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Polymerase engineering

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https://www.readbyqxmd.com/read/28915003/the-evolution-of-dna-templated-synthesis-as-a-tool-for-materials-discovery
#1
Rachel K O'Reilly, Andrew J Turberfield, Thomas R Wilks
Precise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible. The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify "successful" structures...
September 15, 2017: Accounts of Chemical Research
https://www.readbyqxmd.com/read/28911121/engineering-human-primpol-into-an-efficient-rna-dependent-dna-primase-polymerase
#2
Rubén Agudo, Patricia A Calvo, María I Martínez-Jiménez, Luis Blanco
We have developed a straightforward fluorometric assay to measure primase-polymerase activity of human PrimPol (HsPrimPol). The sensitivity of this procedure uncovered a novel RNA-dependent DNA priming-polymerization activity (RdDP) of this enzyme. In an attempt to enhance HsPrimPol RdDP activity, we constructed a smart mutant library guided by prior sequence-function analysis, and tested this library in an adapted screening platform of our fluorometric assay. After screening less than 500 variants, we found a specific HsPrimPol mutant, Y89R, which displays 10-fold higher RdDP activity than the wild-type enzyme...
September 6, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28906473/engineered-disease-resistance-in-cotton-using-rna-interference-to-knock-down-cotton-leaf-curl-kokhran-virus-burewala-and-cotton-leaf-curl-multan-betasatellite-expression
#3
Aftab Ahmad, Muhammad Zia-Ur-Rehman, Usman Hameed, Abdul Qayyum Rao, Ammara Ahad, Aneela Yasmeen, Faheem Akram, Kamran Shahzad Bajwa, Jodi Scheffler, Idrees Ahmad Nasir, Ahmad Ali Shahid, Muhammad Javed Iqbal, Tayyab Husnain, Muhammad Saleem Haider, Judith K Brown
Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the βC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB)...
September 14, 2017: Viruses
https://www.readbyqxmd.com/read/28903467/development-and-characterization-of-a-synthetic-dna-nuversa-to-be-used-as-a-standard-in-quantitative-polymerase-chain-reactions-for-molecular-pneumococcal-serotyping
#4
Fuminori Sakai, Griffin Sonaty, David Watson, Keith P Klugman, Jorge E Vidal
Identification of Streptococcus pneumoniae and its more than 90 serotypes is routinely conducted by culture and Quellung reactions. Quantitative polymerase chain reactions (qPCRs) have been developed for molecular detection, including a pan-pneumococcus lytA assay, and assays targeting 79 serotypes. Reactions require genomic DNA from every target to prepare standards, which can be time consuming. In this study, we have developed a synthetic DNA molecule as a surrogate for genomic DNA and present new single-plex qPCR reactions to increase molecular detection to 94 pneumococcal serotypes...
September 15, 2017: FEMS Microbiology Letters
https://www.readbyqxmd.com/read/28901837/minimal-requirements-for-reverse-polymerization-and-trna-repair-by-trna-his-guanylyltransferase
#5
Riddhi Desai, Kunmo Kim, Hanna C Buechsenschuetz Allan W Chen, Yumin Bi, Mitchell R Mann, Matthew A Turk, Christina Z Chung, Ilka U Heinemann
tRNA(His) guanylyltransferase (Thg1) has unique reverse (3'-5') polymerase activity occurring in all three domains of life. Most eukaryotic Thg1 homologs are essential genes involved in tRNA(His) maturation. These enzymes normally catalyze a single 5' guanylation of tRNA(His) lacking the essential G(-1) identity element required for aminoacylation. Recent studies suggest that archaeal type Thg1, which includes most archaeal and bacterial Thg1 enzymes is phylogenetically distant from eukaryotic Thg1. Thg1 is evolutionarily related to canonical 5'-3' forward polymerases but catalyzes reverse 3'-5'polymerization...
September 13, 2017: RNA Biology
https://www.readbyqxmd.com/read/28872853/new-insights-into-dna-polymerase-function-revealed-by-phosphonoacetic-acid-sensitive-t4-dna-polymerases
#6
Likui Zhang
The bacteriophage T4 DNA polymerase (pol) and the closely related RB69 DNA pol have been developed into model enzymes to study family B DNA pols. While all family B DNA pols have similar structures and share conserved protein motifs, the molecular mechanism underlying natural drug resistance of non-herpes family B DNA pols and drug sensitivity of herpes DNA pols remains unknown. In the present study, we constructed T4 phages containing G466S, Y460F, G466S/Y460F, P469S and V475W mutations in DNA pol. These amino acid substitutions replace the residues in drug-resistant T4 DNA pol with residues found in drug-sensitive herpes family DNA pols...
