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Antigen-specific antibodies are generated by antibody-secreting cells (ASCs). How RNA post-transcriptional modification affects antibody homeostasis remains unclear. Here, we found that mRNA polyadenylations and N6-methyladenosine (m6A) modifications maintain IgG1 antibody production in ASCs. IgG heavy-chain transcripts (Ighg) possessed a long 3' UTR with m6A sites, targeted by the m6A reader YTHDF1. B cell-specific deficiency of YTHDF1 impaired IgG production upon antigen immunization through reducing Ighg1 mRNA abundance in IgG1 + ASCs. Disrupting either the m6A modification of a nuclear-localized splicing intermediate Ighg1 or the nuclear localization of YTHDF1 reduced Ighg1 transcript stability. Single-cell RNA sequencing identified an ASC subset with excessive YTHDF1 expression in systemic lupus erythematosus patients, which was decreased upon therapy with immunosuppressive drugs. In a lupus mouse model, inhibiting YTHDF1-m6A interactions alleviated symptoms. Thus, we highlight a mechanism in ASCs to sustain the homeostasis of IgG antibody transcripts by integrating Ighg1 mRNA polyadenylation and m6A modification., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Industrial fermentation applications typically require enzymes that exhibit high stability and activity at high temperatures. However, efforts to simultaneously improve these properties are usually limited by a trade-off between stability and activity. This report describes a computational strategy to enhance both activity and thermal stability of the mesophilic organophosphate-degrading enzyme, methyl parathion hydrolase (MPH). To predict hotspot mutation sites, we assembled a library of features associated with the target properties for each residue and then prioritized candidate sites by hierarchical clustering. Subsequent in silico screening with multiple algorithms to simulate selective pressures yielded a subset of 23 candidate mutations. Iterative parallel screening of mutations that improved thermal stability and activity yielded, MPHase-m5b, which exhibited 13.3 °C higher T m and 4.2 times higher catalytic activity than wild-type (WT) MPH over a wide temperature range. Systematic analysis of crystal structures, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations revealed a wider entrance to the active site that increased substrate access with an extensive network of interactions outside the active site that reinforced αβ/βα sandwich architecture to improve thermal stability. This study thus provides an advanced, rational design framework to improve efficiency in engineering highly active, thermostable biocatalysts for industrial applications., (© 2024 Wiley-VCH GmbH.)

Dhilirane-type meroterpenoids (DMs) featuring a 6/6/6/5/5 ring system represent a rare group of fungal meroterpenoids. To date, merely 11 DMs have been isolated or derived, leaving their chemical diversity predominantly unexplored. Herein, we leverage an understanding of biosynthesis to develop a workflow for discovery of DMs by genome mining, metabolite analysis, and tailoring enzyme catalysis. Twenty-three new DMs, including seven unprecedented scaffolds, were consequently identified. An α-ketoglutarate (α-KG)-dependent oxygenase DhiD was found to catalyze the stereodivergent ring contraction of dhilirolide D to form the dhilirane skeleton; while the cytochrome P450 DhiH reshaped the structural diversity by establishing diverse C-C bonds and oxidation. Crystallographic and mutagenesis experiments provide a molecular basis for the DhiD reaction and its stereodivergent products. Notably, DhiD exhibits substrate-controlled catalytic versatility in the chemical expansion of DMs through ring contraction, hydroxylation, dehydrogenation, epoxidation, isomerization, epimerization, and α-ketol cleavage. Bioassay results demonstrated that the obtained meroterpenoids exhibited anti-inflammatory and insecticidal activities. Our work provides insight into nature's arsenal for DM biosynthesis and the functional versatility of α-KG-dependent oxygenase and P450, which can be applied for target discovery and diversification of DM-type natural products.

