid droplet assembly and then discuss how droplets respond to stress and in particular nutrient starvation...
May 22, 2018: EMBO Journal
https://www.readbyqxmd.com/read/29782215/detection-of-a-vita-pamp-stings-cells-into-reticulophagy
#3
Julien Moretti, J Magarian Blander
Phagocytes cope with the threat of living bacteria via detection of vita-PAMPs, a specific class of pathogen-associated molecular patterns (PAMPs) that denotes microbial viability and trigger a commensurate innate response. Prokaryotic mRNA and cyclic-di-adenosine monophosphate (c-di-AMP) serve as vita-PAMPs for Gram-negative and Gram-positive bacteria, respectively, and elicit heightened proinflammatory responses not warranted for dead bacteria. The innate sensor TMEM173/STING detects c-di-AMP produced by internalized live Gram-positive bacteria, and quickly mobilizes interdependent pre-formed cell-autonomous responses including endoplasmic reticulum (ER) stress, MTOR inactivation, and reticulophagy...
May 21, 2018: Autophagy
https://www.readbyqxmd.com/read/29782206/active-transport-of-cytoophidia-in-schizosaccharomyces-pombe
#4
Hui Li, Fangfu Ye, Jing-Yi Ren, Peng-Ye Wang, Li-Lin Du, Ji-Long Liu
The metabolic enzyme cytidine triphosphate synthase has recently been found to form micron-sized filamentous structures termed cytoophidia, which are evolutionarily conserved across prokaryotes and eukaryotes. The cytoophidium represents a novel type of membraneless organelle and behaves dynamically inside the cell. The question of how cytoophidia transport is mediated, however, remains unanswered. For the first time, we detected in this study the active transport of cytoophidia, taking advantage of the fission yeast Schizosaccharomyces pombe as an excellent model for studying membraneless organelles...
May 21, 2018: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/29781985/testing-the-role-of-multicopy-plasmids-in-the-evolution-of-antibiotic-resistance
#5
Jose Antonio Escudero, R Craig MacLean, Alvaro San Millan
Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid...
May 2, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29779778/identification-of-tb-e12-as-a-novel-ftsz-inhibitor-with-anti-tuberculosis-activity
#6
Yuan Lin, Hongjuan Zhang, Ningyu Zhu, Xia Wang, Yanxing Han, Minghua Chen, Jiandong Jiang, Shuyi Si
The global pandemic of multidrug-resistant (MDR) Mycobacterium tuberculosis (TB) drives for more effective anti-TB drugs with new drug target. Filamentous temperature sensitive protein Z (FtsZ), a GTP dependent prokaryotic cell division protein, forms a dynamic Z-ring in the center of the cell. Differences between bacterial FtsZ and eukaryotic tubulin make FtsZ a highly attractive drug target. In this study, we used phenotype screening of M. smegmatis and model screening targeting M. tuberculosis FtsZ (Mtb-FtsZ) to identify a hit compound TB-E12...
May 2018: Tuberculosis
https://www.readbyqxmd.com/read/29778212/reactive-cysteine-persulphides-occurrence-biosynthesis-antioxidant-activity-methodologies-and-bacterial-persulphide-signalling
#7
Tomohiro Sawa, Katsuhiko Ono, Hiroyasu Tsutsuki, Tianli Zhang, Tomoaki Ida, Motohiro Nishida, Takaaki Akaike
Cysteine hydropersulphide (CysSSH) is a cysteine derivative having one additional sulphur atom bound to a cysteinyl thiol group. Recent advances in the development of analytical methods for detection and quantification of persulphides and polysulphides have revealed the biological presence, in both prokaryotes and eukaryotes, of hydropersulphides in diverse forms such as CysSSH, homocysteine hydropersulphide, glutathione hydropersulphide, bacillithiol hydropersulphide, coenzyme A hydropersulphide, and protein hydropersulphides...
2018: Advances in Microbial Physiology
https://www.readbyqxmd.com/read/29777837/bacteriophage-research-what-we-have-learnt-and-what-still-needs-to-be-addressed
#8
Andrew M Kropinski
Research on bacteriophages has significantly enhanced our understanding of molecular biology, the genomes of prokaryotic cells, and viral ecology. Phages and lysins offer a viable alternative to the declining utility of antibiotics in this post-antibiotic era. They also provide ideal teaching tools for genomics and bioinformatics. This article touches on the first 100 years of phage research with the author commenting on what he thinks are the highlights, and what needs to be addressed.
May 16, 2018: Research in Microbiology
https://www.readbyqxmd.com/read/29775273/the-role-of-clpp-protease-in-bacterial-pathogenesis-and-human-diseases
#9
Vaibhav Bhandari, Keith S Wong, Jin Lin Zhou, Mark F Mabanglo, Robert A Batey, Walid A Houry
In prokaryotic cells and eukaryotic organelles, the ClpP protease plays an important role in proteostasis. The disruption of ClpP function has been shown to influence the infectivity and virulence of a number of bacterial pathogens. More recently, ClpP has been found to be involved in various forms of carcinomas and in Perrault syndrome, which is an inherited condition characterized by hearing loss in males and females and by ovarian abnormalities in females. Hence, targeting ClpP is a potentially viable, attractive option for the treatment of different ailments...