September 5, 2017: Chemical Research in Toxicology
https://www.readbyqxmd.com/read/28868218/optimization-of-the-secretory-expression-of-recombinant-human-c-reactive-protein-in-pichia-pastoris
#7
Junming Li, Chengming Sun, Lei Chen, Lihui Sun, Lijun Duan, Qing Zheng, Xuejun Hu
Human C-reactive protein (CRP), a classical human acute-phase plasma protein, is not only a sensitive systemic inflammatory marker but also an independent risk predictor of cardiovascular diseases. However, existing heterologous expression systems for expressing CRP is not efficient and cost-effective for large-scale industrial production of CRP to meet the growing market demand for CRP. This study aims to improve the secretion of recombinant CRP by Pichia pastoris via optimizing signal peptides, promoters and carbon sources...
October 2017: 3 Biotech
https://www.readbyqxmd.com/read/28864123/effects-of-glucose-lactate-and-basic-fgf-as-limiting-factors-on-the-expansion-of-human-induced-pluripotent-stem-cells
#8
Ikki Horiguchi, Yusuke Urabe, Keiichi Kimura, Yasuyuki Sakai
Pluripotent stem cells (PSCs) are one of the promising cell sources for tissue engineering and drug screening. However, mass production of induced pluripotent stem cells (iPSCs) is still developing. Especially, a huge amount of culture medium usage causes expensive cost in the mass production process. In this report, we reduced culture medium usage by extending interval of changing culture medium. In parallel, we also increased glucose concentration and supplied heparan sulfate to avoid depletion of glucose and bFGF respectively...
August 29, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28863324/quantification-of-viable-and-non-viable-legionella-spp-by-heterogeneous-asymmetric-recombinase-polymerase-amplification-harpa-on-a-flow-based-chemiluminescence-microarray
#9
Catharina Kober, Reinhard Niessner, Michael Seidel
Increasing numbers of legionellosis outbreaks within the last years have shown that Legionella are a growing challenge for public health. Molecular biological detection methods capable of rapidly identifying viable Legionella are important for the control of engineered water systems. The current gold standard based on culture methods takes up to 10 days to show positive results. For this reason, a flow-based chemiluminescence (CL) DNA microarray was developed that is able to quantify viable and non-viable Legionella spp...
August 24, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/28863186/fusion-of-taq-dna-polymerase-with-single-stranded-dna-binding-like-protein-of-nanoarchaeum-equitans-expression-and-characterization
#10
Marcin Olszewski, Marta Śpibida, Maciej Bilek, Beata Krawczyk
DNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the Taq DNA Stoffel domain and a single-stranded DNA binding-like protein of Nanoarchaeum equitans in order to significantly improve the properties of DNA polymerase...
2017: PloS One
https://www.readbyqxmd.com/read/28840962/biomarkers-and-therapeutic-advances-in-glioblastoma-multiforme
#11
Andrew Octavian Sasmita, Ying Pei Wong, Anna Pick Kiong Ling
Glioblastoma multiforme (GBM) is a malignant tumor within the brain. Generally classified as primary and secondary with several different subtypes, ample molecular biomarkers have risen throughout the years which have garnered the attention of researchers. The advancements in genomics and proteomics have allowed researchers to gather prominent molecular biomarkers. All these biomarkers are gathered by means of biopsy or bodily fluid sample collection and are quantitatively analyzed by polymerase chain reaction coupled with other computational technologies...
August 25, 2017: Asia-Pacific Journal of Clinical Oncology
https://www.readbyqxmd.com/read/28828504/tissue-engineering-relevant-characteristics-of-ex-vivo-and-monolayer-expanded-chondrocytes-from-the-notch-versus-trochlea-of-human-knee-joints
#12
Matthias Aurich, Gunther Olaf Hofmann, Bernd Rolauffs
PURPOSE: The aim was to analyse the biological characteristics of chondrocytes from the two biopsy sites notch vs. trochlea of human knee joints. The question was whether tissue engineering-relevant characteristics such as viability and mRNA expression profile would be comparable ex vivo and after monolayer expansion, as these are parts of routine autologous chondrocyte implantation (ACI). METHODS: Biopsies from the intercondylar notch and the lateral aspect of the trochlea from 20 patients with ICRS grades 3 and 4 cartilage defects were harvested during arthroscopy...
August 21, 2017: International Orthopaedics
https://www.readbyqxmd.com/read/28821270/calmodulin-like-protein-3-is-an-estrogen-receptor-alpha-coregulator-for-gene-expression-and-drug-response-in-a-snp-estrogen-and-serm-dependent-fashion
#13
Sisi Qin, James N Ingle, Mohan Liu, Jia Yu, D Lawrence Wickerham, Michiaki Kubo, Richard M Weinshilboum, Liewei Wang
BACKGROUND: We previously performed a case-control genome-wide association study in women treated with selective estrogen receptor modulators (SERMs) for breast cancer prevention and identified single nucleotide polymorphisms (SNPs) in ZNF423 as potential biomarkers for response to SERM therapy. The ZNF423rs9940645 SNP, which is approximately 200 bp away from the estrogen response elements, resulted in the SNP, estrogen, and SERM-dependent regulation of ZNF423 expression and, "downstream", that of BRCA1...