TNF inhibitors have been used to treat autoimmune and autoinflammatory diseases. Here we report an unexpected mechanism underlying the therapeutic effects of TNF inhibitors in Behçet's disease (BD), an autoimmune inflammatory disorder. Using serum metabolomics and peripheral immunocyte transcriptomics, we find that polymorphonuclear neutrophil (PMN) from patients with BD (BD-PMN) has dysregulated mevalonate pathway and subsequently increased farnesyl pyrophosphate (FPP) levels. Mechanistically, FPP induces TRPM2-calcium signaling for neutrophil extracellular trap (NET) and proinflammatory cytokine productions, leading to vascular endothelial inflammation and damage. TNF, but not IL-1β, IL-6, IL-18, or IFN-γ, upregulates TRPM2 expression on BD-PMN, while TNF inhibitors have opposite effects. Results from mice with PMN-specific FPP synthetase or TRPM2 deficiency show reduced experimental vasculitis. Meanwhile, analyses of public datasets correlate increased TRPM2 expressions with the clinical benefits of TNF inhibitors. Our results thus implicate FPP-TRPM2-TNF/NETs feedback loops for inflammation aggravation, and novel insights for TNF inhibitor therapies on BD., (© 2024. The Author(s).)

Evolutionary pressures sculpt population genetics, whereas immune adaptation fortifies humans against life-threatening organisms. How the evolution of selective genetic variation in adaptive immune receptors orchestrates the adaptation of human populations to contextual perturbations remains elusive. Here, we show that the G396R coding variant within the human immunoglobulin G1 (IgG1) heavy chain presents a concentrated prevalence in Southeast Asian populations. We uncovered a 190-kb genomic linkage disequilibrium block peaked in close proximity to this variant, suggestive of potential Darwinian selection. This variant confers heightened immune resilience against various pathogens and viper toxins in mice. Mechanistic studies involving severe acute respiratory syndrome coronavirus 2 infection and vaccinated individuals reveal that this variant enhances pathogen-specific IgG1 + memory B cell activation and antibody production. This G396R variant may have arisen on a Neanderthal haplotype background. These findings underscore the importance of an IGHG1 variant in reinforcing IgG1 antibody responses against life-threatening organisms, unraveling the intricate interplay between human evolution and immune adaptation., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The enzymes 3-methylcrotonyl-coenzyme A (CoA) carboxylase (MCC), pyruvate carboxylase and propionyl-CoA carboxylase belong to the biotin-dependent carboxylase family located in mitochondria. They participate in various metabolic pathways in human such as amino acid metabolism and tricarboxylic acid cycle. Many human diseases are caused by mutations in those enzymes but their structures have not been fully resolved so far. Here we report an optimized purification strategy to obtain high-resolution structures of intact human endogenous MCC, propionyl-CoA carboxylase and pyruvate carboxylase in different conformational states. We also determine the structures of MCC bound to different substrates. Analysis of MCC structures in different states reveals the mechanism of the substrate-induced, multi-element synergistic activation of MCC. These results provide important insights into the catalytic mechanism of the biotin-dependent carboxylase family and are of great value for the development of new drugs for the treatment of related diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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 (<100 kDa) at near-atomic resolution. This study expands the understanding of SAMs and provides a key to better control the interaction of protein with air-water interface., (© 2024. The Author(s).)

Photosynthesis converting solar energy to chemical energy is one of the most important chemical reactions on earth. In cyanobacteria, light energy is captured by antenna system phycobilisomes (PBSs) and transferred to photosynthetic reaction centers of photosystem II (PSII) and photosystem I (PSI). While most of the protein complexes involved in photosynthesis have been characterized by in vitro structural analyses, how these protein complexes function together in vivo is not well understood. Here we implemented STAgSPA, an in situ structural analysis strategy, to solve the native structure of PBS-PSII supercomplex from the cyanobacteria Arthrospira sp. FACHB439 at resolution of ~3.5 Å. The structure reveals coupling details among adjacent PBSs and PSII dimers, and the collaborative energy transfer mechanism mediated by multiple super-PBS in cyanobacteria. Our results provide insights into the diversity of photosynthesis-related systems between prokaryotic cyanobacteria and eukaryotic red algae but are also a methodological demonstration for high-resolution structural analysis in cellular or tissue samples., (© 2024. The Author(s).)

