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https://www.readbyqxmd.com/read/29333573/simultaneous-site-directed-mutagenesis-of-duplicated-loci-in-soybean-using-a-single-guide-rna
#1
Yuhei Kanazashi, Aya Hirose, Ippei Takahashi, Masafumi Mikami, Masaki Endo, Sakiko Hirose, Seiichi Toki, Akito Kaga, Ken Naito, Masao Ishimoto, Jun Abe, Tetsuya Yamada
Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD)...
January 15, 2018: Plant Cell Reports
https://www.readbyqxmd.com/read/29332217/a-novel-glucuronoyl-esterase-from-aspergillus-fumigatus-the-role-of-conserved-lys-residue-in-the-preference-for-4-o-methyl-glucuronoyl-esters
#2
Hung Hiep Huynh, Nozomi Ishii, Ichiro Matsuo, Manabu Arioka
Cellulose in plant cell walls is mainly covered by hemicellulose and lignin, and thus efficient removal of these components is thought to be a key step in the optimal utilization of lignocellulose. The recently discovered carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) which cleave the linkages between the free carboxyl group of D-glucuronic acid in hemicellulose and the benzyl groups in lignin residues could contribute to this process. Herein, we report the identification, functional expression, and enzymatic characterization of a GE, AfGE, from the filamentous fungus Aspergillus fumigatus...
January 14, 2018: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29332168/efficient-crispr-cas9-based-genome-editing-in-carrot-cells
#3
Magdalena Klimek-Chodacka, Tomasz Oleszkiewicz, Levi G Lowder, Yiping Qi, Rafal Baranski
The first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot-a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome...
January 13, 2018: Plant Cell Reports
https://www.readbyqxmd.com/read/29331223/structure-based-design-and-application-of-a-nucleotide-coenzyme-mimetic-ligand-application-to-the-affinity-purification-of-nucleotide-dependent-enzymes
#4
Marigianna Marinou, Dimitrios Platis, Farid S Ataya, Evangelia Chronopoulou, Dimitrios Vlachakis, Nikolaos E Labrou
In the present study, a structure-based approach was exploited for the in silico design of a nucleotide coenzyme mimetic ligand. The enzyme formate dehydrogenase (FDH) was employed as a model in our study. The biomimetic ligand was designed and synthesized based on a tryptamine/3-aminopropylphosphonic acid bi-substituted 1,3,5-triazine (Trz) scaffold (Tra-Trz-3APP), which potentially mimics the interactions of NAD+-FDH complex. Molecular docking studies of the biomimetic ligand predicted that it can occupy the same binding site as the natural coenzyme...
January 3, 2018: Journal of Chromatography. A
https://www.readbyqxmd.com/read/29331085/verification-of-dna-motifs-in-arabidopsis-using-crispr-cas9-mediated-mutagenesis
#5
Chenlong Li, Chen Chen, Huhui Chen, Suikang Wang, Xuemei Chen, Yuhai Cui
Transcription factors (TFs) and chromatin-modifying factors (CMFs) access chromatin by recognizing specific DNA motifs in their target genes. Chromatin Immunoprecipitation followed by next generation sequencing (ChIP-seq) has been widely used to discover the potential DNA binding motifs for both TFs and CMFs. Yet, an in vivo method for verifying DNA motifs captured by ChIP-seq is lacking in plants. Here, we describe the use of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) to verify DNA motifs in their native genomic context in Arabidopsis...
January 13, 2018: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/29331077/optimized-paired-sgrna-cas9-cloning-and-expression-cassette-triggers-high-efficiency-multiplex-genome-editing-in-kiwifruit
#6
Zupeng Wang, Shuaibin Wang, Dawei Li, Qiong Zhang, Li Li, Caihong Zhong, Yifei Liu, Hongwen Huang
Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing...
