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https://www.readbyqxmd.com/read/29332168/efficient-crispr-cas9-based-genome-editing-in-carrot-cells
#1
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/29323243/expression-of-cry2ah1-and-two-domain-ii-mutants-in-transgenic-tobacco-confers-high-resistance-to-susceptible-and-cry1ac-resistant-cotton-bollworm
#2
Shengyan Li, Zeyu Wang, Yiyao Zhou, Changhui Li, Guiping Wang, Hai Wang, Jie Zhang, Gemei Liang, Zhihong Lang
To improve the novel Bacillus thuringiensis insecticidal gene cry2Ah1 toxicity, two mutants cry2Ah1-vp (V354VP) and cry2Ah1-sp (V354SP) were performed. SWISS-MODEL analysis showed two mutants had a longer loop located between β-4 and β-5 of domain II, resulting in higher binding affinity with brush border membrane vesicles (BBMV) of Helicoverpa armigera comparing with Cry2Ah1. The cry2Ah1, cry2Ah1-vp, and cry2Ah1-sp were optimized codon usage according to plant codon bias, and named mcry2Ah1, mcry2Ah1-vp, and mcry2Ah1-sp...
January 11, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29317625/click-beetle-luciferase-mutant-and-near-infrared-naphthyl-luciferins-for-improved-bioluminescence-imaging
#3
Mary P Hall, Carolyn C Woodroofe, Monika G Wood, Ivo Que, Moniek Van't Root, Yanto Ridwan, Ce Shi, Thomas A Kirkland, Lance P Encell, Keith V Wood, Clemens Löwik, Laura Mezzanotte
The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain...
January 9, 2018: Nature Communications
https://www.readbyqxmd.com/read/29313103/plant-synthetic-gp4-and-gp5-proteins-from-porcine-reproductive-and-respiratory-syndrome-virus-elicit-immune-responses-in-pigs
#4
Chul Han An, Salik Nazki, Sung-Chul Park, Yu Jeong Jeong, Ju Huck Lee, Su-Jin Park, Amina Khatun, Won-Il Kim, Youn-Il Park, Jae Cheol Jeong, Cha Young Kim
We demonstrated successful overexpression of porcine reproductive and respiratory syndrome virus (PRRSV)-derived GP4D and GP5D antigenic proteins in Arabidopsis. Pigs immunized with transgenic plants expressing GP4D and GP5D proteins generated both humoral and cellular immune responses to PRRSV. Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, the most economically significant disease affecting the swine industry worldwide. However, current commercial PRRSV vaccines (killed virus or modified live vaccines) show poor efficacy and safety due to concerns such as reversion of virus to wild type and lack of cross protection...
January 8, 2018: Planta
https://www.readbyqxmd.com/read/29305178/high-yield-production-of-fungal-manganese-peroxidases-by-e-coli-through-soluble-expression-and-examination-of-the-activities
#5
Meng-I Lin, Takashi Nagata, Masato Katahira
Oxidative enzymes of white-rot fungi play a key role in lignin biodegradation. Among those fungus, Ceriporiopsis subvermispora degrades lignin before cellulose in wood; C. subvermispora is the only fungus that secretes all known types of manganese peroxidases (CsMnPs). Utilization of lignin-degrading peroxidases has been limited so far due to the lack of efficient preparation methods and intensive characterization. In this study, we developed a highly efficient method to prepare active CsMnPs through soluble expression by E...
January 2, 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/29275953/translational-research-to-enable-personalized-treatment-of-cystic-fibrosis
#6
Marne C Hagemeijer, Daniel J Siegwart, Lisa J Strug, Liudmila Cebotaru, Michael J Torres, Aderonke Sofoluwe, Jeffrey M Beekman
Translational research efforts in cystic fibrosis (CF) aim to develop therapies for all subjects with CF. To reach this goal new therapies need to be developed that target multiple aspects of the disease. To enable individuals to benefit maximally from these treatments will require improved methods to tailor these therapies specifically to individuals who suffer from CF. This report highlights current examples of translational CF research efforts to reach this goal. The use of intestinal organoids and genetics to better understand individual assessment of CFTR modulator treatment effects to ultimately enable a better personalized treatment for CF subjects will be discussed...
December 21, 2017: Journal of Cystic Fibrosis: Official Journal of the European Cystic Fibrosis Society
https://www.readbyqxmd.com/read/29262333/systemic-messenger-rna-therapy-as-a-treatment-for-methylmalonic-acidemia
#7
Ding An, Jessica L Schneller, Andrea Frassetto, Shi Liang, Xuling Zhu, Ji-Sun Park, Matt Theisen, Sue-Jean Hong, Jenny Zhou, Raj Rajendran, Becca Levy, Rebecca Howell, Gilles Besin, Vladimir Presnyak, Staci Sabnis, Kerry E Murphy-Benenato, E Sathyajith Kumarasinghe, Timothy Salerno, Cosmin Mihai, Christine M Lukacs, Randy J Chandler, Lin T Guey, Charles P Venditti, Paolo G V Martini
Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs)...
