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Polymerase engineering

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https://www.readbyqxmd.com/read/28628207/engineered-polymerases-with-altered-substrate-specificity-expression-and-purification
#1
Ali Nikoomanzar, Matthew R Dunn, John C Chaput
Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from E...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28625978/the-alkylating-chemotherapeutic-temozolomide-induces-metabolic-stress-in-idh1-mutant-cancers-and-potentiates-nad-depletion-mediated-cytotoxicity
#2
Kensuke Tateishi, Fumi Higuchi, Julie Miller, Mara V A Koerner, Nina Lelic, Ganesh M Shankar, Shota Tanaka, David E Fisher, Tracy Batchelor, A John Iafrate, Hiroaki Wakimoto, Andrew S Chi, Daniel P Cahill
IDH1-mutant gliomas are dependent upon the canonical coenzyme nicotinamide adenine dinucleotide (NAD+) for survival. It is known that Poly(ADP-ribose) polymerase (PARP) activation consumes NAD+ during base excision repair (BER) of chemotherapy-induced DNA damage. We therefore hypothesized that a strategy combining NAD+ biosynthesis inhibitors with the alkylating chemotherapeutic agent temozolomide (TMZ) could potentiate NAD+ depletion-mediated cytotoxicity in mutant IDH1 cancer cells. To investigate the impact of TMZ on NAD+ metabolism, patient-derived xenografts and engineered mutant IDH1-expressing cell lines were exposed to TMZ, in vitro and in vivo, both alone and in combination with nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, which block NAD+ biosynthesis...
June 16, 2017: Cancer Research
https://www.readbyqxmd.com/read/28605670/ssb-recruitment-of-exonuclease-i-aborts-template-switching-in-escherichia-coli
#3
Laura T Laranjo, Stephen J Gross, Danna M Zeiger, Susan T Lovett
Misalignment of a nascent strand and the use of an alternative template during DNA replication, a process termed "template-switching", can give rise to frequent mutations and genetic rearrangements. Mutational hotspots are frequently found associated with imperfect inverted repeats ("quasipalindromes" or "QPs") in many organisms, including bacteriophage, bacteria, yeast and mammals. Evidence suggests that QPs mutate by a replication template-switch whereby one copy of the inverted repeat templates synthesis of the other...
June 3, 2017: DNA Repair
https://www.readbyqxmd.com/read/28601700/engineering-and-application-of-polymerases-for-synthetic-genetics
#4
REVIEW
Gillian Houlihan, Sebastian Arangundy-Franklin, Philipp Holliger
Organic chemistry has systematically probed the chemical determinants of function in nucleic acids by variation to the nucleobase, sugar ring and backbone moieties to build synthetic genetic polymers. Concomitantly, protein engineering has advanced to allow the discovery of polymerases capable of utilizing modified nucleotide analogs. A conjunction of these two lines of investigation in nucleotide chemistry and molecular biology has given rise to a new field of synthetic genetics dedicated to the exploration of the capacity of these novel, synthetic nucleic acids for the storage and propagation of genetic information, for evolution and for crosstalk, that is, information exchange with the natural genetic system...
June 8, 2017: Current Opinion in Biotechnology
https://www.readbyqxmd.com/read/28598722/improvement-of-mechanical-strength-and-osteogenic-potential-of-calcium-sulfate-based-hydroxyapatite-3-dimensional-printed-scaffolds-by-%C3%AE%C2%B5-polycarbonate-coating
#5
Beom-Su Kim, Sun-Sik Yang, Ho Park, Se-Hwan Lee, Young-Sam Cho, Jun Lee
Powder-based three-dimensional (3D) printing is an excellent method to fabricate complex-shaped scaffolds for tissue engineering. However, their lower mechanical strength restricts their application in bone tissue engineering. Here, we created a 3D-printed scaffold coated with a ε-polycaprolactone (PCL) polymer solution (5 and 10 w/v %) to improve the mechanical strength of the scaffold. The 3D scaffold was fabricated from calcium sulfate hemihydrate powder (CaSO4-1/2 H2O), transformed into hydroxyapatite (HAp) by treatment with a hydrothermal reaction in an NH4H2PO4 solution...
June 21, 2017: Journal of Biomaterials Science. Polymer Edition
https://www.readbyqxmd.com/read/28576439/serum-icam-1-level-and-icam-1-gene-1462a-g-k469e-polimorphism-on-microalbuminuria-in-nondiabetic-nonhypertensive-and-normolipidemic-obese-patients-genetical-background-of-microalbuminuria-in-obesity
#6
Ahmet Engin Atay, Bennur Esen, Halit Akbas, Emel Saglam Gokmen, Saadet Pilten, Hale Guler, Dilek Gogas Yavuz
BACKGROUND: A growing body of evidence suggest that obese individuals are under risk of renal parenchymal disorders when compared to nonobese counterparts. Microalbuminuria is the early marker of renal involvement. Although most of obese patients carries multiple risk factors for microalbuminuria, some obese individuals without risk factor may progress to microalbuminuria. The present study was performed to examine the role of ICAM-1 gene 1462A>G (K469E) polymorphism on microalbuminuria in obese subjects without diabetes mellitus, hypertension, hiperlipidemia and older age...
