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Journal articles on the topic 'Isothermal denaturation'

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Author: Grafiati
Published: 6 September 2023

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1

Hinrichs, Jörg, and Britta Rademacher. "High pressure thermal denaturation kinetics of whey proteins." Journal of Dairy Research 71, no. 4 (November 2004): 480–88. http://dx.doi.org/10.1017/s0022029904000238.

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Pressure processing of foodstuff has been applied to produce or modify proteinaceous gel structures. In real pressure processing the treatment is non-isothermal, due to the adiabatic nature of the process and the heat loss from the product to the vessel. In order to estimate the effect of pressurization on milk constituents pressure and temperature dependent kinetics were determined separately from each other. In a detailed kinetic study whey protein isolate was treated under isobaric (200 to 800 MPa) and isothermal conditions (−2 to 70 °C), and the resulting degree of denaturation of β-lactoglobulin A and B and α-lactalbumin was analysed. Kinetic parameters of denaturation were estimated using a one step non-linear regression method which allowed a global fit of the whole data set. The isobaric isothermal denaturation of β-lactoglobulin and α-lactalbumin was found to follow third and second order kinetics, respectively. Isothermal pressure denaturation of both β-lactoglobulin fractions do not differ significantly and were characterized by an activation volume decreasing with increasing temperature from −10 to about −30 ml mol−1, which demonstrates that the denaturation rate is accelerated with increasing temperature. The activation energy of about 70 to 100 kJ mol−1 obtained for β-lactoglobulin A and B is not dependent to a great extent on the pressure which indicates that above 200 MPa denaturation rate is limited by the aggregation rate while pressure forces unfolding of the molecule.
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2

Sarver, Ronald W., Joseph M. Rogers, and Dennis E. Epps. "Determination of Ligand-MurB Interactions by Isothermal Denaturation: Applicatio as a Secondary Assay to Complement High Throughput Screening." Journal of Biomolecular Screening 7, no. 1 (February 2002): 21–28. http://dx.doi.org/10.1177/108705710200700104.

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We used a temperature-jump isothermal denaturation procedure with various methods of detection to evaluate the quality of putative inhibitors of MurB discovered by high-throughput screening. Three optical methods of detection—ultraviolet hyperchromicity of absorbance, fluorescence of bound dyes, and circular dichroism—as well as differential scanning calorimetry were used to dissect the effects of two chemical compounds and a natural substrate on the enzyme. The kinetics of the denaturation process and binding of the compounds detected by quenching of flavin fluorescence were used to quantitate the dose dependencies of the ligand effects. We found that the first step in the denaturation of MurB is the rapid loss of flavin from the active site and that the two chemical inhibitors appeared to destabilize the interaction of the cofactor with the enzyme but stabilize the global unfolding. The kinetics of the denaturation process as well as the loss of flavin fluorescence on binding established that both compounds had nanomolar affinities for the enzyme. We showed that coupling of the various detection methods with isothermal denaturation yields a powerful regimen to provide analytical data for assessing inhibitor specificity for a protein target.
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3

NIELSEN, Anders D., Claus C. FUGLSANG, and Peter WESTH. "Effect of calcium ions on the irreversible denaturation of a recombinant Bacillus halmapalus alpha-amylase: a calorimetric investigation." Biochemical Journal 373, no. 2 (July 15, 2003): 337–43. http://dx.doi.org/10.1042/bj20030220.

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The effect of temperature and calcium ions on the denaturation of a recombinant α-amylase from Bacillus halmapalus α-amylase (BHA) has been studied using calorimetry. It was found that thermal inactivation of BHA is irreversible and that calcium ions have a significant effect on stability. Thus an apparent denaturation temperature (Td) of 83 °C in the presence of excess calcium ions was observed, whereas Td decreased to 48 °C when calcium was removed. The difference in thermal stability with and without calcium ions has been used to develop an isothermal titration calorimetric (ITC) procedure that allows simultaneous determination of kinetic parameters and enthalpy changes of the denaturation of calcium-depleted BHA. An activation energy EA of 101 kJ/mol was found for the denaturation of calcium-depleted BHA. The results support a kinetic denaturation mechanism where the calcium-depleted amylase denatures irreversibly at low temperature and if calcium ions are in excess, the amylase denatures irreversibly at high temperatures. The two denaturation reactions are coupled with the calcium-binding equilibrium between calcium-bound and -depleted amylase. A combination of the kinetic denaturation results and calcium-binding constants, determined by isothermal titration calorimetry, has been used to estimate kinetic stability, expressed in terms of the half-life of BHA as a function of temperature and free-calcium-ion concentration. Thus it is estimated that the apparent EA can be increased to approx. 123 kJ/mol by increasing the free-calcium concentration.
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4

CLAEYS, WENDIE L., ANN M. VAN LOEY, and MARC E. HENDRICKX. "Kinetics of alkaline phosphatase and lactoperoxidase inactivation, and of β-lactoglobulin denaturation in milk with different fat content." Journal of Dairy Research 69, no. 4 (November 2002): 541–53. http://dx.doi.org/10.1017/s0022029902005721.

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In the context of identifying intrinsic time temperature integrators (TTIs) for evaluating heat processing of milk, the extent to which milk fat content has an effect on alkaline phosphatase (ALP) and lactoperoxidase (Lpo) inactivation and on β-lactoglobulin (β-lg) denaturation kinetics was studied. Inactivation and denaturation kinetics were analysed in whole, semi-skimmed and skimmed milk. In previous experiments (isothermal and non-isothermal heating conditions), heat inactivation of ALP and Lpo and heat denaturation of β-lg were found to follow first order kinetics. This allowed experimental design to be simplified. Data analysis was performed by non-linear regression and results were evaluated by construction of joint confidence regions. The possible effect of milk fat was illustrated by temperature time tolerance (TTT-) diagrams. Although initial ALP activity was lower in skimmed milk compared with semi-skimmed or whole milk, kinetics were comparable and fat content did not seem to substantially affect the ALP test result for pasteurized milk. Unlike ALP, Lpo inactivation and β-lg denaturation kinetics differed significantly in milk with different fat content. Differences between Lpo inactivation kinetics were relatively small and acceptable in the context of quantifying the process impact. Denaturation of β-lg, on the other hand, seemed to be enhanced at higher milk fat content (>72 °C).
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5

Istrate, Daniel, Crisan Popescu, and Martin Möller. "Non-Isothermal Kinetics of Hardα-Keratin Thermal Denaturation." Macromolecular Bioscience 9, no. 8 (August 11, 2009): 805–12. http://dx.doi.org/10.1002/mabi.200800344.

