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https://www.readbyqxmd.com/read/28231254/rapid-generation-of-drug-resistance-alleles-at-endogenous-loci-using-crispr-cas9-indel-mutagenesis
#1
Jonathan J Ipsaro, Chen Shen, Eri Arai, Yali Xu, Justin B Kinney, Leemor Joshua-Tor, Christopher R Vakoc, Junwei Shi
Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors...
2017: PloS One
https://www.readbyqxmd.com/read/28230828/chromosomal-mutagenesis-in-human-somatic-cells-30-year-cytogenetic-monitoring-after-chornobyl-accident
#2
M A Pilinska, G M Shemetun, O V Shemetun, S S Dybsky, O B Dybska, O O Talan, L R Pedan, D А Kurinnyi
In the lecture we have generalized and analyzed the data of cytogenetic laboratory of National Research Center for Radiation Medicine of the National Academy of Medical Sciences of Ukraine on 30-year selective cytogenetic monitoring among the priority contingents of different ages exposed to radiation after Chornobyl accident in Ukraine. It is highlighted that not only targeted but also untargeted radiation-induced cytogenetic effects should be explored, especially in delayed terms following radiation exposure...
December 2016: Experimental Oncology
https://www.readbyqxmd.com/read/28230769/hypoallergenic-variant-of-the-major-egg-white-allergen-gal-d-1-produced-by-disruption-of-cysteine-bridges
#3
Pathum Dhanapala, Dulashi Withanage-Dona, Mimi L K Tang, Tim Doran, Cenk Suphioglu
Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis will allow the production of a hypoallergenic variant of the protein; Methods: Two cysteine residues at C192 and C210 in domain III of the protein were mutated to alanine using site-directed mutagenesis, to disrupt two separate cysteine-cysteine bridges...
February 21, 2017: Nutrients
https://www.readbyqxmd.com/read/28229167/mutation-in-a-highly-conserved-glycine-residue-in-strand-5b-of-plasminogen-activator-inhibitor-1-causes-polymerisation
#4
Takayuki Iwaki, Kotomi Nagahashi, Katsuhiro Takano, Katsue Suzuki-Inoue, Naohiro Kanayama, Kazuo Umemura, Tetsumei Urano
Serpinopathy is characterised as abnormal accumulation of serine protease inhibitors (SERPINs) in cells and results in clinical symptoms owing to lack of SERPIN function or excessive accumulation of abnormal SERPIN. We recently identified a patient with functional deficiency of plasminogen activator inhibitor-1 (PAI-1), a member of the SERPIN superfamily. The patient exhibited life-threatening bleeding tendencies, which have also been observed in patients with a complete deficiency in PAI-1. Sequence analysis revealed a homozygous single-nucleotide substitution from guanine to cytosine at exon 9, which changed amino acid residue 397 from glycine to arginine (c...
February 23, 2017: Thrombosis and Haemostasis
https://www.readbyqxmd.com/read/28228589/capsid-phosphorylation-state-and-hepadnavirus-virion-secretion
#5
Xiaojun Ning, Suresh H Basagoudanavar, Kuancheng Liu, Laurie Luckenbaugh, Duoqian Wei, Chunyan Wang, Bo Wei, Yingren Zhao, Taotao Yan, William Delaney, Jianming Hu
The C-terminal domain (CTD) of hepadnavirus core protein is involved in multiple steps of viral replication. In particular, CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging into immature nucleocapsids (NCs) and the early stage of viral DNA synthesis. For the avian hepadnavirus, the duck hepatitis B virus (DHBV), CTD is dephosphorylated subsequently to facilitate the late stage of viral DNA synthesis and to stabilize NCs containing mature viral DNA. The role of CTD phosphorylation in virion secretion, if any, has remained unclear...
