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https://www.readbyqxmd.com/read/28934591/combining-traditional-mutagenesis-with-new-high-throughput-sequencing-and-genome-editing-to-reveal-hidden-variation-in-polyploid-wheat
#1
Cristobal Uauy, Brande B H Wulff, Jorge Dubcovsky
Induced mutations have been used to generate novel variation for breeding purposes since the early 1900s. However, the combination of this old technology with the new capabilities of high-throughput sequencing has resulted in powerful reverse genetic approaches in polyploid crops. Sequencing genomes or exomes of large mutant populations can generate extensive databases of mutations for most genes. These mutant collections, together with genome editing, are being used in polyploid species to combine mutations in all copies of a gene (homoeologs), and to expose phenotypic variation that was previously hidden by functional redundancy among homoeologs...
September 20, 2017: Annual Review of Genetics
https://www.readbyqxmd.com/read/28932739/agonist-binding-to-chemosensory-receptors-a-systematic-bioinformatics-analysis
#2
Fabrizio Fierro, Eda Suku, Mercedes Alfonso-Prieto, Alejandro Giorgetti, Sven Cichon, Paolo Carloni
Human G-protein coupled receptors (hGPCRs) constitute a large and highly pharmaceutically relevant membrane receptor superfamily. About half of the hGPCRs' family members are chemosensory receptors, involved in bitter taste and olfaction, along with a variety of other physiological processes. Hence these receptors constitute promising targets for pharmaceutical intervention. Molecular modeling has been so far the most important tool to get insights on agonist binding and receptor activation. Here we investigate both aspects by bioinformatics-based predictions across all bitter taste and odorant receptors for which site-directed mutagenesis data are available...
2017: Frontiers in Molecular Biosciences
https://www.readbyqxmd.com/read/28932265/removal-of-glucuronic-acid-from-xylan-is-a-strategy-to-improve-the-conversion-of-plant-biomass-to-sugars-for-bioenergy
#3
Jan J Lyczakowski, Krzysztof B Wicher, Oliver M Terrett, Nuno Faria-Blanc, Xiaolan Yu, David Brown, Kristian B R M Krogh, Paul Dupree, Marta Busse-Wicher
BACKGROUND: Plant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and biochemicals. The recalcitrance of this plant material is one of the major obstacles in its conversion into sugars. Biomass is primarily composed of secondary cell walls, which is made of cellulose, hemicelluloses and lignin. Xylan, a hemicellulose, binds to the cellulose microfibril and is hypothesised to form an interface between lignin and cellulose. Both softwood and hardwood xylan carry glucuronic acid side branches...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28932220/chalcone-attenuates-staphylococcus-aureus-virulence-by-targeting-sortase-a-and-alpha-hemolysin
#4
Bing Zhang, Zihao Teng, Xianhe Li, Gejin Lu, Xuming Deng, Xiaodi Niu, Jianfeng Wang
Staphylococcus aureus (S.aureus) resistance, considered a dilemma for the clinical treatment of this bacterial infection, is becoming increasingly intractable. Novel anti-virulence strategies will undoubtedly provide a path forward in combating these resistant bacterial infections. Sortase A (SrtA), an enzyme responsible for anchoring virulence-related surface proteins, and alpha-hemolysin (Hla), a pore-forming cytotoxin, have aroused great scientific interest, as they have been regarded as targets for promising agents against S...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28931206/metabolic-engineering-a-yeast-to-produce-astaxanthin
#5
Yu-Ju Lin, Jui-Jen Chang, Hao-Yeh Lin, Caroline Thia, Yi-Ying Kao, Chieh-Chen Huang, Wen-Hsiung Li
In this study, an astaxanthin-biosynthesis Kluyveromyces marxianus strain Sm23 was first constructed, which could produce 31µg/g DCW astaxanthin. Then, repeated genome integration of the key astaxanthin biosynthesis genes Hpchyb and bkt was done to increase gene copy number and astaxanthin yield. Four improved strains were obtained and the yield of astaxanthin and the total yield of carotenoids in a strain increased with the copy numbers of Hpchyb and bkt. To improve the yield further, the gene Hpchyb from Haematococcus pluvialis was modified by site-directed mutagenesis to increase the enzyme efficiency or/and to prevent the heterologous protein degradation by ubiquitination...
