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https://www.readbyqxmd.com/read/28791001/novel-in-vitro-screening-system-based-on-differential-scanning-fluorimetry-to-search-for-small-molecules-against-the-disassembly-or-assembly-of-hiv-1-capsid-protein
#1
Yasuyuki Miyazaki, Naoya Doi, Takaaki Koma, Akio Adachi, Masako Nomaguchi
Varieties of in vitro systems have been used to study biochemical properties of human immunodeficiency virus Gag-capsid protein (HIV Gag-CA). Recently, we have comparatively characterized HIV-1 and HIV-2 Gag-CA proteins using such technology, and have demonstrated that the NaCl-initiated CA-polymerization in vitro and the stability of CA N-terminal domain as judged by differential scanning fluorimetry (DSF) are inversely correlated. In this study, we found that ZnCl2 works as a competent initiator of the in vitro HIV-1 CA-polymerization at much lower concentrations than those of NaCl frequently used for the polymerization initiation...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28740306/comparative-study-of-the-quality-characteristics-of-defatted-soy-flour-treated-by-supercritical-carbon-dioxide-and-organic-solvent
#2
Sung-Won Kang, M Shafiur Rahman, Ah-Na Kim, Kyo-Yeon Lee, Chan-Yang Park, William L Kerr, Sung-Gil Choi
Defatted soy flour is a potential source of food protein, amino acids, ash and isoflavones. The supercritical carbon dioxide (SC-CO2) and a traditional organic solvent extraction methods were used to remove fat from soy flour, and the quality characteristics of a control soy flour (CSF), defatted soy flour by SC-CO2 (DSFSC-CO2) and defatted soy flour by an organic solvent (DSF-OS) were compared. The SC-CO2 process was carried out at a constant temperature of 45 °C, and a pressure of 40 MPa for 3 h with a CO2 flow rate of 30 g/min...
July 2017: Journal of Food Science and Technology
https://www.readbyqxmd.com/read/28637248/expression-and-comparison-of-codon-optimised-aspergillus-tubingensis-amylase-variants-in-saccharomyces-cerevisiae
#3
Rosemary A Cripwell, Shaunita H Rose, Willem H van Zyl
The expression of codon optimised genes is a popular genetic engineering approach for the production of industrially relevant proteins. This study investigates and compares the expression of codon optimised and codon adapted amylase variants. The Aspergillus tubingensis raw starch hydrolysing α-amylase (amyA) and glucoamylase (glaA) encoding genes were redesigned using synonymous codons and expressed in Saccharomyces cerevisiae Y294. Codon optimisation to favour S. cerevisiae codon bias resulted in a decrease in extracellular enzyme activity of 72% (30...
June 1, 2017: FEMS Yeast Research
https://www.readbyqxmd.com/read/28464377/bile-salts-and-alkaline-ph-reciprocally-modulate-the-interaction-between-the-periplasmic-domains-of-vibrio-cholerae-toxr-and-toxs
#4
Charles R Midgett, Salvador Almagro-Moreno, Maria Pellegrini, Ronald K Taylor, Karen Skorupski, F Jon Kull
ToxR is a transmembrane transcription factor that is essential for virulence gene expression and human colonization by Vibrio cholerae. ToxR requires its operon partner ToxS, a periplasmic integral membrane protein, for full activity. These two proteins are thought to interact through their respective periplasmic domains, ToxRp and ToxSp. In addition, ToxR is thought to be responsive to various environmental cues, such as bile salts and alkaline pH, but how these factors influence ToxR is not yet understood...
July 2017: Molecular Microbiology
https://www.readbyqxmd.com/read/28454708/functional-characterisation-of-burkholderia-pseudomallei-biotin-protein-ligase-a-toolkit-for-anti-melioidosis-drug-development
#5
Thomas E H Bond, Alanna E Sorenson, Patrick M Schaeffer
Burkholderia pseudomallei (Bp) is the causative agent of melioidosis. The bacterium is responsible for 20% of community-acquired sepsis cases and 40% of sepsis-related mortalities in northeast Thailand, and is intrinsically resistant to aminoglycosides, macrolides, rifamycins, cephalosporins, and nonureidopenicillins. There is no vaccine and its diagnosis is problematic. Biotin protein ligase (BirA) which is essential for fatty acid synthesis has been proposed as a drug target in bacteria. Very few bacterial BirA have been characterized, and a better understanding of these enzymes is necessary to further assess their value as drug targets...