May 18, 2018: ACS Chemical Biology
https://www.readbyqxmd.com/read/29774213/a-rapid-growth-independent-antibiotic-resistance-detection-test-by-sybr-green-propidium-iodide-viability-assay
#10
Jie Feng, Rebecca Yee, Shuo Zhang, Lili Tian, Wanliang Shi, Wen-Hong Zhang, Ying Zhang
Antibiotic-resistant bacteria have caused huge concerns and demand innovative approaches for their prompt detection. Current antimicrobial susceptibility tests (AST) rely on the growth of the organisms which takes 1-2 days for fast-growing organisms and several weeks for slow growing organisms. Here, we show for the first time the utility of the SYBR Green I/propidium iodide (PI) viability assay for rapidly identifying antibiotic resistance in less than 30 min for major, antibiotic-resistant, fast-growing bacteria, such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae , and Acinetobacter baumannii for bactericidal and bacteriostatic agents and in 16 h for extremely rapid detection of drug resistance for isoniazid and pyrazinamide in slow-growing Mycobacterium tuberculosis ...
2018: Frontiers in Medicine
https://www.readbyqxmd.com/read/29773172/seleno-l-methionine-and-l-ascorbic-acid-differentiate-the-biological-activity-of-doxorubicin-and-its-metal-complexes-as-a-new-anticancer-drugs-candidate
#11
Marzena Matejczyk, Grzegorz Świderski, Renata Świsłocka, Stanisław Józef Rosochacki, Włodzimierz Lewandowski
The most important problems of anti-cancer therapy include the toxicity of the drugs applied to healthy cells and the multi-drug cells resistance to chemotherapeutics. One of the most commonly used anticancer drugs is doxorubicin (DOX) used to treat certain leukemias and non-Hodgkin's lymphomas, as well as bladder, breast, stomach, lung, ovarian, thyroid, multiple myeloma and other cancers. Preliminary studies showed that metal complex with DOX improve its cytostatic activity with changes in their molecular structure and distribution of electrons, resulting in a substantial change of its biological activity (including antitumor activity)...
July 2018: Journal of Trace Elements in Medicine and Biology
https://www.readbyqxmd.com/read/29764913/mapping-and-characterizing-n6-methyladenine-in-eukaryotic-genomes-using-single-molecule-real-time-sequencing
#12
Shijia Zhu, John Beaulaurier, Gintaras Deikus, Tao Wu, Maya Strahl, Ziyang Hao, Guanzheng Luo, James A Gregory, Andrew Chess, Chuan He, Andrew Xiao, Robert Sebra, Eric E Schadt, Gang Fang
N6-methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes, however, methods for high resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes...
May 15, 2018: Genome Research
https://www.readbyqxmd.com/read/29762781/the-tubr-centromere-complex-adopts-a-double-ring-segrosome-structure-in-type-iii-partition-systems
#13
Bárbara Martín-García, Alejandro Martín-González, Carolina Carrasco, Ana M Hernández-Arriaga, Rubén Ruíz-Quero, Ramón Díaz-Orejas, Clara Aicart-Ramos, Fernando Moreno-Herrero, María A Oliva
In prokaryotes, the centromere is a specialized segment of DNA that promotes the assembly of the segrosome upon binding of the Centromere Binding Protein (CBP). The segrosome structure exposes a specific surface for the interaction of the CBP with the motor protein that mediates DNA movement during cell division. Additionally, the CBP usually controls the transcriptional regulation of the segregation system as a cell cycle checkpoint. Correct segrosome functioning is therefore indispensable for accurate DNA segregation...
May 14, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29761109/mesenchymal-stem-cells-differentiate-to-endothelial-cells-using-recombinant-vascular-endothelial-growth-factor-a
#14
Mohsen Khaki, Ali Hatef Salmanian, Hamid Abtahi, Ali Ganji, Ghasem Mosayebi
Background: Vascular endothelial growth factor-A (VEGF-A), an endothelial cell-specific mitogen produced by various cell types, plays important roles in cell differentiation and proliferation. In this study we investigated the effect of recombinant VEGF-A on differentiation of mesenchymal stem cells (MSCs) to endothelial cells (ECs). Methods: VEGF-A was expressed in E. coli BL21 (DE3) and BL21 pLysS competent cells with the pET32a expression vector. Recombinant VEGF-A protein expression was verified by SDS-PAGE and western blotting...
