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https://www.readbyqxmd.com/read/27906496/aerobic-expression-of-vitreoscilla-hemoglobin-improves-the-growth-performance-of-cho-k1-cells
#1
Mariana Juárez, Claudia H González-De la Rosa, Elisa Memún, Juan-Carlos Sigala, Alvaro R Lara
Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO-K1 cell culture was investigated...
December 1, 2016: Biotechnology Journal
https://www.readbyqxmd.com/read/27906450/lovtrap-a-versatile-method-to-control-protein-function-with-light
#2
Hui Wang, Klaus M Hahn
We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by irradiation. The technique relies on binding of an engineered Zdk domain to a LOV2 domain, with affinity <30 nM in the dark and >500 nM upon irradiation between 400 and 500 nm. LOVTRAP can be applied to diverse proteins, as it requires attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered...
December 1, 2016: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27906033/the-role-of-the-backbone-torsion-in-protein-folding
#3
EDITORIAL
Irina Sorokina, Arcady Mushegian
BACKGROUND: The set of forces and sequence of events that govern the transition from an unfolded polypeptide chain to a functional protein with correct spatial structure remain incompletely known, despite the importance of the problem and decades of theory development, computer simulations, and laboratory experiments. Information about the correctly folded state of most proteins is likely to be present in their sequences, and yet many proteins fail to attain native structure after overexpression in a non-native environment or upon experimental denaturation and refolding...
December 1, 2016: Biology Direct
https://www.readbyqxmd.com/read/27905670/induced-pluripotent-stem-cells-as-a-new-getaway-for-bone-tissue-engineering-a-systematic-review
#4
REVIEW
Farshid Bastami, Pantea Nazeman, Hamidreza Moslemi, Maryam Rezai Rad, Kazem Sharifi, Arash Khojasteh
OBJECTIVES: Mesenchymal stem cells (MSCs) are frequently used for bone regeneration, however, they are limited in quantity. Moreover, their proliferation and differentiation capabilities reduce during cell culture expansion. Potential application of induced pluripotent stem cells (iPSCs) has been reported as a promising alternative source for bone regeneration. This study aimed to systematically review the available literature on osteogenic potential of iPSCs and to discuss methods applied to enhance their osteogenic potential...
December 1, 2016: Cell Proliferation
https://www.readbyqxmd.com/read/27905529/efficient-targeted-mutagenesis-of-rice-and-tobacco-genomes-using-cpf1-from-francisella-novicida
#5
Akira Endo, Mikami Masafumi, Hidetaka Kaya, Seiichi Toki
CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM. Cpf1 could be used as a sequence-specific nuclease to target AT-rich regions of a genome that Cas9 had difficulty accessing. Second, Cpf1 generates DNA ends with a 5' overhang, whereas Cas9 creates blunt DNA ends after cleavage...
December 1, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27905091/recent-advances-of-molecular-toolbox-construction-expand-pichia-pastoris-in-synthetic-biology-applications
#6
REVIEW
Zhen Kang, Hao Huang, Yunfeng Zhang, Guocheng Du, Jian Chen
Pichia pastoris: (reclassified as Komagataella phaffii), a methylotrophic yeast strain has been widely used for heterologous protein production because of its unique advantages, such as readily achievable high-density fermentation, tractable genetic modifications and typical eukaryotic post-translational modifications. More recently, P. pastoris as a metabolic pathway engineering platform has also gained much attention. In this mini-review, we addressed recent advances of molecular toolboxes, including synthetic promoters, signal peptides, and genome engineering tools that established for P...
January 2017: World Journal of Microbiology & Biotechnology
https://www.readbyqxmd.com/read/27904922/impacts-of-sediment-organic-matter-content-and-ph-on-ecotoxicity-of-coexposure-of-tio2-nanoparticles-and-cadmium-to-freshwater-snails-bellamya-aeruginosa
#7
Taowu Ma, Meng Wang, Shuangjiao Gong, Bin Tian
The environmental factors are expected to affect the ecotoxicity of heavy metals in the presence of engineered nanoparticles (NPs) in aquatic ecosystems. However, in sediment scenario, little is known regarding their impacts on the ecotoxicity of co-exposure of sediment-associated heavy metals and NPs. This study evaluated the impacts of different levels of organic matter (OM) (4.8-11.6%) and pH (6-9) on the ecotoxicological effects of co-exposure of sediment-associated titanium dioxide nanoparticles (TiO2-NPs) and cadmium (Cd) to a freshwater gastropod Bellamya aeruginosa...
