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Cryo-electron microscopy (cryo-EM) has been widely used to reveal the structures of proteins at atomic resolution. One key challenge is that almost all proteins are predominantly adsorbed to the air-water interface during standard cryo-EM specimen preparation. The interaction of proteins with air-water interface will significantly impede the success of reconstruction and achievable resolution. Here, we highlight the critical role of impenetrable surfactant monolayers in passivating the air-water interface problems, and develop a robust effective method for high-resolution cryo-EM analysis, by using the superstructure GSAMs which comprises surfactant self-assembled monolayers (SAMs) and graphene membrane. The GSAMs works well in enriching the orientations and improving particle utilization ratio of multiple proteins, facilitating the 3.3-Å resolution reconstruction of a 100-kDa protein complex (ACE2-RBD), which shows strong preferential orientation using traditional specimen preparation protocol. Additionally, we demonstrate that GSAMs enables the successful determinations of small proteins (

In order to explore the research hotspots and development trends of protein deamidation, this paper systematically searched 133 Chinese literatures and 337 English literatures on protein deamidation in the CNKI and WOS databases, and the CiteSpace software was used to draw knowledge graphs and visual analysis of publication trends, author collaboration networks, national collaboration networks, institutional cooperation network, keywords and other content. The results showed that the volume of Chinese publications was relatively low and showed a steady trend, while the volume of English publications showed a wave-like upward trend, with a rapid increase from 2020 to 2023, exceeding 30 publications per year. In addition, both Chinese and English literature formed relatively fixed author clusters with close collaborations within the groups, but limited collaborations between clusters. The research hotspots in this field were generally consistent domestically and internationally, mainly focusing on the influence of deamidation on the structure and functional characteristics of proteins. Some research has also been conducted on the mechanisms underlying the influence on functional characteristics and the sensitization of proteins. In recent years, the application of protein glutaminase in the field of protein deamidation has become a new research hotspot.

In this paper, wheat flour was irradiated at different doses (0, 5, 10, 15, 20 and 25 kGy) to investigate the effects of 60Co-γirradiation on the structural and functional properties of its gluten. The disulfide bond was broken by irradiation, and the content of the free sulfhydryl group increased. The results of Fourier infrared spectroscopy showed that the secondary structure of gluten protein was rearranged with decrease in a-helix content and an increase in β-sheet content. Raman spectroscopy and surface hydrophobicity showed that the microenvironment of aromatic amino acid side chains changed. Irradiation treatment resulted in the tryptophan and tyrosine residues tending more toward the exposed state, and the surface hydrophobicity increased. Meanwhile, irradiation increased the solubility, water and oil holding, and emulsifying properties of gluten proteins, and the maximum value reached at 15 kGy（solubility is 0.46 mg/mL, water holding 3.13 g/g, oil holding 4.33 g/g, emulsification 6.669 m2/g）. In conclusion, the proper dose of gamma-irradiation can change the secondary and tertiary structure of gluten, and improve its functional properties such as solubility, water retention and emulsification, which is conducive to expanding the application space of gluten protein and providing theoretical references for industrial applications.

With the development of cryo-electron microscopy (cryo-EM), high-resolution structures of macromolecules can be reconstructed by the single particle method efficiently. However, challenges may still persist during the specimen preparation stage. Specifically, proteins tend to adsorb at the air-water interface and exhibit a preferred orientation in vitreous ice. To overcome these challenges, we have explored dual-affinity graphene (DAG) modified with two different affinity ligands as a supporting material for cryo-EM sample preparation. The ligands can bind to distinct sites on the corresponding tagged particles, which in turn generates various orientation distributions of particles and prevents the adsorption of protein particles onto the air-water interface. As expected, the DAG exhibited high binding specificity and affinity to target macromolecules, resulting in more balanced particle Euler angular distributions compared to single functionalized graphene on two different protein cases, including the SARS -CoV-2 spike glycoprotein. We anticipate that the DAG grids will enable facile and efficient three-dimensional (3D) reconstruction for cryo-EM structural determination, providing a robust and general technique for future studies.

Background/Aim. High-grade glioma (HGG) is an interstitial cell-derived primary tumor of the nervous system. The current guidelines for the diagnosis and treatment of glioma recommend the maximum safe range of tumor resection for treatment methods. Adjuvant concurrent chemoradiotherapy is recommended after surgery, followed by six cycles of single-drug chemotherapy, temozolomide. Evaluation of the early efficacy of concurrent chemoradio-therapy after HGG surgery, especially for patients with a high risk of recurrence, is a crucial step in enhancing the treatment efficiency for patients diagnosed with HGG. In this study, we investigated the clinical utility of magnetic resonance (MR) spectroscopy (MRS) in assessing the early curing impact of concurrent chemoradiotherapy following HGG surgery. Methods. A total of 50 patients with incomplete resection or suspected residual postoperative HGG, treated in the radiotherapy department of our hospital between January 2016 and June 2021, were selected for routine concurrent chemoradiotherapy. Conventional MR imaging and MRS were performed one week prior to treatment and one month after treatment to assess changes in specific brain metabolites. All 50 patients were followed up for 6 to 12 months. Based on the follow-up results, the patients were divided into two groups: the tumor recurrence group and the tumor suppression group. One month after the end of the treatment, the differences in levels of brain metabolites between the two groups were analyzed using MRS. Results. The levels of N-acetylaspartate (NAA) and creatine (Cr) increased after radiotherapy, while choline (Cho) peak value, and Cho/Cr, NAA/Cr, and Cho/NAA ratios decreased compared to pre-treatment levels. There were statistically significant differences in the NAA peak value, and Cho/Cr, and Cho/NAA ratios in the tumor enhancement area before and after treatment (p < 0.05). There were also statistically significant differences in Cho/Cr ratio in the peritumoral edema area before and after treatment (p < 0.05). Conclusion. After concurrent chemoradiotherapy, MRS can be used to detect early metabolic changes in the tumor enhancement and peritumoral edema areas of HGG.

Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air-water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.