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6

Shi, Chao, Fanjin Shang, Meiling Zhou, Pansong Zhang, Yifan Wang, and Cuiping Ma. "Triggered isothermal PCR by denaturation bubble-mediated strand exchange amplification." Chemical Communications 52, no. 77 (2016): 11551–54. http://dx.doi.org/10.1039/c6cc05906f.

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7

Huang, Mengting, Fang Yang, Jiye Fu, Pengfeng Xiao, Jing Tu, and Zuhong Lu. "Reaction parameter comparison and optimization of multiple displacement amplification." Analytical Methods 12, no. 1 (2020): 46–53. http://dx.doi.org/10.1039/c9ay01922g.

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After analysed MDA under different conditions, we found that different DNA denaturation methods before isothermal incubation can influence the amplification speed of MDA, and genome coverage uniformity was correlated with the amplification temperature.
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8

Wafer, Lucas, Marek Kloczewiak, Sharon M. Polleck, and Yin Luo. "Isothermal chemical denaturation of large proteins: Path-dependence and irreversibility." Analytical Biochemistry 539 (December 2017): 60–69. http://dx.doi.org/10.1016/j.ab.2017.10.001.

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9

Epps, Dennis E., Ronald W. Sarver, Joseph M. Rogers, John T. Herberg, and Paul K. Tomich. "The Ligand Affinity of Proteins Measured by Isothermal Denaturation Kinetics." Analytical Biochemistry 292, no. 1 (May 2001): 40–50. http://dx.doi.org/10.1006/abio.2001.5047.

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10

Maltzeva, Yulia I., Daria A. Gorbenko, Ekaterina V. Nikitina, Maria S. Rubel, and Dmitry M. Kolpashchikov. "Visual Detection of Stem-Loop Primer Amplification (SPA) Products without Denaturation Using Peroxidase-like DNA Machines (PxDM)." International Journal of Molecular Sciences 24, no. 9 (April 25, 2023): 7812. http://dx.doi.org/10.3390/ijms24097812.

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Rapid, inexpensive, and accurate determination of nucleic acids is a decisive factor in evaluating population’s health and monitoring treatment at point-of-care (POC) settings. Testing systems with visual outputs can provide instrument-free signal detection. Isothermal amplification technologies can substitute conventional polymerase chain reaction (PCR) testing due to compatibility with the POC diagnostic. Here, we have visually detected DNA fragments obtained by stem-loop-primer-assisted isothermal amplification (SPA), but not those obtained by PCR or LAMP amplification using DNA nanomachines with peroxidase-like activity (PxDM) with sensitivity to a single nucleotide substitution. Compared to the diagnostics with conventional loop-mediated isothermal amplification (LAMP), the PxDM method produces no false positive signals with the non-specific amplification products. The study suggests that PxDM, in conjunction with SPA isothermal amplification, can become a valid platform for POC testing systems.
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11

Augustijn, Dillen, Alina Kulakova, Sujata Mahapatra, Pernille Harris, and Åsmund Rinnan. "Isothermal Chemical Denaturation: Data Analysis, Error Detection, and Correction by PARAFAC2." Analytical Chemistry 92, no. 10 (April 23, 2020): 6958–67. http://dx.doi.org/10.1021/acs.analchem.9b05748.

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12

OWUSU-APENTEN, RICHARD K. "REVISED CONFORMATIONAL STABILITY PARAMETERS FOR BETA-LACTOGLOBULIN ISOTHERMAL DENATURATION BY UREA." Journal of Food Biochemistry 26, no. 3 (July 2002): 181–90. http://dx.doi.org/10.1111/j.1745-4514.2002.tb00851.x.

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13

Schön, Arne, Benjamin R. Clarkson, Maria Jaime, and Ernesto Freire. "Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry." Proteins: Structure, Function, and Bioinformatics 85, no. 11 (August 8, 2017): 2009–16. http://dx.doi.org/10.1002/prot.25354.

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14

CHEN, FUR-CHI, Y. H. PEGGY HSIEH, ROGER C. BRIDGMAN, and AGNES KILONZO-NTHENGE. "Kinetics of Tropomyosin Denaturation as a Predictive Model for Verifying Thermal Processing of Beef Products." Journal of Food Protection 69, no. 10 (October 1, 2006): 2447–53. http://dx.doi.org/10.4315/0362-028x-69.10.2447.

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An enzyme-linked immunosorbent assay (ELISA) was developed to study thermal denaturation of tropomyosin (Tm) using the time-temperature requirements for cooked beef. The ELISA employed a monoclonal antibody (MAb 2C9) raised against bovine Tm for quantifying residual Tm in muscle extracts. The specificity of MAb 2C9 to bovine Tm was demonstrated by Western blot and the analytical validity of ELISA was confirmed by dot blot. Thermal denaturation of Tm, in the temperature range between 54.4 and 70.0°C, showed first-order dependency. Kinetic parameters of Tm denaturation were derived from isothermal heating of beef muscle extract at 54.4, 57.2, 60.0, and 62.8°C. Temperature dependency of the rate constant (k) was demonstrated by Arrhenius plot; the activation energy (Ea) of Tm denaturation was determined to be 484 kJ·mol−1.A mathematic model describing the impact of the heating time-temperature on Tm denaturation was developed. Predicted Tm from the integrated time-temperature model agreed closely with the measured Tm in dynamically heat-processed beef samples. Percent errors between the measured and the predicted values ranged from −5.1 to 5.3%. The kinetic model provides an accurate and reproducible prediction of the impact of actual heating time-temperature on residual Tm in cooked beef. The MAb-based ELISA and kinetic model developed in this study have the potential to be adapted by the meat industry as a quality control tool.
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15

Chen, S. S., N. T. Wright, and J. D. Humphrey. "Heat-Induced Changes in the Mechanics of a Collagenous Tissue: Isothermal, Isotonic Shrinkage." Journal of Biomechanical Engineering 120, no. 3 (June 1, 1998): 382–88. http://dx.doi.org/10.1115/1.2798005.