November 30, 2016: Archives of Environmental Contamination and Toxicology
https://www.readbyqxmd.com/read/27903891/cloning-independent-markerless-gene-editing-in-streptococcus-sanguinis-novel-insights-in-type-iv-pilus-biology
#8
Ishwori Gurung, Jamie-Lee Berry, Alexander M J Hall, Vladimir Pelicic
Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27903819/towards-designing-new-nano-scale-protein-architectures
#9
REVIEW
Jana Aupič, Fabio Lapenta, Žiga Strmšek, Roman Jerala
The complexity of designed bionano-scale architectures is rapidly increasing mainly due to the expanding field of DNA-origami technology and accurate protein design approaches. The major advantage offered by polypeptide nanostructures compared with most other polymers resides in their highly programmable complexity. Proteins allow in vivo formation of well-defined structures with a precise spatial arrangement of functional groups, providing extremely versatile nano-scale scaffolds. Extending beyond existing proteins that perform a wide range of functions in biological systems, it became possible in the last few decades to engineer and predict properties of completely novel protein folds, opening the field of protein nanostructure design...
November 30, 2016: Essays in Biochemistry
https://www.readbyqxmd.com/read/27902801/achieving-plant-crispr-targeting-that-limits-off-target-effects
#10
Jeffrey D Wolt, Kan Wang, Dipali Sashital, Carolyn J Lawrence-Dill
The CRISPR-Cas9 system (clustered regularly interspaced short palindromic repeats with associated Cas9 protein) has been used to generate targeted changes for direct modification of endogenous genes in an increasing number of plant species; but development of plant genome editing has not yet fully considered potential off-target mismatches that may lead to unintended changes within the genome. Assessing the specificity of CRISPR-Cas9 for increasing editing efficiency as well as the potential for unanticipated downstream effects from off-target mutations is an important regulatory consideration for agricultural applications...
November 2016: Plant Genome
https://www.readbyqxmd.com/read/27902325/targeting-naturally-occurring-epitope-variants-of-hepatitis-c-virus-with-high-affinity-t-cell-receptors
#11
Huajun Zhang, Jianbing Zhang, Lei Chen, Zhiming Weng, Ye Tian, Haifeng Zhao, Youjia Li, Lin Chen, Zhaoduan Liang, Hongjun Zheng, Wenzhuo Zhao, Shi Zhong, Yi Li
Hepatitis C virus (HCV) readily establishes chronic infection, which is characterized by failure of virus-specific CD8+ T cells. HCV uses epitope mutation and T-cell exhaustion to escape from the host immune response. Previously, we engineered high-affinity T-cell receptors (HATs) targeting HIV escape mutants. In this study, the affinity of a T-cell receptor specific for the human leukocyte antigen (HLA)-A2-restricted HCV immunodominant epitope NS3 1406-1415 (KLVALGINAV) was improved from a KD of 6.6 µM to 40 pM...
November 11, 2016: Journal of General Virology
https://www.readbyqxmd.com/read/27900555/x-ray-crystallography-as-a-tool-to-determine-three-dimensional-structures-of-commercial-enzymes-subjected-to-treatment-in-pressurized-fluids
#12
REVIEW
Mirian Cristina Feiten, Marco Di Luccio, Karine F Santos, Débora de Oliveira, J Vladimir Oliveira
The study of enzyme function often involves a multi-disciplinary approach. Several techniques are documented in the literature towards determining secondary and tertiary structures of enzymes, and X-ray crystallography is the most explored technique for obtaining three-dimensional structures of proteins. Knowledge of three-dimensional structures is essential to understand reaction mechanisms at the atomic level. Additionally, structures can be used to modulate or improve functional activity of enzymes by the production of small molecules that act as substrates/cofactors or by engineering selected mutants with enhanced biological activity...
November 29, 2016: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/27899674/cdd-sparcle-functional-classification-of-proteins-via-subfamily-domain-architectures
#13
Aron Marchler-Bauer, Yu Bo, Lianyi Han, Jane He, Christopher J Lanczycki, Shennan Lu, Farideh Chitsaz, Myra K Derbyshire, Renata C Geer, Noreen R Gonzales, Marc Gwadz, David I Hurwitz, Fu Lu, Gabriele H Marchler, James S Song, Narmada Thanki, Zhouxi Wang, Roxanne A Yamashita, Dachuan Zhang, Chanjuan Zheng, Lewis Y Geer, Stephen H Bryant
NCBI's Conserved Domain Database (CDD) aims at annotating biomolecular sequences with the location of evolutionarily conserved protein domain footprints, and functional sites inferred from such footprints. An archive of pre-computed domain annotation is maintained for proteins tracked by NCBI's Entrez database, and live search services are offered as well. CDD curation staff supplements a comprehensive collection of protein domain and protein family models, which have been imported from external providers, with representations of selected domain families that are curated in-house and organized into hierarchical classifications of functionally distinct families and sub-families...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899671/repeatsdb-2-0-improved-annotation-classification-search-and-visualization-of-repeat-protein-structures
#14
Lisanna Paladin, Layla Hirsh, Damiano Piovesan, Miguel A Andrade-Navarro, Andrey V Kajava, Silvio C E Tosatto
RepeatsDB 2.0 (URL: http://repeatsdb.bio.unipd.it/) is an update of the database of annotated tandem repeat protein structures. Repeat proteins are a widespread class of non-globular proteins carrying heterogeneous functions involved in several diseases. Here we provide a new version of RepeatsDB with an improved classification schema including high quality annotations for ∼5400 protein structures. RepeatsDB 2.0 features information on start and end positions for the repeat regions and units for all entries...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899664/integration-defective-lentiviral-vector-mediates-efficient-gene-editing-through-homology-directed-repair-in-human-embryonic-stem-cells
#15
Yebo Wang, Yingjia Wang, Tammy Chang, He Huang, Jiing-Kuan Yee
Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09...