May 30, 2017: Nefrología: Publicación Oficial de la Sociedad Española Nefrologia
https://www.readbyqxmd.com/read/28562645/characterization-of-single-cell-derived-cultures-of-periosteal-progenitor-cells-to-ensure-the-cell-quality-for-clinical-application
#7
Stefan Stich, Alexander Loch, Su-Jin Park, Thomas Häupl, Jochen Ringe, Michael Sittinger
For clinical applications of cells and tissue engineering products it is of importance to characterize the quality of the used cells in detail. Progenitor cells from the periosteum are already routinely applied in the clinics for the regeneration of the maxillary bone. Periosteal cells have, in addition to their potential to differentiate into bone, the ability to develop into cartilage and fat. However, the question arises whether all cells isolated from periosteal biopsies are able to differentiate into all three tissue types, or whether there are subpopulations...
2017: PloS One
https://www.readbyqxmd.com/read/28550925/use-of-a-pedicled-omental-flap-to-reduce-inflammation-and-vascularize-an-abdominal-wall-patch
#8
Takafumi Uchibori, Keisuke Takanari, Ryotaro Hashizume, Nicholas J Amoroso, Yuzuru Kamei, William R Wagner
BACKGROUND: Although a variety of synthetic materials have been used to reconstruct tissue defects, these materials are associated with complications such as seromas, fistulas, chronic patient discomfort, and surgical site infection. While alternative, degradable materials that facilitate tissue growth have been examined. These materials can still trigger a foreign body inflammatory response that can lead to complications and discomfort. MATERIALS AND METHODS: In this report, our objective was to determine the effect of placing a pedicled omental flap under a biodegradable, microfibrous polyurethane scaffold serving as a full-wall thickness replacement of the rat abdominal wall...
May 15, 2017: Journal of Surgical Research
https://www.readbyqxmd.com/read/28532101/sonochemical-synthesis-of-fructose-1-6-bisphosphate-dicalcium-porous-microspheres-and-their-application-in-promotion-of-osteogenic-differentiation
#9
Chao Qi, Ding Zhou, Ying-Jie Zhu, Tuan-Wei Sun, Feng Chen, Chang-Qing Zhang
Human bone mesenchymal stem cells (hBMSCs) have the ability to differentiate into bone and cartilage for clinical bone regeneration. Biomaterials with an innate ability to stimulate osteogenic differentiation of hBMSCs into bone and cartilage are considered attractive candidates for the applications in bone tissue engineering and regeneration. In this paper, we synthesized fructose 1,6-bisphosphate dicalcium (Ca2FBP) porous microspheres by the sonochemical method, and investigated the ability of Ca2FBP for the promotion of the osteogenic differentiation of hBMSCs...
August 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
https://www.readbyqxmd.com/read/28522548/a-multi-purpose-toolkit-to-enable-advanced-genome-engineering-in-plants
#10
Tomas Cermak, Shaun J Curtin, Javier Gil-Humanes, Radim Čegan, Thomas J Y Kono, Eva Konečná, Joseph J Belanto, Colby G Starker, Jade W Mathre, Rebecca L Greenstein, Daniel F Voytas
We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on TALENs and the CRISPR/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination...
May 18, 2017: Plant Cell
https://www.readbyqxmd.com/read/28515784/improving-saccharomyces-cerevisiae-ethanol-production-and-tolerance-via-rna-polymerase-ii-subunit-rpb7
#11
Zilong Qiu, Rongrong Jiang
BACKGROUND: Classical strain engineering methods often have limitations in altering multigenetic cellular phenotypes. Here we try to improve Saccharomyces cerevisiae ethanol tolerance and productivity by reprogramming its transcription profile through rewiring its key transcription component RNA polymerase II (RNAP II), which plays a central role in synthesizing mRNAs. This is the first report on using directed evolution method to engineer RNAP II to alter S. cerevisiae strain phenotypes...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28515297/attenuation-of-foot-and-mouth-disease-virus-by-engineering-viral-polymerase-fidelity
#12
Devendra K Rai, Fayna Diaz-San Segundo, Grace Campagnola, Anna Keith, Elizabeth A Schafer, Anna Kloc, Teresa de Los Santos, Olve Peersen, Elizabeth Rieder
Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp or 3D(pol)) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allows FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3D(pol) in combination with prior results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon...
May 17, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28512144/tog-tubulin-binding-specificity-promotes-microtubule-dynamics-and-mitotic-spindle-formation
#13
Amy E Byrnes, Kevin C Slep
XMAP215, CLASP, and Crescerin use arrayed tubulin-binding tumor overexpressed gene (TOG) domains to modulate microtubule dynamics. We hypothesized that TOGs have distinct architectures and tubulin-binding properties that underlie each family's ability to promote microtubule polymerization or pause. As a model, we investigated the pentameric TOG array of a Drosophila melanogaster XMAP215 member, Msps. We found that Msps TOGs have distinct architectures that bind either free or polymerized tubulin, and that a polarized array drives microtubule polymerization...
