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Polymerase engineering

Maryam Bahrami, Ali Valiani, Noushin Amirpour, Mohammad Zamani Ra Rani, Batool Hashemibeni
Background: Nowadays, cartilage tissue engineering is the best candidate for regeneration of cartilage defects. This study evaluates the function of herbal extracts icariin (ICA), the major pharmacological constituent of herba Epimedium , compared with transforming growth factor β3 (TGFβ3) to prove its potential effect for cartilage tissue engineering. Materials and Methods: ICA, TGFβ3, and TGFβ3 + ICA were added fibrin-cell constructions derived from adipose tissue stem cells...
2018: Advanced Biomedical Research
Jinjuan Yang, Qianjun Zhao, Kunfu Wang, Caiyun Ma, Hao Liu, Yingjie Liu, Weijun Guan
Tendon-derived stem cells (TDSCs), a postulated multi-potential stem cell population, play significant role in the postnatal replenishment of tendon injuries. However, the majority of experimental materials were obtained from horse, rat, human and rabbit, but rarely from pig. In this research, 1‑day‑old pig was chosen as experimental sample source to isolate and culture TDSCs in vitro. Specific markers of TDSCs were then characterized by immunofluorescence and reverse transcription polymerase chain reaction (RT‑PCR) assays...
March 7, 2018: International Journal of Molecular Medicine
Bing-Li Bai, Zhong-Jie Xie, She-Ji Weng, Zong-Yi Wu, Hang Li, Zhou-Shan Tao, Viraj Boodhun, De-Yi Yan, Zi-Jian Shen, Jia-Hao Tang, Lei Yang
Chitosan Oligosaccharide (COS) has been widely used for the systemic treatment of clinical diseases such as bone tissue engineering. However, its influence on osteoclast formation, which plays a critical role in bone homeostasis, has never been investigated. The aim of this study was to investigate the effect of chitosan oligosaccharide on differentiation of osteoclast. Using cell counting kit-8, tartrate-resistant acid phosphatase staining, reverse transcription‑quantitative polymerase chain reaction assay and western blot analysis, we demonstrated that chitosan oligosaccharide cannot inhibit RANKL-induced osteoclast precursor proliferation but does promote osteoclast differentiation by stimulating the activation of p38/mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK)/MAPK, extracellular signal-regulated kinase (ERK)/MAPK and protein kinase B (AKT) without affecting nuclear factor kappaB (NF-kB) signaling pathways...
March 3, 2018: Journal of Biomaterials Science. Polymer Edition
Usman Aslam, Bushra Tabassum, Idrees Ahmad Nasir, Anwar Khan, Tayyab Husnain
RNA interference (RNAi) is commonly used to produce virus tolerant transgenic plants. The objective of the current study was to generate transgenic sugarcane plants expressing a short hairpin RNAs (shRNA) targeting the coat protein (CP) gene of sugarcane mosaic virus (SCMV). Based on multiple sequence alignment, including genomic sequences of four SCMV strains, a conserved region of ~ 456 bp coat protein (CP) gene was selected as target gene and amplified through polymerase chain reaction (PCR). Subsequently, siRNAs2 and siRNA4 were engineered as stable short hairpin (shRNA) transgenes of 110 bp with stem and loop sequences derived from microRNA (sof-MIR168a; an active regulatory miRNA in sugarcane)...
February 28, 2018: Transgenic Research
Karl P Hodel, Richard de Borja, Erin E Henninger, Brittany B Campbell, Nathan Ungerleider, Nicholas Light, Tong Wu, Kimberly G LeCompte, A Yasemin Goksenin, Bruce A Bunnell, Uri Tabori, Adam Shlien, Zachary F Pursell
Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations...
February 28, 2018: ELife
Seyed Ehsan Enderami, Mousa Kehtari, Mohammad Foad Abazari, Pegah Ghoraeian, Maryam Nouri Aleagha, Fatemeh Soleimanifar, Masoud Soleimani, Yousef Mortazavi, Samad Nadri, Hossein Mostafavi, Hassan Askari
Pancreatic tissue engineering as a therapeutic option for restoring and maintenance of damaged pancreas function has a special focus to using synthetic Scaffolds. This study was designed to evaluate pancreatic differentiation of human induced pluripotent stem cells (hiPSCs) on poly-L-lactic acid and polyvinyl alcohol (PLLA/PVA) scaffolds as 3 D matrix. During differentiation process, morphology of cells gradually changed and iPSCs derived insulin producing cells (iPSCs-IPCs) formed spherical shaped cell aggregation that was the typical shape of islets of pancreas...