The frequent occurrence of viral infections poses a serious threat to human life. Identifying effective antiviral components is urgent. In China, pearls have been important traditional medicinal ingredients since ancient times, exhibiting various therapeutic properties, including detoxification properties. In this study, a peptide, KKCH, which acts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was derived from Pinctada fucata pearls. Molecular docking showed that it bound to the same pocket of the SARS-CoV-2 S protein and cell surface target angiotensin-converting enzyme II (ACE2). The function of KKCH was analyzed through surface plasmon resonance (SPR), Enzyme-Linked Immunosorbent Assays, immunofluorescence, and simulation methods using the SARS-CoV-2 pseudovirus and live virus. The results showed that KKCH had a good affinity for ACE2 (KD = 6.24 × 10 -7 M) and could inhibit the binding of the S1 protein to ACE2 via competitive binding. As a natural peptide, KKCH inhibited the binding of the SARS-CoV-2 S1 protein to the surface of human BEAS-2B and HEK293T cells. Moreover, viral experiments confirmed the antiviral activity of KKCH against both the SARS-CoV-2 spike pseudovirus and SARS-CoV-2 live virus, with half-maximal inhibitory concentration (IC 50 ) values of 398.1 μM and 462.4 μM, respectively. This study provides new insights and potential avenues for the prevention and treatment of SARS-CoV-2 infections.

Addressing interfacial effects during specimen preparation in cryogenic electron microscopy remains challenging. Here we introduce ESI-cryoPrep, a specimen preparation method based on electrospray ionization in native mass spectrometry, designed to alleviate issues associated with protein denaturation or preferred orientation induced by macromolecule adsorption at interfaces. Through fine-tuning spraying parameters, we optimized protein integrity preservation and achieved the desired ice thickness for analyzing target macromolecules. With ESI-cryoPrep, we prepared high-quality cryo-specimens of five proteins and obtained three-dimensional reconstructions at near-atomic resolution. Our findings demonstrate that ESI-cryoPrep effectively confines macromolecules within the middle of the thin layer of amorphous ice, facilitating the preparation of blotting-free vitreous samples. The protective mechanism, characterized by the uneven distribution of charged biomolecules of varying sizes within charged droplets, prevents the adsorption of target biomolecules at air-water or graphene-water interfaces, thereby avoiding structural damage to the protein particles or the introduction of dominant orientation issues., (© 2024. The Author(s).)

Specimen preparation is a critical but challenging step in high-resolution cryogenic electron microscopy (cryo-EM) structural analysis of macromolecules. In the past decade, graphene has gained much recognition as the supporting substrate to optimize cryo-EM specimen preparation. It improves macromolecule embedding in ice, reduces beam-induced motion, while imposing negligible background noise. Various types of graphene-coated cryo-EM grids were implemented to improve the robustness and efficiency of specimen preparation. Graphene functionalization by different means has been proved specifically useful in addressing challenges related to the air-water interface (AWI), such as preferential orientation and sample denaturation. Graphene sandwich specimen preparation sets a new direction to explore in cryo-EM analysis of biological specimens. In this review, we discuss the current challenges and future prospects of graphene application in cryo-EM analysis of macromolecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Small extracellular vesicles were shown to have similar functional roles to their parent cells without the defect of potential tumorigenicity, which made them a great candidate for regenerative medicine. The last twenty years have witnessed the rapid development of research on small extracellular vesicles. In this paper, we employed a scientometric synthesis method to conduct a retrospective analysis of small extracellular vesicles in the field of bone-related diseases. The overall background analysis consisted the visualization of the countries, institutions, journals, and authors involved in research. The current status of the research direction and future trends were presented through the analysis of references and keywords, which showed that engineering strategies, mesenchymal stem cell derived exosomes, and cartilage damage were the most concerning topics, and scaffold, osteoarthritis, platelet-rich plasma, and senescence were the future trends. We also discussed the current problems and challenges in practical applications, including the in-sight mechanisms, the building of relevant animal models, and the problems in clinical trials. By using CiteSpace, VOSviewer, and Bibliometrix, the presented data avoided subjective selectivity and tendency well, which made the conclusion more reliable and comprehensive. We hope that the findings can provide new perspectives for researchers to understand the evolution of this field over time and to search for novel research directions.

