scholarly journals Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1183
Author(s):  
Márió Miczi ◽  
Ádám Diós ◽  
Beáta Bozóki ◽  
József Tőzsér ◽  
János András Mótyán
Keyword(s):  
Fusion Protein ◽  
Real Time ◽  
Optical Measurement ◽  
Proteolytic Enzymes ◽  
Monitoring Methods ◽  
Kinetic Measurements ◽  
Protein Substrates ◽  
Volume Measurements ◽  
Protease Assay ◽  
Hiv 1

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR’s specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.

scholarly journals Data supporting Ni-NTA magnetic bead-based fluorescent protease assay using recombinant fusion protein substrates

Data in Brief ◽  
2018 ◽  
Vol 18 ◽  
pp. 203-208 ◽  
Author(s):  
János András Mótyán ◽  
Márió Miczi ◽  
Beáta Bozóki ◽  
József Tőzsér
Keyword(s):  
Fusion Protein ◽  
Magnetic Bead ◽  
Recombinant Fusion Protein ◽  
Protein Substrates ◽  
Protease Assay

scholarly journals GLM-Based Flexible Monitoring Methods: An Application to Real-Time Highway Safety Surveillance

Symmetry ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 362
Author(s):  
Arshad Jamal ◽  
Tahir Mahmood ◽  
Muhamad Riaz ◽  
Hassan M. Al-Ahmadi
Keyword(s):  
Real Time ◽  
Control Charts ◽  
Negative Binomial ◽  
Highway Safety ◽  
Monitoring Methods ◽  
Count Data Models ◽  
Crash Data ◽  
Safety Surveillance ◽  
Study Results ◽  
Best Fit

Statistical modeling of historical crash data can provide essential insights to safety managers for proactive highway safety management. While numerous studies have contributed to the advancement from the statistical methodological front, minimal research efforts have been dedicated to real-time monitoring of highway safety situations. This study advocates the use of statistical monitoring methods for real-time highway safety surveillance using three years of crash data for rural highways in Saudi Arabia. First, three well-known count data models (Poisson, negative binomial, and Conway–Maxwell–Poisson) are applied to identify the best fit model for the number of crashes. Conway–Maxwell–Poisson was identified as the best fit model, which was used to find the significant explanatory variables for the number of crashes. The results revealed that the road type and road surface conditions significantly contribute to the number of crashes. From the perspective of real-time highway safety monitoring, generalized linear model (GLM)-based exponentially weighted moving average (EWMA) and cumulative sum (CUSUM) control charts are proposed using the randomized quantile residuals and deviance residuals of Conway–Maxwell (COM)–Poisson regression. A detailed simulation-based study is designed for predictive performance evaluation of the proposed control charts with existing counterparts (i.e., Shewhart charts) in terms of the run-length properties. The study results showed that the EWMA type control charts have better detection ability compared with the CUSUM type and Shewhart control charts under small and/or moderate shift sizes. Finally, the proposed monitoring methods are successfully implemented on actual traffic crash data to highlight the efficacy of the proposed methods. The outcome of this study could provide the analysts with insights to plan sound policy recommendations for achieving desired safety goals.

Kinetics of interaction of HIV fusion protein (gp41) with lipid membranes studied by real-time AFM imaging

2010 ◽  
Vol 110 (6) ◽  
pp. 694-700 ◽  
Author(s):  
Arkady Bitler ◽  
Naama Lev ◽  
Yael Fridmann-Sirkis ◽  
Lior Blank ◽  
Sidney R. Cohen ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Real Time ◽  
Lipid Membranes ◽  
Afm Imaging ◽  
Protein Gp41 ◽  
Kinetics Of

LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01)

2009 ◽  
Vol 81 (2) ◽  
pp. 217-223 ◽  
Author(s):  
Avettand-Fènoël Véronique ◽  
Chaix Marie-Laure ◽  
Blanche Stéphane ◽  
Burgard Marianne ◽  
Floch Corinne ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Blood Cells ◽  
Dna Quantitation ◽  
Hiv 1

A New Real-Time Process Monitor Method Based on MRC

2014 ◽  
Vol 971-973 ◽  
pp. 1481-1484
Author(s):  
Ke He Wu ◽  
Long Chen ◽  
Yi Li
Keyword(s):  
Information System ◽  
Real Time ◽  
Process Monitoring ◽  
Time Process ◽  
Monitoring Methods ◽  
Traditional Process ◽  
Kernel Level ◽  
Process Monitor

In order to ensure safe and stable running of applications, this paper analyses the limitation of traditional process-monitoring methods, and then designs a new real-time process monitor method based on Mandatory Running Control (MRC) technology. This method not only can monitor the processes, but also can control them from system kernel level to improve the reliability and safety of applications, so as to ensure the security and stability of information system.