February 27, 2018: Artificial Cells, Nanomedicine, and Biotechnology
Annette Niehl, Marjukka Soininen, Minna M Poranen, Manfred Heinlein
Pathogens induce severe damages on cultivated plants and represent a serious threat to global food security. Emerging strategies for crop protection involve the external treatment of plants with double-stranded (ds)RNA to trigger RNA interference. However, applying this technology in greenhouses and fields depends on dsRNA quality, stability, and efficient large-scale production. Using components of the bacteriophage phi6, we engineered a stable and accurate in vivo dsRNA production system in Pseudomonas syringae bacteria...
February 26, 2018: Plant Biotechnology Journal
L F Mendes, H Katagiri, W L Tam, Y C Chai, L Geris, S J Roberts, F P Luyten
BACKGROUND: Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models. METHODS: hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2...
February 21, 2018: Stem Cell Research & Therapy
Dan Ding, Kaiyuan Chen, Yuedan Chen, Hong Li, Kabin Xie
The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system has emerged as the revolutionary platform for DNA targeting. This system uses a site-specific RNA guide to direct a CRISPR effector (e.g., Cas9 and Cpf1) to a DNA target. Here, we elaborate a general strategy to simultaneously express multiple guide RNAs (gRNA) and CRISPR RNAs (crRNA) from introns of Cas9 and Cpf1. This method utilizes the endogenous tRNA processing system or crRNA processing activity of Cpf1 to cleave the spliced intron which contains tRNA-gRNA polycistron or crRNA-crRNA array...
February 17, 2018: Molecular Plant
Long V Pham, Santseharay Ramirez, Judith M Gottwein, Ulrik Fahnøe, Yi-Ping Li, Jannie Pedersen, Jens Bukh
BACKGROUND & AIMS: Chronic liver diseases caused by hepatitis C virus (HCV) genotype 6 are prevalent in Asia, and millions of people require treatment with direct-acting antiviral regimens, such as NS5A inhibitor velpatasvir combined with the NS5B polymerase inhibitor sofosbuvir. We developed infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. METHODS: The consensus sequences of prototype strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined by Sanger sequencing of genomes amplified by reverse transcription-PCR...
February 15, 2018: Gastroenterology
Fiyinfoluwa A Adesioye, Thulani P Makhalanyane, Surendra Vikram, Bryan T Sewell, Wolf-Dieter Schubert, Don A Cowan
A hot desert hypolith metagenomic DNA sequence dataset was screened in-silico for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded a ∼36 kDa protein (Axe1 NaM1 ). The synthesised gene was cloned, expressed and the resulting protein, purified. NaM1 was optimally active at pH 8.5 and 30°C, and functionally stable at salt concentrations up to 5 M. The specific activity and catalytic efficiency were 488.9 Umg -1 and 3.26x10 6 M -1 s -1 , respectively. The crystal structure of wild type NaM1 was solved at a resolution of 2...
February 16, 2018: Applied and Environmental Microbiology
Jens Waldmann, Volker Fendrich, Martin Reichert, Andreas Hecker, Detlef K Bartsch, Winfried Padberg, Julia P N Holler
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is among the most dismal of human malignancies. Neuropeptides have shown to be implicated in angiogenesis, tumor growth, and formation of distant metastases in various solid tumors. In the present study, we used a genetically engineered mouse model of pancreatic cancer to evaluate the impact of neuropeptide Y (NPY) and its receptors 1 (Y1) and 2 (Y2) in preneoplastic lesions and pancreatic cancer as a potential target with antiproliferative properties...
March 2018: Journal of Surgical Research
Joshua D Eaton, Lee Davidson, David L V Bauer, Toyoaki Natsume, Masato T Kanemaki, Steven West
Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5' → 3' exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3' flanking region RNA and transcriptional termination...