January 13, 2018: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/29330525/distinct-modulation-of-inactivation-by-a-residue-in-the-pore-domain-of-voltage-gated-na-channels-mechanistic-insights-from-recent-crystal-structures
#7
Rene Cervenka, Peter Lukacs, Vaibhavkumar S Gawali, Song Ke, Xaver Koenig, Lena Rubi, Touran Zarrabi, Karlheinz Hilber, Walter Sandtner, Anna Stary-Weinzinger, Hannes Todt
Inactivation of voltage-gated Na+ channels (VGSC) is essential for the regulation of cellular excitability. The molecular rearrangement underlying inactivation is thought to involve the intracellular linker between domains III and IV serving as inactivation lid, the receptor for the lid (domain III S4-S5 linker) and the pore-lining S6 segements. To better understand the role of the domain IV S6 segment in inactivation we performed a cysteine scanning mutagenesis of this region in rNav 1.4 channels and screened the constructs for perturbations in the voltage-dependence of steady state inactivation...
January 12, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29327725/whole-genome-sequencing-reveals-principles-of-brain-retrotransposition-in-neurodevelopmental-disorders
#8
Jasmine Jacob-Hirsch, Eran Eyal, Binyamin A Knisbacher, Jonathan Roth, Karen Cesarkas, Chen Dor, Sarit Farage-Barhom, Vered Kunik, Amos J Simon, Moran Gal, Michal Yalon, Sharon Moshitch-Moshkovitz, Rick Tearle, Shlomi Constantini, Erez Y Levanon, Ninette Amariglio, Gideon Rechavi
Neural progenitor cells undergo somatic retrotransposition events, mainly involving L1 elements, which can be potentially deleterious. Here, we analyze the whole genomes of 20 brain samples and 80 non-brain samples, and characterized the retrotransposition landscape of patients affected by a variety of neurodevelopmental disorders including Rett syndrome, tuberous sclerosis, ataxia-telangiectasia and autism. We report that the number of retrotranspositions in brain tissues is higher than that observed in non-brain samples and even higher in pathologic vs normal brains...
January 12, 2018: Cell Research
https://www.readbyqxmd.com/read/29327289/study-of-adenylyl-cyclase-g%C3%AE-s-interactions-and-identification-of-novel-ac-ligands
#9
Appalaraju Jaggupilli, Premnath Dhanaraj, Alexander Pritchard, John L Sorensen, Shyamala Dakshinamurti, Prashen Chelikani
Adenylyl cyclases (ACs) are membrane bound enzymes that catalyze the production of cAMP from ATP in response to the activation by G-protein Gαs. Different isoforms of ACs are ubiquitously expressed in different tissues involved in regulatory mechanisms in response to specific stimulants. There are 9 AC isoforms present in humans, with AC5 and AC6 proposed to play a vital role in cardiac functions. The activity of AC6 is sensitive to nitric oxide, such that nitrosylation of the protein might regulate its function...
January 11, 2018: Molecular and Cellular Biochemistry
https://www.readbyqxmd.com/read/29327130/main-steps-in-dna-double-strand-break-repair-an-introduction-to-homologous-recombination-and-related-processes
#10
REVIEW
Lepakshi Ranjha, Sean M Howard, Petr Cejka
DNA double-strand breaks arise accidentally upon exposure of DNA to radiation and chemicals or result from faulty DNA metabolic processes. DNA breaks can also be introduced in a programmed manner, such as during the maturation of the immune system, meiosis, or cancer chemo- or radiotherapy. Cells have developed a variety of repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly, vegetative cells employ mechanisms that restore the integrity of broken DNA with the highest efficiency at the lowest cost of mutagenesis...
