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Polymerase engineering

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
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
Jezamine Lim, Zainul Rashid Mohamad Razi, Jiaxian Law, Azmawati Mohammed Nawi, Ruszymah Binti Haji Idrus, Min Hwei Ng
BACKGROUND AIMS: Human Wharton's jelly-derived mesenchymal stromal cells (hWJMSCs) are possibly the most suitable allogeneic cell source for stromal cell therapy and tissue engineering applications because of their hypo-immunogenic and non-tumorigenic properties, easy availability and minimal ethical concerns. Furthermore, hWJMSCs possess unique properties of both adult mesenchymal stromal cells and embryonic stromal cells. The human umbilical cord (UC) is approximately 50-60 cm long and the existing studies in the literature have not provided information on which segment of the UC was studied...
October 7, 2016: Cytotherapy
Mei-Chi Chang, Hsiao-Hua Chang, Po-Shuan Lin, Yu-An Huang, Chiu-Po Chan, Yi-Ling Tsai, Shen-Yang Lee, Po-Yuan Jeng, Han-Yueh Kuo, Sin-Yuet Yeung, Jiiang-Huei Jeng
Transforming growth factor-β1 (TGF-β1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-β1 on PA system of dental pulp cells and its signaling pathways. Dental pulp cells were treated with different concentrations of TGF-β1. MTT assay, reverse transcription-polymerase chain reaction (RT-PCR), western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-β1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion...
October 9, 2016: Journal of Tissue Engineering and Regenerative Medicine
Juan Chen, Lei Fan, Ya Du, Weining Zhu, Ziqin Tang, Na Li, Dapeng Zhang, Linsheng Zhang
Drought stress is a major factor limiting wheat growth and productivity. Late embryogenesis abundant (LEA) proteins are tolerant to water-related stress. To reveal the regulatory mechanisms of LEA proteins under drought stress, we cloned a novel group 3 LEA gene, namely, TaDlea3, from wheat (Triticum aestivum L.) Shaanhe 6. Subcellular localization assay showed that TaDlea3 protein accumulated in the cytoplasm. Quantitative real-time polymerase chain reaction results revealed that TaDlea3 expression was induced by drought stress...
November 2016: Plant Science: An International Journal of Experimental Plant Biology
Charles E Vejnar, Miguel A Moreno-Mateos, Daniel Cifuentes, Ariel A Bazzini, Antonio J Giraldez
This protocol describes how to generate and genotype mutants using an optimized CRISPR-Cas9 genome-editing system in zebrafish (CRISPRscan). Because single guide RNAs (sgRNAs) have variable efficiency when targeting specific loci, our protocol starts by explaining how to use the web tool CRISPRscan to design highly efficient sgRNAs. The CRISPRscan algorithm is based on the results of an integrated analysis of more than 1000 sgRNAs in zebrafish, which uncovered highly predictive factors that influence Cas9 activity...
October 3, 2016: Cold Spring Harbor Protocols
Pouria HosseinNia, Mehdi Hajian, Mojtaba Tahmoorespur, Sayyed Morteza Hosseini, Somayyeh Ostadhosseini, Mohammad Reza Nasiri, Mohammad Hossein Nasr-Esfahani
BACKGROUND: Little is understood about the regulation of gene expression during early goat embryo development. This study investigated the expression profile of 19 genes, known to be critical for early embryo development in mouse and human, at five different stages of goat in vitro embryo development (oocyte, 8-16 cell, morula, day-7 blastocyst, and day 14 blastocyst). MATERIALS AND METHODS: In this experimental study, stage-specific profiling using real time-quantitative polymerase chain reaction (RT-qPCR) revealed robust and dynamic patterns of stage-specific gene activity that fall into four major clusters depending on their respective mRNA profiles...