Real-time volume measurements in the isolated beating rat heart

1994 ◽  
Vol 41 (4) ◽  
pp. 383-387
Author(s):  
D.G. Waksman ◽  
J.J. McInerney ◽  
M.D. Herr
Keyword(s):  
Real Time ◽  
Rat Heart ◽  
Volume Measurements

Expression in Escherichia coli and in vitro processing of HIV-1 p24 fusion protein

1993 ◽  
Vol 31 (2) ◽  
pp. 225-232 ◽  
Author(s):  
Ilona Marczinovits ◽  
Imre Boros ◽  
Fouad El Jarrah ◽  
György Füst ◽  
János Molnár
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Expression In Escherichia Coli ◽  
In Vitro Processing ◽  
Hiv 1

scholarly journals Real-time monitoring of superoxide anion radical generation in response to wounding: electrochemical study

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3050 ◽  
Author(s):  
Ankush Prasad ◽  
Aditya Kumar ◽  
Ryo Matsuoka ◽  
Akemi Takahashi ◽  
Ryo Fujii ◽  
...  
Keyword(s):  
Real Time ◽  
Superoxide Anion ◽  
Anion Radical ◽  
Spinacia Oleracea ◽  
Superoxide Anion Radical ◽  
Stress Factors ◽  
Iron Porphyrin ◽  
Real Time Monitoring ◽  
Kinetic Measurements ◽  
Oxidation Current

Background The growth and development of plants is deleteriously affected by various biotic and abiotic stress factors. Wounding in plants is caused by exposure to environmental stress, mechanical stress, and via herbivory. Typically, oxidative burst in response to wounding is associated with the formation of reactive oxygen species, such as the superoxide anion radical (O2•−), hydrogen peroxide (H2O2) and singlet oxygen; however, few experimental studies have provided direct evidence of their detection in plants. Detection of O2•− formation in plant tissues have been performed using various techniques including electron paramagnetic resonance spin-trap spectroscopy, epinephrine-adrenochrome acceptor methods, staining with dyes such as tetrazolium dye and nitro blue tetrazolium (NBT); however, kinetic measurements have not been performed. In the current study, we provide evidence of O2•− generation and its kinetics in the leaves of spinach (Spinacia oleracea) subjected to wounding. Methods Real-time monitoring of O2•− generation was performed using catalytic amperometry. Changes in oxidation current for O2•− was monitored using polymeric iron-porphyrin-based modified carbon electrodes (φ = 1 mm) as working electrode with Ag/AgCl as the reference electrode. Result The results obtained show continuous generation of O2•− for minutes after wounding, followed by a decline. The exogenous addition of superoxide dismutase, which is known to dismutate O2•− to H2O2, significantly suppressed the oxidation current. Conclusion Catalytic amperometric measurements were performed using polymeric iron-porphyrin based modified carbon electrode. We claim it to be a useful tool and a direct method for real-time monitoring and precise detection of O2•− in biological samples, with the potential for wide application in plant research for specific and sensitive detection of O2•−.

Real-time optical measurement of biologically relevant thermal damage in tissue-mimicking hydrogels containing bovine serum albumin

2010 ◽  
Vol 26 (5) ◽  
pp. 456-464 ◽  
Author(s):  
Sacha D. Nandlall ◽  
Heiko A. Schiffter ◽  
Sebastian Vonhoff ◽  
Miriam Bazán-Peregrino ◽  
Manish Arora ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Real Time ◽  
Bovine Serum ◽  
Thermal Damage ◽  
Optical Measurement ◽  
Biologically Relevant
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