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Protein engineering

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https://www.readbyqxmd.com/read/28544881/mammalian-systems-biotechnology-reveals-global-cellular-adaptations-in-a-recombinant-cho-cell-line
#1
Faraaz Noor Khan Yusufi, Meiyappan Lakshmanan, Ying Swan Ho, Bernard Liat Wen Loo, Pramila Ariyaratne, Yuansheng Yang, Say Kong Ng, Tessa Rui Min Tan, Hock Chuan Yeo, Hsueh Lee Lim, Sze Wai Ng, Ai Ping Hiu, Chung Ping Chow, Corrine Wan, Shuwen Chen, Gavin Teo, Gao Song, Ju Xin Chin, Xiaoan Ruan, Ken Wing Kin Sung, Wei-Shou Hu, Miranda Gek Sim Yap, Muriel Bardor, Niranjan Nagarajan, Dong-Yup Lee
Effective development of host cells for therapeutic protein production is hampered by the poor characterization of cellular transfection. Here, we employed a multi-omics-based systems biotechnology approach to elucidate the genotypic and phenotypic differences between a wild-type and recombinant antibody-producing Chinese hamster ovary (CHO) cell line. At the genomic level, we observed extensive rearrangements in specific targeted loci linked to transgene integration sites. Transcriptional re-wiring of DNA damage repair and cellular metabolism in the antibody producer, via changes in gene copy numbers, was also detected...
May 24, 2017: Cell Systems
https://www.readbyqxmd.com/read/28544448/using-pymol-to-explore-the-effects-of-ph-on-noncovalent-interactions-between-immunoglobulin-g-and-protein-a-a-guided-inquiry-biochemistry-activity
#2
Zahilyn D Roche Allred, Heeyoung Tai, Stacey Lowery Bretz, Richard C Page
Students' understandings of foundational concepts such as noncovalent interactions, pH and pKa are crucial for success in undergraduate biochemistry courses. We developed a guided-inquiry activity to aid students in making connections between noncovalent interactions and pH/pKa . Students explore these concepts by examining the primary and tertiary structures of immunoglobulin G (IgG) and Protein A. Students use PyMOL, an open source molecular visualization application, to (1) identify hydrogen bonds and salt bridges between and within the proteins at physiological pH and (2) apply their knowledge of pH/pKa to association rate constant data for these proteins at pH 4 and pH 11...
May 24, 2017: Biochemistry and Molecular Biology Education
https://www.readbyqxmd.com/read/28543738/crispr-editing-in-biological-and-biomedical-investigation
#3
Xing-Da Ju, Jing Xu, Zhong Sheng Sun
The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR--associated protein) system, a prokaryotic RNA-based adaptive immune system against viral infection, is emerging as a powerful genome editing tool in broad research areas. To further improve and expand its functionality, various CRISPR delivery strategies have been tested and optimized, and key CRISPR system components such as Cas protein have been engineered with different purposes. Benefiting from more in-depth understanding and further development of CRISPR, versatile CRISPR-based platforms for genome editing have been rapidly developed to advance investigations in biology and biomedicine...
May 20, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/28543403/decellularized-human-liver-is-too-heterogeneous-for-designing-a-generic-extracellular-matrix-mimic-hepatic-scaffold
#4
Giorgio Mattei, Chiara Magliaro, Andrea Pirone, Arti Ahluwalia
Decellularized human livers are considered the perfect extracellular matrix (ECM) surrogate because both three-dimensional architecture and biological features of the hepatic microenvironment are thought to be preserved. However, donor human livers are in chronically short supply, both for transplantation or as decellularized scaffolds, and will become even scarcer as life expectancy increases. It is hence of interest to determine the structural and biochemical properties of human hepatic ECM to derive design criteria for engineering biomimetic scaffolds...
