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Polymerase engineering

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https://www.readbyqxmd.com/read/28230059/synergistic-effects-of-dimethyloxallyl-glycine-and-recombinant-human-bone-morphogenetic-protein-2-on-repair-of-critical-sized-bone-defects-in-rats
#1
Xin Qi, Yang Liu, Zhen-Yu Ding, Jia-Qing Cao, Jing-Huan Huang, Jie-Yuan Zhang, Wei-Tao Jia, Jing Wang, Chang-Sheng Liu, Xiao-Lin Li
In bone remodeling, osteogenesis is closely coupled to angiogenesis. Bone tissue engineering using multifunctional bioactive materials is a promising technique which has the ability to simultaneously stimulate osteogenesis and angiogenesis for repair of bone defects. We developed mesoporous bioactive glass (MBG)-doped poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) composite scaffolds as delivery vehicle. Two bioactive molecules, dimethyloxalylglycine (DMOG), a small-molecule angiogenic drug, and recombinant human bone morphogenetic protein-2 (rhBMP-2), an osteoinductive growth factor, were co-incorporated into the scaffold...
February 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28218004/synthesis-and-characterization-of-some-abundant-nanoparticles-their-antimicrobial-and-enzyme-inhibition-activity
#2
Shams T Khan, Ajmaluddin Malik, Rizwan Wahab, Omar H Abd-Elkader, Maqusood Ahamed, Javed Ahmad, Javed Musarrat, Maqsood A Siddiqui, Abdulaziz A Al-Khedhairy
Although the antimicrobial activity of the engineered nanoparticles (NPs) is well known, the biochemical mechanisms underlying this activity are not clearly understood. Therefore, four NPs with the highest global production, namely SiO2, TiO2, ZnO, and Ag, were synthesized and characterized. The synthesized SiO2, TiO2, ZnO, and Ag NPs exhibit an average size of 11.12, 13.4, 35, and 50 nm, respectively. The antimicrobial activity of the synthesized NPs against bacteria and fungi were also determined. NPs-mediated inhibition of two very important enzymes, namely urease and DNA polymerase, is also reported...
February 20, 2017: Acta Microbiologica et Immunologica Hungarica
https://www.readbyqxmd.com/read/28208291/growth-based-bacterial-viability-assay-for-interference-free-and-high-throughput-toxicity-screening-of-nanomaterials
#3
Tian A Qiu, Thu Ha Thi Nguyen, Natalie V Hudson-Smith, Peter L Clement, Dona-Carla Forester, Hilena Frew, Mimi N Hang, Catherine J Murphy, Robert J Hamers, Z Vivian Feng, Christy L Haynes
Current high-throughput approaches evaluating toxicity of chemical agents toward bacteria typically rely on optical assays, such as luminescence and absorbance, to probe the viability of the bacteria. However, when applied to toxicity induced by nanomaterials, scattering and absorbance from the nanomaterials act as interferences that complicate quantitative analysis. Herein, we describe a bacterial viability assay that is free of optical interference from nanomaterials and can be performed in a high-throughput format on 96-well plates...
February 7, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28205142/stepwise-morphological-changes-and-cytoskeletal-reorganization-of-human-mesenchymal-stem-cells-treated-by-short-time-cyclic-uniaxial-stretch
#4
Azim Parandakh, Mohammad Tafazzoli-Shadpour, Mohammad-Mehdi Khani
This study aimed to investigate stepwise remodeling of human mesenchymal stem cells (hMSCs) in response to cyclic stretch through rearrangement and alignment of cells and cytoskeleton regulation toward smooth muscle cell (SMC) fate in different time spans. Image analysis techniques were utilized to calculate morphological parameters. Cytoskeletal reorganization was observed by investigating F-actin filaments using immunofluorescence staining, and expression level of contractile SMC markers was followed by a quantitative polymerase chain reaction method...