November 28, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899650/a-cas9-based-toolkit-to-program-gene-expression-in-saccharomyces-cerevisiae
#16
Amanda Reider Apel, Leo d'Espaux, Maren Wehrs, Daniel Sachs, Rachel A Li, Gary J Tong, Megan Garber, Oge Nnadi, William Zhuang, Nathan J Hillson, Jay D Keasling, Aindrila Mukhopadhyay
Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags...
November 28, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899587/temporal-self-regulation-of-transposition-through-host-independent-transposase-rodlet-formation
#17
Lauren E Woodard, Laura M Downes, Yi-Chien Lee, Aparna Kaja, Eyuel S Terefe, Matthew H Wilson
Transposons are highly abundant in eukaryotic genomes, but their mobilization must be finely tuned to maintain host organism fitness and allow for transposon propagation. Forty percent of the human genome is comprised of transposable element sequences, and the most abundant cut-and-paste transposons are from the hAT superfamily. We found that the hAT transposase TcBuster from Tribolium castaneum formed filamentous structures, or rodlets, in human tissue culture cells, after gene transfer to adult mice, and ex vivo in cell-free conditions, indicating that host co-factors or cellular structures were not required for rodlet formation...
November 28, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899282/bindprofx-assessing-mutation-induced-binding-affinity-change-by-protein-interface-profiles-with-pseudo-counts
#18
Peng Xiong, Chengxin Zhang, Wei Zheng, Yang Zhang
Understanding how gene-level mutations affect the binding affinity of protein-protein interactions is a key issue of protein engineering. Due to the complexity of the problem, using physical force field to predict the mutation-induced binding free-energy change remains challenging. In this work, we present a renewed approach to calculate the impact of gene mutations on the binding affinity through the structure-based profiling of protein-protein interfaces, where the binding free-energy change (ΔΔG) is counted as the logarithm of relative probability of mutant amino acids over wild-type ones in the interface alignment matrix; three pseudo counts are introduced to alleviate the limit of the current interface library...
November 26, 2016: Journal of Molecular Biology
https://www.readbyqxmd.com/read/27899064/gdf5-mediated-enhancement-of-chondrocyte-phenotype-is-inhibited-by-heparin-implication-for-the-use-of-heparin-in-the-clinic-and-in-tissue-engineering-applications
#19
Bethanie Imogen Ayerst, Raymond A A Smith, Victor Nurcombe, Anthony J Day, Catherine L R Merry, Simon Cool
The highly sulfated glycosaminoglycan (GAG) heparin is widely used in the clinic as an anticoagulant, and researchers are now using it to enhance stem cell expansion/ differentiation protocols, as well as to improve the delivery of growth factors for tissue engineering strategies. Growth differentiation factor 5 (GDF5) belongs to the bone morphogenetic protein family of proteins and is vital for skeletal formation, however, its interaction with heparin and heparan sulfate (HS) has not been studied. We identify GDF5 as a novel heparin/ HS-binding protein, and show that HS proteoglycans are vital in localizing GDF5 to the cell surface...
November 29, 2016: Tissue Engineering. Part A
https://www.readbyqxmd.com/read/27898188/modern-approaches-to-discovering-new-hydroxynitrile-lyases-for-biocatalysis
#20
REVIEW
Santosh Kumar Padhi
Hydroxynitrile lyases (HNLs) have grown in importance from laboratory to industry due to their potential to catalyze stereoselective C-C bond-formation reactions in the synthesis of several chiral intermediates, such as enantiopure α-cyanohydrins, β-nitro alcohols, and their derivatives with multiple functional groups. With these wide applications, the demand for finding new HNLs has increased, and this has led to exploration not only of new HNLs but also of new ways to discover them. An exclusive review article on HNLs by Asano et al...
November 29, 2016: Chembiochem: a European Journal of Chemical Biology
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