June 2017: Microbiological Research
https://www.readbyqxmd.com/read/28427840/aav-id-a-rapid-and-robust-assay-for-batch-to-batch-consistency-evaluation-of-aav-preparations
#6
Simon Pacouret, Mohammed Bouzelha, Rajani Shelke, Eva Andres-Mateos, Ru Xiao, Anna Maurer, Mathieu Mevel, Heikki Turunen, Trisha Barungi, Magalie Penaud-Budloo, Frédéric Broucque, Véronique Blouin, Philippe Moullier, Eduard Ayuso, Luk H Vandenberghe
Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level...
June 7, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/28371560/differential-scanning-calorimetry-and-fluorimetry-measurements-of-monoclonal-antibodies-and-reference-proteins-effect-of-scanning-rate-and-dye-selection
#7
Brian E Lang, Kenneth D Cole
Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) were used to measure the transition temperatures of four proteins: RNase A, invertase, rituximab, and the NISTmAb (NIST Reference Material, RM 8671). The proteins were combined with several different fluorescent dyes for the DSF measurements. This study compares the results of DSC and DSF measurements of transition temperatures with different types of proteins, dye combinations, and thermal scan rates. As protein unfolding is often influenced by kinetic effects, we measured the transition temperatures of the proteins using DSC over a range of temperature scan rates and compared them to the data obtained from DSF over comparable temperature scan rates...
April 3, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/28355693/-influence-of-occupational-aluminum-exposure-on-cognitive-function-and-glutamate-receptor-protein-expression-in-workers
#8
P Ren, R Li, Y Z Yuan, X T Lu, Q Niu
Objective: To investigate the influence of occupational aluminum exposure on cognitive function and glutamate receptor protein expression in peripheral blood lymphocytes in workers and the possibility of glutamate receptor being used as a biomarker for cognitive impairment in aluminum workers. Methods: From October to December, 2014, cluster sampling was performed to select 121 workers in aluminum electrolysis workshop as exposure group and 231 workers in thermoelectric workshop and logistics department as control group...
February 20, 2017: Chinese Journal of Industrial Hygiene and Occupational Diseases
https://www.readbyqxmd.com/read/28340172/repositioning-disulfiram-as-a-radiosensitizer-against-atypical-teratoid-rhabdoid-tumor
#9
Young Eun Lee, Seung Ah Choi, Pil Ae Kwack, Hak Jae Kim, Il Han Kim, Kyu-Chang Wang, Ji Hoon Phi, Ji Yeoun Lee, Sangjoon Chong, Sung-Hye Park, Kyung Duk Park, Do Won Hwang, Kyeung Min Joo, Seung-Ki Kim
Background: Atypical teratoid/rhabdoid tumor (AT/RT) is one of the most common malignant brain tumors in infants. Although cancer stem cells of AT/RT express aldehyde dehydrogenase (ALDH), effective chemotherapies against AT/RT have not been established. Here, we examined radiosensitizing effects of disulfiram (DSF), an irreversible inhibitor of ALDH against AT/RT for a novel therapeutic method. Methods: Patient-derived primary cultured AT/RT cells (SNU.AT/RT-5 and SNU...
