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Polymerase engineering

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https://www.readbyqxmd.com/read/29331090/enhanced-chondrogenesis-of-mesenchymal-stem-cells-over-silk-fibroin-chitosan-chondroitin-sulfate-three-dimensional-scaffold-in-dynamic-culture-condition
#1
Parinita Agrawal, Krishna Pramanik, Varshini Vishwanath, Amit Biswas, Akalabya Bissoyi, Pradeep Kumar Patra
Chondroitin sulfate (Ch) is one of the main structural components of cartilage tissue, therefore, its presence in tissue engineered scaffold is expected to enhance cartilage regeneration. Previously, silk fibroin/chitosan (SF/CS) blend was proven to be a potential biomaterial for tissue development. In this study, the effect of Ch on physicochemical and biological properties of SF/CS blend was investigated and scaffolds with 0.8 wt% Ch was found to be favorable. The scaffolds possess pore size of 37-212 µm, contact angle 46...
January 13, 2018: Journal of Biomedical Materials Research. Part B, Applied Biomaterials
https://www.readbyqxmd.com/read/29315015/tumor-specific-t-cells-engineered-to-overcome-tumor-immune-evasion-induce-clinical-responses-in-patients-with-relapsed-hodgkin-lymphoma
#2
Catherine M Bollard, Tamara Tripic, Conrad Russell Cruz, Gianpietro Dotti, Stephen Gottschalk, Vicky Torrano, Olga Dakhova, George Carrum, Carlos A Ramos, Hao Liu, Meng-Fen Wu, Andrea N Marcogliese, Cecilia Barese, Youli Zu, Daniel Y Lee, Owen O'Connor, Adrian P Gee, Malcolm K Brenner, Helen E Heslop, Cliona M Rooney
Purpose Transforming growth factor-β (TGF-β) production in the tumor microenvironment is a potent and ubiquitous tumor immune evasion mechanism that inhibits the expansion and function of tumor-directed responses; therefore, we conducted a clinical study to discover the effects of the forced expression of a dominant-negative TGF-β receptor type 2 (DNRII) on the safety, survival, and activity of infused tumor-directed T cells. Materials and Methods In a dose escalation study, eight patients with Epstein Barr virus-positive Hodgkin lymphoma received two to 12 doses of between 2 × 107 and 1...
January 9, 2018: Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology
https://www.readbyqxmd.com/read/29304078/the-endocannabinoid-gene-faah2a-modulates-stress-associated-behavior-in-zebrafish
#3
Randall G Krug, Han B Lee, Louis Y El Khoury, Ashley N Sigafoos, Morgan O Petersen, Karl J Clark
The ability to orchestrate appropriate physiological and behavioral responses to stress is important for survival, and is often dysfunctional in neuropsychiatric disorders that account for leading causes of global disability burden. Numerous studies have shown that the endocannabinoid neurotransmitter system is able to regulate stress responses and could serve as a therapeutic target for the management of these disorders. We used quantitative reverse transcriptase-polymerase chain reactions to show that genes encoding enzymes that synthesize (abhd4, gde1, napepld), enzymes that degrade (faah, faah2a, faah2b), and receptors that bind (cnr1, cnr2, gpr55-like) endocannabinoids are expressed in zebrafish (Danio rerio)...
2018: PloS One
https://www.readbyqxmd.com/read/29261807/characterization-of-host-proteins-interacting-with-the-lymphocytic-choriomeningitis-virus-l-protein
#4
Kseniya Khamina, Alexander Lercher, Michael Caldera, Christopher Schliehe, Bojan Vilagos, Mehmet Sahin, Lindsay Kosack, Anannya Bhattacharya, Peter Májek, Alexey Stukalov, Roberto Sacco, Leo C James, Daniel D Pinschewer, Keiryn L Bennett, Jörg Menche, Andreas Bergthaler
RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics...
December 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/29246156/the-tunable-prex-expression-vector-enables-optimizing-the-t7-based-production-of-membrane-and-secretory-proteins-in-e-coli
#5
Grietje Kuipers, Alexandros Karyolaimos, Zhe Zhang, Nurzian Ismail, Gianluca Trinco, David Vikström, Dirk Jan Slotboom, Jan-Willem de Gier
BACKGROUND: To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme...