January 11, 2018: Chromosoma
https://www.readbyqxmd.com/read/29326437/insertional-mutagenesis-in-a-her2-positive-breast-cancer-model-reveals-eras-as-a-driver-of-cancer-and-therapy-resistance
#11
Gerjon J Ikink, Mandy Boer, Elvira R M Bakker, Annabel Vendel-Zwaagstra, Chris Klijn, Jelle Ten Hoeve, Jos Jonkers, Lodewyk F Wessels, John Hilkens
Personalized medicine for cancer patients requires a deep understanding of the underlying genetics that drive cancer and the subsequent identification of predictive biomarkers. To discover new genes and pathways contributing to oncogenesis and therapy resistance in HER2+ breast cancer, we performed Mouse Mammary Tumor Virus (MMTV)-induced insertional mutagenesis screens in ErbB2/cNeu-transgenic mouse models. The screens revealed 34 common integration sites (CIS) in mammary tumors of MMTV-infected mice, highlighting loci with multiple independent MMTV integrations in which potential oncogenes are activated, most of which had never been reported as MMTV CIS...
January 12, 2018: Oncogene
https://www.readbyqxmd.com/read/29326169/dual-cyclooxygenase-fatty-acid-amide-hydrolase-inhibitor-exploits-novel-binding-interactions-in-the-cyclooxygenase-active-site
#12
Michael C Goodman, Shu Xu, Carol A Rouzer, Surajit Banerjee, Kebreab Ghebreselasie, Marco Migliore, Daniele Piomelli, Lawrence J Marnett
The cyclooxygenases COX-1 and COX-2 oxygenate arachidonic acid (AA) to prostaglandin H2 (PGH2). COX-2 also oxygenates the endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA), to the corresponding PGH2 analogs. Both enzymes are targets of nonsteroidal anti-inflammatory drugs (NSAIDs), but NSAID-mediated COX inhibition is associated with gastrointestinal toxicity. One potential strategy to counter this toxicity is to also inhibit fatty acid amide hydrolase (FAAH), which hydrolyzes bioactive fatty acid ethanolamides (FAEs) into fatty acids and ethanolamine...
January 11, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29325885/9-aminoacridine-based-agents-impair-the-bovine-viral-diarrhea-virus-bvdv-replication-targeting-the-rna-dependent-rna-polymerase-rdrp
#13
Roberta Loddo, Valeria Francesconi, Erik Laurini, Silvia Boccardo, Suzana Aulic, Maurizio Fermeglia, Sabrina Pricl, Michele Tonelli
Bovine viral diarrhea virus (BVDV) infection is still a plague that causes important livestock pandemics. Despite the availability of vaccines against BVDV, and the implementation of massive eradication or control programs, this virus still constitutes a serious agronomic burden. Therefore, the alternative approach to combat Pestivirus infections, based on the development of antiviral agents that specifically inhibit the replication of these viruses, is of preeminent actuality and importance. Capitalizing from a long-standing experience in antiviral drug design and development, in this work we present and characterize a series of small molecules based on the 9-aminoacridine scaffold that exhibit potent anti-BVDV activity coupled with low cytotoxicity...
January 4, 2018: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/29325795/insertional-mutagenesis-confounds-the-mechanism-of-the-morbid-phenotype-of-a-plnr9c-transgenic-mouse-line
#14
Alexander Kraev
BACKGROUND: A mouse line with heterozygous transgenic expression of phospholamban carrying a substitution of cysteine for arginine 9 (TgPLNR9C) under the control of α-myosin heavy chain (αMHC) promoter features dilated cardiomyopathy, heart failure and premature death. METHODS AND RESULTS: Determination of transgene chromosomal localization by conventional methods shows that in this line the transgenic array of 13 PLNR9C expression cassettes, arranged in a head-to-tail tandem orientation, has integrated into the bi-directional promoter of the αMHC (Myh6) gene and the gene for the regulatory non-coding RNA Myheart (Mhrt), both of which are known to be involved in cardiac development and pathology...