B cells are central players in the immune system, responsible for producing antibodies and modulating immune responses. This review explores the intricate relationship between aberrant B cell activation and the development of autoimmune diseases, emphasizing the essential role of B cells in these conditions. We also summarize B cell receptor signaling and Toll-like receptor signaling in B cell activation, as well as their association with autoimmune diseases, shedding light on the molecular mechanisms behind these associations. Additionally, we explore the clinical observations involving B cell activation and their significance in autoimmune disease management. Various clinical studies related to B cell-targeted therapies are also discussed, offering insights into potential avenues for improving treatment strategies. Overall, this review serves as a resource for researchers and clinicians in the field of immunology and autoimmune diseases, providing a general view of B cell signaling and its role in autoimmunity., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objectives: B10 and B10pro cells suppress immune responses via secreting interleukin (IL)-10. However, their regulators and underlying mechanisms, especially in human autoimmune diseases, are elusive. This study aimed to address these questions in rheumatoid arthritis (RA), one of the most common highly disabling autoimmune diseases., Methods: The frequencies and functions of B10 and B10pro cells in healthy individuals and patients with RA were first analysed. The effects of proinflammatory cytokines, particularly tumour necrosis factor (TNF)-α on the quantity, stability and pathogenic phenotype of these cells, were then assessed in patients with RA before and after anti-TNF therapy. The underlying mechanisms were further investigated by scRNA-seq database reanalysis, transcriptome sequencing, TNF-α -/- and B cell-specific SHIP-1 -/- mouse disease model studies., Results: TNF-α was a key determinant for B10 cells. TNF-α elicited the proinflammatory feature of B10 and B10pro cells by downregulating IL-10, and upregulating interferon-γ and IL-17A. In patients with RA, B10 and B10pro cells were impaired with exacerbated proinflammatory phenotype, while anti-TNF therapy potently restored their frequencies and immunosuppressive functions, consistent with the increased B10 cells in TNF-α -/- mice. Mechanistically, TNF-α diminished B10 and B10pro cells by inhibiting their glycolysis and proliferation. TNF-α also regulated the phosphatidylinositol phosphate signalling of B10 and B10pro cells and dampened the expression of SHIP-1, a dominant phosphatidylinositol phosphatase regulator of these cells., Conclusions: TNF-α provoked the proinflammatory phenotype of B10 and B10pro cells by disturbing SHIP-1 in RA, contributing to the disease development. Reinstating the immunosuppressive property of B10 and B10pro cells might represent novel therapeutic approaches for RA., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

The function of transient receptor potential vanilloid (TRPV) cation channels governing B cell activation remains to be explored. We present evidence that TRPV2 is highly expressed in B cells and plays a crucial role in the formation of the B cell immunological synapse and B cell activation. Physiologically, TRPV2 expression level is positively correlated to influenza-specific antibody production and is low in newborns and seniors. Pathologically, a positive correlation is established between TRPV2 expression and the clinical manifestations of systemic lupus erythematosus (SLE) in adult and child SLE patients. Correspondingly, mice with deficient TRPV2 in B cells display impaired antibody responses following immunization. Mechanistically, the pore and N-terminal domains of TRPV2 are crucial for gating cation permeation and executing mechanosensation in B cells upon antigen stimulation. These processes synergistically contribute to membrane potential depolarization and cytoskeleton remodeling within the B cell immunological synapse, fostering efficient B cell activation. Thus, TRPV2 is critical in augmenting B cell activation and function., (© 2024 Li et al.)

