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Polymerase engineering

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https://www.readbyqxmd.com/read/27918898/roles-of-l-type-calcium-channels-cav1-2-and-the-distal-c-terminus-dct-in-differentiation-and-mineralization-of-rat-dental-apical-papilla-stem-cells-rscaps
#1
Qianqian Gao, Jianping Ge, Yanqin Ju, Changsheng Li, Jian Gao, Meisheng Wu, Shouliang Zhao
OBJECTIVE: Voltage-gated inward Ca(2+) currents (ICa) are triggered by cell depolarization and commonly produce transient increases in the cytoplasmic free Ca(2+) concentration. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt or DCT). Stem cells from the apical papilla (SCAPs) has a capacity for differentiation into the odontoblastic-like cells in vitro and dentin forming in vivo, which makes SCAPs advantages in tissue engineering and regenerative endodontic...
November 4, 2016: Archives of Oral Biology
https://www.readbyqxmd.com/read/27902845/cloning-and-heterologous-expression-of-chlorophyll-a-synthase-in-rhodobacter-sphaeroides
#2
Emre M Ipekoğlu, Koray Göçmen, Mehmet T Öz, Muazzez Gürgan, Meral Yücel
Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm...
November 30, 2016: Journal of Basic Microbiology
https://www.readbyqxmd.com/read/27895039/region-specific-effect-of-the-decellularized-meniscus-extracellular-matrix-on-mesenchymal-stem-cell-based-meniscus-tissue-engineering
#3
Kazunori Shimomura, Benjamin B Rothrauff, Rocky S Tuan
BACKGROUND: The meniscus is the most commonly injured knee structure, and surgical repair is often ineffective. Tissue engineering-based repair or regeneration may provide a needed solution. Decellularized, tissue-derived extracellular matrices (ECMs) have received attention for their potential use as tissue-engineered scaffolds. In considering meniscus-derived ECMs (mECMs) for meniscus tissue engineering, it is noteworthy that the inner and outer regions of the meniscus have different structural and biochemical features, potentially directing the differentiation of cells toward region-specific phenotypes...
November 28, 2016: American Journal of Sports Medicine
https://www.readbyqxmd.com/read/27889295/novel-t7-like-expression-systems-used-for-halomonas
#4
Han Zhao, Haoqian M Zhang, Xiangbin Chen, Teng Li, Qiong Wu, Qi Ouyang, Guo-Qiang Chen
To engineer non-model organisms, suitable genetic parts must be available. However, biological parts are often host strain sensitive. It is therefore necessary to develop genetic parts that are functional regardless of host strains. Here we report several novel phage-derived expression systems used for transcriptional control in non-model bacteria. Novel T7-like RNA polymerase-promoter pairs were obtained by mining phage genomes, followed by in vivo characterization in non-model strains Halomonas spp TD01 and Pseudomonas entomophila...
November 24, 2016: Metabolic Engineering
https://www.readbyqxmd.com/read/27886065/dual-function-of-glucosamine-in-gelatin-hyaluronic-acid-cryogel-to-modulate-scaffold-mechanical-properties-and-to-maintain-chondrogenic-phenotype-for-cartilage-tissue-engineering
#5
Chih-Hao Chen, Chang-Yi Kuo, Yan-Jie Wang, Jyh-Ping Chen
Glucosamine (GlcN) fulfills many of the requirements as an ideal component in scaffolds used in cartilage tissue engineering. The incorporation of GlcN in a gelatin/hyaluronic acid (GH) cryogel scaffold could provide biological cues in maintaining the phenotype of chondrocytes. Nonetheless, substituting gelatin with GlcN may also decrease the crosslinking density and modulate the mechanical properties of the cryogel scaffold, which may be beneficial as physical cues for chondrocytes in the scaffold. Thus, we prepared cryogel scaffolds containing 9% GlcN (GH-GlcN9) and 16% GlcN (GH-GlcN16) by carbodiimide-mediated crosslinking reactions at -16 °C...
November 23, 2016: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/27884105/structural-features-of-dna-that-determine-rna-polymerase-ii-core-promoter
#6
Irina A Il'icheva, Mingian V Khodikov, Maria S Poptsova, Dmitry Yu Nechipurenko, Yury D Nechipurenko, Sergei L Grokhovsky
BACKGROUND: The general structure and action of all eukaryotic and archaeal RNA polymerases machinery have an astonishing similarity despite the diversity of core promoter sequences in different species. The goal of our work is to find common characteristics of DNA region that define it as a promoter for the RNA polymerase II (Pol II). RESULTS: The profiles of a large number of physical and structural characteristics, averaged over representative sets of the Pol II minimal core promoters of the evolutionary divergent species from animals, plants and unicellular fungi were analysed...
November 25, 2016: BMC Genomics
https://www.readbyqxmd.com/read/27878977/silk-fibroin-scaffolds-with-muscle-like-elasticity-support-in-vitro-differentiation-of-human-skeletal-muscle-cells
#7
Vishal Chaturvedi, Deboki Naskar, Beverley F Kinnear, Elizabeth Grenik, Danielle E Dye, Miranda D Grounds, Subhas C Kundu, Deirdre R Coombe
Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three-dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myoblasts. Human skeletal muscle myoblasts (HSMMs) adhered, spread and deposited extensive ECM on all the scaffolds, but immunofluorescence and quantitative polymerase chain reaction analysis of gene expression revealed that myotube formation occurred differently on the various scaffolds...