December 16, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/29227944/effects-of-starch-coating-of-magnetite-nanoparticles-on-cellular-uptake-toxicity-and-gene-expression-profiles-in-adult-zebrafish
#6
Min Zheng, Jianguo Lu, Dongye Zhao
Engineered magnetite nanoparticles (Fe3O4 NPs) have been used in many fields. To prevent particle agglomeration, stabilizers or coatings are often required. While such coatings have been shown to enhance performances, the environmental impact or toxicity of stabilized or coated Fe3O4 NPs remain poorly understood. In an effort to understand the impacts of such coatings on the toxicity of Fe3O4 NPs, we used the transcriptome sequencing (RNA-seq) technique to characterize the gill and liver transcriptomes from adult zebrafish when exposed to bare and starch-stabilized Fe3O4 NPs for 7days, demonstrating remarkable differences in gene expression profiles, also known as differentially expressed genes (DEGs) profiles, in both tissues...
December 8, 2017: Science of the Total Environment
https://www.readbyqxmd.com/read/29225080/second-shell-basic-residues-expand-the-two-metal-ion-architecture-of-dna-and-rna-processing-enzymes
#7
Vito Genna, Matteo Colombo, Marco De Vivo, Marco Marcia
Synthesis and scission of phosphodiester bonds in DNA and RNA regulate vital processes within the cell. Enzymes that catalyze these reactions operate mostly via the recognized two-metal-ion mechanism. Our analysis reveals that basic amino acids and monovalent cations occupy structurally conserved positions nearby the active site of many two-metal-ion enzymes for which high-resolution (<3 Å) structures are known, including DNA and RNA polymerases, nucleases such as Cas9, and splicing ribozymes. Integrating multiple-sequence and structural alignments with molecular dynamics simulations, electrostatic potential maps, and mutational data, we found that these elements always interact with the substrates, suggesting that they may play an active role for catalysis, in addition to their electrostatic contribution...
December 1, 2017: Structure
https://www.readbyqxmd.com/read/29214500/enhancing-cellulase-and-hemicellulase-production-in-trichoderma-orientalis-eu7-22-via-knockout-of-the-crea
#8
Chuannan Long, Yijin Cheng, Jingjing Cui, Jian Liu, Lihui Gan, Bin Zeng, Minnan Long
The role of the transcription factor creA-mediating carbon catabolite repression in Trichoderma orientalis EU7-22 was investigated for cellulase and hemicellulase production. The binary vector pUR5750G/creA::hph was constructed to knock out creA by homologous integration, generating the ΔcreA mutant Trichoderma orientalis CF1D. For strain CF1D, the filter paper activities (FPA), endoglucanase activities (CMC), cellobiohydrolase activity(CBH), β-glucosidase activity (BG), xylanase activity (XYN), and extracellular protein concentration were 1...
December 6, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29208279/collagen-alginate-as-bioink-for-three-dimensional-3d-cell-printing-based-cartilage-tissue-engineering
#9
Xingchen Yang, Zhenhui Lu, Huayu Wu, Wei Li, Li Zheng, Jinmin Zhao
Articular cartilage repair is still a huge challenge for researchers and clinicians. 3D bioprinting could be an innovative technology for cartilage tissue engineering. In this study, we used collagen type I (COL) or agarose (AG) mixed with sodium alginate (SA) to serve as 3D bioprinting bioinks and incorporated chondrocytes to construct in vitro 3D printed cartilage tissue. Swelling ratio, mechanical properties, scanning electron microscopy (SEM), cell viability and cytoskeleton, biochemistry analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to investigate the function of different bioinks in 3D printing cartilage tissue engineering applications...
February 1, 2018: Materials Science & Engineering. C, Materials for Biological Applications
https://www.readbyqxmd.com/read/29207049/coordinated-microrna-mrna-expression-profiles-reveal-a-putative-mechanism-of-corneal-epithelial-cell-transdifferentiation-from-skin-epidermal-stem-cells
#10
Yanjie Guo, Xiya Ma, Weini Wu, Mingyan Shi, Junlong Ma, Yaping Zhang, Erkang Zhao, Xueyi Yang
Skin epidermal stem cells (SESCs), which share a single origin with corneal epithelial cells (CECs), are considered to be one of the most ideal seed cells for the construction of tissue engineered corneas. However, the mechanism underlying the transdifferentiation of SESCs to CECs has not been fully elucidated. In the present study, to identify critical microRNAs (miRNAs/miRs) and genes that regulate the transdifferentiation of SESCs to CECs, SESCs and CECs were collected from sheep and used for small RNA sequencing and mRNA microarray analyses...