December 19, 2017: Cell Reports
https://www.readbyqxmd.com/read/29259248/aav-vector-meditated-expression-of-hla-g-reduces-injury-induced-corneal-vascularization-immune-cell-infiltration-and-fibrosis
#8
Matthew L Hirsch, Laura M Conatser, Sara M Smith, Jacklyn H Salmon, Jerry Wu, Nicholas E Buglak, Rich Davis, Brian C Gilger
Over 1.5 million individuals suffer from cornea vascularization due to genetic and/or environmental factors, compromising visual acuity and often resulting in blindness. Current treatments of corneal vascularization are limited in efficacy and elicit undesirable effects including, ironically, vision loss. To develop a safe and effective therapy for corneal vascularization, adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by human leukocyte antigen G (HLA-G), was investigated...
December 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29250754/high-level-production-of-dna-specific-endonuclease-asendi-with-synonymous-codon-and-its-potential-utilization-for-removing-dna-contamination
#9
Yuan Wang, Peng Jia, Rahat Sharif, Zhengchen Li, Yuhong Li, Peng Chen
Endonuclease I is a widely distributed periplasmic or extracellular enzyme. A method for the high-level production of recombinant AsEndI (endonuclease I from Aliivibrio salmonicida) in Escherichia coli with secretion expression is investigated. The coding sequence of AsEndI gene was assembled according to the E. coli codon usage bias, and AsEndI was expressed in the periplasm of E. coli TOP10 with a C-terminal 6× His-tagged fusion. The recombinant AsEndI (His-AsEndI) was purified by Ni-NTA resin with a yield of 1...
December 18, 2017: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/29247671/efficient-expression-of-cyclodextrin-glycosyltransferase-from-geobacillus-stearothermophilus-in-escherichia-coli-by-promoter-engineering-and-downstream-box-evolution
#10
Chen Deng, Jianghua Li, Hyun-Dong Shin, Guocheng Du, Jian Chen, Long Liu
Cyclodextrin glycosyltransferase (CGTase) catalyzes hydrolysis, cyclization, coupling, and disproportionation reactions and is widely used in the starch processing industry. In this work, the expression of CGTase from Geobacillus stearothermophilus in Escherichia coli BL21 (DE3) was significantly improved by promoter engineering and downstream box evolution. Firstly, the effects of the promoter type (PT7, Ptrp, PlacUV5, and the hybrid promoters PtacI and PtacII) and spacer sequence on the expression of CGTase were examined...
December 13, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/29246900/gene-therapy-with-adeno-associated-virus-vector-5-human-factor-ix-in-adults-with-hemophilia-b
#11
Wolfgang Miesbach, Karina Meijer, Michiel Coppens, Peter Kampmann, Robert Klamroth, Roger Schutgens, Marco Tangelder, Giancarlo Castaman, Joachim Schwäble, Halvard Bonig, Erhard Seifried, Federica Cattaneo, Christian Meyer, Frank W G Leebeek
Hemophilia B gene therapy aims to ameliorate bleeding risk and provide endogenous factor IX (FIX) activity/synthesis through a single treatment, eliminating the requirement for FIX concentrate. AMT-060 combines an adeno-associated virus-5 (AAV5) vector with a liver-specific promoter driving expression of a codon-optimized wild-type human FIX gene. This multi-national, open-label study included ten adults with hemophilia B (FIX ≤2% of normal) and severe-bleeding phenotype. No participants tested positive for AAV5-neutralizing antibodies using a green-fluorescent protein-based assay and all 10 were enrolled...
December 15, 2017: Blood
https://www.readbyqxmd.com/read/29235217/heterologous-protein-expression-in-pichia-pastoris-latest-research-progress-and-applications
#12
REVIEW
Veeresh Juturu, Jin Chuan Wu
Pichia pastoris is a well-known platform strain for heterologous protein expression. Over the past five years, different strategies to improve the efficiency of recombinant protein expression by this yeast strain have been developed; these include a patent-free protein expression kit, construction of the P. pastoris CBS7435Ku70 platform strain with its high efficiency in site-specific recombination of plasmid DNA into the genomic DNA, the design of synthetic promoters and their variants by combining different core promoters with multiple putative transcription factors, the generation of mutant GAP promoter variants with various promoter strengths, codon optimization, engineering the α-factor signal sequence by replacing the native glutamic acid at the Kex2 cleavage site with the other 19 natural amino acids and the addition of mammalian signal sequence to the yeast signal sequence, and the co-expression of single chaperones, multiple chaperones or helper proteins that aid in recombinant protein folding...
December 13, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/29232624/genomic-form-of-rhodopsin-dna-nanoparticles-rescued-autosomal-dominant-retinitis-pigmentosa-in-the-p23h-knock-in-mouse-model
#13
Rajendra Narayan Mitra, Min Zheng, Ellen R Weiss, Zongchao Han
Retinitis pigmentosa (RP) is a group of inherited retinal degenerative conditions and a leading cause of irreversible blindness. 25%-30% of RP cases are caused by inherited autosomal dominant (ad) mutations in the rhodopsin (Rho) protein of the retina, which impose a barrier for developing therapeutic treatments for this genetically heterogeneous disorder, as simple gene replacement is not sufficient to overcome dominant disease alleles. Previously, we have explored using the genomic short-form of Rho (sgRho) for gene augmentation therapy of RP in a Rho knockout mouse model...
