Journal articles: 'Bio-layer interferometry'

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Laigre, Eugénie, David Goyard, Claire Tiertant, Jérôme Dejeu, and Olivier Renaudet. "The study of multivalent carbohydrate–protein interactions by bio-layer interferometry." Organic & Biomolecular Chemistry 16, no. 46 (2018): 8899–903. http://dx.doi.org/10.1039/c8ob01664j.

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Волкова, М. В., В. В. Бояринцев, А. В. Трофименко, С. А. Бирюков, Е. В. Горина, Г. И. Фильков, and М. О. Дурыманов. "АДАПТАЦИЯ МЕТОДА ИНТЕРФЕРОМЕТРИИ СЛОЯ БИОМОЛЕКУЛ ДЛЯ КОЛИЧЕСТВЕННОЙ ОЦЕНКИ СОДЕРЖАНИЯ ФАКТОРА РОСТА ЭНДОТЕЛИЯ СОСУДОВ В КОНДИЦИОНИРОВАННОЙ КЛЕТОЧНОЙ СРЕДЕ." Биофизика 65, no. 6 (2020): 1099–106. http://dx.doi.org/10.31857/s0006302920060083.

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Quantitative analysis of cytokines, chemokines, growth factors and other soluble proteins in different biological liquids is routinely performed in contemporary diagnostics and biomedicine research. However, current methods of analysis are time-consuming and include multiple steps. In this study, we have developed a protocol that describes how bio-layer interferometry can be applied to quantify an analyte in several minutes. Conditioned growth media collected from mouse mesenchymal stem cells grown in normoxia or hypoxic conditions in a monolayer fashion, MSC-derived 3D cell sheets and 3D spheroids were used as a model system in which we determined a concentration of vascular endothelial growth factor (VEGF-A). This technique displayed a high sensitivity (down to 0.1 ng of VEGF-A per mL as a minimum). The measured concentrations of VEGF-A in the conditioned media from mesenchymal stem cells turned out to be similar with values determined by the enzyme-linked immunosorbent assay. Using bio-layer interferometry, it was shown that as compared to mesenchymal stem cells grown in monolayer, spheroids and 3D sheets of mesenchymal stem cells produce significantly more VEGF-A (by 2.5-3.0-fold). Thus, due to the developed protocol it was possible to adapt bio-layer interferometry for rapid quantification of growth factors in conditioned media.

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Sun, Tingwan, Felicia Reid, Yuqi Liu, Yuan Cao, Patricia Estep, Claire Nauman, and Yingda Xu. "High throughput detection of antibody self-interaction by bio-layer interferometry." mAbs 5, no. 6 (November 2013): 838–41. http://dx.doi.org/10.4161/mabs.26186.

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Petersen, Rejane. "Strategies Using Bio-Layer Interferometry Biosensor Technology for Vaccine Research and Development." Biosensors 7, no. 4 (October 31, 2017): 49. http://dx.doi.org/10.3390/bios7040049.

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Mostafa, Mahmoud, Nehal E. Elsadek, Sherif E. Emam, Hidenori Ando, Taro Shimizu, Hamdy Abdelkader, Yu Ishima, Usama Farghaly Aly, Hatem A. Sarhan, and Tatsuhiro Ishida. "Using Bio-Layer Interferometry to Evaluate Anti-PEG Antibody-Mediated Complement Activation." Biological and Pharmaceutical Bulletin 45, no. 1 (January 1, 2022): 129–35. http://dx.doi.org/10.1248/bpb.b21-00772.

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Wallner, Jakob, Gabriele Lhota, Dominik Jeschek, Alexander Mader, and Karola Vorauer-Uhl. "Application of Bio-Layer Interferometry for the analysis of protein/liposome interactions." Journal of Pharmaceutical and Biomedical Analysis 72 (January 2013): 150–54. http://dx.doi.org/10.1016/j.jpba.2012.10.008.

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Gut, Kazimierz. "Broadband differential interference in a waveguide with a gradient refractive index distribution." Photonics Letters of Poland 14, no. 3 (September 30, 2022): 53. http://dx.doi.org/10.4302/plp.v14i3.1157.

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The paper presents a model of a planar broadband differential waveguide interferometer with a gradient refractive index distribution. Its response to the change in the refractive index of the waveguide cover layer is presented. The analysis was performed for the wavelength range from 0.5um to 0.7um. The orthogonal TE0 and TM0 modes propagating in this wavelength range are considered. The influence of the coverage refractive index change on the output characteristics of the system is shown. Full Text: PDF ReferencesP. Kozma, F. Kehl, E.Ehrentreich-Forster, C. Stamm and F.F. Bier, "Integrated planar optical waveguide interferometer biosensors: A comparative review", Biosens. Bioelectron. 58, 287 (2014), CrossRef M. Kitsara, K. Misiakos, I. Raptis, and E. Makarona, "Integrated optical frequency-resolved Mach-Zehnder interferometers for label-free affinity sensing", Opt. Express 18, 8193 (2010). CrossRef K. Misiakos, I. Raptis, A. Salapatas, E. Makarona, A. Bostials, et al., "Broad-band Mach-Zehnder interferometers as high performance refractive index sensors: Theory and monolithic implementation", Opt. Express 22, 8856 (2014). CrossRef K. Misiakos, I. Raptis, E. Makarona, A. Botsialas, A. Salapatas, et al, "All-silicon monolithic Mach-Zehnder interferometer as a refractive index and bio-chemical sensor", Opt. Express 22, 26803 (2014) CrossRef K. Misiakos, E. Makarona, M. Hoekman, R. Fyrogenis, K. Tukkiniemi, et al., "All-Silicon Spectrally Resolved Interferometric Circuit for Multiplexed Diagnostics: A Monolithic Lab-on-a-Chip Integrating All Active and Passive Components", ACS Photonics 6, 1694 (2019). CrossRef E. Makarona, A. Salapatas, I. Raptis, P. Petrou, S. Kakabakos, et al., "Broadband Young interferometry for simultaneous dual polarization bioanalytics", J Opt Soc Am B 34, 1691 (2017). CrossRef K. Gut, "Broad-band difference interferometer as a refractive index sensor", Opt. Express 25, 3111 (2017), CrossRef K. Gut, "Study of a Broadband Difference Interferometer Based on Low-Cost Polymer Slab Waveguides", Nanomaterials 9, 729 (2019), CrossRef T. Pustelny, J. Ignac-Nowacka and Z. Opilski, "Optical investigations on layered metalphthalocyanine nanostructures affected by NO2 applying the surface plasmon resonance method", Opt. Appl. 34, 563 (2004). CrossRef W. Lukosz, Sensor Actuat. B-Chem. "Integrated optical chemical and direct biochemical sensors", 29, 37 (1995). CrossRef Z. Qi, S. Xia and N. Matsuda, "Spectropolarimetric interferometer based on single-mode glass waveguides", Opt. Express, 16, 2245 (2008). CrossRef K. Gut, A. Zakrzewski, T. Pustelny, "Sensitivity of Polarimetric Waveguide Interferometer for Different Wavelengths", Acta Phys. Pol. 118, 1140 (2010). CrossRef J.E. Broquin, S. Honkanen, "Integrated Photonics on Glass: A Review of the Ion-Exchange Technology Achievements", Appl.Sci. 11, 4472 (2021). CrossRef G.C. Righini, J. Linares, "Active and Quantum Integrated Photonic Elements by Ion Exchange in Glass", Appl.Sci. 11, 5222 (2021). CrossRef

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Groner, Myles, Taryn Ng, Weidong Wang, and Andrew K. Udit. "Bio-layer interferometry of a multivalent sulfated virus nanoparticle with heparin-like anticoagulant activity." Analytical and Bioanalytical Chemistry 407, no. 19 (May 10, 2015): 5843–47. http://dx.doi.org/10.1007/s00216-015-8735-x.