May 23, 2017: Artificial Organs
https://www.readbyqxmd.com/read/28542638/a-single-amino-acid-mutation-affects-elicitor-and-expansins-like-activities-of-cerato-platanin-a-non-catalytic-fungal-protein
#5
Simone Luti, Federica Martellini, Francesco Bemporad, Lorenzo Mazzoli, Paolo Paoli, Luigia Pazzagli
Cerato-platanin (CP) is a non-catalytic, cysteine-rich protein, the first member of the cerato-platanin family. It is a single-domain protein with a double Ψ/β barrel domain resembling the D1 domain of plant and bacterial expansins. Similarly to expansins, CP shows a cell wall-loosening activity on cellulose and can be defined as an expanisin-like protein, in spite of the missing D2 domain, normally present in plant expansins. The weakening activity shown on cellulose may facilitate the CP-host interaction, corroborating the role of CP in eliciting plant defence response...
2017: PloS One
https://www.readbyqxmd.com/read/28542609/potent-and-selective-inhibition-of-pathogenic-viruses-by-engineered-ubiquitin-variants
#6
Wei Zhang, Ben A Bailey-Elkin, Robert C M Knaap, Baldeep Khare, Tim J Dalebout, Garrett G Johnson, Puck B van Kasteren, Nigel J McLeish, Jun Gu, Wenguang He, Marjolein Kikkert, Brian L Mark, Sachdev S Sidhu
The recent Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola and Zika virus outbreaks exemplify the continued threat of (re-)emerging viruses to human health, and our inability to rapidly develop effective therapeutic countermeasures. Many viruses, including MERS-CoV and the Crimean-Congo hemorrhagic fever virus (CCHFV) encode deubiquitinating (DUB) enzymes that are critical for viral replication and pathogenicity. They bind and remove ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) from cellular proteins to suppress host antiviral innate immune responses...
May 18, 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/28542539/a-leishmania-secretion-system-for-the-expression-of-major-ampullate-spidroin-mimics
#7
Todd A Lyda, Elizabeth L Wagner, Andre X Bourg, Congyue Peng, Golnaz Najaf Tomaraei, Delphine Dean, Marian S Kennedy, William R Marcotte
Spider major ampullate silk fibers have been shown to display a unique combination of relatively high fracture strength and toughness compared to other fibers and show potential for tissue engineering scaffolds. While it is not possible to mass produce native spider silks, the potential ability to produce fibers from recombinant spider silk fibers could allow for an increased innovation rate within tissue engineering and regenerative medicine. In this pilot study, we improved upon a prior fabrication route by both changing the expression host and additives to the fiber pulling precursor solution to improve the performance of fibers...
2017: PloS One
https://www.readbyqxmd.com/read/28542473/stably-engineered-nanobubbles-and-ultrasound-an-effective-platform-for-enhanced-macromolecular-delivery-to-representative-cells-of-the-retina
#8
Sachin S Thakur, Micheal S Ward, Amirali Popat, Nicole B Flemming, Marie-Odile Parat, Nigel L Barnett, Harendra S Parekh
Herein we showcase the potential of ultrasound-responsive nanobubbles in enhancing macromolecular permeation through layers of the retina, ultimately leading to significant and direct intracellular delivery; this being effectively demonstrated across three relevant and distinct retinal cell lines. Stably engineered nanobubbles of a highly homogenous and echogenic nature were fully characterised using dynamic light scattering, B-scan ultrasound and transmission electron microscopy (TEM). The nanobubbles appeared as spherical liposome-like structures under TEM, accompanied by an opaque luminal core and darkened corona around their periphery, with both features indicative of efficient gas entrapment and adsorption, respectively...
2017: PloS One
https://www.readbyqxmd.com/read/28542462/a-human-monocytic-nf-%C3%AE%C2%BAb-fluorescent-reporter-cell-line-for-detection-of-microbial-contaminants-in-biological-samples
#9
Claire Battin, Annika Hennig, Patrick Mayrhofer, Renate Kunert, Gerhard J Zlabinger, Peter Steinberger, Wolfgang Paster
Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs), which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations...