November 22, 2016: Journal of Tissue Engineering and Regenerative Medicine
https://www.readbyqxmd.com/read/27877186/selection-of-reference-genes-for-rt-qpcr-analysis-in-coccinella-septempunctata-to-assess-un-intended-effects-of-rnai-transgenic-plants
#8
Chunxiao Yang, Evan L Preisser, Hongjun Zhang, Yong Liu, Liangying Dai, Huipeng Pan, Xuguo Zhou
The development of genetically engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is reverse transcriptase-quantitative polymerase chain reaction, a technique for quantifying gene expression using a suite of reference genes for normalization...
2016: Frontiers in Plant Science
https://www.readbyqxmd.com/read/27871370/enhanced-detection-of-rna-by-mmlv-reverse-transcriptase-coupled-with-thermostable-dna-polymerase-and-dna-rna-helicase
#9
Hiroyuki Okano, Yuta Katano, Misato Baba, Ayako Fujiwara, Ryota Hidese, Shinsuke Fujiwara, Itaru Yanagihara, Tsukasa Hayashi, Kenji Kojima, Teisuke Takita, Kiyoshi Yasukawa
Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4polL329A from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis...
January 2017: Enzyme and Microbial Technology
https://www.readbyqxmd.com/read/27865598/suppression-of-cytochrome-p450-4b1-an-early-event-in-adrenocortical-tumorigenesis
#10
Timothy D Murtha, Reju Korah, Tobias Carling
BACKGROUND: Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Conversely, adrenocortical adenomas are common and benign. Despite their shared histologic origin, little evidence exists to suggest that adrenocortical adenoma arises from adrenocortical carcinoma. Recent genetic analyses of adrenocortical carcinoma have shown recurrent gene copy deletion of CYP4B1, a cytochrome P450 isozyme. This study investigates a potential role for CYP4B1 in modulating adrenocortical tumorigenesis and/or conferring chemoresistance to adrenocortical carcinomas...
November 16, 2016: Surgery
https://www.readbyqxmd.com/read/27855193/transcriptome-wide-identification-and-expression-analysis-of-the-nac-gene-family-in-tea-plant-camellia-sinensis-l-o-kuntze
#11
Yong-Xin Wang, Zhi-Wei Liu, Zhi-Jun Wu, Hui Li, Jing Zhuang
In plants, the NAC (NAM-ATAF1/2-CUC) family of proteins constitutes several transcription factors and plays vital roles in diverse biological processes, such as growth, development, and adaption to adverse factors. Tea, as a non-alcoholic drink, is known for its bioactive ingredients and health efficacy. Currently, knowledge about NAC gene family in tea plant remains very limited. In this study, a total of 45 CsNAC genes encoding NAC proteins including three membrane-bound members were identified in tea plant through transcriptome analysis...
2016: PloS One
https://www.readbyqxmd.com/read/27847750/in-vitro-characterization-of-human-dental-pulp-stem-cells-isolated-by-three-different-methods
#12
Ji-Hyun Jang, Hyeon-Woo Lee, Kyu Min Cho, Hee-Woong Shin, Mo Kwan Kang, Sang Hyuk Park, Euiseong Kim
OBJECTIVES: In this study, we characterized human dental pulp cells (HDPCs) obtained by different culture methods to establish the most suitable methodology for dental tissue engineering and regenerative endodontic applications. MATERIALS AND METHODS: HDPCs were isolated by the outgrowth method (HDPCs-OG), the enzymatic digestion method (collagenase/dispase/trypsin, HDPCs-ED), or the combination of both methods (HDPCs-Combined). The expression of mesenchymal stem cell markers (CD105, CD90, and CD73) was investigated...
November 2016: Restorative Dentistry & Endodontics
https://www.readbyqxmd.com/read/27842921/suppression-of-osteoblast-related-genes-during-osteogenic-differentiation-of-adipose-tissue-derived-stromal-cells
#13
Yahya Açil, Amir Alexander Ghoniem, Aydin Gülses, Tobias Kisch, Felix Stang, Jörg Wiltfang, Matthias Gierloff
Recent studies indicated a lower osteogenic differentiation potential of adipose tissue-derived stromal cells (ASCs) compared to bone marrow derived mesenchymal stromal cells. The aim of this study was to evaluate the effects of potent combinations of highly osteogenic bone morphogenetic proteins (BMPs) in order to enhance the osteogenic differentiation potential of ASCs. Human ASCs were cultured for 10 days in the presence of osteogenic medium consisting of dexamethasone, ß-glycerophosphate and ascorbat-2-phosphate (OM) supplemented with BMP-2, BMP-6, BMP-9+IGF-2 and BMP-2,-6,-9 (day 1+2: 50 ng/ml, days 3-6: 100 ng/ml, days 7-10: 200 ng/ml)...