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Wu, Songhua, Weibiao Chen, Junwu Tang, Chaofang Zhao, and Ge Chen. "Lidar Concept of “Guanlan” Mission for Space Oceanography." EPJ Web of Conferences 237 (2020): 01012. http://dx.doi.org/10.1051/epjconf/202023701012.

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Among the various challenges in spaceborne radar observation of the ocean, the following two issues are probably of a higher priority: inadequate dynamic resolution, and ineffective vertical penetration. Two highly anticipated breakthroughs in the coming decade are likely to be associated with radar interferometry and ocean lidar technology, which are expected to make a substantial contribution to a sub-mesoscale-resolving and depth-resolving observation of the ocean. The planned “Guanlan” science mission comprises a dual-frequency (Ku and Ka) interferometric altimetry (IA) and a near-nadir pointing ocean lidar (OL). The spaceborne active OL will ensure a deeper penetration depth and an all-time detection which leads to a layered characterization of the optical properties of the subsurface ocean. The simultaneous functioning of the OL and a dual-frequency (Ku and Ka) interferometric altimetry system will allow an enhanced understanding of contributions of the atmosphere and the air-sea interface which in turn considerably reduce the error budgets of the two sensors. The OL payload is expected to partially reveal the marine food chain and ecosystem with 10-m vertical interval in the euphotic layer, moving a significant step down to the oceanic mixed layer both dynamically and bio-optically.

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Choy, Cindy J., and Clifford E. Berkman. "A method to determine the mode of binding for GCPII inhibitors using bio-layer interferometry." Journal of Enzyme Inhibition and Medicinal Chemistry 31, no. 6 (February 12, 2016): 1690–93. http://dx.doi.org/10.3109/14756366.2015.1132208.

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Lad, Latesh, Sheila Clancy, Maria Kovalenko, Chian Liu, Terence Hui, Victoria Smith, and Nikos Pagratis. "High-Throughput Kinetic Screening of Hybridomas to Identify High-Affinity Antibodies Using Bio-Layer Interferometry." Journal of Biomolecular Screening 20, no. 4 (November 25, 2014): 498–507. http://dx.doi.org/10.1177/1087057114560123.

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Kinetic analysis of antibodies is crucial in both clone selection and characterization. Historically, antibodies in supernatants from hybridomas are selected based on a solid-phase enzyme-linked immunosorbent assay (ELISA) in which the antigen is immobilized on the assay plate. ELISA selects clones based on a combination of antibody concentration in the supernatant and affinity. The antibody concentration in the supernatant can vary significantly and is typically unknown. Using the ELISA method, clones that express high levels of a low-affinity antibody can give an equivalent signal as clones that express low levels of a high-affinity antibody. As a consequence, using the ELISA method, superior clones can be overshadowed by inferior clones. In this study, we have applied Bio-Layer Interferometry to screen hybridoma clones based on disassociation rates using the OctetRED 384 platform. Using the OctetRED platform, we were able to screen 2000 clones within 24 hours and select clones containing high-affinity antibodies for further expansion and subsequent characterization. Using this method, we were able to identify several clones producing high-affinity antibodies that were missed by ELISA.

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Piesanen, Jaakko, Jarkko Valjakka, Sanna Niemelä, Marjut Borgenström, Seppo Nikkari, Vesa Hytönen, Juha Määttä, and Tarja Kunnas. "Hepcidin is potential regulator for renin activity." PLOS ONE 17, no. 4 (April 20, 2022): e0267343. http://dx.doi.org/10.1371/journal.pone.0267343.

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An association between genetic variants in the genes HFE, HJV, BMP4 and arterial hypertension has been shown earlier. Proteins encoded by these genes participate in the signalling routes leading eventually to the production of the peptide hormone hepcidin. Mutations in these genes have been associated with the abnormal production of hepcidin in the body. This finding led to studies exploring the possible role of hepcidin in regulating the activity of blood pressure related renin-angiotensin system enzymes. We used molecular modelling to find out if it is possible for hepcidin to bind to the active site of the renin-angiotensin system enzymes, especially renin. Fluorometric assays were used to evaluate the inhibitory effect of hepcidin on renin as well as angiotensin converting enzymes 1 and 2. Finally, bio-layer interferometry technique was used to study hepcidin binding to renin. The molecular modelling showed that hepcidin seems to have similar binding properties to the renin active site as angiotensinogen does. Based on fluorometric enzyme activity assay, hepcidin has an inhibitory effect on renin in vitro, too. However, angiotensin converting enzymes 1 and 2 were not inhibited remarkably by hepcidin-25. In bio-layer interferometry analysis hepcidin-renin binding was concentration dependent. Our results suggest that hepcidin could act as an inhibitor to the renin. Nowadays, there is no known biological inhibitor for renin in vivo and our finding may thus have important clinical implications.

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Miczi, Márió, Ádám Diós, Beáta Bozóki, József Tőzsér, and János András Mótyán. "Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model." Viruses 13, no. 6 (June 21, 2021): 1183. http://dx.doi.org/10.3390/v13061183.

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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.

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Kamat, Vishal, and Ashique Rafique. "Designing binding kinetic assay on the bio-layer interferometry (BLI) biosensor to characterize antibody-antigen interactions." Analytical Biochemistry 536 (November 2017): 16–31. http://dx.doi.org/10.1016/j.ab.2017.08.002.

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Dysinger, Mark, and Lindsay E. King. "Practical quantitative and kinetic applications of bio-layer interferometry for toxicokinetic analysis of a monoclonal antibody therapeutic." Journal of Immunological Methods 379, no. 1-2 (May 2012): 30–41. http://dx.doi.org/10.1016/j.jim.2012.02.017.

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Naik, Subhashchandra, Ozan S. Kumru, Melissa Cullom, Srivalli N. Telikepalli, Elizabeth Lindboe, Taylor L. Roop, Sangeeta B. Joshi, et al. "Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry." Protein Science 23, no. 10 (July 28, 2014): 1461–78. http://dx.doi.org/10.1002/pro.2515.

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Ullah, Sadia Fida, Geisianny Moreira, Shoumen Palit Austin Datta, Eric McLamore, and Diana Vanegas. "An Experimental Framework for Developing Point-of-Need Biosensors: Connecting Bio-Layer Interferometry and Electrochemical Impedance Spectroscopy." Biosensors 12, no. 11 (October 29, 2022): 938. http://dx.doi.org/10.3390/bios12110938.