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We present data from isothermal, isotonic-shrinkage tests wherein bovine chordae tendineae were subjected to well-defined constant temperatures (from 65 to 90°C), durations of heating (from 180 to 3600 s), and isotonic uniaxial stresses during heating (from 100 to 650 kPa). Tissue response during heating and “recovery” at 37°C following heating was evaluated in terms of the axial shrinkage, a gross indicator of underlying heat-induced denaturation. There were three key findings. First, scaling the heating time via temperature and load-dependent characteristic times for the denaturation process collapsed all shrinkage data to a single curve, and thereby revealed a time-temperature-load equivalency. Second, the characteristic times exhibited an Arrhenius-type behavior with temperature wherein the slopes were nearly independent of applied load—this suggested that applied loads during heating affect the activation entropy, not energy. Third, all specimens exhibited a time-dependent, partial recovery when returned to 37°C following heating, but the degree of recovery decreased with increases in the load imposed during heating. These new findings on heat-induced changes in tissue behavior will aid in the design of improved clinical heating protocols and provide guidance for the requisite constitutive formulations.
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16

Mahran, R., N. Vello, A. Komulainen, H. Härmä, and K. Kopra. "64P Isothermal chemical KRAS denaturation assay for monitoring stability and inhibitors interactions." ESMO Open 8, no. 1 (February 2023): 100922. http://dx.doi.org/10.1016/j.esmoop.2023.100922.

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17

Chandran, Harish, Nikhil Gopalkrishnan, Bernard Yurke, and John Reif. "Meta-DNA: synthetic biology via DNA nanostructures and hybridization reactions." Journal of The Royal Society Interface 9, no. 72 (January 11, 2012): 1637–53. http://dx.doi.org/10.1098/rsif.2011.0819.

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Can a wide range of complex biochemical behaviour arise from repeated applications of a highly reduced class of interactions? In particular, can the range of DNA manipulations achieved by protein enzymes be simulated via simple DNA hybridization chemistry? In this work, we develop a biochemical system which we call meta-DNA (abbreviated as mDNA), based on strands of DNA as the only component molecules. Various enzymatic manipulations of these mDNA molecules are simulated via toehold-mediated DNA strand displacement reactions. We provide a formal model to describe the required properties and operations of our mDNA, and show that our proposed DNA nanostructures and hybridization reactions provide these properties and functionality. Our meta-nucleotides are designed to form flexible linear assemblies (single-stranded mDNA ( ss mDNA)) analogous to single-stranded DNA. We describe various isothermal hybridization reactions that manipulate our mDNA in powerful ways analogous to DNA–DNA reactions and the action of various enzymes on DNA. These operations on mDNA include (i) hybridization of ss mDNA into a double-stranded mDNA ( ds mDNA) and heat denaturation of a ds mDNA into its component ss mDNA, (ii) strand displacement of one ss mDNA by another, (iii) restriction cuts on the backbones of ss mDNA and ds mDNA, (iv) polymerization reactions that extend ss mDNA on a template to form a complete ds mDNA, (v) synthesis of mDNA sequences via mDNA polymerase chain reaction, (vi) isothermal denaturation of a ds mDNA into its component ss mDNA, and (vii) an isothermal replicator reaction that exponentially amplifies ss mDNA strands and may be modified to allow for mutations.
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18

FRANCO, Ricardo, Guangyue BAI, Vesna PROSINECKI, Filipa ABRUNHOSA, Gloria C. FERREIRA, and Margarida BASTOS. "Porphyrin-substrate binding to murine ferrochelatase: effect on the thermal stability of the enzyme." Biochemical Journal 386, no. 3 (March 8, 2005): 599–605. http://dx.doi.org/10.1042/bj20040921.

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Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the chelation of Fe(II) into the protoporphyrin IX ring. The energetics of the binding between murine ferrochelatase and mesoporphyrin were determined using isothermal titration calorimetry, which revealed a stoichiometry of one molecule of mesoporphyrin bound per protein monomer. The binding is strongly exothermic, with a large intrinsic enthalpy (ΔH=−97.1 kJ · mol−1), and is associated with the uptake of two protons from the buffer. This proton transfer suggests that hydrogen bonding between ferrochelatase and mesoporphyrin is a key factor in the thermodynamics of the binding reaction. Differential scanning calorimetry thermograms indicated a co-operative two-state denaturation process with a single transition temperature of 56 °C for wild-type murine ferrochelatase. An increase in the thermal stability of ferrochelatase is dependent upon mesoporphyrin binding. Similarly, murine ferrochelatase variants, in which the active site Glu-289 was replaced by either glutamine or alanine and, when purified, contained specifically-bound protoporphyrin, exhibited enhanced protein stability when compared with wild-type ferrochelatase. However, in contrast with the wild-type enzyme, the thermal denaturation of ferrochelatase variants was best described as a non-co-operative denaturation process.
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19

BEHBAHANI, G. REZAEI, M. SHALBAFAN, N. GHEIBI, L. BARZEGAR, H. REZAEI BEHBAHANI, N. YAGHDAVAEI, and Z. REZAEI BEHBAHANI. "DENATURATION OF HUMAN SERUM ALBUMIN BY CERIUM (III) CHLORIDE." Biophysical Reviews and Letters 08, no. 01n02 (June 2013): 59–71. http://dx.doi.org/10.1142/s1793048013500033.

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Cerium (III) Chloride-induced conformational changes of human serum albumin, HSA, in phosphate buffer, 10 mM at pH 7.4 was investigated, using isothermal titration calorimetry (ITC), UV and fluorescence emission spectroscopic methods. The results indicate that CeCl3, Ce3+, induces irreversible denaturation of the HSA structure. The UV absorption intensity of HSA + Ce3+ shows a slight blueshift in the absorbance wavelength with increasing Ce3+ concentration. The fluorescence intensity was increased regularly and a slight redshift was observed in the emission wavelength. The HSA + Ce3+ complex quenches the fluorescence of HSA and changes the microenvironment of tryptophan residue. The emission intensity increases suggesting the loss of the tertiary structure of HSA. The results obtained from the ITC data are in agreement with the spectroscopic methods. The strong negative cooperativity of Ce3+ binding with HSA (Table 1) recovered from the extended solvation model, indicates that HSA has been denatured as a result of its interaction with Ce3+ ions.
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20

Suzuki, Ryota, Masaru Ihira, Yoshihiko Enomoto, Hiroaki Yano, Fumio Maruyama, Nobuhiko Emi, Yoshizo Asano, and Tetsushi Yoshikawa. "Heat denaturation increases the sensitivity of the cytomegalovirus loop-mediated isothermal amplification method." Microbiology and Immunology 54, no. 8 (June 1, 2010): 466–70. http://dx.doi.org/10.1111/j.1348-0421.2010.00236.x.