February 22, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28228480/a-stable-but-reversible-integrated-surrogate-reporter-for-assaying-crispr-cas9-stimulated-homology-directed-repair
#6
Yahong Wen, Grace Liao, Thomas Pritchard, Ting-Ting Zhao, Jon P Connelly, Shondra M Pruett-Miller, Valerie Blanc, Nicholas O Davidson, Blair B Madison
The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) following aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway, or alternatively, for enhancing homology directed repair (HDR) to facilitate the generation of a specific mutation (or knock-in). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA (gRNA) activity...
February 22, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28225819/lipid-interactions-and-angle-of-approach-to-the-hiv-1-viral-membrane-of-broadly-neutralizing-antibody-10e8-insights-for-vaccine-and-therapeutic-design
#7
Adriana Irimia, Andreia M Serra, Anita Sarkar, Ronald Jacak, Oleksandr Kalyuzhniy, Devin Sok, Karen L Saye-Francisco, Torben Schiffner, Ryan Tingle, Michael Kubitz, Yumiko Adachi, Robyn L Stanfield, Marc C Deller, Dennis R Burton, William R Schief, Ian A Wilson
Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity...
February 22, 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/28225256/analysis-and-control-of-chain-mobility-in-protein-hydrogels
#8
Peter B Rapp, Ahmad K Omar, Jeff J Shen, Maren E Buck, Zhen-Gang Wang, David A Tirrell
Coiled-coil domains can direct the assembly of protein block copolymers into physically crosslinked, viscoelastic hydrogels. Here we describe the use of fluorescence recovery after photobleaching (FRAP) to probe chain mobility in reversible hydrogels assembled from engineered proteins bearing terminal coiled-coil domains. We show that chain mobility can be related to the underlying dynamics of the coiled-coil domains by application of a 3-state "hopping" model of chain migration. We further show that genetic programming allows the effective mobility of network chains to be varied 500-fold through modest changes in protein sequence...
February 22, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28223526/hypermutation-signature-reveals-a-slippage-and-realignment-model-of-translesion-synthesis-by-rev3-polymerase-in-cisplatin-treated-yeast
#9
Romulo Segovia, Yaoqing Shen, Scott A Lujan, Steven J M Jones, Peter C Stirling
Gene-gene or gene-drug interactions are typically quantified using fitness as a readout because the data are continuous and easily measured in high throughput. However, to what extent fitness captures the range of other phenotypes that show synergistic effects is usually unknown. Using Saccharomyces cerevisiae and focusing on a matrix of DNA repair mutants and genotoxic drugs, we quantify 76 gene-drug interactions based on both mutation rate and fitness and find that these parameters are not connected. Independent of fitness defects, we identified six cases of synthetic hypermutation, where the combined effect of the drug and mutant on mutation rate was greater than predicted...
February 21, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28223515/rational-design-of-adjuvants-targeting-the-c-type-lectin-mincle
#10
Alexiane Decout, Sandro Silva-Gomes, Daniel Drocourt, Sophie Barbe, Isabelle André, Francisco J Cueto, Thierry Lioux, David Sancho, Eric Pérouzel, Alain Vercellone, Jacques Prandi, Martine Gilleron, Gérard Tiraby, Jérôme Nigou
The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cell-mediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6'-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle...
February 21, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28223460/mechanism-of-h2s-oxidation-by-the-dissimilatory-perchlorate-reducing-microorganism-azospira-suillum-ps
#11
Misha G Mehta-Kolte, Dana Loutey, Ouwei Wang, Matthew D Youngblut, Christopher G Hubbard, Kelly M Wetmore, Mark E Conrad, John D Coates
The genetic and biochemical basis of perchlorate-dependent H2S oxidation (PSOX) was investigated in the dissimilatory perchlorate-reducing microorganism (DPRM) Azospira suillum PS (PS). Previously, it was shown that all known DPRMs innately oxidize H2S, producing elemental sulfur (S(o)). Although the process involving PSOX is thermodynamically favorable (ΔG°' = -206 kJ ⋅ mol(-1) H2S), the underlying biochemical and genetic mechanisms are currently unknown. Interestingly, H2S is preferentially utilized over physiological electron donors such as lactate or acetate although no growth benefit is obtained from the metabolism...