Rationally modulating the binding strength of reaction intermediates on surface sites of copper-based catalysts could facilitate C-C coupling to generate multicarbon products in an electrochemical CO 2 reduction reaction. Herein, theoretical calculations reveal that cascade Ag-Cu dual sites could synergistically increase local CO coverage and lower the kinetic barrier for CO protonation, leading to enhanced asymmetric C-C coupling to generate C 2 H 4 . As a proof of concept, the Cu 3 N-Ag nanocubes (NCs) with Ag located in partial Cu sites and a Cu 3 N unit center are successfully synthesized. The Faraday efficiency and partial current density of C 2 H 4 over Cu 3 N-Ag NCs are 7.8 and 9.0 times those of Cu 3 N NCs, respectively. In situ spectroscopies combined with theoretical calculations confirm that Ag sites produce CO and Cu sites promote asymmetric C-C coupling to *COCHO, significantly enhancing the generation of C 2 H 4 . Our work provides new insights into the cascade catalysis strategy at the atomic scale for boosting CO 2 to multicarbon products.

High-quality specimen preparation plays a crucial role in cryo-electron microscopy (cryo-EM) structural analysis. In this study, we have developed a reliable and convenient technique called the graphene sandwich method for preparing cryo-EM specimens. This method involves using two layers of graphene films that enclose macromolecules on both sides, allowing for an appropriate ice thickness for cryo-EM analysis. The graphene sandwich helps to mitigate beam-induced charging effect and reduce particle motion compared to specimens prepared using the traditional method with graphene support on only one side, therefore improving the cryo-EM data quality. These advancements may open new opportunities to expand the use of graphene in the field of biological electron microscopy., Competing Interests: Competing interests statement:The authors declare no competing interest.

High-entropy-alloy nanoparticles (HEA-NPs) show great potential as electrocatalysts for water splitting, fuel cells, CO 2 conversion, etc. However, fine-tuning the surface, morphology, structure, and crystal phase of HEA remains a great challenge. Here, the high-temperature liquid shock (HTLS) technique is applied to produce HEA-NPs, e.g., PtCoNiRuIr HEA-NPs, with tunable elemental components, ultrafine particle size, controlled crystal phases, and lattice strains. HTLS directly applied Joule heating on the liquid mixture of metal precursors, capping agents, and reducing agents, which is feasible for controlling the morphology and structure such as the atomic arrangement of the resulting products, thereby facilitating the rationally designed nanocatalysts. Impressively, the as-obtained PtCoNiRuIr HEA-NPs delivered superior activity and long-term stability for the hydrogen evolution reaction (HER), with low overpotentials at 10 mA cm -2 and 1 A cm -2 of only 18 and 408 mV, respectively, and 10000 CV stable cycles in 0.5 M H 2 SO 4 . Furthermore, in the near future, by combining the HTLS method with artificial intelligence (AI) and theoretical calculations, it is promising to provide an advanced platform for the high-throughput synthesis of HEA nanocatalysts with optimized performance for various energy applications, which is of great significance for achieving a carbon-neutral society with an effective and environmentally friendly energy system.

The structural engineering of metastable nanomaterials with abundant defects has attracted much attention in energy-related fields. The high-temperature shock (HTS) technique, as a rapidly developing and advanced synthesis strategy, offers significant potential for the rational design and fabrication of high-quality nanocatalysts in an ultrafast, scalable, controllable and eco-friendly way. In this review, we provide an overview of various metastable micro- and nanomaterials synthesized via HTS, including single metallic and bimetallic nanostructures, high entropy alloys, metal compounds (e.g. metal oxides) and carbon nanomaterials. Note that HTS provides a new research dimension for nanostructures, i.e. kinetic modulation. Furthermore, we summarize the application of HTS-as supporting films for transmission electron microscopy grids-in the structural engineering of 2D materials, which is vital for the direct imaging of metastable materials. Finally, we discuss the potential future applications of high-throughput and liquid-phase HTS strategies for non-equilibrium micro/nano-manufacturing beyond energy-related fields. It is believed that this emerging research field will bring new opportunities to the development of nanoscience and nanotechnology in both fundamental and practical aspects., (© The Author(s) 2024. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd.)