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21

Nasreen, Khalida, Zahoor Ahmad Parray, Shahzaib Ahamad, Faizan Ahmad, Anwar Ahmed, Salman Freeh Alamery, Tajamul Hussain, Md Imtaiyaz Hassan, and Asimul Islam. "Interactions Under Crowding Milieu: Chemical-Induced Denaturation of Myoglobin is Determined by the Extent of Heme Dissociation on Interaction with Crowders." Biomolecules 10, no. 3 (March 23, 2020): 490. http://dx.doi.org/10.3390/biom10030490.

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Generally, in vivo function and structural changes are studied by probing proteins in a dilute solution under in vitro conditions, which is believed to be mimicking proteins in intracellular milieu. Earlier, thermal-induced denaturation of myoglobin, in the milieu of crowder molecule showed destabilization of the metal protein. Destabilization of protein by thermal-induced denaturation involves a large extrapolation, so, the reliability is questionable. This led us to measure the effects of macromolecular crowding on its stability by chemical-induced denaturation of the protein using probes like circular dichroism and absorption spectroscopy in the presence of dextran 70 and ficoll 70 at various pHs (acidic: 6.0, almost neutral: 7.0 and basic: 8.0). Observations showed that the degree of destabilization of myoglobin was greater due to ficoll 70 as compared to that of dextran 70 so it can be understood that the nature of the crowder or the shape of the crowder has an important role towards the stability of proteins. Additionally, the degree of destabilization was observed as pH dependent, however the pH dependence is different for different crowders. Furthermore, isothermal titration calorimetry and molecular docking studies confirmed that both the crowders (ficoll and dextran) bind to heme moiety of myoglobin and a single binding site was observed for each.
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22

Senisterra, Guillermo A., Bum Soo Hong, Hee-Won Park, and Masoud Vedadi. "Application of High-Throughput Isothermal Denaturation to Assess Protein Stability and Screen for Ligands." Journal of Biomolecular Screening 13, no. 5 (June 2008): 337–42. http://dx.doi.org/10.1177/1087057108317825.

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Many diseases in humans are caused by mutations that decrease the stability of specific proteins or increase their susceptibility to aggregation. Consequently, the availability of high-throughput methods for assessing protein stability and aggregation properties under physiological conditions (e.g., 37 °C) is necessary to analyze physicochemical properties under conditions that are closer to in vivo models. Therefore, the authors have explored the use of isothermal denaturation (ITD) in a 384-well format to evaluate the reproducibility of the method in assessing the stability of proteins at temperatures below the melting temperature and detecting the binding of ligands. Under the conditions tested, the authors were able to assess the stability of citrate synthase and malate dehydrogenase at different constant temperatures and detect the binding of oxaloacetate and nicotinamide adenine dinucleotide to these 2 enzymes, respectively, using the 384-well format. The ITD experiments detected ligand binding to these proteins at about 4 times lower concentration compared with techniques that measure changes in melting temperature. The data show that ITD can be applied to screen libraries of a relatively large number of compounds or detect small stability differences between protein variants. ( Journal of Biomolecular Screening 2008:337-342)
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23

Shahrajabian, Mohamad H., Wenli Sun, and Qi Cheng. "Different Methods for Molecular and Rapid Detection of Human Novel Coronavirus." Current Pharmaceutical Design 27, no. 25 (September 15, 2021): 2893–903. http://dx.doi.org/10.2174/1381612827666210604114411.

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Introduction: While PCR has been recognized as one of the appropriate ways to diagnose infectious diseases, Loop-mediated isothermal amplification (LAMP) which is a nucleic acid amplification method, can be considered as an alternative to PCR, and it is faster, cost-effective, and easier to perform than nested PCR. Patients and Methods: Keywords were searched in PubMed/MEDLINE, Scopus and Institute for Scientific Information Web of Science, as well as the search engine of Google Scholar. Keywords included PCR, LAMP, RAA, RPA, Virus and COVID-19. Results: LAMP technology has been extensively applied for the detection of human pathogenic bacteria, crop pests, pathogenic organisms and components in meat products. A new isotheral method, Recombinase polymerase amplification (RPA), can amplify the DNA as well as RPA. RPA involves benefits of isothermal PCR as well as simplicity and rapid amplification. Recombinase aided amplification (RAA) assay has been favorably used in the detection of bacterial and viral pathogens and solved the technical difficulties posed by DNA amplification methods because it does not need thermal denaturation of the template and involves a low and constant temperature. Conclusions: Reverse transcription polymerase chain reaction, digital PCR, LAMP, nicking endonuclease amplification reaction, recombinase polymerase amplification, and clustered regularly interspaced short palindromic repeats are different nucleic acid amplification tests of COVID-19. LAMP methods can be more specific than qPCR and immunoassays. The LAMP assay can be applied for rapid detection of SARS-CoV, MERS-CoV, SARS-CoV-2, and influenza, because LAMP is a highly sensitive and specific DNA/RNA amplification technique.
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24

BAJAJ, Kanika, Ghadiyaram CHAKSHUSMATHI, Kiran BACHHAWAT-SIKDER, Avadhesha SUROLIA, and Raghavan VARADARAJAN. "Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)." Biochemical Journal 380, no. 2 (June 1, 2004): 409–17. http://dx.doi.org/10.1042/bj20031528.

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The protein CcdB (controller of cell division or death B) is an F-plasmid-encoded toxin that acts as an inhibitor of Escherichia coli DNA gyrase. The stability and aggregation state of CcdB have been characterized as a function of pH and temperature. Size-exclusion chromatography revealed that the protein is a dimer at pH 7.0, but a monomer at pH 4.0. CD analysis and fluorescence spectroscopy showed that the monomer is well folded, and has similar tertiary structure to the dimer. Hence intersubunit interactions are not required for folding of individual subunits. The stability of both forms was characterized by isothermal denaturant unfolding and calorimetry. The free energies of unfolding were found to be 9.2 kcal·mol−1 (1 cal≈4.184 J) and 21 kcal·mol−1 at 298 K for the monomer and dimer respectively. The denaturant concentration at which one-half of the protein molecules are unfolded (Cm) of the dimer is dependent on protein concentration, whereas the Cm of the monomer is independent of protein concentration, as expected. Although thermal unfolding of the protein in aqueous solution is irreversible at neutral pH, it was found that thermal unfolding is reversible in the presence of GdmCl (guanidinium chloride). Differential scanning calorimetry in the presence of low concentrations of GdmCl in combination with isothermal denaturation melts as a function of temperature were used to derive the stability curve for the protein. The value of ΔCp (representing the change in excess heat capacity upon protein denaturation) is 2.8±0.2 kcal·mol−1·K−1 for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration.
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25

Ross, Patrick, Wilhelm Weihofen, Fai Siu, Amy Xie, Hetal Katakia, S. Kirk Wright, Ian Hunt, Richard K. Brown, and Ernesto Freire. "Isothermal chemical denaturation to determine binding affinity of small molecules to G-protein coupled receptors." Analytical Biochemistry 473 (March 2015): 41–45. http://dx.doi.org/10.1016/j.ab.2014.11.019.