June 5, 2017: Journal of Cell Biology
https://www.readbyqxmd.com/read/28500952/isolation-and-characterization-of-lymphoid-enhancer-factor-1-positive-deciduous-dental-pulp-stem-like-cells-after-transfection-with-a-piggybac-vector-containing-lef1-promoter-driven-selection-markers
#14
Tomoya Murakami, Issei Saitoh, Masahiro Sato, Emi Inada, Miki Soda, Masataka Oda, Hisanori Domon, Yoko Iwase, Tadashi Sawami, Kazunari Matsueda, Yutaka Terao, Hayato Ohshima, Hirofumi Noguchi, Haruaki Hayasaki
OBJECTIVE: Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B and T cells. LEF1 is also an important member of the Wnt/β-catenin signaling pathway that plays important roles in the self-renewal and differentiation of embryonic stem cells. We speculated that LEF1 might function in the stem cells from human exfoliated deciduous teeth (SHED). In this study, we attempted to isolate such LEF1-positive cells from human deciduous dental pulp cells (HDDPCs) by genetic engineering technology, using the human LEF1 promoter...
May 7, 2017: Archives of Oral Biology
https://www.readbyqxmd.com/read/28467855/use-of-ice-nucleating-proteins-to-improve-the-performance-of-freeze-thaw-valves-in-microfluidic-devices
#15
Joseph C Gaiteri, W Hampton Henley, Nathan A Siegfried, Thomas H Linz, J Michael Ramsey
Currently, reliable valving on integrated microfluidic devices fabricated from rigid materials is confined to expensive and complex methods. Freeze-thaw valves (FTVs) can provide a low cost, low complexity valving mechanism, but reliable implementation of them has been greatly hindered by the lack of ice nucleation sites within the valve body's small volume. Work to date has required very low temperatures (on the order of -40 °C or colder) to induce freezing without nucleation sites, making FTVs impractical due to instrument engineering challenges...
June 6, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28464223/casting-eppcr-ceppcr-a-simple-random-mutagenesis-method-to-generate-high-quality-mutant-libraries
#16
Jianhua Yang, Anna J Ruff, Marcus Arlt, Ulrich Schwaneberg
During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error-prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance...
May 2, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28463494/sort-seq-approach-to-engineering-a-formaldehyde-inducible-promoter-for-dynamically-regulated-escherichia-coli-growth-on-methanol
#17
Julia Rohlhill, Nicholas R Sandoval, Eleftherios T Papoutsakis
Tight and tunable control of gene expression is a highly desirable goal in synthetic biology for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence-function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence-function relationships in a robust and rapid way...
May 9, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28462820/short-dna-hairpins-compromise-recombinant-adeno-associated-virus-genome-homogeneity
#18
Jun Xie, Qin Mao, Phillip W L Tai, Ran He, Jianzhong Ai, Qin Su, Ye Zhu, Hong Ma, Jia Li, Shoufang Gong, Dan Wang, Zhen Gao, Mengxin Li, Li Zhong, Heather Zhou, Guangping Gao
Short hairpin (sh)RNAs delivered by recombinant adeno-associated viruses (rAAVs) are valuable tools to study gene function in vivo and a promising gene therapy platform. Our data show that incorporation of shRNA transgenes into rAAV constructs reduces vector yield and produces a population of truncated and defective genomes. We demonstrate that sequences with hairpins or hairpin-like structures drive the generation of truncated AAV genomes through a polymerase redirection mechanism during viral genome replication...
June 7, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/28459860/nucleotide-time-alignment-for-molecular-recorders
#19
Thaddeus R Cybulski, Edward S Boyden, George M Church, Keith E J Tyo, Konrad P Kording
Using a DNA polymerase to record intracellular calcium levels has been proposed as a novel neural recording technique, promising massive-scale, single-cell resolution monitoring of large portions of the brain. This technique relies on local storage of neural activity in strands of DNA, followed by offline analysis of that DNA. In simple implementations of this scheme, the time when each nucleotide was written cannot be determined directly by post-hoc DNA sequencing; the timing data must be estimated instead...
May 2017: PLoS Computational Biology
https://www.readbyqxmd.com/read/28447733/gene-expression-profile-in-human-induced-pluripotent-stem-cells-chondrogenic-differentiation-in%C3%A2-vitro-part-b
#20
Ewelina Augustyniak, Wiktoria Maria Suchorska, Tomasz Trzeciak, Magdalena Richter
The development of human induced pluripotent stem cells (hiPSCs) is considered a turning point in tissue engineering. However, more data are required to improve understanding of key aspects of the cell differentiation process, including how specific chondrogenic processes affect the gene expression profile of chondrocyte‑like cells and the relative value of cell differentiation markers. The main aims of the present study were as follows: To determine the gene expression profile of chondrogenic‑like cells derived from hiPSCs cultured in mediums conditioned with HC‑402‑05a cells or supplemented with transforming growth factor β3 (TGF‑β3), and to assess the relative utility of the most commonly‑used chondrogenic markers as indicators of cell differentiation...
May 2017: Molecular Medicine Reports
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