Monocarboxylate transporter 1 (MCT1) exhibits essential roles in cellular metabolism and energy supply. Although MCT1 is highly expressed in activated B cells, it is not clear how MCT1-governed monocarboxylates transportation is functionally coupled to antibody production during the glucose metabolism. Here, we report that B cell-lineage deficiency of MCT1 significantly influences the class-switch recombination (CSR), rendering impaired IgG antibody responses in Mct1 f/f Mb1 Cre mice after immunization. Metabolic flux reveals that glucose metabolism is significantly reprogrammed from glycolysis to oxidative phosphorylation in Mct1-deficient B cells upon activation. Consistently, activation-induced cytidine deaminase (AID), is severely suppressed in Mct1-deficient B cells due to the decreased level of pyruvate metabolite. Mechanistically, MCT1 is required to maintain the optimal concentration of pyruvate to secure the sufficient acetylation of H3K27 for the elevated transcription of AID in activated B cells. Clinically, we found that MCT1 expression levels are significantly upregulated in systemic lupus erythematosus patients, and Mct1 deficiency can alleviate the symptoms of bm12-induced murine lupus model. Collectively, these results demonstrate that MCT1-mediated pyruvate metabolism is required for IgG antibody CSR through an epigenetic dependent AID transcription, revealing MCT1 as a potential target for vaccine development and SLE disease treatment., (© 2024. The Author(s).)

Pinctada fucata is an important pearl production shellfish in aquaculture. The formation of shells and pearls is a hot research topic in biomineralization, and matrix proteins secreted by the mantle tissues play the key role in this process. However, upstream regulatory mechanisms of transcription factors on the matrix protein genes remain unclear. Previous studies have shown that NF-κB signaling pathway regulated biomineralization process through expression regulation of specific matrix proteins, including Nacrein, Prismalin-14 and MSI60. In this study, we systematically investigated the regulatory effect of the NF-κB signaling pathway key factor Pf-Rel and inhibitory protein poI-κB on the biomineralization and shell regeneration process. We applied RNA interference and antibody injection assays to study in vivo function of transcription factor Pf-Rel and characterized shell morphology changes using scanning electron microscopy and Raman spectroscopy. We found that transcription factor Pf-Rel plays a positive regulatory role in the growth regulation of the prismatic and nacreous layers, while the function of inhibitory protein poI-κB is to prevent excessive growth and accumulation of both layers. RNA-seq was conducted based on RNA interference animal model to identify potential regulatory genes by transcription factor Pf-Rel. Shell damage repair experiments were performed to simulate shell regeneration process, and observations of newly formed shells revealed that NF-κB signaling pathway had different functions at different times. This study provides us with a more macroscopic perspective based on transcription factors to investigate biomineralization and shell regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated RNAi pathway plays important roles in defending against viral infections and protecting genome integrity. During the maturation of siRNAs, two cofactors can regulate DmDcr-2's functions: Loqs-PD that is required for dsRNA processing, and R2D2 that is essential for the subsequent loading of siRNAs into effector Ago2 to form RISC complexes. However, due to the lack of structural information, it is still unclear whether R2D2 and Loqs-PD affect the functions of DmDcr-2 simultaneously. Here we present several cryo-EM structures of DmDcr-2/R2D2/Loqs-PD complex bound to dsRNAs with various lengths by the Helicase domain. These structures revealed that R2D2 and Loqs-PD can bind to different regions of DmDcr-2 without interfering with each other. Furthermore, the cryo-EM results demonstrate that these complexes can form large oligomers and assemble into fibers. The formation and depolymerization of these oligomers are associated with ATP hydrolysis. These findings provide insights into the structural mechanism of DmDcr-2 and its cofactors during siRNA processing., (© 2023. Springer Nature Limited.)