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26

Soroka, Marianna, Barbara Wasowicz, and Anna Rymaszewska. "Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?" Cells 10, no. 8 (July 29, 2021): 1931. http://dx.doi.org/10.3390/cells10081931.

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In 1998, when the PCR technique was already popular, a Japanese company called Eiken Chemical Co., Ltd. designed a method known as the loop-mediated isothermal amplification of DNA (LAMP). The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets. Furthermore, the Bst DNA polymerase most used in LAMP shows a high strand displacement activity, which eliminates the DNA denaturation stage. One of the most significant advantages of LAMP is that it can be conducted at a stable temperature, for instance, in a dry block heater or an incubator. The products of LAMP can be detected much faster than in standard techniques, sometimes only requiring analysis with the naked eye. The following overview highlights the usefulness of LAMP and its effectiveness in various fields; it also considers the superiority of LAMP over PCR and presents RT-LAMP as a rapid diagnostic tool for SARS-CoV-2.
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27

Taneja, S., and F. Ahmad. "Increased thermal stability of proteins in the presence of amino acids." Biochemical Journal 303, no. 1 (October 1, 1994): 147–53. http://dx.doi.org/10.1042/bj3030147.

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This study is a systematic attempt to understand the roles of osmolytes in protecting proteins against denaturing stress. Thermal denaturation of cytochrome c has been studied in the presence of various concentrations of all L-amino acids that are more hydrophobic than glycine and have a solubility of 0.1 M or higher in water at 25 degrees C. The basic observations are as follows. (1) Arginine and histidine destabilize the native protein; both Tm (the midpoint of thermal transition) and delta GDH2O (25 degrees C) (the Gibbs energy of stabilization) decrease with increasing amino acid concentration. (2) Isoleucine, leucine and phenylalanine have no effect on Tm and deltaGDH2O (25 degrees C). (3) Valine and less hydrophobic amino acids stabilize the protein in terms of Tm but deltaGDH2O (25 degrees C) is unchanged. This observation was confirmed by the study of isothermal denaturation of cytochrome c by guanidinium chloride which suggested that delta GDH2O is independent of osmolyte concentration, but Cm (the midpoint of transition) is increased in their presence. (4) In the case of stabilizers, change in Tm/mol of amino acid decreases with increasing hydrophobicity of these osmolytes.
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Chen, S. S., N. T. Wright, and J. D. Humphrey. "Heat-Induced Changes in the Mechanics of a Collagenous Tissue: Isothermal Free Shrinkage." Journal of Biomechanical Engineering 119, no. 4 (November 1, 1997): 372–78. http://dx.doi.org/10.1115/1.2798281.

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We present data from isothermal free-shrinkage tests (i.e., performed in the absence of mechanical loads) wherein bovine chordae tendineae were subjected to temperatures from 65 to 85°C for 120 to 1200 s. These data reveal four new insights into heat-induced denaturation of a collagenous tissue. First, a characteristic time for the free shrinkage appears to exhibit an Arrhenius-type relationship with temperature. Second, scaling the actual heating time via the characteristic time results in a single correlation between free shrinkage and the duration of heating; this correlation suggests a time-temperature equivalence. Third, it is the cumulative, not current, heating time that governs the free shrinkage. And fourth, heat-induced free shrinkage is partially recovered when the tissue is returned to 37°C, this recovery also being time-dependent. Although these findings will help guide future experimentation and constitutive modeling, as well as the design of new heat-based clinical therapies, there is a pressing need to collect additional isothermal data, particularly in the presence of well-defined mechanical loads.
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29

CLAEYS, WENDIE L., LINDA R. LUDIKHUYZE, ANN M. VAN LOEY, and MARC E. HENDRICKX. "Inactivation kinetics of alkaline phosphatase and lactoperoxidase, and denaturation kinetics of β-lactoglobulin in raw milk under isothermal and dynamic temperature conditions." Journal of Dairy Research 68, no. 1 (February 2001): 95–107. http://dx.doi.org/10.1017/s002202990000460x.

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A detailed kinetic study of alkaline phosphatase, lactoperoxidase and β-lactoglobulin was carried out in the context of identifying intrinsic time–temperature indicators for controlling the heat processing of milk. The heat inactivation or denaturation of alkaline phosphatase, lactoperoxidase and β-lactoglobulin under isothermal conditions was found to follow first order kinetics. Experimental results were analysed using both a two step linear regression and a one step non-linear regression method. Results obtained using the two statistical techniques were comparable, but the 95% confidence interval for the predicted values was smaller when the one step non-linear regression method was used, indicating its superiority for estimating kinetic parameters. Thermal inactivation of alkaline phosphatase and lactoperoxidase was characterized by z values of 5·3 deg C (D60 °C = 24·6 min) and 4·3 deg C (D71 °C = 38·6 min) respectively. For the denaturation of β-lactoglobulin we found z values of 7·9 deg C (D75 °C = 49·9 min) in the temperature range 70–80 °C and 24·2 deg C (D85 °C = 3·53 min) in the range 83-95 °C. Dref and z were evaluated under dynamic temperature conditions. To estimate the statistical accuracy of the parameters, 90% joint confidence regions were constructed.
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30

Datta, Nivedita, Maurice G. Hayes, Hilton C. Deeth, and Alan L. Kelly. "Significance of frictional heating for effects of high pressure homogenisation on milk." Journal of Dairy Research 72, no. 4 (May 23, 2005): 393–99. http://dx.doi.org/10.1017/s0022029905001056.