July 23, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28928732/facile-affinity-maturation-of-antibody-variable-domains-using-natural-diversity-mutagenesis
#6
Kathryn E Tiller, Ratul Chowdhury, Tong Li, Seth D Ludwig, Sabyasachi Sen, Costas D Maranas, Peter M Tessier
The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/28928454/a-comprehensive-characterization-of-pnca-polymorphisms-that-confer-resistance-to-pyrazinamide
#7
Adam N Yadon, Kashmeel Maharaj, John H Adamson, Yi-Pin Lai, James C Sacchettini, Thomas R Ioerger, Eric J Rubin, Alexander S Pym
Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being threatened by emerging drug resistance. Resistance is mediated through mutations in the bacterial gene pncA. Methods for testing pyrazinamide susceptibility are difficult and rarely performed, and this means that the full spectrum of pncA alleles that confer clinical resistance to pyrazinamide is unknown. Here, we performed in vitro saturating mutagenesis of pncA to generate a comprehensive library of PncA polymorphisms resultant from a single-nucleotide polymorphism...
September 19, 2017: Nature Communications
https://www.readbyqxmd.com/read/28928381/crispr-cas9-induced-targeted-mutagenesis-and-gene-replacement-to-generate-long-shelf-life-tomato-lines
#8
Qing-Hui Yu, Baike Wang, Ning Li, Yaping Tang, Shengbao Yang, Tao Yang, Juan Xu, Chunmiao Guo, Peng Yan, Qiang Wang, Patiguli Asmutola
Quickly and precisely gain genetically enhanced breeding elites with value-adding performance traits is desired by the crop breeders all the time. The present of gene editing technologies, especially the CRISPR/Cas9 system with the capacities of efficiency, versatility and multiplexing provides a reasonable expectation towards breeding goals. For exploiting possible application to accelerate the speed of process at breeding by CRISPR/Cas9 technology, in this study, the Agrobacterium tumefaciens-mediated CRISPR/Cas9 system transformation method was used for obtaining tomato ALC gene mutagenesis and replacement, in absence and presence of the homologous repair template...
September 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28927827/crispr-cas9-mediated-blos2-knockout-resulting-in-disappearance-of-yellow-strips-and-white-spots-on-the-larval-integument-in-spodoptera-litura
#9
Guan-Heng Zhu, Ying-Chuan Peng, Mei-Yan Zheng, Xiao-Qing Zhang, Jia-Bin Sun, Yongping Huang, Shuang-Lin Dong
The custom-design bacterial CRISPR/Cas9 system has been recently used in some insects, indicating a powerful technique for studies on gene function and transgenic insects. However, its use in lepidopteran pests is scarce. Here, we reported a CRISPR/Cas9 system mediated mutagenesis of biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene in a noctuid pest Spodoptera litura. A fragment of SlitBLOS2 was identified by analyzing a S. litura transcriptome database by local basic BLAST, and the full length cDNA was acquired by RACE strategy...
September 16, 2017: Journal of Insect Physiology
https://www.readbyqxmd.com/read/28926242/structure-function-studies-of-artemisia-tridentata-farnesyl-diphosphate-synthase-and-chrysanthemyl-diphosphate-synthase-by-site-directed-mutagenesis-and-morphogenesis
#10
J Scott Lee, Jian-Jung Pan, Gurusankar Ramamoorthy, C Dale Poulter
The amino acid sequences of farnesyl diphosphate synthase (FPPase) and chrysanthemyl diphosphate synthase (CPPase) from Artemisia tridentata ssp. Spiciformis, minus their chloroplast targeting regions, are 71% identical and 90% similar. FPPase efficiently and selectively synthesizes the "regular" sesquiterpenoid farnesyl diphosphate (FPP) by coupling isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) and then to geranyl diphosphate (GPP). In contrast, CPPase is an inefficient promiscuous enzyme, which synthesizes the "irregular" monoterpenes chrysanthemyl diphosphate (CPP), lavandulyl diphosphate (LPP), and trace quantities of maconelliyl diphosphate (MPP) from two molecules of DMAPP, in addition to coupling IPP to DMAPP...