February 21, 2017: MBio
https://www.readbyqxmd.com/read/28223458/mutation-of-the-n-terminal-region-of-chikungunya-virus-capsid-protein-implications-for-vaccine-design
#12
Adam Taylor, Xiang Liu, Ali Zaid, Lucas Y H Goh, Jody Hobson-Peters, Roy A Hall, Andres Merits, Suresh Mahalingam
Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV...
February 21, 2017: MBio
https://www.readbyqxmd.com/read/28223379/mutations-in-blakpc-3-that-confer-ceftazidime-avibactam-resistance-encode-novel-kpc-3-variants-that-function-as-extended-spectrum-%C3%AE-lactamases
#13
Ghady Haidar, Cornelius J Clancy, Ryan K Shields, Binghua Hao, Shaoji Cheng, M Hong Nguyen
We identified four blaKPC-3 mutations in ceftazidime-avibactam resistant clinical Klebsiella pneumoniae isolates, corresponding to D179Y, T243M, D179Y/T243M, and EL165 KPC-3 variants. Using site-directed mutagenesis and transforming vectors into Escherichia coli, we conclusively demonstrated that mutant blaKPC-3 encoded enzymes that functioned as extended-spectrum β-lactamases; mutations directly conferred higher MICs of ceftazidime-avibactam MICs, and decreased MICs of carbapenems and other β-lactams. Impact was strongest for the D179Y mutant, highlighting the importance of the KPC Ω-loop...
February 21, 2017: Antimicrobial Agents and Chemotherapy
https://www.readbyqxmd.com/read/28223356/molecular-insights-into-hydrogen-peroxide-sensing-mechanism-of-the-metalloregulator-mntr-in-controlling-bacterial-resistance-to-oxidative-stresses
#14
Zhaoyuan Chen, Xinhui Wang, Fan Yang, Qingqing Hu, Huichun Tong, Xiuzhu Dong
Manganese contributes to anti-oxidative stress particularly in catalase-devoid bacteria, and DtxR family metalloregulators, through sensing cellular Mn(2+) content, regulate its homeostasis. Here, we show that metalloregulator MntR (So-MntR) functions dually as Mn(2+) and H2O2 sensors in mediating H2O2 resistance by an oral streptococcus. H2O2 disrupted So-MntR's binding to Mn(2+) transporter mntABC promoter, and induced disulfide-linked dimerization of the protein. Mass spectrometry identified Cys11-Cys156 and Cys11-Cys11 disulfide-linked peptides in H2O2 treated So-MntR...
February 21, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28222378/fetal-hemoglobin-is-much-less-prone-to-dna-cleavage-compared-to-the-adult-protein
#15
Sandeep Chakane, Tiago Matos, Karin Kettisen, Leif Bulow
Hemoglobin (Hb) is well protected inside the red blood cells (RBCs). Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA). Hb induced cleavage of supercoiled pDNA (sc pDNA) which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10µM of Hb in 12h...
February 12, 2017: Redox Biology
https://www.readbyqxmd.com/read/28220528/enzymatic-characterization-and-functional-implication-of-two-structurally-different-isocitrate-dehydrogenases-from-xylella-fastidiosa
#16
Peipei Lv, Wanggang Tang, Peng Wang, Zhengyu Cao, Guoping Zhu
Isocitrate dehydrogenase (IDH) is a key enzyme at the critical junction between the tricarboxylic acid cycle and the glyoxylate cycle. Most bacteria have only one IDH, while a few of bacteria contain two IDH isozymes. The coexistence of two different type IDHs in one organism was little known. Xylella fastidiosa is a nutritionally fastidious plant pathogen that contains two structurally different IDHs, an NAD(+) -dependent homodimeric IDH (diXfIDH) and an NADP(+) -dependent monomeric IDH (monoXfIDH). Kinetic characterization showed that diXfIDH displayed 206-fold preferences for NAD(+) over NADP(+) , while monoXfIDH showed 13800-fold preferences for NADP(+) over NAD(+) ...