Salt bridges are important factors in maintaining the stability of proteins, and their contribution to protein folding has received much attention. Although the interaction energies, or stabilizing contributions, of individual salt bridges have been measured in various proteins, a systematic assessment of various types of salt bridges in a relatively uniform environment is still a valuable analysis. Here, we used a collagen heterotrimer as a host-guest platform to construct 48 heterotrimers with the same charge pattern. A variety of salt bridges were formed between the oppositely charged residues Lys, Arg, Asp, and Glu. The melting temperature (T m ) of the heterotrimers was measured with circular dichroism. The atomic structures of 10 salt bridges were shown in three x-ray crystals of heterotrimer. Molecular dynamics simulation based on the crystal structures indicated that strong, intermediate, and weak salt bridges have distinctive N-O distances. A linear regression model was used to predict the stability of heterotrimers with high accuracy (R 2  = 0.93). We developed an online database to help readers understand how a salt bridge stabilizes collagen. This work will help us better understand the stabilizing mechanism of salt bridges in collagen folding and provide a new strategy to design collagen heterotrimers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Biomolecular condensates play key roles in various biological processes. However, specific condensation modulators are currently lacking. PROTAC is a new technology that can use small molecules to degrade target proteins specifically. PROTAC molecules are expected to regulate biomolecular condensates dynamically by degrading/recovering key molecules in biomolecular condensates. In this study, we employed a BRD4-targeting PROTAC molecule to regulate the super-enhancer (SE) condensate and monitored the changes of SE condensate under PROTAC treatment using live-cell imaging and high-throughput sequencing technologies. As a result, we found that BRD4-targeting PROTACs can significantly reduce the BRD4 condensates, and we established a quantitative method for tracking BRD4 condensates by PROTAC and cellular imaging. Surprisingly and encouragingly, BRD4 condensates were observed to preferentially form and play specialized roles in biological process regulation for the first time. Additionally, BRD4 PROTAC makes it possible to observe the dynamics of other condensate components under the continued disruption of BRD4 condensates. Together, these results shed new light on research methods for liquid-liquid phase separation (LLPS), and specifically demonstrate that PROTAC presents a powerful and distinctive tool for the study of biomolecular condensates., (© 2023. The Author(s).)

Cryogenic electron microscopy (cryo-EM) is now one of the most powerful and widely used methods to determine high-resolution structures of macromolecules. A major bottleneck of cryo-EM is to prepare high-quality vitrified specimen, which still faces many practical challenges. During the conventional vitrification process, macromolecules tend to adsorb at the air-water interface (AWI), which is known unfriendly to biological samples. In this review, we outline the nature of AWI and the problems caused by it, such as unpredictable or uneven particle distribution, protein denaturation, dissociation of complex and preferential orientation. We review and discuss the approaches and underlying mechanisms to deal with AWI: 1) Additives, exemplified by detergents, forming a protective layer at AWI and thus preserving the native folds of target macromolecules. 2) Fast vitrification devices based on the idea to freeze in-solution macromolecules before their touching of AWI. 3) Thin layer of continuous supporting films to adsorb macromolecules, and when functionalized with affinity ligands, to specifically anchor the target particles away from the AWI. Among these supporting films, graphene, together with its derivatives, with negligible background noise and mechanical robustness, has emerged as a new generation of support. These strategies have been proven successful in various cases and enable us a better handling of the problems caused by the AWI in cryo-EM specimen preparation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

In oxygenic photosynthetic organisms, light energy is captured by antenna systems and transferred to photosystem II (PSII) and photosystem I (PSI) to drive photosynthesis 1,2 . The antenna systems of red algae consist of soluble phycobilisomes (PBSs) and transmembrane light-harvesting complexes (LHCs) 3 . Excitation energy transfer pathways from PBS to photosystems remain unclear owing to the lack of structural information. Here we present in situ structures of PBS-PSII-PSI-LHC megacomplexes from the red alga Porphyridium purpureum at near-atomic resolution using cryogenic electron tomography and in situ single-particle analysis 4 , providing interaction details between PBS, PSII and PSI. The structures reveal several unidentified and incomplete proteins and their roles in the assembly of the megacomplex, as well as a huge and sophisticated pigment network. This work provides a solid structural basis for unravelling the mechanisms of PBS-PSII-PSI-LHC megacomplex assembly, efficient energy transfer from PBS to the two photosystems, and regulation of energy distribution between PSII and PSI., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