December 5, 2017: Biomaterials
https://www.readbyqxmd.com/read/29224506/aav5-factor-viii-gene-transfer-in-severe-hemophilia-a
#14
Savita Rangarajan, Liron Walsh, Will Lester, David Perry, Bella Madan, Michael Laffan, Hua Yu, Christian Vettermann, Glenn F Pierce, Wing Y Wong, K John Pasi
Background Patients with hemophilia A rely on exogenous factor VIII to prevent bleeding in joints, soft tissue, and the central nervous system. Although successful gene transfer has been reported in patients with hemophilia B, the large size of the factor VIII coding region has precluded improved outcomes with gene therapy in patients with hemophilia A. Methods We infused a single intravenous dose of a codon-optimized adeno-associated virus serotype 5 (AAV5) vector encoding a B-domain-deleted human factor VIII (AAV5-hFVIII-SQ) in nine men with severe hemophilia A...
December 9, 2017: New England Journal of Medicine
https://www.readbyqxmd.com/read/29223161/cell-free-expression-purification-and-characterization-of-the-functional-%C3%AE-2-adrenergic-receptor-for-multianalyte-detection-of-%C3%AE-agonists
#15
Jian Wang, Yuan Liu, Junhua Zhang, Zhengzheng Han, Wei Wang, Yang Liu, Dong Wei, Wei Huang
Large-scale expression of β2-adrenergic receptor (β2-AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have manycritical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β2-AR in a cell-free system based on Escherichia coli extracts...
November 2017: Biochemistry. Biokhimii︠a︡
https://www.readbyqxmd.com/read/29222764/optimization-of-multi-omic-genome-scale-models-methodologies-hands-on-tutorial-and-perspectives
#16
Supreeta Vijayakumar, Max Conway, Pietro Lió, Claudio Angione
Genome-scale metabolic models are valuable tools for assessing the metabolic potential of living organisms. Being downstream of gene expression, metabolism is increasingly being used as an indicator of the phenotypic outcome for drugs and therapies. We here present a review of the principal methods used for constraint-based modelling in systems biology, and explore how the integration of multi-omic data can be used to improve phenotypic predictions of genome-scale metabolic models. We believe that the large-scale comparison of the metabolic response of an organism to different environmental conditions will be an important challenge for genome-scale models...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29222449/detection-of-prions-in-blood-of-cervids-at-the-asymptomatic-stage-of-chronic-wasting-disease
#17
Carlos Kramm, Sandra Pritzkow, Adam Lyon, Tracy Nichols, Rodrigo Morales, Claudio Soto
Chronic wasting disease (CWD) is a rapidly spreading prion disorder affecting captive and free-ranging cervids. The zoonotic potential of CWD is unknown, as well as the mechanism for its highly efficient transmission. A top priority to minimize further spreading of this disease and its potential impact on environmental prion contamination is the development of a non-invasive, sensitive, and specific test for ante-mortem detection of infected animals. Here, we optimized the protein misfolding cyclic amplification (PMCA) assay for highly efficient detection of CWD prions in blood samples...
December 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29221829/constitutive-expression-of-the-active-fragment-of-human-vasostatin-vs30-in-pichia-pastoris-smd1168
#18
Sergio Calderon-Salais, Prisiliana Velazquez-Bernardino, Victor E Balderas-Hernandez, Ana P Barba-de la Rosa, Antonio De Leon-Rodriguez
Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135-164aa) of human calreticulin (hCALR) and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a multi-copy strain was obtained using an in vitro multimerization approach...
December 5, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/29221807/ex%C3%A2-vivo-selection-of-transduced-hematopoietic-stem-cells-for-gene-therapy-of-%C3%AE-hemoglobinopathies
#19
Kanit Bhukhai, Edouard de Dreuzy, Marie Giorgi, Charlotte Colomb, Olivier Negre, Maria Denaro, Béatrix Gillet-Legrand, Joëlle Cheuzeville, Anaïs Paulard, Hélène Trebeden-Negre, Suparerk Borwornpinyo, Karine Sii-Felice, Leila Maouche, Julian D Down, Phillippe Leboulch, Emmanuel Payen
Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications...
October 24, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/29218897/codon-bias-among-synonymous-rare-variants-is-associated-with-alzheimer-s-disease-imaging-biomarker
#20
Jason E Miller, Manu K Shivakumar, Shannon L Risacher, Andrew J Saykin, Seunggeun Lee, Kwangsik Nho, Dokyoon Kim
Alzheimer's disease (AD) is a neurodegenerative disorder with few biomarkers even though it impacts a relatively large portion of the population and is predicted to affect significantly more individuals in the future. Neuroimaging has been used in concert with genetic information to improve our understanding in relation to how AD arises and how it can be potentially diagnosed. Additionally, evidence suggests synonymous variants can have a functional impact on gene regulatory mechanisms, including those related to AD...
2018: Pacific Symposium on Biocomputing
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