February 21, 2017: Biotechnology and Applied Biochemistry
https://www.readbyqxmd.com/read/28220356/overexpression-of-line-1-retrotransposons-in-autism-brain
#17
Svitlana Shpyleva, Stepan Melnyk, Oleksandra Pavliv, Igor Pogribny, S Jill James
Long interspersed nuclear elements-1 (LINE-1 or L1) are mobile DNA sequences that are capable of duplication and insertion (retrotransposition) within the genome. Recently, retrotransposition of L1 was shown to occur within human brain leading to somatic mosaicism in hippocampus and cerebellum. Because unregulated L1 activity can promote genomic instability and mutagenesis, multiple mechanisms including epigenetic chromatin condensation have evolved to effectively repress L1 expression. Nonetheless, L1 expression has been shown to be increased in patients with Rett syndrome and schizophrenia...
February 20, 2017: Molecular Neurobiology
https://www.readbyqxmd.com/read/28219662/fumarase-deficiency-causes-protein-and-metabolite-succination-and-intoxicates-mycobacterium-tuberculosis
#18
Nadine Ruecker, Robert Jansen, Carolina Trujillo, Susan Puckett, Pradeepa Jayachandran, Gerardo G Piroli, Norma Frizzell, Henrik Molina, Kyu Y Rhee, Sabine Ehrt
Enzymes of central carbon metabolism are essential mediators of Mycobacterium tuberculosis (Mtb) physiology and pathogenicity, but are often perceived to lack sufficient species selectivity to be pursued as potential drug targets. Fumarase (Fum) is an enzyme of the canonical tricarboxylic acid cycle and is dispensable in many organisms. Transposon mutagenesis studies in Mtb, however, indicate that Fum is required for optimal growth. Here, we report the generation and characterization of a genetically engineered Mtb strain in which Fum expression is conditionally regulated...
February 9, 2017: Cell Chemical Biology
https://www.readbyqxmd.com/read/28219523/combinatorial-effect-of-mutagenesis-and-medium-component-optimization-on-bacillus-amyloliquefaciens-antifungal-activity-and-efficacy-in-eradicating-botrytis-cinerea
#19
Fatma Masmoudi, Saoussen Ben Khedher, Amel Kamoun, Nabil Zouari, Slim Tounsi, Mohamed Trigui
This work is directed towards Bacillus amyloliquefaciens strain BLB371 metabolite production for biocontrol of fungal phytopathogens. In order to maximise antifungal metabolite production by this strain, two approaches were combined: random mutagenesis and medium component optimization. After three rounds of mutagenesis, a hyper active mutant, named M3-7, was obtained. It produces 7 fold more antifungal metabolites (1800AU/mL) than the wild strain in MC medium. A hybrid design was applied to optimise a new medium to enhance antifungal metabolite production by M3-7...
April 2017: Microbiological Research
https://www.readbyqxmd.com/read/28219448/a-computational-protocol-to-evaluate-the-effects-of-protein-mutants-in-the-kinase-gatekeeper-position-on-the-binding-of-atp-substrate-analogues
#20
Valentina Romano, Tjaart A P de Beer, Torsten Schwede
BACKGROUND: The determination of specific kinase substrates in vivo is challenging due to the large number of protein kinases in cells, their substrate specificity overlap, and the lack of highly specific inhibitors. In the late 90s, Shokat and coworkers developed a protein engineering-based method addressing the question of identification of substrates of protein kinases. The approach was based on the mutagenesis of the gatekeeper residue within the binding site of a protein kinase to change the co-substrate specificity from ATP to ATP analogues...
February 20, 2017: BMC Research Notes
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