December 1, 2017: International Journal of Molecular Medicine
https://www.readbyqxmd.com/read/29204151/an-alcohol-dehydrogenase-gene-from-synechocystis-sp-confers-salt-tolerance-in-transgenic-tobacco
#11
So Young Yi, Seong Sub Ku, Hee-Jung Sim, Sang-Kyu Kim, Ji Hyun Park, Jae Il Lyu, Eun Jin So, So Yeon Choi, Jonghyun Kim, Myung Suk Ahn, Suk Weon Kim, Hyunwoo Park, Won Joong Jeong, Yong Pyo Lim, Sung Ran Min, Jang Ryol Liu
Synechocystis salt-responsive gene 1 (sysr1) was engineered for expression in higher plants, and gene construction was stably incorporated into tobacco plants. We investigated the role of Sysr1 [a member of the alcohol dehydrogenase (ADH) superfamily] by examining the salt tolerance of sysr1-overexpressing (sysr1-OX) tobacco plants using quantitative real-time polymerase chain reactions, gas chromatography-mass spectrometry, and bioassays. The sysr1-OX plants exhibited considerably increased ADH activity and tolerance to salt stress conditions...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/29203175/impact-of-hpv-16-18-infection-on-clinical-outcomes-in-locally-advanced-cervical-cancers-treated-with-radical-radio-chemo-therapy-a-prospective-observational-study
#12
Umesh Mahantshetty, Tanuja Teni, Pushpa Naga, Chandni Hotwani, Sumuki Umesh, Sadhana Kannan, Vinod Hande, Sagar Pawar, Reena Engineer, Supriya Chopra, Kedar Deodhar, Amita Maheshwari, Lavanya Gurram, Sudeep Gupta, Shyam Kishore Shrivastava
OBJECTIVE: With an aim to investigate the impact of Human Papilloma Virus (HPV) 16/18 infection on clinical outcomes in locally advanced cervical cancers treated with radical radio (chemo) therapy, we undertook this prospective study. METHODS: Between May 2010 and April 2012, 150 histologically proven cervical cancer patients treated with radio (chemo) therapy were accrued. Cervical biopsies/brushings were collected at pre-treatment, end of treatment and at 3 monthly intervals up to 24months...
December 1, 2017: Gynecologic Oncology
https://www.readbyqxmd.com/read/29202279/cancer-specific-inhibitory-effects-of-genetically-engineered-stem-cells-expressing-cytosine-deaminase-and-interferon-%C3%AE-against-choriocarcinoma-in-xenografted-metastatic-mouse-models
#13
Gyu-Sik Kim, Jae-Rim Heo, Seung U Kim, Kyung-Chul Choi
Cancer treatments using stem cells expressing therapeutic genes have been identified for various types of cancers. In this study, we investigated inhibitory effects of HB1.F3.CD and HB1.F3.CD.IFN-β cells expressing Escherichia coli cytosine deaminase (CD) and human interferon-β (IFN-β) genes in intravenously (i.v.) injected mice with a metastasis model. In this treatment, pro-drug 5-fluorocytosine (5-FC) is converted to cytotoxic drug 5-fluorouracil by hNSCs expressing the CD gene, which inhibits DNA synthesis in cancer cells...
December 1, 2017: Translational Oncology
https://www.readbyqxmd.com/read/29202217/dna-modulates-the-interaction-of-genetically-engineered-dna-binding-proteins-and-gold-nanoparticles-diagnosis-of-high-risk-hpv-infection
#14
Ju-Yi Mao, Han-Wei Li, Shih-Chun Wei, Scott G Harroun, Ming-Ying Lee, Hung-Yun Lin, Chih-Yu Chung, Chun-Hua Hsu, Yet-Ran Chen, Han-Jia Lin, Chih-Ching Huang
Gene detection has an important role in diagnosing several serious diseases and genetic defects in modern clinical medicine. Herein, we report a fast and convenient gene detection method based on the modulation of the interaction between a heat-resistant double-stranded DNA (dsDNA)-binding protein (Sso7d) and gold nanoparticles (Au NPs). We prepared a recombinant Cys-Sso7d, which is Sso7d with an extra cysteine (Cys) residue in the N-terminus, through protein engineering to control the interaction between Sso7d and Au NPs...