February 15, 2017: In Vitro Cellular & Developmental Biology. Animal
https://www.readbyqxmd.com/read/28192413/evolution-of-a-split-rna-polymerase-as-a-versatile-biosensor-platform
#5
Jinyue Pu, Julia Zinkus-Boltz, Bryan C Dickinson
Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies are often limited by a low signal-to-noise ratio, the extensive optimization required for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE)...
February 13, 2017: Nature Chemical Biology
https://www.readbyqxmd.com/read/28186723/bioresponsive-dna-hydrogels-beyond-the-conventional-stimuli-responsiveness
#6
Dong Wang, Yue Hu, Peifeng Liu, Dan Luo
Bioresponsive hydrogels can respond to various biological stimuli by a macroscopic change of physical state or by converting biochemical inputs into biological or mechanical outputs. These materials are playing an increasingly important role in a wide variety of applications, especially in the biological and biomedical fields. However, the design and engineering of intriguing bioresponsive materials with adequate biocompatibility and biodegradability have proven to be a great challenge. DNA, on the other hand, possesses many unique and fascinating properties, including its indispensable genetic function, broad biocompatibility, precise molecular recognition capability, tunable multifunctionality, and convenient programmability...
February 10, 2017: Accounts of Chemical Research
https://www.readbyqxmd.com/read/28177825/enhancement-of-single-guide-rna-transcription-for-efficient-crispr-cas-based-genomic-engineering
#7
Kumiko Ui-Tei, Shohei Maruyama, Yuko Nakano
Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein...
January 26, 2017: Genome Génome / Conseil National de Recherches Canada
https://www.readbyqxmd.com/read/28148824/biochemical-and-genetic-functional-dissection-of-the-p38-viral-suppressor-of-rna-silencing
#8
Taichiro Iki, Marie-Aude Tschopp, Olivier Voinnet
Phytoviruses encode viral suppressors of RNA-silencing (VSRs) to counteract the plant antiviral silencing response, which relies on virus-derived small interfering (si)RNAs processed by Dicer RNaseIII enzymes and subsequently loaded into ARGONAUTE (AGO) effector proteins. Here, a tobacco cell-free system was engineered to recapitulate the key steps of antiviral RNA-silencing and, in particular, the most upstream double-stranded (ds)RNA processing reaction, not kinetically investigated thus far in the context of plant VSR studies...
February 1, 2017: RNA
https://www.readbyqxmd.com/read/28131814/molecular-characterization-of-genetically-modified-crops-challenges-and-strategies
#9
REVIEW
Rong Li, Sheng Quan, Xiaofang Yan, Sukumar Biswas, Dabing Zhang, Jianxin Shi
Molecular characterization lays a foundation for safety assessment and subsequent monitoring of genetically modified (GM) crops. Due to the target-specific nature, conventional polymerase chain reaction (PCR)-based methods cannot comprehensively detect unintended gene insertions, let alone unknown GM events. As more and more new developed GM crops including new plant breeding technology (NPBT) generated crops are in the pipeline for commercialization, alternative -omics approaches, particularly next generation sequencing, have been developed for molecular characterization of authorized or unauthorized GM (UGM) crops...
March 2017: Biotechnology Advances
https://www.readbyqxmd.com/read/28128417/construction-and-transformation-of-expression-vector-containing-panax-japonicus-ss-gene
#10
L Zhang, T T Wang
Panax japonicus C.A. Meyer, a perennial herb belonging to the Araliaceae ginseng genus, is one of the seven rare and endangered Chinese medical herbs. By cloning the SS segment, the expression vectors pCXSN-PjSS and pCXSN-antiPjSS were constructed and introduced into Agrobactria LBA4404, which is used for engineering bacteria. Polymerase chain reaction (PCR) showed that the PjSS, antiPjSS, and Hyg were integrated in Nicotiana tabacum. Reverse transcription-PCR indicated that the PjSS was transcribed into mRNA in N...
