Epoxidized graphene grid for highly efficient high-resolution cryoEM structural analysis.

Functionalization of graphene is one of the most important fundamental technologies in a wide variety of fields including industry and biochemistry. We have successfully achieved a novel oxidative modification of graphene using photoactivated ClO2· as a mild oxidant and confirmed the oxidized graphene grid is storable with its functionality for at least three months under N2 atmosphere. Subsequent chemical functionalization enabled us to develop an epoxidized graphene grid (EG-grid™), which effectively adsorbs protein particles for electron cryomicroscopy (cryoEM) image analysis. The EG-grid dramatically improved the particle density and orientation distribution. The density maps of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were reconstructed at 1.99 and 2.16 Å resolution from only 504 and 241 micrographs, respectively. A sample solution of 0.1 mg ml-1 was sufficient to reconstruct a 3.10 Å resolution map of SARS-CoV-2 spike protein from 1163 micrographs. The map resolutions of ß-galactosidase and apoferritin easily reached 1.81 Å and 1.29 Å resolution, respectively, indicating its atomic-resolution imaging capability. Thus, the EG-grid will be an extremely powerful tool for highly efficient high-resolution cryoEM structural analysis of biological macromolecules.

Structural insights into the rational design of a nanobody that binds with high affinity to the SARS-CoV-2 spike variant.

The continuous emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants associated with the adaptive evolution of the virus is prolonging the global coronavirus disease 2019 (COVID-19) pandemic. The modification of neutralizing antibodies based on structural information is expected to be a useful approach to rapidly combat emerging variants. A dimerized variable domain of heavy chain of heavy chain antibody (VHH) P17 that has highly potent neutralizing activity against SARS-CoV-2 has been reported but the mode of interaction with the epitope remains unclear. Here, we report the X-ray crystal structure of the complex of monomerized P17 bound to the SARS-CoV-2 receptor binding domain (RBD) and investigated the binding activity of P17 toward various variants of concern (VOCs) using kinetics measurements. The structure revealed details of the binding interface and showed that P17 had an appropriate linker length to have an avidity effect and recognize a wide range of RBD orientations. Furthermore, we identified mutations in known VOCs that decrease the binding affinity of P17 and proposed methods for the acquisition of affinity toward the Omicron RBD because Omicron is currently the most predominant VOC. This study provides information for the rational design of effective VHHs for emerging VOCs.

Efficacy of Lactococcus lactis strain plasma (LC-Plasma) in easing symptoms in patients with mild COVID-19: protocol for an exploratory, multicentre, double-blinded, randomised controlled trial (PLATEAU study).

INTRODUCTION: The COVID-19 pandemic has been a major concern worldwide; however, easily accessible treatment options for patients with mild COVID-19 remain limited. Since the oral intake of Lactococcus lactis strain plasma (LC-Plasma) enhances both the innate and acquired immune systems through the activation of plasmacytoid dendritic cells (pDCs), we hypothesised that the oral intake of LC-Plasma could aid the relief or prevention of symptoms in patients with asymptomatic or mild COVID-19. METHODS AND ANALYSIS: This is an exploratory, multicentre, double-blinded, randomised, placebo-controlled trial. This study was initiated in December 2021 and concludes in April 2023. The planned number of enrolled subjects is 100 (50 subjects×2 groups); subject enrolment will be conducted until October 2022. Patients with asymptomatic or mild COVID-19 will be enrolled and randomly assigned in a 1:1 ratio to group A (oral intake of LC-Plasma-containing capsule, 200 mg/day, for 14 days) or group B (oral intake of placebo capsule, for 14 days). The primary endpoint is the change in subjective symptoms measured by the severity score. Secondary endpoints include SARS-CoV-2 viral loads, biomarkers for pDC activation, serum SARS-CoV-2-specific antibodies, serum cytokines, interferon and interferon-inducible antiviral effectors and the proportion of subjects with emergency room visits to medical institutions or who are hospitalised. ETHICS AND DISSEMINATION: The study protocol was approved by the Clinical Research Review Board of Nagasaki University, in accordance with the Clinical Trials Act of Japan. The study will be conducted in accordance with the Declaration of Helsinki, the Clinical Trials Act, and other current legal regulations in Japan. Written informed consent will be obtained from all the participants. The results of this study will be reported in journal publications. TRIAL REGISTRATION NUMBER: Japan Registry of Clinical Trials (registration number: jRCTs071210097).

A panel of nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants including Omicron.

We are amid the historic coronavirus infectious disease 2019 (COVID-19) pandemic. Imbalances in the accessibility of vaccines, medicines, and diagnostics among countries, regions, and populations, and those in war crises, have been problematic. Nanobodies are small, stable, customizable, and inexpensive to produce. Herein, we present a panel of nanobodies that can detect the spike proteins of five SARS-CoV-2 variants of concern (VOCs) including Omicron. Here we show via ELISA, lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays that our nanobodies can quantify the spike variants. This panel of nanobodies broadly neutralizes viral infection caused by pseudotyped and authentic SARS-CoV-2 VOCs. Structural analyses show that the P86 clone targets epitopes that are conserved yet unclassified on the receptor-binding domain (RBD) and contacts the N-terminal domain (NTD). Human antibodies rarely access both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins that go undetected by conventional antibodies.

Safety evaluation of MA-T after ingestion in mice.

MA-T (Matching Transformation System®) is a proprietary chemical mixture for on-demand production of aqueous chlorine dioxide that is used for the treatment of oral malodor. MA-T is also an effective disinfectant against at least 39 pathological microorganisms, including severe acute respiratory syndrome coronavirus 2, and therefore may be useful as a disinfectant mouthwash to prevent the spread of infection. Accidental ingestion is the putative worst hazard scenario associated with mouthwash use; therefore, here we investigated the safety of MA-T ingestion in mice. Mice were provided drinking water containing 0-3000 µg/ml MA-T for 7 days followed by non-spiked drinking water for an additional 14 days. At day 7, mice ingesting 1000 or 3000 µg/ml MA-T showed significantly decreased body weight and significantly increased liver, kidney, and heart tissue injury biomarkers compared with control. However, at 14 days after stopping MA-T ingestion, body weight and tissue injury biomarkers had returned to normal. Histological analysis revealed that MA-T-induced injuries in liver, kidney, spleen, stomach, duodenum, colon, and rectum had also recovered at 14 days after stopping MA-T ingestion; however, mild vascular endothelial injuries remained in heart, jejunum, and ileum in the worst-case scenario. Taken together, MA-T may be potentially safety for further development as a disinfectant mouthwash by risk management, such as placing a caution of the label and adding a distinctive flavor.

Empirical Study of Virtual Reality to Promote Intergenerational Communication: Taiwan Traditional Glove Puppetry as Example

Based on glove puppetry, a traditional cultural pursuit of Taiwan, this study designed a virtual reality (VR) intergenerational game to bring together the elderly and young participants. In the game, a system of cooperation and sharing was constructed which would lead to an exchange between experience and knowledge of traditional culture and digital technology and result in intergenerational interaction and communication learning. Through interviews with eight subjects after the empirical study, this study explored the operation and experience of this game, the perception of interaction and dialogue, and the cultural heritage and learning. According to the research findings, VR game cultural elements and technology learning positively influence intergenerational relations and communication. Key factors of VR intergenerational games include the following: (1) the game content must be attractive for the elderly;(2) the operating procedure of the game and affordance of the interface for the elderly must be simplified;and (3) the game must establish a sense of achievement for players.