2017: PloS One
https://www.readbyqxmd.com/read/28541275/engineering-the-haemogenic-niche-mitigates-endogenous-inhibitory-signals-and-controls-pluripotent-stem-cell-derived-blood-emergence
#10
Nafees Rahman, Patrick M Brauer, Lilian Ho, Tatiana Usenko, Mukul Tewary, Juan Carlos Zúñiga-Pflücker, Peter W Zandstra
Efforts to recapitulate haematopoiesis, a process guided by spatial and temporal inductive signals, to generate haematopoietic progenitors from human pluripotent stem cells (hPSCs) have focused primarily on exogenous signalling pathway activation or inhibition. Here we show haemogenic niches can be engineered using microfabrication strategies by micropatterning hPSC-derived haemogenic endothelial (HE) cells into spatially-organized, size-controlled colonies. CD34+VECAD+ HE cells were generated with multi-lineage potential in serum-free conditions and cultured as size-specific haemogenic niches that displayed enhanced blood cell induction over non-micropatterned cultures...
May 25, 2017: Nature Communications
https://www.readbyqxmd.com/read/28540926/entorhinal-cortical-deep-brain-stimulation-rescues-memory-deficits-in-both-young-and-old-mice-genetically-engineered-to-model-alzheimer-s-disease
#11
Frances Xia, Adelaide Yiu, Scellig Sd Stone, Soojin Oh, Andres M Lozano, Sheena A Josselyn, Paul W Frankland
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline. Deep brain stimulation (DBS) has been used to treat a variety of brain disorders and shows promise in alleviating cognitive symptoms in some AD patients (Laxton et al., 2010). We previously showed that DBS of the entorhinal cortex (EC) enhances spatial memory formation in normal (wild-type) mice (Stone et al., 2011). Here we tested the effects of EC-DBS on the progressive cognitive deficits in a genetically-based mouse model of AD...
May 25, 2017: Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology
https://www.readbyqxmd.com/read/28540701/inverse-pcr-for-point-mutation-introduction
#12
Diogo Silva, Gustavo Santos, Mário Barroca, Tony Collins
Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28539590/abcc2-is-associated-with-bacillus-thuringiensis-cry1ac-toxin-oligomerization-and-membrane-insertion-in-diamondback-moth
#13
Josue Ocelotl, Jorge Sánchez, Isabel Gómez, Bruce E Tabashnik, Alejandra Bravo, Mario Soberón
Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. Cry1Ac interaction with cadherin triggers oligomerization. However, a mutation in an ABC transporter gene (ABCC2) is linked to Cry1Ac resistance in Plutella xylostella. Cry1AcMod, engineered to lack helix α-1, was able to form oligomers without cadherinbinding and effectively countered Cry1Ac resistance linked to ABCC2. Here we analyzed Cry1Ac and Cry1AcMod binding and oligomerization by western blots using brush border membrane vesicles (BBMV) from a strain of P...
May 24, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28538671/engineering-exosomes-for-cancer-therapy
#14
REVIEW
Katie E Gilligan, Róisín M Dwyer
There remains an urgent need for novel therapeutic strategies to treat metastatic cancer, which results in over 8 million deaths annually worldwide. Following secretion, exosomes are naturally taken up by cells, and capable of the stable transfer of drugs, therapeutic microRNAs and proteins. As knowledge of the biogenesis, release and uptake of exosomes continues to evolve, and thus also has interest in these extracellular vesicles as potential tumor-targeted vehicles for cancer therapy. The ability to engineer exosome content and migratory itinerary holds tremendous promise...