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Biolayer interferometry (BLI) is a well-established laboratory technique for studying biomolecular interactions important for applications such as drug development. Currently, there are interesting opportunities for expanding the use of BLI in other fields, including the development of rapid diagnostic tools. To date, there are no detailed frameworks for implementing BLI in target-recognition studies that are pivotal for developing point-of-need biosensors. Here, we attempt to bridge these domains by providing a framework that connects output(s) of molecular interaction studies with key performance indicators used in the development of point-of-need biosensors. First, we briefly review the governing theory for protein-ligand interactions, and we then summarize the approach for real-time kinetic quantification using various techniques. The 2020 PRISMA guideline was used for all governing theory reviews and meta-analyses. Using the information from the meta-analysis, we introduce an experimental framework for connecting outcomes from BLI experiments (KD, kon, koff) with electrochemical (capacitive) biosensor design. As a first step in the development of a larger framework, we specifically focus on mapping BLI outcomes to five biosensor key performance indicators (sensitivity, selectivity, response time, hysteresis, operating range). The applicability of our framework was demonstrated in a study of case based on published literature related to SARS-CoV-2 spike protein to show the development of a capacitive biosensor based on truncated angiotensin-converting enzyme 2 (ACE2) as the receptor. The case study focuses on non-specific binding and selectivity as research goals. The proposed framework proved to be an important first step toward modeling/simulation efforts that map molecular interactions to sensor design.

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Gut, Kazimierz. "Model of the planar broadband differential waveguide interferometer as a humidity sensor." Photonics Letters of Poland 12, no. 2 (July 1, 2020): 55. http://dx.doi.org/10.4302/plp.v12i2.1022.

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The paper presents a model of the planar broadband differential waveguide interferometer. Its response to the change in thickness and refractive index of the waveguide layer due to the change in humidity is presented. The analysis was carried out for the wavelength range from 450 nm to 850 nm. The orthogonal modes TE0 and TM0, which propagate in this wavelength range, are considered. It is shown that by using light near the maximum of the system characteristic, instead of the spectrometer, the total power at the system output can be measured. Full Text: PDF ReferencesM. Kitsara, K. Misiakos, I. Raptis, and E. Makarona, "Integrated optical frequency-resolved Mach-Zehnder interferometers for label-free affinity sensing", Opt. Express 18, 8193 (2010). CrossRef K. Misiakos, I. Raptis, A. Salapatas, E. Makarona, A. Bostials, et al., "Broad-band Mach-Zehnder interferometers as high performance refractive index sensors: Theory and monolithic implementation", Opt. Express 22, 8856 (2014). CrossRef K. Misiakos, I. Raptis, E. Makarona, A. Botsialas, A. Salapatas, et al "All-silicon monolithic Mach-Zehnder interferometer as a refractive index and bio-chemical sensor", Opt. Express 22, 26803 (2014). CrossRef K. Misiakos, E. Makarona, M. Hoekman, R. Fyrogenis, K. Tukkiniemi, et al., "All-Silicon Spectrally Resolved Interferometric Circuit for Multiplexed Diagnostics: A Monolithic Lab-on-a-Chip Integrating All Active and Passive Components", ACS Photonics 6, 1694 (2019). CrossRef E. Makarona, A. Salapatas, I. Raptis, P. Petrou, S. Kakabakos, et al., "Broadband Young interferometry for simultaneous dual polarization bioanalytics", J Opt Soc Am B 34, 1691 (2017). CrossRef K. Gut, "Broad-band difference interferometer as a refractive index sensor", Opt. Express 25, 3111 (2017). CrossRef K. Gut, "Study of a Broadband Difference Interferometer Based on Low-Cost Polymer Slab Waveguides", Nanomaterials 9, 729 (2019). CrossRef W. Lukosz, "Integrated optical chemical and direct biochemical sensors", Sensor Actuat. B-Chem. 29, 37 (1995). CrossRef W. Knoll, O. Azzaroni, H. Duran, J. Kunze-Liebhäuser, K. Lau, et al. "Nanoporous thin films in optical waveguide spectroscopy for chemical analytics", Analytical and Bioanalytical Chemistry 412, 3299 (2020). CrossRef A. Bucciarellia, V. Mullonib, D. Maniglio, R.K. Pal, V.K. Yadavalli, at al., "A comparative study of the refractive index of silk protein thin films towards biomaterial based optical devices", Optical Materials 78, 407 (2018). CrossRef V.Prajzler, K. Min, S. Kim, and P. Nekvindova, "The Investigation of the Waveguiding Properties of Silk Fibroin from the Visible to Near-Infrared Spectrum", Materials 11, 112 (2018). CrossRef Q. Li, N. Qi, Y. Peng, Y. Zhange, L.Shi, et al. "Aggregation induced red shift emission of phosphorus doped carbon dots", RSC Advances 7, 178889 (2017). CrossRef P. Giovanni, Z. Yuji, N. Deboki, P. Nereus, D. Kaplan, et al. "The optical properties of regenerated silk fibroin films obtained from different sources", App. Phys. Lett. 111, 103702 (2017). CrossRef M. Procek, Z. Opilski, A. M. Maquenda, X.M. Berbel, S.Aznar-Cervantes et al., "Silk fibroin thin films for optical humidity sensing", Proceedings of SPIE 11204,1120409 (2019). CrossRef https://www.thorlabs.com/thorproduct.cfm?partnumber=M595F2 DirectLink

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Shaw, Andrew E., Alison Burman, Amin Asfor, Emiliana Brocchi, Santina Grazioli, Clare Browning, Anna Ludi, Tobias J. Tuthill, and Donald P. King. "Avidity of Polyclonal Antibodies to Foot-and-Mouth Disease Virus in Bovine Serum Measured Using Bio-Layer Interferometry." Viruses 14, no. 4 (March 29, 2022): 714. http://dx.doi.org/10.3390/v14040714.

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Foot-and-mouth disease (FMD) is a disease of cloven-hoofed livestock caused by FMD virus (FMDV). FMD can be controlled through the use of inactivated vaccines, and it is well established that the protection afforded by FMD vaccines correlates strongly with neutralising antibody titres. However, the overall strength of binding, referred to as avidity, is also an important parameter with respect to the ability of antibodies to neutralise virus infection, and there is evidence that avidity can affect the level of protection afforded by FMDV vaccines. Here, as an alternative to modified enzyme-linked immunosorbent assays (avidity ELISAs) incorporating a chaotropic wash step, we used bio-layer interferometry (BLI) to measure the avidity of bovine polyclonal antibodies against FMDV capsids. We conducted preliminary experiments using recombinant FMDV capsids, as well as peptides representing antigenic loops, to demonstrate that the binding of monoclonal antibodies targeting specific antigenic sites could be detected using BLI. Subsequent experiments using polyclonal sera derived from FMD vaccinated cattle provided evidence of a positive correlation between the neutralising titre of the serum and the avidity as measured by BLI. Furthermore, we observed an increase in BLI avidity, as well as in the titre, in vaccinated animals upon challenge with the live virus.

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Wallner, Jakob, Manfred Kühleitner, Norbert Brunner, Gabriele Lhota, and Karola Vorauer-Uhl. "Application of the log-normal model for long term high affinity antibody/antigen interactions using Bio-Layer Interferometry." Journal of Mathematical Chemistry 52, no. 2 (December 6, 2013): 575–87. http://dx.doi.org/10.1007/s10910-013-0278-9.