January 23, 2017: Genetics and Molecular Research: GMR
https://www.readbyqxmd.com/read/28114966/tgf-%C3%AE-1-induced-differentiation-of-shed-into-functional-smooth-muscle-cells
#11
Jian Guang Xu, Shao Yue Zhu, Boon Chin Heng, Waruna Lakmal Dissanayaka, Cheng Fei Zhang
BACKGROUND: Adequate vascularization is crucial for supplying nutrition and discharging metabolic waste in freshly transplanted tissue-engineered constructs. Obtaining the appropriate building blocks for vascular tissue engineering (i.e. endothelial and mural cells) is a challenging task for tissue neovascularization. Hence, we investigated whether stem cells from human exfoliated deciduous teeth (SHED) could be induced to differentiate into functional vascular smooth muscle cells (vSMCs)...
January 23, 2017: Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/28110800/resource-reallocation-in-bacteria-by-reengineering-the-gene-expression-machinery
#12
REVIEW
Hidde de Jong, Johannes Geiselmann, Delphine Ropers
Bacteria have evolved complex regulatory networks to control the activity of transcription and translation, and thus the growth rate, over a range of environmental conditions. Reengineering RNA polymerase and ribosomes allows modifying naturally evolved regulatory networks and thereby profoundly reorganizing the manner in which bacteria allocate resources to different cellular functions. This opens new opportunities for our fundamental understanding of microbial physiology and for a variety of applications...
January 16, 2017: Trends in Microbiology
https://www.readbyqxmd.com/read/28109755/construction-of-agrobacterium-tumefaciens-mediated-tomato-black-ring-virus-infectious-cdna-clones
#13
Aleksandra Zarzyńska-Nowak, Inmaculada Ferriol, Bryce W Falk, Natasza Borodynko-Filas, Beata Hasiów-Jaroszewska
Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS)...
January 18, 2017: Virus Research
https://www.readbyqxmd.com/read/28109156/-cloning-and-sequence-analysis-of-variable-region-genes-of-mouse-anti-human-s100a9-monoclonal-antibody
#14
Min Yan, Danyang Mi, Xiangdong Sun, Zhenqiang Zhang, Huahui Zeng, Wendi Liu
Objective To clone and analyze variable region gene sequences of mouse monoclonal antibody against human S100A9 (anti-human S100A9 mAb). Methods Total RNA was extracted from the hybridoma cell line (IID4B10) secreting mouse anti-human S100A9 mAb. Furthermore, the heavy chain variable region (VH) and light chain variable region (VL) genes were amplified using reverse transcription-polymerase chain reaction (RT-PCR). After the cloning and sequencing of the VH and VL genes, the sequence analyses were performed by BLAST program...
February 2017: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/28108511/biomarker-based-pet-imaging-of-diffuse-intrinsic-pontine-glioma-in-mouse-models
#15
Susanne Kossatz, Brandon Carney, Melanie E Schweitzer, Giuseppe Carlucci, Vesselin Miloushev, Uday B Maachani, Prajwal Rajappa, Kayvan Keshari, David J Pisapia, Wolfgang A Weber, Mark M Souweidane, Thomas Reiner
Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a universally poor prognosis. Here, we characterize on a positron emission tomography (PET) probe for imaging DIPG in vivo. In human histological tissues, the probes target, poly(ADP)ribose polymerase 1 (PARP1), was highly expressed in DIPG compared to normal brain. PET imaging allowed for the sensitive detection of DIPG in a genetically engineered mouse model (GEMM), and probe uptake correlated to histologically determined tumor infiltration...
January 20, 2017: Cancer Research
https://www.readbyqxmd.com/read/28105130/biological-characterization-of-sheep-kidney-derived-mesenchymal-stem-cells
#16
Meng Ji, Chunyu Bai, Lu Li, Ya'Nan Fan, Caiyun Ma, Xiangchen Li, Weijun Guan
The aim of the present study was to isolate, culture and characterize sheep metanephric mesenchymal stem cells (MMSCs). The MMSCs were isolated from the kidney tissue of six-week-old sheep fetus. This study investigated whether primary MMSCs could be grown for 26 passages and expressed Oct-4, which is involved in the self-renewal of undifferentiated pluripotent stem cells. The MMSCs also expressed the renal lineage marker gene PAX2, and mesenchymal cell marker genes CD44, FN1 and VIM. Expression of these genes was detected using immunofluorescence and reverse transcription-polymerase chain reaction assays...