October 2016: International Journal of Fertility & Sterility
Wei-Zhen Lew, Yu-Chih Huang, Kuen-Yu Huang, Che-Tong Lin, Ming-Tzu Tsai, Haw-Ming Huang
Dental pulp stem cells (DPSCs) can be a potential stem cell resource for clinical cell therapy and tissue engineering. However, obtain a sufficient number of DPSCs for repairing defects is still an issue in clinical applications. Static magnetic fields (SMFs) enhance the proliferation of several cell types. Whether or not SMFs have a positive effect on DPSC proliferation is unknown. Therefore, the aim of this study was to investigate the effect of SMFs on DPSC proliferation and its possible intracellular mechanism of action...
September 29, 2016: Journal of Tissue Engineering and Regenerative Medicine
Gangyi Chen, Juan Dong, Yi Yuan, Na Li, Xin Huang, Xin Cui, Zhuo Tang
Nucleic acid amplification is the core technology of molecular biology and genetic engineering. Various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). However, most of these methods can only detect single stranded nucleic acid. Herein, we put forward a simple solution for opening double-stranded DNA for isothermal detection methods. The strategy employs recombination protein from E. coli (RecA) to form nucleoprotein complex with single-stranded DNA, which could scan double-stranded template for homologous sites...
September 30, 2016: Scientific Reports
Vishnu Krishnamurthy, Kai Zhang
Precise DNA manipulation is a key enabling technology for synthetic biology. Approaches based on restriction digestion are often limited by the presence of certain restriction enzyme recognition sites. Recent development of restriction-free cloning approaches has greatly enhanced the flexibility and speed of molecular cloning. Most restriction-free cloning methods focus on DNA assembly. Much less work has been dedicated towards DNA removal. Here we introduce a protocol that allows simultaneous removal of multiple DNA segments from a plasmid using polymerase chain reactions (PCR)...
2017: Methods in Molecular Biology
Shu He, Kai-Feng Lin, Jun-Jun Fan, Gang Hu, Xin Dong, Yi-Nan Zhao, Yue Song, Zhong-Shang Guo, Long Bi, Jian Liu
A microsphere composite made of poly(DL-lactic-co-glycolic acid) (PLGA), mesoporous silica nanoparticle (MSN), and nanohydroxyapatite (nHA) (PLGA-MSN/nHA) was prepared and evaluated as bone tissue engineering materials. The objective of this study was to investigate the synergistic effect of MSN/nHA on biocompatibility as well as its potential ability for bone formation. First, we found that this PLGA-MSN/nHA composite performed good characteristics on microstructure, mechanical strength, and wettability. By cell culture experiments, the adhesion and proliferation rate of the cells seeded on PLGA-MSN/nHA composite was higher than those of the controls and high levels of osteogenetic factors such as ALP and Runx-2 were detected by reverse transcriptase polymerase chain reaction...
2016: BioMed Research International
Marco-Antonio Mendoza-Parra, Valeriya Malysheva, Mohamed Ashick Mohamed Saleem, Michele Lieb, Aurelie Godel, Hinrich Gronemeyer
Cell lineages, which shape the body architecture and specify cell functions, derive from the integration of a plethora of cell intrinsic and extrinsic signals. These signals trigger a multiplicity of decisions at several levels to modulate the activity of dynamic gene regulatory networks (GRNs), which ensure both general and cell-specific functions within a given lineage, thereby establishing cell fates. Significant knowledge about these events and the involved key drivers comes from homogeneous cell differentiation models...
September 20, 2016: Genome Research
Christian B Winiger, Ryan W Shaw, Myong-Jung Kim, Jennifer D Moses, Mariko F Matsuura, Steven A Benner
2,4-Diaminopyrimidine (trivially K) and imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (trivially X) form a nucleobase pair with Watson-Crick geometry as part of an artificially expanded genetic information system (AEGIS). Neither K nor X can form a Watson-Crick pair with any natural nucleobase. Further, neither K nor X has an accessible tautomeric form or a protonated/deprotonated state that can form a Watson-Crick pair with any natural nucleobase. In vitro experiments show how DNA polymerase I from E. coli manages replication of DNA templates with one K:X pair, but fails with templates containing two adjacent K:X pairs...