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Levina, Aviva, and Peter A. Lay. "Influence of an anti-metastatic ruthenium(iii) prodrug on extracellular protein–protein interactions: studies by bio-layer interferometry." Inorg. Chem. Front. 1, no. 1 (2014): 44–48. http://dx.doi.org/10.1039/c3qi00054k.

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Brenac Brochier, Virginie, and Vincent Ravault. "High throughput development of a non protein A monoclonal antibody purification process using mini-columns and bio-layer interferometry." Engineering in Life Sciences 16, no. 2 (October 20, 2015): 152–59. http://dx.doi.org/10.1002/elsc.201400244.

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Maffei, Mariano, Linda Celeste Montemiglio, Grazia Vitagliano, Luigi Fedele, Shaila Sellathurai, Federica Bucci, Mirco Compagnone, et al. "The Nuts and Bolts of SARS-CoV-2 Spike Receptor-Binding Domain Heterologous Expression." Biomolecules 11, no. 12 (December 2, 2021): 1812. http://dx.doi.org/10.3390/biom11121812.

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COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful “tool” to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.

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Yun, Xiaoyun, Yuhan Xie, Jerome P. L. Ng, Betty Yuen Kwan Law, Vincent Kam Wai Wong, and Paolo Coghi. "2-Bromo-3-((1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)-methoxy)-benzaldehyde." Molbank 2022, no. 1 (March 9, 2022): M1351. http://dx.doi.org/10.3390/m1351.

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The 1,2,3-triazole ring system can be easily obtained by copper-catalyzed click reaction of azides with alkynes. 1,2,3-Triazole exhibits a myriad of biological activities, including antimalarial, antibacterial, and antiviral activities. We herein reported the synthesis of quinoline-based [1,2,3]-triazole hybrid via Cu(I)-catalyzed click reaction of 4-azido-7-chloroquinoline with alkyne derivative of 2-bromobenzaldehyde. The compound was fully characterized by proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), heteronuclear single quantum coherence (HSQC), ultraviolet (UV), and high-resolution mass spectroscopies (HRMS). This compound was screened in vitro against two different normal cell lines. Preliminary studies attempted to evaluate its interaction with Delta RBD of spike protein of SARS-CoV-2 by bio-layer interferometry. Finally, the drug-likeness of the compound was also investigated by predicting its pharmacokinetic properties.

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Volkova, M. V., V. V. Boyarintsev, A. V. Trofimenko, S. A. Biryukov, E. V. Gorina, G. I. Filkov, and M. O. Durymanov. "Adaptation of Bio-Layer Interferometry for Quantitative Assessment of the Vascular Endothelial Growth Factor Content in Cell-Conditioned Culture Medium." Biophysics 65, no. 6 (November 2020): 935–41. http://dx.doi.org/10.1134/s0006350920060226.

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Zhang, Dingqi, Sami Hamdoun, Ruihong Chen, Lijun Yang, Chi Kio Ip, Yuanqing Qu, Runfeng Li, et al. "Identification of natural compounds as SARS-CoV-2 entry inhibitors by molecular docking-based virtual screening with bio-layer interferometry." Pharmacological Research 172 (October 2021): 105820. http://dx.doi.org/10.1016/j.phrs.2021.105820.

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Wallner, Jakob, Gabriele Lhota, Markus Schosserer, and Karola Vorauer-Uhl. "An approach for liposome immobilization using sterically stabilized micelles (SSMs) as a precursor for bio-layer interferometry-based interaction studies." Colloids and Surfaces B: Biointerfaces 154 (June 2017): 186–94. http://dx.doi.org/10.1016/j.colsurfb.2017.03.015.

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Singh, Chingakham Ranjit, Rahul Jaiswal, Carlos R. Escalante, and Katsura Asano. "Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein." STAR Protocols 3, no. 3 (September 2022): 101615. http://dx.doi.org/10.1016/j.xpro.2022.101615.

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Domnowski, M., B. Hackner, T. Neuber, J. Jaehrling, and W. Frieß. "Analysis of antibody self-interaction by bio-layer interferometry as tool to support lead candidate selection during preformulation and developability assessments." International Journal of Pharmaceutics 589 (November 2020): 119854. http://dx.doi.org/10.1016/j.ijpharm.2020.119854.

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Shang, Jiaqi, Minhe Liao, Ritian Jin, Xiangyu Teng, Hao Li, Yan Xu, Ligang Zhang, and Ning Liu. "Molecular Properties of Flammulina velutipes Polysaccharide–Whey Protein Isolate (WPI) Complexes via Noncovalent Interactions." Foods 10, no. 1 (December 22, 2020): 1. http://dx.doi.org/10.3390/foods10010001.

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Whey protein isolate (WPI) has a variety of nutritional benefits. The stability of WPI beverages has attracted a large amount of attention. In this study, Flammulina velutipes polysaccharides (FVPs) interacted with WPI to improve the stability via noncovalent interactions. Multiple light scattering studies showed that FVPs can improve the stability of WPI solutions, with results of radical scavenging activity assays demonstrating that the solutions of the complex had antioxidant activity. The addition of FVPs significantly altered the secondary structures of WPI, including its α-helix and random coil. The results of bio-layer interferometry (BLI) analysis indicated that FVPs interacted with the WPI, and the equilibrium dissociation constant (KD) was calculated as 1.736 × 10−4 M in this study. The in vitro digestibility studies showed that the FVPs protected WPI from pepsin digestion, increasing the satiety. Therefore, FVPs effectively interact with WPI through noncovalent interactions and improve the stability of WPI, with this method expected to be used in protein-enriched and functional beverages.

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Berthiol, Florian, Joseph Boissieras, Hugues Bonnet, Marie Pierrot, Christian Philouze, Jean-François Poisson, Anton Granzhan, Jérôme Dejeu, and Eric Defrancq. "Novel Synthesis of IMC-48 and Affinity Evaluation with Different i-Motif DNA Sequences." Molecules 28, no. 2 (January 10, 2023): 682. http://dx.doi.org/10.3390/molecules28020682.

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During the last decade, the evidence for the biological relevance of i-motif DNA (i-DNA) has been accumulated. However, relatively few molecules were reported to interact with i-DNA, and a controversy concerning their binding mode, affinity, and selectivity persists in the literature. In this context, the cholestane derivative IMC-48 has been reported to modulate bcl-2 gene expression by stabilizing an i-motif structure in its promoter. In the present contribution, we report on a novel, more straightforward, synthesis of IMC-48 requiring fewer steps compared to the previous approach. Furthermore, the interaction of IMC-48 with four different i-motif DNA sequences was thoroughly investigated by bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy. Surprisingly, our results show that IMC-48 is a very weak ligand of i-DNA as no quantifiable interaction or significant stabilization of i-motif structures could be observed, stimulating a quest for an alternative mechanism of its biological activity.

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Coghi, Paolo, Li Jun Yang, Jerome P. L. Ng, Richard K. Haynes, Maurizio Memo, Alessandra Gianoncelli, Vincent Kam Wai Wong, and Giovanni Ribaudo. "A Drug Repurposing Approach for Antimalarials Interfering with SARS-CoV-2 Spike Protein Receptor Binding Domain (RBD) and Human Angiotensin-Converting Enzyme 2 (ACE2)." Pharmaceuticals 14, no. 10 (September 23, 2021): 954. http://dx.doi.org/10.3390/ph14100954.

