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Polymerase engineering

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https://www.readbyqxmd.com/read/28737162/silk-fiber-reinforcement-modulates-in-vitro-chondrogenesis-in-3d-composite-scaffolds
#1
Yogendra Pratap Singh, Mimi Adhikary, Nandana Bhardwaj, Bibhas Kumar Bhunia, Biman B Mandal
The limited self-regenerative capacity of adult cartilage has steered the upsurge in tissue engineered replacements to combat the problem of osteoarthritis. In the present study, the potential of fiber-reinforced silk composites from mulberry (Bombyx mori) and non-mulberry (Antheraea assamensis) silk has been investigated for cartilage tissue engineering. The fabricated composites were physico-chemically characterized and analyzed for cellular viability, proliferation, extracellular matrix formation and immunocompatibility...
July 24, 2017: Biomedical Materials
https://www.readbyqxmd.com/read/28718514/aldehyde-dehydrogenase-1-a1-regulates-energy-metabolism-in-adipocytes-from-different-species
#2
Kefeng Yang, Christopher Adin, Qiwen Shen, Ly James Lee, Lianbo Yu, Paolo Fadda, Arpad Samogyi, Kathleen Ham, Lu Xu, Chen Gilor, Ouliana Ziouzenkova
BACKGROUND: Survival and longevity of xenotransplants depend on immune function and ability to integrate energy metabolism between cells from different species. However, mechanisms for interspecies cross talk in energy metabolism are not well understood. White adipose tissue stores energy and is capable of mobilization and dissipation of energy as heat (thermogenesis) by adipocytes expressing uncoupling protein 1 (Ucp1). Both pathways are under the control of vitamin A metabolizing enzymes...
July 17, 2017: Xenotransplantation
https://www.readbyqxmd.com/read/28715443/far-infrared-radiation-promotes-rabbit-renal-proximal-tubule-cell-proliferation-and-functional-characteristics-and-protects-against-cisplatin-induced-nephrotoxicity
#3
I-Ni Chiang, Yeong-Shiau Pu, Chao-Yuan Huang, Tai-Horng Young
Far infrared radiation, a subdivision of the electromagnetic spectrum, is beneficial for long-term tissue healing, anti-inflammatory effects, growth promotion, sleep modulation, acceleration of microcirculation, and pain relief. We investigated if far infrared radiation is beneficial for renal proximal tubule cell cultivation and renal tissue engineering. We observed the effects of far infrared radiation on renal proximal tubules cells, including its effects on cell proliferation, gene and protein expression, and viability...
2017: PloS One
https://www.readbyqxmd.com/read/28710267/increased-processivity-misincorporation-and-nucleotide-incorporation-efficiency-in-sulfolobus-solfataricus-dpo4-thumb-domain-mutations
#4
Li Wang, Chenchen Liang, Jing Wu, Liming Liu, Keith E J Tyo
The present study aimed to increase the processivity of Sulfolobus solfataricus DNA polymerase Dpo4. Protein engineering and bioinformatics were used to compile a library of potential Dpo4 mutation sites. Ten potential mutants were identified and constructed. A primer extension assay was used to evaluate the processivity of Dpo4 mutants. Thumb (A181D) and finger (E63K) domain mutants showed a processivity of 20 and 19 nucleotides (nt), respectively. A little finger domain mutant (I248Y) exhibited a processivity of 17 nt, only 1 nt more than wild-type Dpo4...
July 14, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28704177/morphology-and-contractile-gene-expression-of-adipose-derived-mesenchymal-stem-cells-in-response-to-short-term-cyclic-uniaxial-strain-and-tgf-%C3%AE-1
#5
Neda Rashidi, Mohammad Tafazzoli-Shadpour, Nooshin Haghighipour, Mohammad-Mehdi Khani
Previous studies have shown smooth muscle induction in adipose-derived mesenchymal stem cells (ASCs) caused by long-term cyclic stretch. Here we examined the capability of the short-term straining with time steps of 4, 8, 16 and 24 h alone or combined with TGF-β1 on smooth muscle induction of rabbit ASCs. Alterations in cell morphology were quantified through the cell shape index and orientation angle, and expression levels of α-SMA, SM22-α, h-caldesmon and calponin3 markers were examined using the real-time polymerase chain reaction (PCR) method...