January 8, 2018: Journal of Cardiac Failure
https://www.readbyqxmd.com/read/29325769/the-structural-determinants-of-the-bitopic-binding-mode-of-a-negative-allosteric-modulator-of-the-dopamine-d2-receptor
#15
Christopher J Draper-Joyce, Mayako Michino, Ravi Kumar Verma, Carmen Klein Herenbrink, Jeremy Shonberg, Anitha Kopinathan, Peter J Scammells, Ben Capuano, David M Thal, Jonathan A Javitch, Arthur Christopoulos, Lei Shi, J Robert Lane
SB269652 is a negative allosteric modulator of the dopamine D2 receptor (D2R) yet possesses structural similarity to ligands with a competitive mode of interaction. In this study, we aimed to understand the ligand-receptor interactions that confer its allosteric action. We combined site-directed mutagenesis with molecular dynamics simulations using both SB269652 and derivatives from our previous structure activity studies. We identify residues within the conserved orthosteric binding site (OBS) and a secondary binding pocket (SBP) that determine affinity and cooperativity...
January 8, 2018: Biochemical Pharmacology
https://www.readbyqxmd.com/read/29325723/double-function-hydroperoxide-lyases-epoxyalcohol-synthases-cyp74c-of-higher-plants-identification-and-conversion-into-allene-oxide-synthases-by-site-directed-mutagenesis
#16
Yana Y Toporkova, Svetlana S Gorina, Elena K Bessolitsyna, Elena O Smirnova, Valeria S Fatykhova, Fredi Brühlmann, Tatiana M Ilyina, Lucia S Mukhtarova, Alexander N Grechkin
The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(SS)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids...
January 8, 2018: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29325088/the-comet-assay-ready-for-30-more-years
#17
Peter Møller
During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a comet assay guideline for in vivo testing of DNA strand breaks in animals. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay...
January 6, 2018: Mutagenesis
https://www.readbyqxmd.com/read/29324969/apobec-mutagenesis-in-drug-resistance-and-immune-escape-in-hiv-and-cancer-evolution
#18
S Venkatesan, R Rosenthal, N Kanu, N McGranahan, J Bartek, S A Quezada, J Hare, R S Harris, C Swanton
The APOBEC mutational signature has only recently been detected in a multitude of cancers through next-generation sequencing. In contrast, APOBEC has been a focus of virology research for over a decade. Many lessons learnt regarding APOBEC within virology are likely to be applicable to cancer. In this review, we explore the parallels between the role of APOBEC enzymes in HIV and cancer evolution. We discuss data supporting the role of APOBEC mutagenesis in creating HIV genome heterogeneity, drug resistance, and immune escape variants...
January 8, 2018: Annals of Oncology: Official Journal of the European Society for Medical Oncology
https://www.readbyqxmd.com/read/29324742/functional-characterization-of-adaptive-variation-within-a-cis-regulatory-element-influencing-drosophila-melanogaster-growth
#19
Amanda Glaser-Schmitt, John Parsch
Gene expression variation is a major contributor to phenotypic diversity within species and is thought to play an important role in adaptation. However, examples of adaptive regulatory polymorphism are rare, especially those that have been characterized at both the molecular genetic level and the organismal level. In this study, we perform a functional analysis of the Drosophila melanogaster CG9509 enhancer, a cis-regulatory element that shows evidence of adaptive evolution in populations outside the species' ancestral range in sub-Saharan Africa...
January 11, 2018: PLoS Biology
https://www.readbyqxmd.com/read/29323742/a-molecular-dynamics-investigation-into-the-mechanisms-of-alectinib-resistance-of-three-alk-mutants
#20
Muyang He, Weikang Li, Qingchuan Zheng, Hongxing Zhang
Alectinib, a highly selective next-genetation anaplastic lymphoma kinase (ALK) inhibitor, has demonstrated promising antitumor activity in patients with ALK-positive non-small cell lung carcinomas (NSCLC). However, the therapeutic benefits of alectinib is inescapably hampered by the development of acquired resistant mutations in ALK. Despite the availability of ample experimental mutagenesis data, the molecular origin and the structural motifs under alectinib binding affinity deficiencies are still ambiguous...
January 11, 2018: Journal of Cellular Biochemistry
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