October 7, 2016: ACS Synthetic Biology
Wendong Ruan, Yuan Xue, Yaqi Zong, Chao Sun
In the present study, third‑generation autologous‑inactivated bone morphogenic protein 2 (BMP2), BMP4, BMP6, BMP7, BMP9 and Wnt3a lentiviral vectors were constructed and integrated into the genome of MC3T3‑E1 murine mesenchymal stem cells (MMSCs) to produce osteoinductive factor gene‑modified MMSCs. The transfection efficiency of each osteoinductive factor was then determined by detecting the expression levels of runt related transcription factor 2 (Runx2) mRNA. The cotransfection with combinations of two lentiviruses was performed, and the expression levels of bone γ‑carboxyglutamate protein and alkaline phosphatase in the MC3T3‑E1 cell culture supernatant were detected...
September 12, 2016: Molecular Medicine Reports
Elham Ahmadi, Hoorieh Soleimanjahi, Majid Sadeghizadeh, Ali Teimoori
BACKGROUND: Group A Rotaviruses (GARV) are the main viral cause of acute gastroenteritis, leading to 870,000 deaths annually in the developing world and representing a major health problem. Therefore, diagnosis and treatment of this disease are crucial. Gene rearrangement within segmented viruses as well as rotavirus is seen throughout chronic rotavirus infection in immunodeficient young children and through serial passage of rotavirus in cell culture at a high multiplicity of infection...
September 2016: Archives of Iranian Medicine
Zongli Chu, Junying Chen, Haixia Xu, Zhongdong Dong, Feng Chen, Dangqun Cui
Feasible and efficient tissue culture plays an important role in plant genetic engineering. Wheat (Triticum aestivum L.) immature embryos (IMEs) are preferred for tissue culture to mature embryos (MEs) because IMEs easily generate embryogenic callus, producing large number of plants. The molecular mechanisms of regulation and the biological pathways involved in embryogenic callus formation in wheat remain unclear. Here, microRNAs (miRNAs) potentially involved in embryogenic callus formation and somatic embryogenesis were identified through deep sequencing of small RNAs (sRNAs) and analyzed with bioinformatics tools...
2016: Frontiers in Plant Science
Zhen Zhang, Ming Li, Feixue Chen, Lixiang Li, Jun Liu, Zhen Li, Rui Ji, Xiuli Zuo, Yanqing Li
INTRODUCTION: Mesenchymal stem cells (MSCs) can serve as vehicles for therapeutic genes. However, little is known about MSC behavior in vivo. Here, we demonstrated that probe-based confocal laser endomicroscopy (pCLE) can be used to track MSCs in vivo and individually monitor tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) gene expression within carcinomas. METHODS: Isolated BALB/c nu/nu mice MSCs (MSCs) were characterized and engineered to co-express the TRAIL and enhanced green fluorescent protein (EGFP) genes...
2016: PloS One
Tetsuya Suzuki, Petr Grúz, Masamitsu Honma, Noritaka Adachi, Takehiko Nohmi
Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i...
September 2016: Mutation Research
R J Bevacqua, R Fernandez-Martín, V Savy, N G Canel, M I Gismondi, W A Kues, D F Carlson, S C Fahrenkrug, H Niemann, O A Taboga, S Ferraris, D F Salamone
The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos...
November 2016: Theriogenology
Henning Hanken, Friedemann Göhler, Ralf Smeets, Max Heiland, Alexander Gröbe, Reinhard E Friedrich, Philipp Busch, Marco Blessmann, Lan Kluwe, Philip Hartjen
BACKGROUND/AIM: For application of stem cells and progenitor cells in regenerative medicine, scaffolds for carrying the cells play a key role. One promising biomaterial for scaffold generation is silk because of its mechanical strength, good cytocompatibility and low immunogenicity. Furthermore, bioengineering of silk proteins enable co-expression of various growth, differentiation and angiogenic factors on silk fibers, which may promote cell growth, differentiation and angiogenesis. This study aimed to test cytocompatibility and growth/differentiation of pre-adipose cells on scaffolds with and without expressed growth factors fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF)...
September 2016: In Vivo
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