September 19, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28926145/a-crispr-reimagining-new-twists-and-turns-of-crispr-beyond-the-genome-engineering-revolution
#11
Robert J Plummer, Yi Guo, Ying Peng
Despite its explosive applications in genome engineering, CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) has been developed into a versatile tool beyond its well-known nuclease function. In this prospect article, we summarize a few exciting "off-label" applications of CRISPR including manipulating DNA sequences, visualizing chromosomal loci in living cells, and modulating transcription and chromatin structures. These novel applications will likely elevate CRISPR tools into yet another level of sophistication and diversity, leading to many more exciting cell biological discoveries...
September 19, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/28925012/a-new-negative-allosteric-modulator-ap14145-for-the-study-of-small-conductance-calcium-activated-potassium-channels
#12
Rafel Simó-Vicens, Jeppe E Kirchhoff, Bernardo Dolce, Lea Abildgaard Jensen, Tobias Speerschneider, Ulrik S Sørensen, Morten Grunnet, Jonas G Diness, Bo H Bentzen
BACKGROUND AND PURPOSE: Small conductance Ca(2+) -activated K(+) (KCa 2) channels represent a promising atrial-selective target for treatment of atrial fibrillation (AF). Here, we establish the mechanism of KCa 2 inhibition by the new compound AP14145. EXPERIMENTAL APPROACH: Using site directed mutagenesis binding determinants for AP14145 inhibition were explored. AP14145 selectivity and mechanism of action were investigated by patch clamp recordings of heterologously expressed KCa 2 channels...
September 19, 2017: British Journal of Pharmacology
https://www.readbyqxmd.com/read/28924866/keratinolytic-protease-a-green-biocatalyst-for-leather-industry
#13
REVIEW
Zhen Fang, Yang-Chun Yong, Juan Zhang, Guocheng Du, Jian Chen
Depilation/unhairing is the crucial but heavy pollution process in leather industry. Traditional inorganic sulfide treatment was the most widely used depilation technique in the past decades, which was usually detrimental to leather quality and resulted in serious environmental pollution. Using biocatalysts to substitute inorganic sulfide showed great advantages in environment protection and unhairing efficiency. Keratinolytic protease is one of the excellent biocatalysts to hydrolyze disulfide bond-rich proteins of hair and has little damage to leather...
September 18, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28924049/specific-electrostatic-interactions-between-charged-amino-acid-residues-regulate-binding-of-von-willebrand-factor-to-blood-platelets
#14
Gianluca Interlandi, Olga Yakovenko, An-Yue Tu, Jeff Harris, Jennie Le, Junmei Chen, José A López, Wendy E Thomas
The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet binding A1 domain of VWF is connected to the VWF's N-terminally located D'D3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ibα (GpIbα). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated...
September 18, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28924009/site-specific-regulation-of-p2x7-receptor-function-in-microglia-gates-morphine-analgesic-tolerance
#15
Heather Leduc-Pessah, Nicholas L Weilinger, Churmy Y Fan, Nicole E Burma, Roger J Thompson, Tuan Trang
Tolerance to the analgesic effects of opioids is a major problem in chronic pain management. Microglia are implicated in opioid tolerance, but the core mechanisms regulating their response to opioids remain obscure. By selectively ablating microglia in the spinal cord using a saporin-conjugated antibody to Mac1, we demonstrate a causal role for microglia in the development, but not maintenance, of morphine tolerance in male rats. Increased P2X7 receptor (P2X7R) activity is a cardinal feature of microglial activation, and in this study we found that morphine potentiates P2X7R-mediated Ca(2+) responses in resident spinal microglia acutely isolated from morphine tolerant rats...