October 18, 2016: Journal of Cranio-maxillo-facial Surgery
https://www.readbyqxmd.com/read/27837351/biomanufacturing-history-and-perspective
#14
REVIEW
Yi-Heng Percival Zhang, Jibin Sun, Yanhe Ma
Biomanufacturing is a type of manufacturing that utilizes biological systems (e.g., living microorganisms, resting cells, animal cells, plant cells, tissues, enzymes, or in vitro synthetic (enzymatic) systems) to produce commercially important biomolecules for use in the agricultural, food, material, energy, and pharmaceutical industries. History of biomanufacturing could be classified into the three revolutions in terms of respective product types (mainly), production platforms, and research technologies...
November 11, 2016: Journal of Industrial Microbiology & Biotechnology
https://www.readbyqxmd.com/read/27808482/crystal-structure-of-ralstonia-eutropha-polyhydroxyalkanoate-synthase-c-terminal-domain-and-reaction-mechanisms
#15
Jieun Kim, Yeo-Jin Kim, So Young Choi, Sang Yup Lee, Kyung-Jin Kim
Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by numerous microorganisms as energy and reducing power storage materials, and have attracted much attention as substitutes for petroleum-based plastics. Here, we report the first crystal structure of Ralstonia eutropha PHA synthase at 1.8 Å resolution and structure-based mechanisms for PHA polymerization. RePhaC1 contains two distinct domains, the N-terminal (RePhaC1ND ) and C-terminal domains (RePhaC1CD ), and exists as a dimer. RePhaC1CD catalyzes polymerization via non-processive ping-pong mechanism using a Cys-His-Asp catalytic triad...
November 3, 2016: Biotechnology Journal
https://www.readbyqxmd.com/read/27803268/library-quantification-using-sybr-green-quantitative-polymerase-chain-reaction-qpcr
#16
Elaine Mardis, W Richard McCombie
To quantify complex DNA libraries, Kapa Biosystems engineered a DNA polymerase specifically for SYBR Green-based qPCR, enabling efficient amplification of targets such as GC-rich DNAs that present a challenge to wild-type DNA polymerases. Kapa Library Quantification Kits contain this engineered polymerase to ensure robust amplification of longer fragments, across a broad range of GC content.
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27797358/t-dna-integration-in-plants-results-from-polymerase-%C3%AE-mediated-dna-repair
#17
Maartje van Kregten, Sylvia de Pater, Ron Romeijn, Robin van Schendel, Paul J J Hooykaas, Marcel Tijsterman
Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown(1). Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration...
October 31, 2016: Nature Plants
https://www.readbyqxmd.com/read/27794600/engineered-photoactivatable-genetic-switches-based-on-the-bacterium-phage-t7-rna-polymerase
#18
Tiyun Han, Quan Chen, Haiyan Liu
Genetic switches in which the activity of T7 RNA polymerase (RNAP) is directly regulated by external signals are obtained with an engineering strategy of splitting the protein into fragments and using regulatory domains to modulate their reconstitutions. Robust switchable systems with excellent dark-off/light-on properties are obtained with the light-activatable VVD domain and its variants as regulatory domains. For the best split position found, working switches exploit either the light-induced interactions between the VVD domains or allosteric effects...
October 30, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27761639/current-advances-in-molecular-methods-for-detection-of-nitrite-dependent-anaerobic-methane-oxidizing-bacteria-in-natural-environments
#19
Jing Chen, Richard Dick, Jih-Gaw Lin, Ji-Dong Gu
Nitrite-dependent anaerobic methane oxidation (n-damo) process uniquely links microbial nitrogen and carbon cycles. Research on n-damo bacteria progresses quickly with experimental evidences through enrichment cultures. Polymerase chain reaction (PCR)-based methods for detecting them in various natural ecosystems and engineered systems play a very important role in the discovery of their distribution, abundance, and biodiversity in the ecosystems. Important characteristics of n-damo enrichments were obtained and their key significance in microbial nitrogen and carbon cycles was investigated...
October 20, 2016: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/27730213/tunable-and-label-free-virus-enrichment-for-ultrasensitive-virus-detection-using-carbon-nanotube-arrays
#20
Yin-Ting Yeh, Yi Tang, Aswathy Sebastian, Archi Dasgupta, Nestor Perea-Lopez, Istvan Albert, Huaguang Lu, Mauricio Terrones, Si-Yang Zheng
Viral infectious diseases can erupt unpredictably, spread rapidly, and ravage mass populations. Although established methods, such as polymerase chain reaction, virus isolation, and next-generation sequencing have been used to detect viruses, field samples with low virus count pose major challenges in virus surveillance and discovery. We report a unique carbon nanotube size-tunable enrichment microdevice (CNT-STEM) that efficiently enriches and concentrates viruses collected from field samples. The channel sidewall in the microdevice was made by growing arrays of vertically aligned nitrogen-doped multiwalled CNTs, where the intertubular distance between CNTs could be engineered in the range of 17 to 325 nm to accurately match the size of different viruses...
October 2016: Science Advances
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