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Host cell invasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by the interaction of the viral spike protein (S) with human angiotensin-converting enzyme 2 (ACE2) through the receptor-binding domain (RBD). In this work, computational and experimental techniques were combined to screen antimalarial compounds from different chemical classes, with the aim of identifying small molecules interfering with the RBD-ACE2 interaction and, consequently, with cell invasion. Docking studies showed that the compounds interfere with the same region of the RBD, but different interaction patterns were noted for ACE2. Virtual screening indicated pyronaridine as the most promising RBD and ACE2 ligand, and molecular dynamics simulations confirmed the stability of the predicted complex with the RBD. Bio-layer interferometry showed that artemisone and methylene blue have a strong binding affinity for RBD (KD = 0.363 and 0.226 μM). Pyronaridine also binds RBD and ACE2 in vitro (KD = 56.8 and 51.3 μM). Overall, these three compounds inhibit the binding of RBD to ACE2 in the μM range, supporting the in silico data.

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Sivaccumar, Jwala Priyadarsini, Antonio Leonardi, Emanuela Iaccarino, Giusy Corvino, Luca Sanguigno, Angela Chambery, Rosita Russo, et al. "Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry." International Journal of Molecular Sciences 22, no. 6 (March 20, 2021): 3166. http://dx.doi.org/10.3390/ijms22063166.

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Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

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Su, Mei-Tzu, Masanori Inui, Yi Li Wong, Maika Takahashi, Akiko Sugahara-Tobinai, Karin Ono, Shotaro Miyamoto, et al. "Blockade of checkpoint ILT3/LILRB4/gp49B binding to fibronectin ameliorates autoimmune disease in BXSB/Yaa mice." International Immunology 33, no. 8 (June 5, 2021): 447–58. http://dx.doi.org/10.1093/intimm/dxab028.

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Abstract The extracellular matrix (ECM) is the basis for virtually all cellular processes and is also related to tumor metastasis. Fibronectin (FN), a major ECM macromolecule expressed by different cell types and also present in plasma, consists of multiple functional modules that bind to ECM-associated, plasma, and cell-surface proteins such as integrins and FN itself, thus ensuring its cell-adhesive and modulatory role. Here we show that FN constitutes an immune checkpoint. Thus, FN was identified as a physiological ligand for a tumor/leukemia/lymphoma- as well as autoimmune-associated checkpoint, ILT3/LILRB4 (B4, CD85k). Human B4 and the murine ortholog, gp49B, bound FN with sub-micromolar affinities as assessed by bio-layer interferometry. The major B4-binding site in FN was located at the N-terminal 30-kDa module (FN30), which is apart from the major integrin-binding site present at the middle of the molecule. Blockade of B4–FN binding such as with B4 antibodies or a recombinant FN30-Fc fusion protein paradoxically ameliorated autoimmune disease in lupus-prone BXSB/Yaa mice. The unexpected nature of the B4–FN checkpoint in autoimmunity is discussed, referring to its potential role in tumor immunity.

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Yao, Xuejing, Guiping Qi, Yaocheng Qu, Shasha Yun, Wenlong Sun, Chungang Liang, Mupeng Du, and Zhuanglin Li. "Structural Characterization of RC28-E, a Recombinant Fusion Protein With Dual Targets on VEGF and FGF2." Natural Product Communications 17, no. 3 (March 2022): 1934578X2210869. http://dx.doi.org/10.1177/1934578x221086989.

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Vascular endothelial growth factor (VEGF) and fibroblast growthfactor (FGF) play important roles in angiogenesis-related diseases. RC28-E is a soluble fusion protein composed of the human VEGF receptor 1 (VEGFR1) extracellular domain 2 (ECD 2), VEGFR2 ECD 3, FGFR1 ECDs 2 and 3, and the Fc regions of human immunoglobulin G1. By targeting both VEGF and FGF2, RC28-E may represent a useful antiangiogenetic agent, but structural and functional characterizations of this fusion protein are needed. Liquid chromatography–tandem mass spectrometry, size exclusion high-performance liquid chromatography, capillary electrophoresis-sodium dodecyl sulfate, imaged capillary isoelectric focusing, and bio-layer interferometry were used to characterize the properties of RC28-E. The purity of RC28-E was confirmed to be 98% or greater. The glycosylation modification of RC28-E was found to be very complicated, with 11 potential N-linked glycosylation points and 23 types of N-glycans, causing high heterogeneity of the protein. The primary modifications of the amino acid sequence of RC28-E protein included C-terminal K truncation, N-deamidation, and M-oxidation modification. Notably, RC28-E demonstrated a higher affinity for both VEGF and FGF2 than VEGF trap or FGF trap for their respective targets.

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Ling, Wei-Li, Joshua Yi Yeo, Yuen-Ling Ng, Anil Wipat, and Samuel Ken-En Gan. "More Than Meets the Kappa for Antibody Superantigen Protein L (PpL)." Antibodies 11, no. 1 (February 11, 2022): 14. http://dx.doi.org/10.3390/antib11010014.

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Immunoglobulin superantigens play an important role in affinity purification of antibodies and the microbiota-immune axis at mucosal areas. Based on current understanding, Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG) and Finegoldia Protein L (PpL) are thought to only bind specific regions of human antibodies, allowing for selective purification of antibody isotypes and chains. Clinically, these superantigens are often classified as toxins and increase the virulence of the producing pathogen through unspecific interactions with immune proteins. To perform an in-depth interaction study of these three superantigens with antibodies, bio-layer interferometry (BLI) measurements of their interactions with a permutation panel of 63 IgG1 variants of Pertuzumab and Trastuzumab CDRs grafted to the six human Vκ and seven human VH region families were tested. Through this holistic and systemic analysis of IgG1 variants with various antibody regions modified, comparisons revealed novel PpL–antibody interactions influenced by other non-canonical antibody known light-chain framework regions, whereas SpA and SpG showed relatively consistent interactions. These findings have implications on PpL-based affinity antibody purification and design that can guide the engineering and understanding of PpL-based microbiota-immune effects.

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Li, Mengjia, Yongzheng Zhang, Amir Zeb, Yang Wu, and Lufeng Cheng. "Bergamottin and PAP-1 Induced ACE2 Degradation to Alleviate Infection of SARS-CoV-2." International Journal of Molecular Sciences 23, no. 20 (October 19, 2022): 12565. http://dx.doi.org/10.3390/ijms232012565.

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Angiotensin-converting enzyme 2 (ACE2), a functional receptor for SARS-CoV, now appears likely to mediate 2019-nCoV entry into human cells. However, inhibitors such as PAP-1 and bergamottin have been discovered; both of them can preferentially bind to ACE2, prevent RBD Spike S protein from binding to ACE2, and reduce the binding sites for RBD Spike S protein. In addition, we investigated the binding energy of PAP-1 and bergamottin with ACE2 through molecular docking with bio-layer interferometry (BLI) and found relatively high binding affinity (KD = 48.5 nM, 53.1 nM) between the PAP-1 and bergamottin groups. In addition, the nanomolar fraction had no effect on growth of the AT-II cell, but 150 µM PAP-1 and 75 µM bergamottin inhibited the proliferation of AT-II cells in vitro by 75% and 68%, respectively. Meanwhile, they significantly reduced ACE2 mRNA and proteins by 67%, 58% and 55%, 41%, respectively. These results indicate that psoralen compounds PAP-1 and bergamottin binding to ACE2 protein could be further developed in the fight against COVID-19 infection during the current pandemic. However, attention should be paid to the damage to human alveolar type II epithelial cells.