December 18, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/29188330/metabolic-pathway-of-6-aminohexanoate-in-the-nylon-oligomer-degrading-bacterium-arthrobacter-sp-ki72-identification-of-the-enzymes-responsible-for-the-conversion-of-6-aminohexanoate-to-adipate
#15
Ikki Takehara, Tsubasa Fujii, Yuuki Tanimoto, Dai-Ichiro Kato, Masahiro Takeo, Seiji Negoro
Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD 1 and nylE 1 , responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies...
November 29, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29187601/efficient-solid-phase-synthesis-of-meningococcal-capsular-oligosaccharides-enables-simple-and-fast-chemoenzymatic-vaccine-production
#16
Timm Fiebig, Christa Litschko, Friedrich Freiberger, Andrea Bethe, Monika Berger, Rita Gerardy-Schahn
Neisseria meningitidis serogroups A (NmA) and X (NmX) are among the leading causes of bacterial meningitis in the African meningitis belt. Glycoconjugate vaccines, consisting of an antigenic carrier protein coupled to the capsular polysaccharide of the bacterial pathogen, are the most effective strategy for prevention of meningococcal disease. However, the distribution of effective glycoconjugate vaccines in this region is limited by high cost of cultivating pathogens and purification of their capsular polysaccharides...
November 29, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29183117/isothermal-amplification-on-a-structure-switchable-symmetric-toehold-dumbbell-template-a-strategy-enabling-microrna-analysis-at-the-single-cell-level-with-ultrahigh-specificity-and-accuracy
#17
Jun Chen, Taixue An, Yingjun Ma, Bo Situ, Danping Chen, Yuzhi Xu, Li Zhang, Zong Dai, Xiaoyong Zou
Accurate analysis of microRNAs (miRNAs) at the single-cell level seriously requires analytical methods possessing extremely high sensitivity, specificity and precision. By rational engineering of a structure-switchable symmetric toehold dumbbell-template (STD-template), we propose a novel isothermal Symmetric EXPonential Amplification Reaction (SEXPAR) method. The sealed and symmetric structure of the STD-template allows exponential amplification reaction (EXPAR) to occur upon every annealing of target miRNA without loss of amplification efficiency...
November 28, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/29180809/structural-basis-for-tna-synthesis-by-an-engineered-tna-polymerase
#18
Nicholas Chim, Changhua Shi, Sujay P Sau, Ali Nikoomanzar, John C Chaput
Darwinian evolution experiments carried out on xeno-nucleic acid (XNA) polymers require engineered polymerases that can faithfully and efficiently copy genetic information back and forth between DNA and XNA. However, current XNA polymerases function with inferior activity relative to their natural counterparts. Here, we report five X-ray crystal structures that illustrate the pathway by which α-(L)-threofuranosyl nucleic acid (TNA) triphosphates are selected and extended in a template-dependent manner using a laboratory-evolved polymerase known as Kod-RI...
November 27, 2017: Nature Communications
https://www.readbyqxmd.com/read/29175708/engineering-of-biomolecules-by-bacteriophage-directed-evolution
#19
REVIEW
Andreas K Brödel, Mark Isalan, Alfonso Jaramillo
Conventional in vivo directed evolution methods have primarily linked the biomolecule's activity to bacterial cell growth. Recent developments instead rely on the conditional growth of bacteriophages (phages), viruses that infect and replicate within bacteria. Here we review recent phage-based selection systems for in vivo directed evolution. These approaches have been applied to evolve a wide range of proteins including transcription factors, polymerases, proteases, DNA-binding proteins, and protein-protein interactions...
November 22, 2017: Current Opinion in Biotechnology
https://www.readbyqxmd.com/read/29169271/differentiation-of-mesenchymal-stem-cells-into-neuron-like-cells-using-composite-3d-scaffold-combined-with-valproic-acid-induction
#20
Sadegh Ghorbani, Taki Tiraihi, Masoud Soleimani
The nervous system has little capacity for self-repair after injury because neurons cannot proliferate owing to lack of suitable microenvironment. Therefore, neural tissue engineering that combines neural stem, scaffolds, and growth factors may improve the chance of restoration of damaged neural tissues. A favorable niche for neural regeneration would be both fibrous and electrically conductive scaffolds. Human Wharton jelly-derived mesenchymal stem cells were seeded on wet-electrospun 3D scaffolds composed of poly lactic acid coated with natural polymers including alginate and gelatin, followed by a multi-wall carbon nanotube coating...
January 1, 2017: Journal of Biomaterials Applications
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