August 1, 2017: Neuro-oncology
https://www.readbyqxmd.com/read/28244030/characterization-of-a-bacillus-thuringiensis-chitinase-that-binds-to-cellulose-and-chitin
#10
Shotaro Honda, Toshiyuki Kunii, Kenta Nohara, Satoshi Wakita, Yasusato Sugahara, Masao Kawakita, Fumitaka Oyama, Masayoshi Sakaguchi
Bacillus thuringiensis is a Gram-positive soil bacterium that is known to be a bacterial biopesticide that produces insecticidal proteins called crystal proteins (Cry). In the insecticidal process, chitinases are suggested to perforate the peritrophic membrane barrier to facilitate the invasion of the Cry proteins into epithelial membranes. A chitinase gene from B. thuringiensis was successfully expressed in a soluble form in Escherichia coli, and the gene product was purified and characterized. The purified recombinant enzyme, BthChi74, hydrolyzed an artificial substrate, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside [4NP-(GlcNAc)2], and the natural substrates, colloidal chitin and crystalline α-chitin, but it did not hydrolyze cellulose...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28151054/the-effect-of-organic-shock-loads-on-the-stability-of-anaerobic-granular-sludge
#11
Yangcheng Ding, Huajun Feng, Dongsheng Shen, Na Li, Meizhen Wang, Yuyang Zhou
This study investigated the effect of organic shock loads on the stability of anaerobic granular sludge (AnGS). At double and triple shock intensity (8 and 12 kg COD/m(3)d organic loading rate (OLR), respectively), the accumulation of volatile fatty acids resulted in the decreasing pH of the reactors. The unstable performances of AnGS also appeared in the macroscopic properties, including the decreasing contents of relative hydrophobicity (RH), granular strength and diameter, and the fluctuation of EPS production...
February 15, 2017: Environmental Technology
https://www.readbyqxmd.com/read/28143565/disulfiram-overcomes-bortezomib-and-cytarabine-resistance-in-down-syndrome-associated-acute-myeloid-leukemia-cells
#12
Ranjan Bista, David W Lee, Oliver B Pepper, David O Azorsa, Robert J Arceci, Eiman Aleem
BACKGROUND: Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL...
February 1, 2017: Journal of Experimental & Clinical Cancer Research: CR
https://www.readbyqxmd.com/read/28088457/preformulation-characterization-stabilization-and-formulation-design-for-the-acrylodan-labeled-glucose-binding-protein-sm4-ac
#13
Neha Sahni, Rajoshi Chaudhuri, John M Hickey, Prakash Manikwar, Ajit D'Souza, Andrew Metters, Sangeeta B Joshi, C Russell Middaugh, David B Volkin
This study describes the physicochemical characterization, stabilization, and formulation design of SM4-AC, an acrylodan-labeled glucose/galactose-binding protein for use in a continuous glucose monitoring device. The physical stability profile of SM4-AC as a function of pH and temperature was monitored using a combination of biophysical techniques and the resulting physical stability profile was visualized using an empirical phase diagram. Forced degradation chemical stability studies (Asn deamidation, Met oxidation) of SM4-AC were performed using a combination of capillary isoelectric focusing, peptide mapping, and reversed-phase HPLC...
January 11, 2017: Journal of Pharmaceutical Sciences
https://www.readbyqxmd.com/read/28067526/extraction-of-thermodynamic-parameters-of-protein-unfolding-using-parallelized-differential-scanning-fluorimetry
#14
Thaiesha A Wright, Jamie M Stewart, Richard C Page, Dominik Konkolewicz
Thermodynamic properties of protein unfolding have been extensively studied; however, the methods used have typically required significant preparation time and high protein concentrations. Here we present a facile, simple, and parallelized differential scanning fluorimetry (DSF) method that enables thermodynamic parameters of protein unfolding to be extracted. This method assumes a two-state, reversible protein unfolding mechanism and provides the capacity to quickly analyze the biophysical mechanisms of changes in protein stability and to more thoroughly characterize the effect of mutations, additives, inhibitors, or pH...
January 12, 2017: Journal of Physical Chemistry Letters
https://www.readbyqxmd.com/read/28003428/evaluation-of-compound-selectivity-of-aldo-keto-reductases-using-differential-scanning-fluorimetry
#15
Aurangazeb Kabir, Satoshi Endo, Naoki Toyooka, Mayuko Fukuoka, Kazuo Kuwata, Yuji O Kamatari
Inhibitors of AKR1B10 belonging to the aldo-keto reductase (AKR) superfamily are considered promising candidates for anti-cancer drugs. AKR1B1, a structurally similar isoform of AKR1B10, is involved in glucose metabolism. Thus, selective inhibition of AKR1B10 is required for the development of anti-cancer drugs. In this study, we first compared correlations between melting temperature and the 50% inhibition concentration obtained from differential scanning fluorimetry (DSF) and an enzyme inhibitory experiment, respectively, and a good correlation was found, except for compounds with low solubility...