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Vallet, Sylvain D., Coline Berthollier, Romain Salza, Laurent Muller, and Sylvie Ricard-Blum. "The Interactome of Cancer-Related Lysyl Oxidase and Lysyl Oxidase-Like Proteins." Cancers 13, no. 1 (December 29, 2020): 71. http://dx.doi.org/10.3390/cancers13010071.

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The members of the lysyl oxidase (LOX) family are amine oxidases, which initiate the covalent cross-linking of the extracellular matrix (ECM), regulate ECM stiffness, and contribute to cancer progression. The aim of this study was to build the first draft of the interactome of the five members of the LOX family in order to determine its molecular functions, the biological and signaling pathways mediating these functions, the biological processes it is involved in, and if and how it is rewired in cancer. In vitro binding assays, based on surface plasmon resonance and bio-layer interferometry, combined with queries of interaction databases and interaction datasets, were used to retrieve interaction data. The interactome was then analyzed using computational tools. We identified 31 new interactions and 14 new partners of LOXL2, including the α5β1 integrin, and built an interactome comprising 320 proteins, 5 glycosaminoglycans, and 399 interactions. This network participates in ECM organization, degradation and cross-linking, cell-ECM interactions mediated by non-integrin and integrin receptors, protein folding and chaperone activity, organ and blood vessel development, cellular response to stress, and signal transduction. We showed that this network is rewired in colorectal carcinoma, leading to a switch from ECM organization to protein folding and chaperone activity.

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39

Zheng, Yun-Xiao, Lei Wang, Wei-Sha Kong, Hong Chen, Xue-Ning Wang, Qingfeng Meng, Hai-Nan Zhang, Shu-Juan Guo, He-Wei Jiang, and Sheng-Ce Tao. "Nsp2 has the potential to be a drug target revealed by global identification of SARS-CoV-2 Nsp2-interacting proteins." Acta Biochimica et Biophysica Sinica 53, no. 9 (January 9, 2021): 1134–41. http://dx.doi.org/10.1093/abbs/gmab088.

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Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health threat since December 2019, and there is still no highly effective drug to control the pandemic. To facilitate drug target identification for drug development, studies on molecular mechanisms, such as SARS-CoV-2 protein interactions, are urgently needed. In this study, we focused on Nsp2, a non-structural protein with largely unknown function and mechanism. The interactome of Nsp2 was revealed through the combination of affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), and 84 proteins of high-confidence were identified. Gene ontology analysis demonstrated that Nsp2-interacting proteins are involved in several biological processes such as endosome transport and translation. Network analysis generated two clusters, including ribosome assembly and vesicular transport. Bio-layer interferometry (BLI) assay confirmed the bindings between Nsp2- and 4-interacting proteins, i.e. STAU2 (Staufen2), HNRNPLL, ATP6V1B2, and RAP1GDS1 (SmgGDS), which were randomly selected from the list of 84 proteins. Our findings provide insights into the Nsp2–host interplay and indicate that Nsp2 may play important roles in SARS-CoV-2 infection and serve as a potential drug target for anti-SARS-CoV-2 drug development.

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Goulet, Dennis R., Soumili Chatterjee, Wai-Ping Lee, Andrew B. Waight, Yi Zhu, and Amanda Nga-Sze Mak. "Engineering an Enhanced EGFR Engager: Humanization of Cetuximab for Improved Developability." Antibodies 11, no. 1 (January 13, 2022): 6. http://dx.doi.org/10.3390/antib11010006.

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The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase whose proliferative effects can contribute to the development of many types of solid tumors when overexpressed. For this reason, EGFR inhibitors such as cetuximab can play an important role in treating cancers such as colorectal cancer and head and neck cancer. Cetuximab is a chimeric monoclonal antibody containing mouse variable regions that bind to EGFR and prevent it from signaling. Although cetuximab has been used clinically since 2004 to successfully control solid tumors, advances in protein engineering have created the opportunity to address some of its shortcomings. In particular, the presence of mouse sequences could contribute to immunogenicity in the form of anti-cetuximab antibodies, and an occupied glycosylation site in FR3 can contribute to hypersensitivity reactions and product heterogeneity. Using simple framework graft or sequence-/structure-guided approaches, cetuximab was humanized onto 11 new frameworks. In addition to increasing humanness and removing the VH glycosylation site, dynamic light scattering revealed increases in stability, and bio-layer interferometry confirmed minimal changes in binding affinity, with patterns emerging across the humanization method. This work demonstrates the potential to improve the biophysical and clinical properties of first-generation protein therapeutics and highlights the advantages of computationally guided engineering.

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Diez, Lisa, Larisa E. Kapinos, Janine Hochmair, Sabrina Huebschmann, Alvaro Dominguez-Baquero, Amelie Vogt, Marija Rankovic, Markus Zweckstetter, Roderick Y. H. Lim, and Susanne Wegmann. "Phosphorylation but Not Oligomerization Drives the Accumulation of Tau with Nucleoporin Nup98." International Journal of Molecular Sciences 23, no. 7 (March 23, 2022): 3495. http://dx.doi.org/10.3390/ijms23073495.

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Tau is a neuronal protein that stabilizes axonal microtubules (MTs) in the central nervous system. In Alzheimer’s disease (AD) and other tauopathies, phosphorylated Tau accumulates in intracellular aggregates, a pathological hallmark of these diseases. However, the chronological order of pathological changes in Tau prior to its cytosolic aggregation remains unresolved. These include its phosphorylation and detachment from MTs, mislocalization into the somatodendritic compartment, and oligomerization in the cytosol. Recently, we showed that Tau can interact with phenylalanine-glycine (FG)-rich nucleoporins (Nups), including Nup98, that form a diffusion barrier inside nuclear pore complexes (NPCs), leading to defects in nucleocytoplasmic transport. Here, we used surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to investigate the molecular details of Tau:Nup98 interactions and determined how Tau phosphorylation and oligomerization impact the interactions. Importantly, phosphorylation, but not acetylation, strongly facilitates the accumulation of Tau with Nup98. Oligomerization, however, seems to inhibit Tau:Nup98 interactions, suggesting that Tau-FG Nup interactions occur prior to oligomerization. Overall, these results provide fundamental insights into the molecular mechanisms of Tau-FG Nup interactions within NPCs, which might explain how stress-and disease-associated posttranslational modifications (PTMs) may lead to Tau-induced nucleocytoplasmic transport (NCT) failure. Intervention strategies that could rescue Tau-induced NCT failure in AD and tauopathies will be further discussed.

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Wang, Jingjing, Qunfang Weng, Fei Yin, and Qiongbo Hu. "Interactions of Destruxin A with Silkworms’ Arginine tRNA Synthetase and Lamin-C Proteins." Toxins 12, no. 2 (February 22, 2020): 137. http://dx.doi.org/10.3390/toxins12020137.