February 8, 2018: Genes & Development
Vito Genna, Paolo Carloni, Marco De Vivo
Polymerases (Pols) synthesize the double-stranded nucleic acids in the Watson-Crick (W-C) conformation, which is critical for DNA and RNA functioning. Yet, the molecular basis to catalyze the W-C base pairing during Pol-mediated nucleic acids biosynthesis remains unclear. Here, through bioinformatics analyses on a large dataset of Pol/DNA structures, we first describe the conserved presence of one positively charged residue (Lys or Arg), which is similarly located near the enzymatic two-metal active site, always interacting directly with the incoming substrate (d)NTP...
February 9, 2018: Journal of the American Chemical Society
Houssein Gbaguidi-Haore, Amélie Varin, Pascal Cholley, Michelle Thouverez, Didier Hocquet, Xavier Bertrand
OBJECTIVE To describe an outbreak of multidrug-resistant Pseudomonas aeruginosa in which the hospital waste-pipe system was the likely source of contamination and to report the bundle of measures that facilitated the long-term control of the outbreak. DESIGN Outbreak investigation. SETTING The hematology unit of a tertiary-care referral center. PATIENTS Patients who were colonized or infected with P. aeruginosa belonging to the clonal outbreak. METHODS Patients admitted to our 15-bed stem-cell transplantation hematology unit were screened for P...
February 2018: Infection Control and Hospital Epidemiology
Maryam Shahi, Mahmood Nadari, Mehdi Sahmani, Ehsan Seyedjafari, Naser Ahmadbeigi, Amir Peymani
The limitation of traditional bone grafts could be overcome by applying engineered bone constructs, which are mainly produced by seeding suitable stem cells on appropriate scaffolds. So far, bone marrow-derived stromal cells have been the most applied cells in bone tissue engineering, but current data show that unrestricted somatic stem cells (USSCs) from human cord blood might actually be a better stem cell source due to the accessibility and noninvasive procedure of collection. In this study, we cultured USSCs on a plasma-treated electrospun polylactic-co-glycolic acid (PLGA) scaffold coated with nanohydroxyapatite (nHA)...
February 8, 2018: Cells, Tissues, Organs
Gabriel Bidaux, Corentin Le Nézet, Mariano Gonzalez Pisfil, Mélanie Henry, Alessandro Furlan, Oliver Bensaude, Bernard Vandenbunder, Laurent Héliot
Biochemical studies have revealed that the RNA Polymerase II (RNAPII) pause release is triggered by phosphorylation of the transcription machinery by the positive transcription elongation factor b (P-TEFb). However, there are no direct report that P-TEFb and RNA polymerase II interact in single living cells and the biophysical mechanisms mediating this association are still unclear. Förster resonance energy transfer (FRET) detects molecular interactions at the subcellular level. Time domain fluorescence lifetime imaging provides an accurate quantification of FRET efficiency, EFRET, because it is fluorochrome concentration-independent and insensitive to fluorescence bleed-through...
February 6, 2018: Biophysical Journal
Christopher Cozens, Vitor B Pinheiro
Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations - particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0...
February 2, 2018: Nucleic Acids Research
Masateru Takahashi, Etsuko Takahashi, Luay I Joudeh, Monica Marini, Gobind Das, Mohamed M Elshenawy, Anastassja Akal, Kosuke Sakashita, Intikhab Alam, Muhammad Tehseen, Mohamed A Sobhy, Ulrich Stingl, Jasmeen S Merzaban, Enzo Di Fabrizio, Samir M Hamdan
The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein's surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing...
January 24, 2018: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
Ling Liu, Jian Pan, Zilong Wang, Xiaohui Yan, Dong Yang, Xiangcheng Zhu, Ben Shen, Yanwen Duan, Yong Huang
Tiancimycin (TNM) A, a recently discovered enediyne natural product from Streptomyces sp. CB03234, showed rapid and complete killing of cancer cells and could be used as a payload in antibody drug conjugates. The low yield of TNM A in the wild-type strain promoted us to use ribosome engineering and fermentation optimization for its yield improvement. The Streptomyces sp. CB03234-R-16 mutant strain with a L422P mutation in RpoB, the RNA polymerase β-subunit, was obtained from the rifamycin-resistant screening...
March 2018: Journal of Industrial Microbiology & Biotechnology
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