Molecular mechanism underlying substrate recognition of the peptide macrocyclase PsnB.

Graspetides, also known as ω-ester-containing peptides (OEPs), are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) bearing side chain-to-side chain macrolactone or macrolactam linkages. Here, we present the molecular details of precursor peptide recognition by the macrocyclase enzyme PsnB in the biosynthesis of plesiocin, a group 2 graspetide. Biochemical analysis revealed that, in contrast to other RiPPs, the core region of the plesiocin precursor peptide noticeably enhanced the enzyme-precursor interaction via the conserved glutamate residues. We obtained four crystal structures of symmetric or asymmetric PsnB dimers, including those with a bound core peptide and a nucleotide, and suggest that the highly conserved Arg213 at the enzyme active site specifically recognizes a ring-forming acidic residue before phosphorylation. Collectively, this study provides insights into the mechanism underlying substrate recognition in graspetide biosynthesis and lays a foundation for engineering new variants.

Development of a neutralization monoclonal antibody with a broad neutralizing effect against SARS-CoV-2 variants.

BACKGROUND: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has challenged the effectiveness of current therapeutic regimens. Here, we aimed to develop a potent SARS-CoV-2 antibody with broad neutralizing effect by screening a scFv library with the spike protein receptor-binding domain (RBD) via phage display. METHODS: SKAI-DS84 was identified through phage display, and we performed pseudovirus neutralization assays, authentic virus neutralization assays, and in vivo neutralization efficacy evaluations. Furthermore, surface plasmon resonance (SPR) analysis was conducted to assess the physical characteristics of the antibody, including binding kinetics and measure its affinity for variant RBDs. RESULTS: The selected clones were converted to human IgG, and among them, SKAI-DS84 was selected for further analyses based on its binding affinity with the variant RBDs. Using pseudoviruses, we confirmed that SKAI-DS84 was strongly neutralizing against wild-type, B.1.617.2, B.1.1.529, and subvariants of SARS-CoV-2. We also tested the neutralizing effect of SKAI-DS84 on authentic viruses, in vivo and observed a reduction in viral replication and improved lung pathology. We performed binding and epitope mapping experiments to understand the mechanisms underlying neutralization and identified quaternary epitopes formed by the interaction between RBDs as the target of SKAI-DS84. CONCLUSIONS: We identified, produced, and tested the neutralizing effect of SKAI-DS84 antibody. Our results highlight that SKAI-DS84 could be a potential neutralizing antibody against SARS-CoV-2 and its variants.

Aptamer-Based Switching System for Communication of Non-Interacting Proteins.

Biological macromolecules, such as DNA, RNA, and proteins in living organisms, form an intricate network that plays a key role in many biological processes. Many attempts have been made to build new networks by connecting non-communicable proteins with network mediators, especially using antibodies. In this study, we devised an aptamer-based switching system that enables communication between non-interacting proteins. As a proof of concept, two proteins, Cas13a and T7 RNA polymerase (T7 RNAP), were rationally connected using an aptamer that specifically binds to T7 RNAP. The proposed switching system can be modulated in both signal-on and signal-off manners and its responsiveness to the target activator can be controlled by adjusting the reaction time. This study paves the way for the expansion of biological networks by mediating interactions between proteins using aptamers.

Effect of lyophilized gelatin-norbornene cryogel size on calvarial bone regeneration.

Molding processes with molds containing topographical structures have been used for fabrication of hydrogel and cryogel particles. However, they can involve difficulties in separation of fabricated particles with complex shape from the molds or repeated fabrication of the particles although the overall processes do not require much skill and equipment. In this study, molds with etched superhydrophobic patterns have been developed by etching polytetrafluoroethylene (PTFE) blocks in user-defined designs with a femtosecond (FS) laser-based etching system. Lyophilized cryogel particles with various designs and sizes were fabricated by molding precursors with these PTFE molds. Additionally, the clean and easy separation of particles from the molds allowed repeated fabrication of the particles. For an application, relatively 'big' gelatin-norbornene (GelNB) cryogel particles prepared via molding with polydimethylsiloxane (PDMS) molds, swelling in phosphate buffered saline (PBS) and slicing height in half and 'small' GelNB cryogel particles fabricated with the PTFE molds were fabricated. Then, they were used to study scaffold size effect on calvarial bone regeneration. The molds generated with the FS laser-based etching system can be useful for various applications that require the mass production of cryogel particles in various geometries.

A Correlation of Overall Mass Transfer Coefficient of Water Transport in a Hollow-Fiber Membrane Module via an Artificial Neural Network Approach.

Water transport in a hollow-fiber membrane depends on mass convection around the tube, mass convection inside the tube, and water diffusion through the membrane tube. The performance of water transport is then explained by the overall mass transfer coefficient in hollow-fiber membranes. This study presents the prediction of overall mass transfer coefficients of water transport in a hollow-fiber membrane module by an artificial neural network (ANN) that is used for a humidifier of a vehicular fuel cell system. The input variables of ANN are collected from water transport experiments of the hollow-fiber membrane module that is composed of inlet flow rates, inlet relative humidity, system pressures, and operating temperatures. The experimental mass transfer coefficients are the targets of the training model, which are determined via the effectiveness analysis. When unknown data are applied to the ANN model, the correlation of the overall mass transfer coefficient predicts precise results with R = 0.99 (correlation coefficient). The ANN model shows good prediction capability of water transport in membrane humidifiers.

Characteristics of Water Transport of Membrane Electrolyte over Selected Temperature for Proton Exchange Membrane Fuel Cell.

The water contents at both the anode and cathode of PEMFCs depend on the water-transport mechanism at the membrane. The humidity at the outside layers of the membrane determines the diffusion of water through it. The operating temperatures and pressures regulate the humidity conditions in the system. Because these parameters are nonlinear, the water-transport mechanism is analyzed via the difference in the water concentration on each side of the membrane. In this work, an experimental configuration is designed to investigate the diffusion mechanism of water through the membrane. A flat membrane module is tested in an isothermal test chamber to test the influence of temperature on the water-absorption and -transport characteristics of Nafion 117 and Nafion 211 membranes. A parametric study is conducted to test the water-transport mechanism at an operating pressure of 1 bar; temperatures of 30 °C, 50 °C, 70 °C and 90 °C; and a relative humidity ranging from 10% to 100%. The results indicate that the water content of Nafion 211 is higher than that of Nafion 117. The water content and diffusion coefficient are proportional to the operating temperature. In addition, the diffusion coefficient reaches its peak at conditions of 1 bar, 100% humidity, and 90 °C for both membrane types.