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Destruxin A (DA), a cyclodepsipeptidic mycotoxin produced by entomopathogenic fungus Metarhizium anisopliae, has good insecticidal activity and potential to be a new pesticide. However, the mechanism of action is still obscure. Our previous experiments showed that DA was involved in regulation of transcription and protein synthesis and suggested that silkworms’ arginine tRNA synthetase (BmArgRS), Lamin-C Proteins (BmLamin-C) and ATP-dependent RNA helicase PRP1 (BmPRP1) were candidates of DA-binding proteins. In this study, we employed bio-layer interferometry (BLI), circular dichroism (CD), cellular thermal shift assay (CETSA), and other technologies to verify the interaction of DA with above three proteins in vitro and in vivo. The results of BLI indicated that BmArgRS and BmLamin-C were binding-protein of DA with KD value 5.53 × 10−5 and 8.64 × 10−5 M, but not BmPRP1. These interactions were also verified by CD and CETSA tests. In addition, docking model and mutants assay in vitro showed that BmArgRS interacts with DA at the pocket including Lys228, His231, Asp434 and Gln437 in its enzyme active catalysis region, while BmLamin-C binds to DA at His524 and Lys528 in the tail domain. This study might provide new insight and evidence in illustrating molecular mechanism of DA in breaking insect.

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Yin, Xuyu, Haitao Peng, Qunfang Weng, Qiongbo Hu, and Jingjing Wang. "Interaction of Destruxin A with Three Silkworm Proteins: BmCRT, BmDPP3, and BmPDIA5." Molecules 27, no. 22 (November 9, 2022): 7713. http://dx.doi.org/10.3390/molecules27227713.

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Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, has insecticidal activity, but its molecular mechanism of action is still not clear. Three proteins with modification-related functions, calreticulin (BmCRT), dipeptidyl peptidase Ⅲ (BmDPP3), and protein disulfide isomerase A5 (BmPDIA5), were selected to verify the interactions with DA in this study. The kinetic data of the interactions were measured by surface plasmon resonance (SPR) and bio-layer interferometry (BLI) in vitro. The KD values of DA with BmCRT, BmDPP3, and BmPDIA5 ranged from 10−4 to 10−5 mol/L, which suggested that the three proteins all had fairly strong interactions with DA. Then, it was found that DA in a dose-dependent manner affected the interactions of the three proteins with their partners in insect two-hybrid tests in SF-9 cells. Furthermore, the results of enzyme activities by ELISA indicated that DA could inhibit the activity of BmDPP3 but had no significant effect on BmPDIA5. In addition, DA induced the upregulation of BmDPP3 and the downregulation of BmCRT. The results prove that BmCRT, BmDPP3, and BmPDIA5 are all binding proteins of DA. This study might provide new insights to elucidate the molecular mechanism of DA.

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Zhao, Linfei, Mingliang Chen, Xiaona Wang, Shoukai Kang, Weiwei Xue, and Zengpeng Li. "Identification of Anti-TNFα VNAR Single Domain Antibodies from Whitespotted Bambooshark (Chiloscyllium plagiosum)." Marine Drugs 20, no. 5 (April 29, 2022): 307. http://dx.doi.org/10.3390/md20050307.

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Tumor necrosis factor α (TNFα), an important clinical testing factor and drug target, can trigger serious autoimmune diseases and inflammation. Thus, the TNFα antibodies have great potential application in diagnostics and therapy fields. The variable binding domain of IgNAR (VNAR), the shark single domain antibody, has some excellent advantages in terms of size, solubility, and thermal and chemical stability, making them an ideal alternative to conventional antibodies. This study aims to obtain VNARs that are specific for mouse TNF (mTNF) from whitespotted bamboosharks. After immunization of whitespotted bamboosharks, the peripheral blood leukocytes (PBLs) were isolated from the sharks, then the VNAR phage display library was constructed. Through phage display panning against mTNFα, positive clones were validated through ELISA assay. The affinity of the VNAR and mTNFα was measured using ELISA and Bio-Layer Interferometry. The binding affinity of 3B11 VNAR reached 16.7 nM. Interestingly, one new type of VNAR targeting mTNF was identified that does not belong to any known VNAR type. To understand the binding mechanism of VNARs to mTNFα, the models of VNARs-mTNFα complexes were predicted by computational modeling combining HawkDock and RosettaDock. Our results showed that four VNARs’ epitopes overlapped in part with that of mTNFR. Furthermore, the ELISA assay shows that the 3B11 potently inhibited mTNFα binding to mTNFR. This study may provide the basis for the TNFα blockers and diagnostics applications.

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Li, Lin, Ran An, and Xingguo Liang. "Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis." Molecules 27, no. 12 (June 9, 2022): 3706. http://dx.doi.org/10.3390/molecules27123706.

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The binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) with an ssDNA loop is produced. The circular ssDNAs are prepared by the hybridization of two ssDNA fragments to form two nicks, followed by nick sealing with T4 DNA ligase. No splint (a scaffold DNA for circularizing ssDNA) is required, and no polymeric byproducts are produced. The ssDNA loop on the LR-chimera can be used to attach it with other molecules by hybridization with another ssDNA. The gel shift binding assay with Z-DNA specific binding antibody (Z22) or Z-DNA binding protein 1 (ZBP1) shows that stable Z-DNA can form under physiological ionic conditions even when the extra ssDNA part is present. Concretely, a 5′-terminal biotin-modified DNA oligonucleotide complementary to the ssDNA loop on the LR-chimera is used to attach it on the surface of a biosensor inlaid with streptavidin molecules, and the binding constant of ZBP1 with Z-DNA is analyzed by BLI (bio-layer interferometry). This approach is convenient for quantitatively analyzing the binding dynamics of Z-DNA with other molecules.

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Yin, Fei, Miaomiao Xiao, Alexander Berestetskiy, and Qiongbo Hu. "The Metarhizium anisopliae Toxin, Destruxin A, Interacts with the SEC23A and TEME214 Proteins of Bombyx mori." Journal of Fungi 7, no. 6 (June 8, 2021): 460. http://dx.doi.org/10.3390/jof7060460.

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Destruxin A (DA), a mycotoxin isolated from the entomopathogenic fungus Metarhizium anisopliae, has good insecticidal and immune-inhibitory activity, but the action mechanism has not yet been elucidated. In order to identify the DA-binding proteins, we conducted drug affinity responsive target stability (DARTS) experiments, which indicated that the silkworm’s (Bombyx mori) transmembrane protein 214 (BmTEME214) and protein transport protein SEC23A isoform X2 (BmSEC23) are the potential DA-binding proteins. The current research was focused on validation of the interaction between DA and these two proteins via bio-layer interferometry (BLI) in vitro, insect two-hybrid (I2H) in Sf9 cells, and RNAi in the insect. The results of the BLI tests showed that DA has strong affinity to bind BmTEME214 and BmSEC23 proteins with a KD value of 0.286 and 0.291 µM, respectively. In the I2H experiments, DA inhibited (at 0.02 µg/mL) and activated (at 0.002–0.0002 µg/mL) the protein interactions of BmSEC23–BmSEC13, but it only inhibited the BmTMEM214–BmSEC13L interaction. Furthermore, in the RNAi tests, an apparent increase in the silkworm’s mortality was recorded in the joint treatment of DA with dsBmSEC23 or dsBmTMEM214 when compared with the single treatment of DA (1.5 µg/g body), 40 µg/g body dsBmSEC23, or dsBmTMEM214. This research confirmed that BmSEC23 and BmTMEM214 are the DA-binding proteins and provided new insights to understand the action mechanism of DA.