April 2018: Reports of Biochemistry & Molecular Biology
https://www.readbyqxmd.com/read/29756604/alkaloids-nitric-oxide-and-nitrite-reductases-evolutionary-coupling-as-key-regulators-of-cellular-bioenergetics-with-special-relevance-to-the-human-microbiome
#15
George B Stefano, Richard M Kream
Typical alkaloids expressed by prokaryotic and eukaryotic cells are small heterocyclic compounds containing weakly basic nitrogen groups that are critically important for mediating essential biological activities. The prototype opiate alkaloid morphine represents a low molecular mass heterocyclic compound that has been evolutionarily fashioned from a relatively restricted role as a secreted antimicrobial phytoalexin into a broad spectrum regulatory molecule. As an essential corollary, positive evolutionary pressure has driven the development of a cognate 6-transmembrane helical (TMH) domain μ3 opiate receptor that is exclusively responsive to morphine and related opiate alkaloids...
May 14, 2018: Medical Science Monitor: International Medical Journal of Experimental and Clinical Research
https://www.readbyqxmd.com/read/29754246/a-rapid-combinatorial-approach-to-assembling-synthetic-prokaryotic-and-eukaryotic-protein-expression-vectors
#16
Rebecca Mullinax, Sarah E Johns, Denise Rhodes, Vivian Zhang, Nancy McKinney, Katherine A Felts, Carsten P Carstens, Peter Sheffield
Vector construction and gene cloning are ubiquitous techniques essential to all fields of biological and medical research. They are the first steps in many endeavors leading to expressing proteins to understand gene function and regulation. However, they can often be rate-limiting, particularly in multi-gene studies, due to the time and effort required to assemble gene constructs and to identify the optimal constructs for protein expression.The SureVector system was developed to address this by enabling the rapid and reliable assembly of multiple DNA modules into a recombinant plasmid containing a gene-of-interest (GOI)...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29753109/expression-of-glyceraldehyde-3-phosphate-dehydrogenase-on-the-surface-of-clostridium-perfringens-cells
#17
Nozomu Matsunaga, Haruka Shimizu, Kanako Fujimoto, Kanako Watanabe, Tsutomu Yamasaki, Naoya Hatano, Eiji Tamai, Seiichi Katayama, Yasuo Hitsumoto
During research to identify fibronectin (Fn)-binding proteins (Fbps) on the surface of Clostridium perfringens cells, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a candidate Fbp. GAPDH is a glycolytic enzyme found in a wide range of prokaryotes and eukaryotes. The Fn-binding activity of recombinant C. perfringens GAPDH (rGAPDH) was investigated using both ligand blotting analysis and enzyme-linked immunosorbent assay (ELISA). rGAPDH strongly bound plasminogen but not laminin or gelatin...
May 9, 2018: Anaerobe
https://www.readbyqxmd.com/read/29746549/circularly-permuted-variants-of-two-cg-specific-prokaryotic-dna-methyltransferases
#18
Pál Albert, Bence Varga, Nikolett Zsibrita, Antal Kiss
The highly similar prokaryotic DNA (cytosine-5) methyltransferases (C5-MTases) M.MpeI and M.SssI share the specificity of eukaryotic C5-MTases (5'-CG), and can be useful research tools in the study of eukaryotic DNA methylation and epigenetic regulation. In an effort to improve the stability and solubility of complementing fragments of the two MTases, genes encoding circularly permuted (CP) variants of M.MpeI and M.SssI were created, and cloned in a plasmid vector downstream of an arabinose-inducible promoter...
2018: PloS One
https://www.readbyqxmd.com/read/29746250/cellular-assays-for-studying-the-fe-s-cluster-containing-base-excision-repair-glycosylase-mutyh-and-homologs
#19
Chandrima Majumdar, Nicole N Nuñez, Alan G Raetz, Cindy Khuu, Sheila S David
Many DNA repair enzymes, including the human adenine glycosylase MUTYH, require iron-sulfur (Fe-S) cluster cofactors for DNA damage recognition and subsequent repair. MUTYH prokaryotic and eukaryotic homologs are a family of adenine (A) glycosylases that cleave A when mispaired with the oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (OG). Faulty OG:A repair has been linked to the inheritance of missense mutations in the MUTYH gene. These inherited mutations can result in the onset of a familial colorectal cancer disorder known as MUTYH-associated polyposis (MAP)...
2018: Methods in Enzymology
https://www.readbyqxmd.com/read/29745002/helicobacter-pylori-shows-asymmetric-and-polar-cell-divisome-assembly-associated-with-dna-replisome
#20
Mohammad Kamran, Priyanka Dubey, Vijay Verma, Santanu Dasgupta, Suman K Dhar
DNA replication and cell division are two fundamental processes in the life cycle of a cell. Majority of the prokaryotic cells undergo division by means of binary fission in coordination with replication of the genome. Both processes, specially their coordination, are poorly understood in Helicobacter pylori. Here, we studied the cell divisome assembly and the subsequent processes of membrane and peptidoglycan syntheses in the bacterium. To our surprise, we found the cell divisome assembly to be polar, as well-corroborated by the asymmetric membrane and peptidoglycan synthesis at the poles...
May 9, 2018: FEBS Journal
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