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High pressure homogenisation (HPH) is a novel dairy processing tool, which has many effects on enzymes, microbes, fat globules and proteins in milk. The effects of HPH on milk are due to a combination of shear forces and frictional heating of the milk during processing; the relative importance of these different factors is unclear, and was the focus of this study. The effect of milk inlet temperature (in the range 10–50 °C) on residual plasmin, alkaline phosphatase, lactoperoxidase and lipase activities in raw whole bovine milk homogenised at 200 MPa was investigated. HPH caused significant heating of the milk; outlet temperature increased in a linear fashion (0·5887 °C/°C, R2=0·9994) with increasing inlet temperature. As milk was held for 20 s at the final temperature before cooling, samples of the same milk were heated isothermally in glass capillary tubes for the same time/temperature combinations. Inactivation profiles of alkaline phosphatase in milk were similar for isothermal heating or HPH, indicating that loss of enzyme activity was due to heating alone. Loss of plasmin and lactoperoxidase activity in HPH milk, however, was greater than that in heated milk. Large differences in residual lipase activities in milks subjected to heating or HPH were observed due to the significant increase in lipase activity in homogenised milk. Denaturation of β-lactoglobulin was more extensive following HPH than the equivalent heat treatment. Inactivation of plasmin was correlated with increasing fat/serum interfacial area but was not correlated with denaturation of β-lactoglobulin. Thus, while some effects of HPH on milk are due to thermal effects alone, many are induced by the combination of forces and heating to which the milk is exposed during HPH.
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31

Tang, Chuanning, Scott Lew, and Dacheng He. "Using a second-order differential model to fit data without baselines in protein isothermal chemical denaturation." Protein Science 25, no. 4 (February 11, 2016): 898–904. http://dx.doi.org/10.1002/pro.2878.

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32

Harris, J. L., P. B. Wells, and J. D. Humphrey. "Altered Mechanical Behavior of Epicardium Due to Isothermal Heating Under Biaxial Isotonic Loads." Journal of Biomechanical Engineering 125, no. 3 (June 1, 2003): 381–88. http://dx.doi.org/10.1115/1.1567754.

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Recent isothermal biaxial isotonic tests suggest that increasing the temperature hastens the rate of denaturation of epicardium whereas increasing the mechanical load during heating delays this process, findings that are consistent with prior uniaxial tests on tendons. Yet, contrary to uniaxial reports, a clear time-temperature-load equivalency was not found in this multiaxial setting. There is, therefore, a need to delineate multiaxial thermomechanical behavior in greater detail, and ultimately, to correlate changes therein with the underlying microstructure. Toward this end, we describe a new experimental approach for quantifying heating-induced changes in the multiaxial mechanical response of thin sheet-like specimens. Illustrative results are presented for bovine epicardium subjected to nine different thermomechanical loading protocols. Among other results, it is shown that thermal damage tends to increase the stiffness at low strains and that overall changes in extensibility correlate well with the degree of thermal damage independent of the specific thermomechanical protocol. Multiaxial changes in behavior are nevertheless complex, and there is a need for significantly more testing before constitutive relations can be formulated.
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33

Saboury, A. A., M. Miroliae, M. Nemat-Gorgani, and A. A. Moosavi-Movahedi. "Kinetics denaturation of yeast alcohol dehydrogenase and the effect of temperature and trehalose. An isothermal microcalorimetry study." Thermochimica Acta 326, no. 1-2 (February 1999): 127–31. http://dx.doi.org/10.1016/s0040-6031(98)00588-7.

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34

Sarver, Ronald W., Joseph M. Rogers, and Dennis E. Epps. "Determination of Ligand-MurB Interactions by Isothermal Denaturation: Application as a Secondary Assay to Complement High Throughput Screening." Journal of Biomolecular Screening 7, no. 1 (February 1, 2002): 21–28. http://dx.doi.org/10.1089/108705702753520305.

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35

Rubio, Ana R., Natalia Busto, José M. Leal, and Begoña García. "Doxorubicin binds to duplex RNA with higher affinity than ctDNA and favours the isothermal denaturation of triplex RNA." RSC Advances 6, no. 103 (2016): 101142–52. http://dx.doi.org/10.1039/c6ra21387a.

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The higher affinity of DOX with AU to give the intercalated complex AU/DOX is responsible for the disproportionation of the groove binding complex, UAU/DOX, to give rise to the AU/DOX and the U/DOX complexes at 25 °C
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36

Haque, M. Amdadul, Peter Aldred, Jie Chen, Colin J. Barrow, and Benu Adhikari. "Comparative study of denaturation of whey protein isolate (WPI) in convective air drying and isothermal heat treatment processes." Food Chemistry 141, no. 2 (November 2013): 702–11. http://dx.doi.org/10.1016/j.foodchem.2013.03.035.

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37

Fleischman, Marianna L., Rajoshi Chaudhuri, Ria T. Caringal, Lisa Kueltzo, K. C. Cheng, and Frank Arnold. "Optimization of Formulation Conditions for Broadly Neutralizing Antibodies (bnAb): Utilizing Isothermal Chemical Denaturation as a High-throughput Screening Method." Biophysical Journal 116, no. 3 (February 2019): 334a. http://dx.doi.org/10.1016/j.bpj.2018.11.1817.

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38

Liu, Mengmeng, Mengzhe Li, Cuiping Ma, and Chao Shi. "Detection of canine parvovirus and feline panleukopenia virus in fecal samples by strand exchange amplification." Journal of Veterinary Diagnostic Investigation 32, no. 6 (September 30, 2020): 880–86. http://dx.doi.org/10.1177/1040638720962067.

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Canine parvovirus 2 (CPV-2) and feline panleukopenia virus (FPLV) often cause acute enteric disease in their hosts. A simple, rapid, and effective method for the on-site detection of these viruses would be useful. We used a denaturation bubble-mediated strand exchange amplification (SEA) method to successfully detect CPV-2 and FPLV in fecal samples. SEA could detect as little as 3.6 pg/μL of CPV-2 and 6.6 pg/μL of FPLV genomic DNA following a 40-min incubation at an isothermal temperature of 61°C. Unlike PCR, SEA does not require complicated equipment, and positive samples produce a color change that can be visualized by the naked eye. Additionally, SEA is simpler than PCR because no extraction is needed, and heating of the fecal sample at 98°C can be performed with a heating block or water bath. This rapid and effective nucleic acid detection platform could be used as a point-of-care test for the detection of CPV-2 and FPLV.
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39

Nye, Dillon B., and Nathan A. Tanner. "Chimeric DNA byproducts in strand displacement amplification using the T7 replisome." PLOS ONE 17, no. 9 (September 19, 2022): e0273979. http://dx.doi.org/10.1371/journal.pone.0273979.