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Teodori, Laura, Marjan Omer, Anders Märcher, Mads K. Skaanning, Veronica L. Andersen, Jesper S. Nielsen, Emil Oldenburg, Yuchen Lin, Kurt V. Gothelf, and Jørgen Kjems. "Site-specific nanobody-oligonucleotide conjugation for super-resolution imaging." Journal of Biological Methods 9, no. 1 (March 1, 2022): e159. http://dx.doi.org/10.14440/jbm.2022.381.

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Camelid single-domain antibody fragments, also called nanobodies, constitute a class of binders that are small in size (~15 kDa) and possess antigen-binding properties similar to their antibody counterparts. Facile production of recombinant nanobodies in several microorganisms has made this class of binders attractive within the field of molecular imaging. Particularly, their use in super-resolution microscopy has improved the spatial resolution of molecular targets due to a smaller linkage error. In single-molecule localization microscopy techniques, the effective spatial resolution can be further enhanced by site-specific fluorescent labeling of nanobodies owing to a more homogeneous protein-to-fluorophore stoichiometry, reduced background staining and a known distance between dye and epitope. Here, we present a protocol for site-specific bioconjugation of DNA oligonucleotides to three distinct nanobodies expressed with an N- or C-terminal unnatural amino acid, 4-azido-L-phenylalanine (pAzF). Using copper-free click chemistry, the nanobody-oligonucleotide conjugation reactions were efficient and yielded highly pure bioconjugates. Target binding was retained in the bioconjugates, as demonstrated by bio-layer interferometry binding assays and the super-resolution microscopy technique, DNA points accumulation for imaging in nanoscale topography (PAINT). This method for site-specific protein-oligonucleotide conjugation can be further extended for applications within drug delivery and molecular targeting where site-specificity and stoichiometric control are required.

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Razai, Amir S., Brendan P. Eckelman, and Guy S. Salvesen. "Selective inhibition of matrix metalloproteinase 10 (MMP10) with a single-domain antibody." Journal of Biological Chemistry 295, no. 8 (January 17, 2020): 2464–72. http://dx.doi.org/10.1074/jbc.ra119.011712.

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Since their discovery, the matrix metalloproteinase (MMP) family proteases have been considered as therapeutic targets in numerous diseases and disorders. Unfortunately, clinical trials with MMP inhibitors have failed to yield any clinical benefits of these inhibitors. These failures were largely due to a lack of MMP-selective agents; accordingly, it has become important to identify a platform with which high selectivity can be achieved. To this end, we propose using MMP-targeting antibodies that can achieve high specificity in interactions with their targets. Using a scaffold of single-domain antibodies, here we raised a panel of MMP10-selective antibodies through immunization of llamas, a member of the camelid family, whose members generate conventional heavy/light-chain antibodies and also smaller antibodies lacking light-chain and CH1 domains. We report the generation of a highly selective and tightly binding MMP10 inhibitor (Ki < 2 nm). Using bio-layer interferometry–based binding assays, we found that this antibody interacts with the MMP10 active site. Activity assays demonstrated that the antibody selectively inhibits MMP10 over its closest relative, MMP3. The ability of a single-domain antibody to discriminate between the most conserved MMP pair via an active site–directed mechanism of inhibition reported here supports the potential of this antibody as a broadly applicable scaffold for the development of selective, tightly binding MMP inhibitors.

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Wang, Jingjing, Qunfang Weng, and Qiongbo Hu. "Effects of Destruxin A on Silkworm’s Immunophilins." Toxins 11, no. 6 (June 18, 2019): 349. http://dx.doi.org/10.3390/toxins11060349.

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Destruxin A (DA), a major secondary metabolite of Metarhizium anisopliae, has anti-immunity to insects. However, the detailed mechanism and its interactions with target proteins are elusive. Previously, three immunophilins, peptidyl–prolyl cis–trans isomerase (BmPPI), FK506 binding-protein 45 (BmFKBP45) and BmFKBP59 homologue, were isolated from the silkworm, Bombyx mori Bm12 cell line following treatment with DA, which suggested that these proteins were possible DA-binding proteins. To validate the interaction between DA and the three immunophilins, we performed bio-layer interferometry (BLI) assay, and the results showed that DA has interaction with BmPPI, whose affinity constant value is 1.98 × 10−3 M and which has no affinity with FKBP45 and FKBP59 homologue in vitro. Furthermore, we investigated the affinity between DA and human PPI protein (HsPPIA) and the affinity constant (KD) value is 2.22 × 10−3 M. Additionally, we compared the effects of silkworm and human PPI proteins produced by DA and immunosuppressants, cyclosporine A (CsA), and tacrolimus (FK506), by employing I2H (insect two-hybrid) in the SF-9 cell line. The results indicated that in silkworm, the effects created by DA and CsA were stronger than FK506. Furthermore, the effects created by DA in silkworm were stronger than those in humans. This study will offer new thinking to elucidate the molecular mechanism of DA in the immunity system of insects.

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Sotokawauchi, Ami, Nobutaka Nakamura, Takanori Matsui, Yuichiro Higashimoto, and Sho-ichi Yamagishi. "Glyceraldehyde-Derived Pyridinium Evokes Renal Tubular Cell Damage via RAGE Interaction." International Journal of Molecular Sciences 21, no. 7 (April 9, 2020): 2604. http://dx.doi.org/10.3390/ijms21072604.

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Glyceraldehyde-derived advanced glycation end products (glycer-AGEs) contribute to proximal tubulopathy in diabetes. However, what glycer-AGE structure could evoke tubular cell damage remains unknown. We first examined if deleterious effects of glycer-AGEs on reactive oxygen species (ROS) generation in proximal tubular cells were blocked by DNA-aptamer that could bind to glyceraldehyde-derived pyridinium (GLAP) (GLAP-aptamer), and then investigated whether and how GLAP caused proximal tubular cell injury. GLAP-aptamer and AGE-aptamer raised against glycer-AGEs were prepared using a systemic evolution of ligands by exponential enrichment. The binding affinity of GLAP-aptamer to glycer-AGEs was measured with a bio-layer interferometry. ROS generation was evaluated using fluorescent probes. Gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). GLAP-aptamer bound to glycer-AGEs with a dissociation constant of 7.7 × 10−5 M. GLAP-aptamer, glycer-AGE-aptamer, or antibodies directed against receptor for glycer-AGEs (RAGE) completely prevented glycer-AGE- or GLAP-induced increase in ROS generation, MCP-1, PAI-1, or RAGE gene expression in tubular cells. Our present results suggest that GLAP is one of the structurally distinct glycer-AGEs, which may mediate oxidative stress and inflammatory reactions in glycer-AGE-exposed tubular cells. Blockade of the interaction of GLAP-RAGE by GLAP-aptamer may be a therapeutic target for proximal tubulopathy in diabetic nephropathy.

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Journal articles on the topic 'Bio-layer interferometry'

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