July 12, 2017: Biomedizinische Technik. Biomedical Engineering
https://www.readbyqxmd.com/read/28697500/cx43-and-smad-mediated-tgf-%C3%AE-bmp-signaling-pathway-promotes-cartilage-differentiation-of-bone-marrow-mesenchymal-stem-cells-and-inhibits-osteoblast-differentiation
#6
Ya-Dong Zhang, Shi-Chang Zhao, Zhong-Sheng Zhu, Yi-Fei Wang, Jian-Xiang Liu, Zhi-Cai Zhang, Feng Xue
BACKGROUND/AIMS: The aim of this study was to investigate the influence of Cx43- and Smad-mediated TGF-β/BMP signaling pathway on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cartilage and inhibition of ossification. METHODS: BMSCs of Wistar rats were cultured and assigned into 5 groups for transfection with adenoviruses. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were employed to detect mRNA and protein expressions of target genes...
July 11, 2017: Cellular Physiology and Biochemistry
https://www.readbyqxmd.com/read/28687761/inactivation-of-pol-%C3%AE-and-c-nhej-eliminates-off-target-integration-of-exogenous-dna
#7
Alex N Zelensky, Joost Schimmel, Hanneke Kool, Roland Kanaar, Marcel Tijsterman
Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration...
July 7, 2017: Nature Communications
https://www.readbyqxmd.com/read/28677757/hypertrophic-differentiation-of-mesenchymal-stem-cells-is-suppressed-by-xanthotoxin-via-the-p38%C3%A2-mapk-hdac4-pathway
#8
Zhen Cao, Yun Bai, Chuan Liu, Ce Dou, Jianmei Li, Junyu Xiang, Chunrong Zhao, Zhao Xie, Qiang Xiang, Shiwu Dong
Chondrocyte hypertrophy is a physiological process in endochondral ossification. However, the hypertrophic‑like alterations of chondrocytes at the articular surface may result in osteoarthritis (OA). In addition, the generation of fibrocartilage with a decreased biological function in tissue engineered cartilage, has been attributed to chondrocyte hypertrophy. Therefore, suppressing chondrocyte hypertrophy in OA and the associated regeneration of non‑active cartilage is of primary concern. The present study examined the effects of xanthotoxin (XAT), which is classified as a furanocoumarin, on chondrocyte hypertrophic differentiation of mesenchymal stem cells...
September 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/28675007/-effect-of-hypoxia-on-the-biological-characteristics-of-human-dental-follicle-cells
#9
Liang Xi, Chen Guoqing, Tian Weidong
OBJECTIVE: This study aimed to investigate the effects of hypoxia on the characteristics of human dental follicle cells (hDFCs). METHODS: The tissue explant collagenase method was used to isolate hDFCs from young permanent teeth. The immunofluorescence technique was used to detect cell surface markers, and the multi-differentiation potential was detected by multilineage differentiation induction assay. Then, the hypoxic microenvironment was physically mimicked, and the cells were divided into the normoxia group (20%O₂) and the hypoxia group (2%O₂)...
June 1, 2017: Hua Xi Kou Qiang Yi Xue za Zhi, Huaxi Kouqiang Yixue Zazhi, West China Journal of Stomatology
https://www.readbyqxmd.com/read/28673853/enhanced-simultaneous-overlap-extension-pcr-by-gold-nanoparticles
#10
Wenchao Yang, Xueyan Cao, Xinhui Li
Methods to fuse multiple DNA fragments are extremely useful in synthetic biology and protein engineering. Here, we report a gold nanoparticle-mediated simultaneous overlap extension-PCR (AuNP-mediated SOE-PCR) method that enables the fusion of multiple DNA fragments simultaneously with their amplification in a single reaction using typical PCR conditions. Using greater concentrations of rTaq DNA polymerase and AuNPs significantly improves the performance of SOE-PCR especially for the fusion of more than three DNA fragments...
July 1, 2017: Nanomedicine: Nanotechnology, Biology, and Medicine
https://www.readbyqxmd.com/read/28672891/osteogenic-potency-of-dedifferentiated-fat-cells-isolated-from-elderly-people-with-osteoporosis
#11
Wei Hao, Chuanqiang Jiang, Ming Jiang, Tian Wang, Xin Wang
Mature adipocytes are the major cell type in adipose tissue. This study aimed to explore the osteogenic potency of dedifferentiated fat cells obtained from osteoporotic patients (opDFATs) in vitro and in vivo. Mature adipocytes and adipose-derived stem cells (opASCs) were harvested from subcutaneous adipose tissue. Mature adipocytes were dedifferentiated to produce opDFATs by the ceiling culture method. OpDFATs were osteogenically induced in vitro with opASCs as a control. Cell growth, alkaline phosphatase (ALPase) activity and cell mineralization were determined, and expression levels of osteogenesis-specific genes (collagen I, osteocalcin and bone sialoprotein) were analyzed using quantitative reverse transcription polymerase chain reaction...