December 2016: Experimental and Therapeutic Medicine
https://www.readbyqxmd.com/read/28088723/discovery-and-metagenomic-analysis-of-an-anammox-bacterial-enrichment-related-to-candidatus-brocadia-caroliniensis-in-a-full-scale-glycerol-fed-nitritation-denitritation-separate-centrate-treatment-process
#17
Hongkeun Park, Ariane C Brotto, Mark C M van Loosdrecht, Kartik Chandran
A distinctive red biofilm was observed in a glycerol-fed digester liquid effluent treatment process coupling partial nitrification (nitritation) and partial denitrification (denitritation) processes. Based on initial phylogenetic screening using 16S rRNA clone libraries and quantitative polymerase chain reaction, the biofilm was enriched in novel anaerobic ammonium oxidizing bacteria (AMX/anammox) closely related to Candidatus "Brocadia caroliniensis". The metabolic functionality of the C. "Brocadia caroliniensis" enrichment was further explored using high-throughput sequencing and de novo metagenome assembly...
January 6, 2017: Water Research
https://www.readbyqxmd.com/read/28077981/dna-polymerase-kappa-protects-human-cells-against-mmc-induced-genotoxicity-through-error-free-translesion-dna-synthesis
#18
Yuki Kanemaru, Tetsuya Suzuki, Akira Sassa, Kyomu Matsumoto, Noritaka Adachi, Masamitsu Honma, Satoshi Numazawa, Takehiko Nohmi
BACKGROUND: Interactions between genes and environment are critical factors for causing cancer in humans. The genotoxicity of environmental chemicals can be enhanced via the modulation of susceptible genes in host human cells. DNA polymerase kappa (Pol κ) is a specialized DNA polymerase that plays an important role in DNA damage tolerance through translesion DNA synthesis. To better understand the protective roles of Pol κ, we previously engineered two human cell lines either deficient in expression of Pol κ (KO) or expressing catalytically dead Pol κ (CD) in Nalm-6-MSH+ cells and examined cytotoxic sensitivity against various genotoxins...
2017: Genes and Environment: the Official Journal of the Japanese Environmental Mutagen Society
https://www.readbyqxmd.com/read/28068880/low-intensity-vibrations-normalize-adipogenesis-induced-morphological-and-molecular-changes-of-adult-mesenchymal-stem-cells
#19
Oznur Baskan, Gulistan Mese, Engin Ozcivici
Bone marrow mesenchymal stem cells that are committed to adipogenesis were exposed daily to high-frequency low-intensity mechanical vibrations to understand molecular, morphological and ultrastructural adaptations to mechanical signals during adipogenesis. D1-ORL-UVA mouse bone marrow mesenchymal stem cells were cultured with either growth or adipogenic medium for 1 week. Low-intensity vibration signals (15 min/day, 90 Hz, 0.1 g) were applied to one group of adipogenic cells, while the other adipogenic group served as a sham control...
February 2017: Proceedings of the Institution of Mechanical Engineers. Part H, Journal of Engineering in Medicine
https://www.readbyqxmd.com/read/28053101/generation-of-a-genetically-stable-high-fidelity-influenza-vaccine-strain
#20
Tadasuke Naito, Kotaro Mori, Hiroshi Ushirogawa, Naoki Takizawa, Eri Nobusawa, Takato Odagiri, Masato Tashiro, Ryosuke L Ohniwa, Kyosuke Nagata, Mineki Saito
: Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth PR8 master virus...
January 4, 2017: Journal of Virology
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