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Recent advances in next generation sequencing technologies enable reading DNA molecules hundreds of kilobases in length and motivate development of DNA amplification methods capable of producing long amplicons. In vivo, DNA replication is performed not by a single polymerase enzyme, but multiprotein complexes called replisomes. Here, we investigate strand-displacement amplification reactions using the T7 replisome, a macromolecular complex of a helicase, a single-stranded DNA binding protein, and a DNA polymerase. The T7 replisome may initiate processive DNA synthesis from DNA nicks, and the reaction of a 48 kilobase linear double stranded DNA substrate with the T7 replisome and nicking endonucleases is shown to produce discrete DNA amplicons. To gain a mechanistic understanding of this reaction, we utilized Oxford Nanopore long-read sequencing technology. Sequence analysis of the amplicons revealed chimeric DNA reads and uncovered a connection between template switching and polymerase exonuclease activity. Nanopore sequencing provides insight to guide the further development of isothermal amplification methods for long DNA, and our results highlight the need for high-specificity, high-turnover nicking endonucleases to initiate DNA amplification without thermal denaturation.
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40

Nai, Yi Heng, Egan H. Doeven, and Rosanne M. Guijt. "An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein." PLOS ONE 17, no. 3 (March 24, 2022): e0265391. http://dx.doi.org/10.1371/journal.pone.0265391.

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The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.
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41

Liu, Wentao, and Guoying Li. "Non-isothermal kinetic analysis of the thermal denaturation of type I collagen in solution using isoconversional and multivariate non-linear regression methods." Polymer Degradation and Stability 95, no. 12 (December 2010): 2233–40. http://dx.doi.org/10.1016/j.polymdegradstab.2010.09.012.

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42

Svilenov, Hristo, Uroš Markoja, and Gerhard Winter. "Isothermal chemical denaturation as a complementary tool to overcome limitations of thermal differential scanning fluorimetry in predicting physical stability of protein formulations." European Journal of Pharmaceutics and Biopharmaceutics 125 (April 2018): 106–13. http://dx.doi.org/10.1016/j.ejpb.2018.01.004.

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43

Cotrina, Ellen Y., Ângela Oliveira, José Pedro Leite, Jordi Llop, Luis Gales, Jordi Quintana, Isabel Cardoso, and Gemma Arsequell. "Repurposing Benzbromarone for Familial Amyloid Polyneuropathy: A New Transthyretin Tetramer Stabilizer." International Journal of Molecular Sciences 21, no. 19 (September 28, 2020): 7166. http://dx.doi.org/10.3390/ijms21197166.

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Transthyretin (TTR) is a homotetrameric protein involved in human amyloidosis, including familial amyloid polyneuropathy (FAP). Discovering small-molecule stabilizers of the TTR tetramer is a therapeutic strategy for these diseases. Tafamidis, the only approved drug for FAP treatment, is not effective for all patients. Herein, we discovered that benzbromarone (BBM), a uricosuric drug, is an effective TTR stabilizer and inhibitor against TTR amyloid fibril formation. BBM rendered TTR more resistant to urea denaturation, similarly to iododiflunisal (IDIF), a very potent TTR stabilizer. BBM competes with thyroxine for binding in the TTR central channel, with an IC50 similar to IDIF and tafamidis. Results obtained by isothermal titration calorimetry (ITC) demonstrated that BBM binds TTR with an affinity similar to IDIF, tolcapone and tafamidis, confirming BBM as a potent binder of TTR. The crystal structure of the BBM-TTR complex shows two molecules binding deeply in the thyroxine binding channel, forming strong intermonomer hydrogen bonds and increasing the stability of the TTR tetramer. Finally, kinetic analysis of the ability of BBM to inhibit TTR fibrillogenesis at acidic pH and comparison with other stabilizers revealed that benzbromarone is a potent inhibitor of TTR amyloidogenesis, adding a new interesting scaffold for drug design of TTR stabilizers.
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44

Jensen, Jaime L., Beau D. Jernberg, Sangita C. Sinha, and Christopher L. Colbert. "Structural basis of cell-surface signaling by a conserved sigma regulator in Gram-negative bacteria." Journal of Biological Chemistry 295, no. 17 (February 26, 2020): 5795–806. http://dx.doi.org/10.1074/jbc.ra119.010697.

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Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. The molecular details of ferric siderophore-mediated activation of the iron import machinery through a sigma regulator are unclear. Here, we present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. The structure revealed that the CCSSD consists of two subdomains: a juxta-membrane subdomain, which has a novel all-β-fold, followed by a secretin/TonB, short N-terminal subdomain at the C terminus of the CCSSD, a previously unobserved topological arrangement of this domain. Using affinity pulldown assays, isothermal titration calorimetry, and thermal denaturation CD spectroscopy, we show that both subdomains are required for binding the NTSD with micromolar affinity and that NTSD binding improves CCSSD stability. Our findings prompt us to present a revised model of CSS wherein the CCSSD:NTSD complex forms prior to ferric-siderophore binding. Upon siderophore binding, conformational changes in the CCSSD enable regulated intramembrane proteolysis of the sigma regulator, ultimately resulting in transcriptional regulation.
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45

Trollope, K. M., J. F. Görgens, and H. Volschenk. "Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production." Applied and Environmental Microbiology 81, no. 20 (August 7, 2015): 7319–29. http://dx.doi.org/10.1128/aem.02134-15.

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ABSTRACTTheAspergillus japonicusβ-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the β-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semirational approach yielded a functionally enriched first-round library of 36 single-amino-acid-substitution variants with 58% retaining activity, and of these, 71% displayed improved activities compared to the parent. The substitutions yielding the five most improved variants subsequently were exhaustively combined and evaluated. A four-substitution combination variant was identified as the most improved and reduced the time to completion of an efficient industrial-like reaction by 22%. Characterization of the top five combination variants by isothermal denaturation assays indicated that these variants displayed improved thermostability, with the most thermostable variant displaying a 5.7°C increased melting temperature. The variants displayed uniquely altered, concentration-dependent substrate and product binding as determined by differential scanning fluorimetry. The altered catalytic activity was evidenced by increased specific activities of all five variants, with the most improved variant doubling that of the parent. Variant homology modeling and computational analyses were used to rationalize the effects of amino acid changes lacking direct interaction with substrates. Data indicated that targeting substitutions to loop regions resulted in improved enzyme thermostability, specific activity, and relief from product inhibition.
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46

Kolli, Vidyalatha, Subhankar Paul, Praveen Kumar Guttula, and Nandini Sarkar. "Elucidating the Role of Val-Asn 95 and Arg-Gly 52 Mutations on Structure and Stability of Fibroblast Growth Factor Homologous Factor 2." Protein & Peptide Letters 26, no. 11 (October 24, 2019): 848–59. http://dx.doi.org/10.2174/0929866526666190503092718.