May 24, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28538198/protein-and-metabolic-engineering-for-the-production-of-organic-acids
#15
Jingjing Liu, Jianghua Li, Hyun-Dong Shin, Long Liu, Guocheng Du, Jian Chen
Organic acids are natural metabolites of living organisms. They have been widely applied in the food, pharmaceutical, and bio-based materials industries. In recent years, biotechnological routes to organic acids production from renewable raw materials have been regarded as very promising approaches. In this review, we provide an overview of current developments in the production of organic acids using protein and metabolic engineering strategies. The organic acids include propionic acid, pyruvate, itaconic acid, succinic acid, fumaric acid, malic acid and citric acid...
April 14, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28537592/programming-molecular-self-assembly-of-intrinsically-disordered-proteins-containing-sequences-of-low-complexity
#16
Joseph R Simon, Nick J Carroll, Michael Rubinstein, Ashutosh Chilkoti, Gabriel P López
Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments...
June 2017: Nature Chemistry
https://www.readbyqxmd.com/read/28537482/programmed-platelet-derived-growth-factor-%C3%AE-%C3%AE-and-bone-morphogenetic-protein-2-delivery-from-a-hybrid-calcium-phosphate-alginate-scaffold
#17
Emily A Bayer, Jahnelle Jordan, Riccardo Gottardi, Morgan Fedorchak, Prashant N Kumta, Steven Little
Bone tissue engineering requires the upregulation of several regenerative stages, including a critical early phase of angiogenesis. Previous studies have suggested that a sequential delivery of PDGF to BMP-2 could promote angiogenic tubule formation when delivered to in vitro co-cultures of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs). However, it was previously unclear that this PDGF to BMP-2 delivery schedule will result in cell migration into the scaffolding system and affect the later expression of bone markers...
May 24, 2017: Tissue Engineering. Part A
https://www.readbyqxmd.com/read/28537381/collagen-gels-with-different-fibrillar-microarchitectures-elicit-different-cellular-responses
#18
Jing Xie, Min Bao, Stephanie M C Bruekers, Wilhelm T S Huck
The extracellular matrix consists of a complex mixture of fibrillar proteins, where the architecture and mechanical properties of the protein fibrils vary considerably in various tissues. Here, we systematically polymerized collagen gels at different temperatures, providing substrates with tunable mechanics and defined local micro-architecture. We studied the dependence of spreading dynamics, proliferation, migration and differentiation of human mesenchymal stem cells (hMSCs) on the fibrillar properties as compared to the bulk properties of the matrix...
May 24, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28537256/design-of-synthetic-epigenetic-circuits-featuring-memory-effects-and-reversible-switching-based-on-dna-methylation
#19
Johannes A H Maier, Raphael Möhrle, Albert Jeltsch
Epigenetic systems store information in DNA methylation patterns in a durable but reversible manner, but have not been regularly used in synthetic biology. Here, we designed synthetic epigenetic memory systems using DNA methylation sensitive engineered zinc finger proteins to repress a memory operon comprising the CcrM methyltransferase and a reporter. Triggering by heat, nutrients, ultraviolet irradiation or DNA damaging compounds induces CcrM expression and DNA methylation. In the induced on-state, methylation in the operator of the memory operon prevents zinc finger protein binding leading to positive feedback and permanent activation...
May 24, 2017: Nature Communications
https://www.readbyqxmd.com/read/28536832/proteomic-profiling-reveals-crucial-retinal-protein-alterations-in-the-early-phase-of-an-experimental-glaucoma-model
#20
Fabian Anders, Julia Teister, Sebstian Funke, Norbert Pfeiffer, Franz Grus, Thanos Solon, Verena Prokosch
PURPOSE: Clinical glaucoma is difficult to assess in terms of molecular pathophysiology, prompting studies in experimental models of glaucoma. The purpose of this study was to investigate quantitative changes in retinal protein expression at the onset of experimental glaucoma in rats. Analyzing the proteome provides a suitable tool to decipher the pathophysiological processes in glaucomatous degeneration. METHODS: Thermic cauterization of episcleral veins was utilized to elevate the intraocular pressure in Sprague Dawley rats...
May 24, 2017: Graefe's Archive for Clinical and Experimental Ophthalmology
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