Capillary Flow-Based One-Minute Quantification of Amyloid Proteolysis.

Quantifying the formation and decomposition of amyloid is a crucial issue in the development of new drugs and therapies for treating amyloidosis. The current technologies for grasping amyloid formation and decomposition include fluorescence analysis using thioflavin-T, secondary structure analysis using circular dichroism, and image analysis using atomic force microscopy or transmission electron microscopy. These technologies typically require spectroscopic devices or expensive nanoscale imaging equipment and involve lengthy analysis, which limits the rapid screening of amyloid-degrading drugs. In this study, we introduce a technology for rapidly assessing amyloid decomposition using capillary flow-based paper (CFP). Amyloid solutions exhibit gel-like physical properties due to insoluble denatured polymers, resulting in a shorter flow distance on CFP compared to pure water. Experimental conditions were established to consistently control the flow distance based on a hen-egg-white lysozyme amyloid solution. It was confirmed that as amyloid is decomposed by trypsin, the flow distance increases on the CFP. Our method is highly useful for detecting changes in the gel properties of amyloid solutions within a minute, and we anticipate its use in the rapid, large-scale screening of anti-amyloid agents in the future.

Bioengineered amyloid peptide for rapid screening of inhibitors against main protease of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has evoked a worldwide pandemic. As the emergence of variants has hampered the neutralization capacity of currently available vaccines, developing effective antiviral therapeutics against SARS-CoV-2 and its variants becomes a significant challenge. The main protease (Mpro) of SARS-CoV-2 has received increased attention as an attractive pharmaceutical target because of its pivotal role in viral replication and proliferation. Here, we generated a de novo Mpro-inhibitor screening platform to evaluate the efficacies of Mpro inhibitors based on Mpro cleavage site-embedded amyloid peptide (MCAP)-coated gold nanoparticles (MCAP-AuNPs). We fabricated MCAPs comprising an amyloid-forming sequence and Mpro-cleavage sequence, mimicking in vivo viral replication process mediated by Mpro. By measuring the proteolytic activity of Mpro and the inhibitory efficacies of various drugs, we confirmed that the MCAP-AuNP-based platform was suitable for rapid screening potential of Mpro inhibitors. These results demonstrated that our MCAP-AuNP-based platform has great potential for discovering Mpro inhibitors and may accelerate the development of therapeutics against COVID-19.

Direct observation of surface charge and stiffness of human metaphase chromosomes.

Metaphase chromosomes in which both polynucleotides and proteins are condensed with hierarchies are closely related to life phenomena such as cell division, cancer development, and cellular senescence. Nevertheless, their nature is rarely revealed, owing to their structural complexity and technical limitations in analytical methods. In this study, we used surface potential and nanomechanics mapping technology based on atomic force microscopy to measure the surface charge and intrinsic stiffness of metaphase chromosomes. We found that extra materials covering the chromosomes after the extraction process were positively charged. With the covering materials, the chromosomes were positively charged (ca. 44.9 ± 16.48 mV) and showed uniform stiffness (ca. 6.23 ± 1.98 MPa). In contrast, after getting rid of the extra materials through treatment with RNase and protease, the chromosomes were strongly negatively charged (ca. -197.4 ± 77.87 mV) and showed relatively non-uniform and augmented stiffness (ca. 36.87 ± 17.56 MPa). The results suggested undulating but compact coordination of condensed chromosomes. Additionally, excessive treatment with RNase and protease could destroy the chromosomal structure, providing an exceptional opportunity for multiscale stiffness mapping of polynucleotides, nucleosomes, chromatin fibers, and chromosomes in a single image. Our approach offers a new horizon in terms of an analytical technique for studying chromosome-related diseases.

Proteolysis-driven proliferation and rigidification of pepsin-resistant amyloid fibrils.

Proteolysis of amyloids is related to prevention and treatment of amyloidosis. What if the conditions for proteolysis were the same to those for amyloid formation? For example, pepsin, a gastric protease is activated in an acidic environment, which, interestingly, is also a condition that induces the amyloid formation. Here, we investigate the competition reactions between proteolysis and synthesis of amyloid under pepsin-activated conditions. The changes in the quantities and nanomechanical properties of amyloids after pepsin treatment were examined by fluorescence assay, circular dichroism and atomic force microscopy. We found that, in the case of pepsin-resistant amyloid, a secondary reaction can be accelerated, thereby proliferating amyloids. Moreover, after this reaction, the amyloid became 32.4 % thicker and 24.2 % stiffer than the original one. Our results suggest a new insight into the proteolysis-driven proliferation and rigidification of pepsin-resistant amyloids.

Colorimetric Nanoparticle-Embedded Hydrogels for a Biosensing Platform.

Hydrogels containing colorimetric nanoparticles have been used for ion sensing, glucose detection, and microbial metabolite analyses. In particular, the rapid chemical reaction owing to both the hydrogel form of water retention and the sensitive color change of nanoparticles enables the rapid detection of target substances. Despite this advantage, the poor dispersibility of nanoparticles and the mechanical strength of nanoparticle-hydrogel complexes have limited their application. In this study, we demonstrate a milliliter agarose gel containing homogeneously synthesized polyaniline nanoparticles (PAni-NPs), referred to as PAni-NP-hydrogel complexes (PNHCs). To fabricate the optimal PNHC, we tested various pH solvents based on distilled water and phosphate-buffered saline and studied the colorimetric response of the PNHC with thickness. The colorimetric response of the prepared PNHC to the changes in the pH of the solution demonstrated excellent linearity, suggesting the possibility of using PNHC as a pH sensor. In addition, it was verified that the PNHC could detect minute pH changes caused by the cancer cell metabolites without cytotoxicity. Furthermore, the PNHC can be stably maintained outside water for approximately 12 h without deformation, indicating that it can be used as a disposable patch-type wearable biosensing platform.