July 2017: Experimental and Therapeutic Medicine
https://www.readbyqxmd.com/read/28667117/reduced-mutation-rate-and-increased-transformability-of-transposon-free-acinetobacter-baylyi-adp1-isx
#12
Gabriel A Suárez, Brian A Renda, Aurko Dasgupta, Jeffrey E Barrick
The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction...
June 30, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28658997/evaluation-of-the-potential-of-kartogenin-encapsulated-poly-l-lactic-acid-co-caprolactone-collagen-nanofibers-for-tracheal-cartilage-regeneration
#13
Haiyue Yin, Juan Wang, Ziqi Gu, Wenhao Feng, Manchen Gao, Yu Wu, Hao Zheng, Xiaomin He, Xiumei Mo
Tracheal stenosis is one of major challenging issues in clinical medicine because of the poor intrinsic ability of tracheal cartilage for repair. Tissue engineering provides an alternative method for the treatment of tracheal defects by generating replacement tracheal structures. In this study, we fabricated coaxial electrospun fibers using poly(L-lactic acid-co-caprolactone) and collagen solution as shell fluid and kartogenin solution as core fluid. Scanning electron microscope and transmission electron microscope images demonstrated that nanofibers had uniform and smooth structure...
January 1, 2017: Journal of Biomaterials Applications
https://www.readbyqxmd.com/read/28653139/spreading-proliferation-and-differentiation-of-human-dental-pulp-stem-cells-on-chitosan-scaffolds-immobilized-with-rgd-or-fibronectin
#14
Farzin Asghari Sana, Merve Çapkın Yurtsever, Gökçe Kaynak Bayrak, Ekin Özge Tunçay, Arlin S Kiremitçi, Menemşe Gümüşderelioğlu
Nowadays, human dental pulp stem cells (hDPSCs) became more attractive for therapeutic purposes because of their high proliferation and differentiation potential. Thus, coupling the desired cellular characteristics of hDPSCs with good biomaterial properties of the chitosan scaffolds provide an interesting approach for tissue engineering applications. On the other hand, scaffold surface modification is also needed to promote stem cell adhesion since chitosan lacks adhesion motifs to support direct cell anchorage...
June 26, 2017: Cytotechnology
https://www.readbyqxmd.com/read/28652865/crispr-cas9-enabled-genetic-disruptions-for-understanding-ethanol-and-ethyl-acetate-biosynthesis-in-kluyveromyces-marxianus
#15
Ann-Kathrin Löbs, Ronja Engel, Cory Schwartz, Andrew Flores, Ian Wheeldon
BACKGROUND: The thermotolerant yeast Kluyveromyces marxianus shows promise as an industrial host for the biochemical production of fuels and chemicals. Wild-type strains are known to ferment high titers of ethanol and can effectively convert a wide range of C5, C6, and C12 sugars into the volatile short-chain ester ethyl acetate. Strain engineering, however, has been limited due to a lack of advanced genome-editing tools and an incomplete understanding of ester and ethanol biosynthesis...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28651051/molecular-precision-at-micrometer-length-scales-hierarchical-assembly-of-dna-protein-nanostructures
#16
Daniel Schiffels, Veronika A Szalai, J Alexander Liddle
Robust self-assembly across length scales is a ubiquitous feature of biological systems but remains challenging for synthetic structures. Taking a cue from biology-where disparate molecules work together to produce large, functional assemblies-we demonstrate how to engineer microscale structures with nanoscale features: Our self-assembly approach begins by using DNA polymerase to controllably create double-stranded DNA (dsDNA) sections on a single-stranded template. The single-stranded DNA (ssDNA) sections are then folded into a mechanically flexible skeleton by the origami method...
July 3, 2017: ACS Nano
https://www.readbyqxmd.com/read/28628207/engineered-polymerases-with-altered-substrate-specificity-expression-and-purification
#17
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
#18
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
#19
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
#20
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 9, 2017: Current Opinion in Biotechnology
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