September 18, 2017: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
https://www.readbyqxmd.com/read/28923954/macroevolutionary-shifts-of-wnta-function-potentiate-butterfly-wing-pattern-diversity
#16
Anyi Mazo-Vargas, Carolina Concha, Luca Livraghi, Darli Massardo, Richard W R Wallbank, Linlin Zhang, Joseph D Papador, Daniel Martinez-Najera, Chris D Jiggins, Marcus R Kronforst, Casper J Breuker, Robert D Reed, Nipam H Patel, W Owen McMillan, Arnaud Martin
Butterfly wing patterns provide a rich comparative framework to study how morphological complexity develops and evolves. Here we used CRISPR/Cas9 somatic mutagenesis to test a patterning role for WntA, a signaling ligand gene previously identified as a hotspot of shape-tuning alleles involved in wing mimicry. We show that WntA loss-of-function causes multiple modifications of pattern elements in seven nymphalid butterfly species. In three butterflies with a conserved wing-pattern arrangement, WntA is necessary for the induction of stripe-like patterns known as symmetry systems and acquired a novel eyespot activator role specific to Vanessa forewings...
September 18, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28923929/exceptionally-tight-membrane-binding-may-explain-the-key-role-of-the-synaptotagmin-7-c2a-domain-in-asynchronous-neurotransmitter-release
#17
Rashmi Voleti, Diana R Tomchick, Thomas C Südhof, Josep Rizo
Synaptotagmins (Syts) act as Ca(2+) sensors in neurotransmitter release by virtue of Ca(2+)-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca(2+)-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca(2+)-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca(2+)-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1...
September 18, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28923910/a-transposon-screen-identifies-loss-of-primary-cilia-as-a-mechanism-of-resistance-to-smo-inhibitors
#18
Xuesong Zhao, Ekaterina Pak, Kimberly J Ornell, Maria F Pazyra-Murphy, Ethan L MacKenzie, Emily J Chadwick, Tatyana Ponomaryov, Joseph F Kelleher, Rosalind A Segal
Drug resistance poses a great challenge to targeted cancer therapies. In Hedgehog pathway-dependent cancers, the scope of mechanisms enabling resistance to Smo-inhibitors is not known. Here, we performed a transposon mutagenesis screen in medulloblastoma and identified multiple modes of resistance. Surprisingly, mutations in ciliogenesis genes represent a frequent cause of resistance, and patient datasets indicate that cilia loss constitutes a clinically relevant category of resistance. Conventionally, primary cilia are thought to enable oncogenic Hedgehog signaling...
September 18, 2017: Cancer Discovery
https://www.readbyqxmd.com/read/28923894/h-ipse-is-a-pathogen-secreted-host-nucleus-infiltrating-protein-infiltrin-expressed-exclusively-by-the-schistosoma-haematobium-egg-stage
#19
Luke F Pennington, Abdulaziz Alouffi, Evaristus C Mbanefo, Debalina Ray, David M Heery, Theodore S Jardetzky, Michael H Hsieh, Franco H Falcone
Urogenital schistosomiasis, caused by the parasitic trematode Schistosoma haematobium, affects over 112 million people worldwide. As with S. mansoni infections, the pathology in urogenital schistosomiasis is mainly related to the egg stage, which induces granulomatous inflammation of affected tissues. Schistosoma eggs and their secretions have been studied extensively for the related S. mansoni organism which is more amenable to laboratory studies. Indeed, we have shown that IPSE/alpha-1 (M-IPSE herein), a major protein secreted from S ...
September 18, 2017: Infection and Immunity
https://www.readbyqxmd.com/read/28923662/selecting-of-a-cytochrome-p450cam-sesam-library-with-3-chloroindole-and-endosulfan-identification-of-mutants-that-dehalogenate-3-chloroindole
#20
Shaima Kammoonah, Brinda Prasad, Priyadarshini Balaraman, Hemanshu Mundhada, Ulrich Schwaneberg, Erika Plettner
Cytochrome P450cam (a camphor hydroxylase) from the soil bacterium Pseudomonas putida shows potential importance in environmental applications such as the degradation of chlorinated organic pollutants. Seven P450cam mutants generated from Sequence Saturation Mutagenesis (SeSaM) and isolated by selection on minimal media with either 3-chloroindole or the insecticide endosulfan were studied for their ability to oxidize of 3-chloroindole to isatin. The wild-type enzyme did not accept 3-chloroindole as a substrate...
September 15, 2017: Biochimica et Biophysica Acta
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