February 1, 2017: Journal of Biochemistry
https://www.readbyqxmd.com/read/27813538/the-use-of-differential-scanning-fluorimetry-in-the-rational-design-of-plastic-antibodies-for-protein-targets
#16
Jon Ashley, Yunus Shukor, Ibtisam E Tothill
The development of molecularly imprinted polymer nanoparticles (MIP-NPs), which specifically bind biomolecules, is of great interest in the area of biosensors, sample purification, therapeutic agents and biotechnology. Polymerisation techniques such as precipitation polymerisation, solid phase synthesis and core shell surface imprinting have allowed for significant improvements to be made in developing MIP-NPs which specifically recognise proteins. However, the development of MIP-NPs for protein templates (targets) still require lengthy optimisation and characterisation using different ratios of monomers in order to control their size, binding affinity and specificity...
November 14, 2016: Analyst
https://www.readbyqxmd.com/read/27794454/characterization-of-aminoacyl-trna-synthetase-stability-and-substrate-interaction-by-differential-scanning-fluorimetry
#17
REVIEW
Jamie A Abbott, Nathan M Livingston, Shawn B Egri, Ethan Guth, Christopher S Francklyn
Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori...
January 15, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/27739422/decrease-in-ph-destabilizes-individual-vault-nanocages-by-weakening-the-inter-protein-lateral-interaction
#18
Aida Llauró, Pablo Guerra, Ravi Kant, Brian Bothner, Núria Verdaguer, Pedro J de Pablo
Vault particles are naturally occurring proteinaceous cages with promising application as molecular containers. The use of vaults as functional transporters requires a profound understanding of their structural stability to guarantee the protection and controlled payload delivery. Previous results performed with bulk techniques or at non-physiological conditions have suggested pH as a parameter to control vault dynamics. Here we use Atomic Force Microscopy (AFM) to monitor the structural evolution of individual vault particles while changing the pH in real time...
October 14, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27541006/influence-of-arginine-salts-on-the-thermal-stability-and-aggregation-kinetics-of-monoclonal-antibody-dominant-role-of-anions
#19
Jifeng Zhang, Vadim Frey, Marta Corcoran, Jian Zhang-van Enk, J Anand Subramony
Thermal stability of the CH2 domain for an IgG1 monoclonal antibody and its aggregation kinetics were systematically studied at pH 4.8, below its pI of 8.8 in individual solutions of arginine salts with acetate, glutamate (Glu(-)), chloride, and sulfate as the anion, in comparison to sodium chloride and sodium sulfate. Thermal unfolding temperature, Tm, an indicator of thermal stability, was measured by both differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF). The aggregation kinetics was determined by assessing reversibility for the CH2 domain in the DSC repetitive scans and then cross-examined by the isothermal aggregation study measured by size exclusion chromatography...
October 3, 2016: Molecular Pharmaceutics
https://www.readbyqxmd.com/read/27452990/insights-in-understanding-aggregate-formation-and-dissociation-in-cation-exchange-chromatography-for-a-structurally-unstable-fc-fusion-protein
#20
Zhiqiang Chen, Chao Huang, Naresh Chennamsetty, Xuankuo Xu, Zheng Jian Li
Cation-exchange chromatography (CEX) of a structurally unstable Fc-fusion protein exhibited multi-peak elution profile upon a salt-step elution due to protein aggregation during intra-column buffer transition where low pH and high salt coexisted. The protein exhibited a single-peak elution behavior during a pH-step elution; nevertheless, the levels of soluble aggregates (i.e. high molecular weight species, HMW) in the CEX eluate were still found up to 12-fold higher than that for the load material. The amount of the aggregates formed upon the pH-step elution was dependent on column loading with maximum HMW achieved at intermediate loading levels, supporting the hypothesis that the aggregation was the result of both the conformational changes of the bound protein and the solution concentration of the aggregation-susceptible proteins during elution...
August 19, 2016: Journal of Chromatography. A
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