August 18, 2017: Breast Cancer Research: BCR
https://www.readbyqxmd.com/read/28817345/reverse-transcription-quantitative-polymerase-chain-reaction-for-detection-of-and-differentiation-between-rna-and-dna-of-hiv-1-based-lentiviral-vectors
#14
Melanie Pavlovic, Nina Koehler, Martina Anton, Anna Dinkelmeier, Maren Haase, Thorsten Stellberger, Ulrich Busch, Armin E Baiker
The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described...
August 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28815907/t7-rna-polymerase-driven-inducible-cell-lysis-for-dna-transfer-from-escherichia-coli-to-bacillus-subtilis
#15
Mario Juhas, James W Ajioka
The majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr-ts-1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E...
August 16, 2017: Microbial Biotechnology
https://www.readbyqxmd.com/read/28815333/comparison-of-different-culture-conditions-for-smooth-muscle-cell-differentiation-of-human-umbilical-cord-vein-cd146-perivascular-cells
#16
Beyza Gökçinar-Yagci, Betül Çelebi-Saltik
Pericytes are CD146+ perivascular cells (PCs) that have multipotential differentiation capacity as mesenchymal stem cells. Beside their crucial roles in vascular development and blood flow regulation, they have ability to differentiate into vascular cell types in vivo. These properties make pericytes preferred cells in the field of vascular tissue engineering. Culture medium for in vitro differentiation of pericytes to vascular smooth muscle cells (SMCs) has not been defined yet. The aim of this study is to try different culture media for SMC differentiation of CD146+ PCs...
August 16, 2017: Cell and Tissue Banking
https://www.readbyqxmd.com/read/28809467/multidimensional-control-of-cas9-by-evolved-rna-polymerase-based-biosensors
#17
Jinyue Pu, Kaitlin Kentala, Bryan C Dickinson
Systems to control Cas9 with spatial and temporal precision offer opportunities to decrease side effects, protect sensitive tissues, and create gene therapies that are only activated at defined times and places. Here, we present the design of new Cas9 controllers based on RNA polymerase (RNAP)-based biosensors that produce gRNAs, thereby regulating target knockout. After development and validation of a new abscisic acid-inducible biosensor to control Cas9, we lowered the background of the system using continuous evolution...
August 15, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28804565/thrombospondin-1-inhibits-ossification-of-tissue-engineered-cartilage-constructed-by-adscs
#18
Aiguo Xie, Jixin Xue, Gan Shen, Lanjun Nie
Cartilage tissue engineering provides a new method in the treatment of cartilage defects, and adipose derived stem cells seem to be an ideal seed cell in cartilage tissue engineering because of its characteristics. However, ossification after in vivo implantation of tissue engineered cartilage remains a challenge. Thrombospondin-1 which has been reported to have an inhibitory effect on angiogenesis, may play an important role in inhibiting the ossification of tissue engineered cartilage constructed by adipose derived stem cells...
2017: American Journal of Translational Research
https://www.readbyqxmd.com/read/28800110/response-of-dermal-fibroblasts-to-biochemical-and-physical-cues-in-aligned-polycaprolactone-silk-fibroin-nanofiber-scaffolds-for-application-in-tendon-tissue-engineering
#19
Chih-Hao Chen, Shih-Hsien Chen, Chang-Yi Kuo, Meng-Lun Li, Jyh-Ping Chen
Silk fibroin (SF) and fiber alignment were introduced into polycaprolactone (PCL)-based electrospun nanofibers as chemical and physical cues for tendon tissue engineering applications. The physicochemical properties of random PCL (RP) nanofibers, random PCL/SF (RPSF) nanofibers and aligned PCL/SF (APSF) nanofibers were characterized for fiber orientation and SF blending effects. An in vitro cell culture with rabbit dermal fibroblasts (RDFBs) on nanofibers indicated that SF promotes cell proliferation to a higher extent than fiber alignment...
August 11, 2017: Nanomaterials
https://www.readbyqxmd.com/read/28792664/collagen-coated-nano-electrospun-pcl-seeded-with-human-endometrial-stem-cells-for-skin-tissue-engineering-applications
#20
Shiva Sharif, Jafar Ai, Mahmoud Azami, Javad Verdi, Mohammad Ali Atlasi, Sadegh Shirian, Ali Samadikuchaksaraei
Human endometrial stem cells (hEnSCs) are known as an attractive source of stem cells for regenerative medicine. hEnSCs are easily isolated and are capable of repairing uterine through their strong ability of creating new capillaries. In this study, a three-dimensional (3D) nanofibrous polycaprolactone (PCL)/collagen scaffold was fabricated and characterized in order to be applied as a new approach for skin reconstruction. Furthermore, the behavior of hEnSCs on this scaffold was investigated. First, a PCL 3D scaffold was constructed using electrospinning technique...
August 9, 2017: Journal of Biomedical Materials Research. Part B, Applied Biomaterials
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