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Background: Fibroblast growth Factor Homologous Factors (FHFs) belong to a subclass of Fibroblast Growth Factor (FGF) family owing to their high sequence and structural similarities with FGFs. However, despite these similarities, there are properties which set them apart from FGFs. FHFs lack the secretion signal sequence unlike other FGF members, except FGF1 and 2. Unlike FGFs, FHFs are not able to bind to FGF Receptors (FGFRs) and instead have been implicated in binding to Voltage-Gated Sodium Channels (VGSCs), neuronal MAP kinase scaffold protein and islet-brain-2 (IB2). The two amino acids Arg-52 and Val95 are conserved in all FHFs and mutation of these residues lead to its inability to bind with VGSC/IB2. However, it is not clear whether the loss of binding is due to destabilization of the protein on mutation or due to involvement of Arg52 and Val95 in conferring functionality to FHFs. Objective: In the present study, we have mutated these two conserved residues of FHF2 with its corresponding FGF counterpart amino acids and studied the effects of the mutations on the structure and stability of the protein. Methods: Several biophysical methods like isothermal equilibrium denaturation study, ANS fluorescence, intrinsic fluorescence, acrylamide quenching, circular dichroism studies as well as using computational approaches were employed. Results: The single mutations were found to affect the overall stability, conformation and functionality of the protein. Conclusion: Thus, the studies throw light on the role of specific amino acids in deciding the stability, structure and functionality of proteins and will be useful for development of therapeutically engineered proteins.
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47

Chen, Jyh-Jian, and Zong-Hong Lin. "Fabrication of an Oscillating Thermocycler to Analyze the Canine Distemper Virus by Utilizing Reverse Transcription Polymerase Chain Reaction." Micromachines 13, no. 4 (April 12, 2022): 600. http://dx.doi.org/10.3390/mi13040600.

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The reverse transcription-polymerase chain reaction (RT-PCR) has been utilized as an effective tool to diagnose the infectious diseases of viruses. In the present work, the oscillating thermocycler is fabricated and performed to carry out the one-step RT-PCR process successfully. The ribonucleic acid (RNA) mixture is pipetted into the fixed sample volume inside an aluminum reaction block. The sample oscillates the pathway onto the linear motion control system and through the specific RT-PCR heating zones with individual homemade thermal control modules. The present oscillating thermocycler combines the merits of the chamber type and the CF type systems. Before PCR, the reaction chamber moves to the low-temperature zone to complete the RT stage and synthesize the complementary deoxyribonucleic acid (DNA). Next, the low-temperature zone is regulated to the annealing zone. Furthermore, the reactive sample is moved back and forth among three isothermal zones to complete PCR. No extra heating zone is required for the RT stage. The total length of the moving displacement of the chamber is within 100 mm. The miniaturization of the oscillating thermocycler can be expected. In our oscillatory device, the denaturation zone located between the annealing and extension zones is suggested as the appropriate arrangement of the heating blocks. Heat management without thermal cross-talk is easy. Finally, an improved oscillating device is demonstrated to execute the RT-PCR process directly, utilized to amplify the canine distemper virus templates successfully, which could be well applied to a low-cost DNA analysis system in the future.
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48

Almeida, Ana, Rui P. P. L. Ribeiro, José P. B. Mota, and Carlos Grande. "Extrusion and Characterization of High Si/Al Ratio ZSM-5 Using Silica Binder." Energies 13, no. 5 (March 5, 2020): 1201. http://dx.doi.org/10.3390/en13051201.

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Biogas upgrading is a key operation for transforming raw biogas into valuable biomethane that can be used as fuel or transported through pipelines. Pressure swing adsorption (PSA) is one possible technique that can be used for upgrading. ZSM-5 with high silica/aluminum (Si/Al) ratio has a reasonable CO2/CH4 selectivity and an almost linear CO2 adsorption isotherm, which can reduce power consumption. Extrusion of zeolites uses Al-based binders which can result in a denaturation and in a decrease of Si/Al ratio, promoting a steeper CO2 isotherm and also impacting the water adsorption. In this work, we have extruded a ZSM-5 (with a Si/Al = 200) using only silica-based binder. Different samples were obtained using different extrusion paste compositions and operating conditions and their textural properties characterized. The mechanical strength of the samples as well as the CO2, CH4, and H2O adsorption equilibrium isotherms at 303–343 K were measured. Our results show that it is possible to produce extrudates with mechanical resistance comparable to (or higher than) commercial zeolite materials with surface area reductions lower than 10% and little or no impact on the CO2/CH4 selectivity.
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49

Biswas, Priyanka, Dillip K. Sahu, Kalyanasis Sahu, and Rajat Banerjee. "Spectroscopic Studies of Asparaginyl-tRNA Synthetase from Entamoeba histolytica." Protein & Peptide Letters 26, no. 6 (July 4, 2019): 435–48. http://dx.doi.org/10.2174/0929866526666190327122419.

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Background: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts. Objective: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties. Methods: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4 µM protein was used for the rest of the experiments. Results: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. Conclusion: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two – state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.
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50

Jin, Yonghui, Qiuju Du, Yanhui Li, Yang Zhang, Bing Chen, Mingzhen Wang, Kewei Chen, Yaohui Sun, Shiyong Zhao, and Zhenyu Jing. "Removal of Methylene Blue by Crosslinked Egg White Protein/Graphene Oxide Bionanocomposite Aerogels." Nanomaterials 12, no. 15 (August 3, 2022): 2659. http://dx.doi.org/10.3390/nano12152659.

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Egg white protein is a non-toxic and biodegradable biopolymer that forms a gel easily via simple thermal denaturation treatment. A novel aerogel on the basis of egg white protein crosslinked with graphene oxide was prepared via a facile freeze-drying method. The structure and physicochemical characteristics of the aerogels were characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and Brunauer–Emmett–Teller (BET) analysis. The adsorption properties of the aerogels were investigated by studying the influencing factors such as the solution pH, dose, temperature and contact time. The adsorption capacity of methylene blue onto the aerogels was tested, whose maximum adsorption capacity, calculated by the Langmuir isotherm equation, reached 91.7 mg/g. Adsorption kinetics studies showed that the adsorption followed the pseudo-second-order kinetic model. Thermodynamic data implied that methylene blue adsorbed by the aerogels was an exothermic and spontaneous process.
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