For both nacre formation and biomineralization in mollusks, understanding the molecular mechanism is imperative. Biomineralization, especially shell formation, is dedicatedly regulated by multiple matrix proteins. However, ACC conversion to stable crystals still lacks positive factors. In this research, we found a novel matrix protein named PNU5 in Pinctada fucata that plays a regulatory role in both prismatic layer and nacreous layer formation. Functional studies in vivo and in vitro have shown that it might be involved in shell formation in a positive manner. RT-qPCR analysis showed that pnu5 was highly expressed in mantle pallial and participated in shell repairing and regeneration. RNAi-mediated repression of pnu5 could affect the normal structure of prismatic layer and nacreous layer. The recombinant protein rPNU5 significantly enhanced the precipitation rate of CaCO 3 both in the calcite and aragonite crystallization systems, as well as altering the morphology of the crystals. Based on ACC transition experiments, the recombinant protein rPNU5 facilitated amorphous calcium carbonate (ACC) transformation into stable calcite or aragonite. This study could provide us with a better understanding of how positive regulatory mechanisms contribute to biomineralization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cryo-electron microscopy (cryo-EM) visualizes the atomic structure of macromolecules that are embedded in vitrified thin ice at their close-to-native state. However, the homogeneity of ice thickness, a key factor to ensure high image quality, is poorly controlled during specimen preparation and has become one of the main challenges for high-resolution cryo-EM. Here we found that the uniformity of thin ice relies on the surface flatness of the supporting film, and developed a method to use ultraflat graphene (UFG) as the support for cryo-EM specimen preparation to achieve better control of vitreous ice thickness. We show that the uniform thin ice on UFG improves the image quality of vitrified specimens. Using such a method we successfully determined the three-dimensional structures of hemoglobin (64 kDa), α-fetoprotein (67 kDa) with no symmetry, and streptavidin (52 kDa) at a resolution of 3.5 Å, 2.6 Å and 2.2 Å, respectively. Furthermore, our results demonstrate the potential of UFG for the fields of cryo-electron tomography and structure-based drug discovery., (© 2022. The Author(s).)

High entropy oxides (HEOs) with fascinating physical and chemical properties have exhibited unprecedented application potential in many fields. However, it remains a huge challenge to realize the precise control of the dimension and morphology at the sub-1 nm scale. Herein, with the aid of polyoxometalate (POM) clusters, we first develop a versatile strategy to realize the controllable incorporation of multiple immiscible metal oxides into sub-1 nm nanowires (SNWs) under 140 °C to obtain a wide range of HEO-POM SNWs with highly ordered structures, where the species of metal oxides and POMs could be regulated flexibly. Meanwhile, these obtained HEO materials are first to be used as anodes in Na-ion batteries. Benefiting from the effect of entropy modulation, these HEO-POM SNWs show better electrochemical properties in Na-ion batteries with the increase of metal oxide species stepwise. A long cycle life with a capacity retention of ∼92% even after 5000 cycles at 10C further confirms the good stability under fast discharging/charging. This method opens a new insight for designing and preparing HEOs at the sub-1 nm scale under facile conditions.

Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot, and mouth disease (HFMD) in many countries, most frequently affecting children, and a small proportion of cases may lead to death. Currently, no vaccine is available in most endemic regions, and no licenced treatments for EV-A71 infection are available. Here, we characterize a human monoclonal antibody (HuMAb), E1, by screening a Fab antibody phage library derived from patients who recovered from EV-A71 infection. E1 exhibits strong neutralizing activity against EV-A71 virus in cells. The cryo-electron microscopy (cryo-EM) structures of the EV-A71 virion in complex with E1 Fab fragments demonstrated that E1 recognized an epitope formed by residues in the BC and HI loops of VP1. In a mouse model, E1 effectively protected against lethal EV-A71 challenge in both prophylactic and therapeutic treatment. In particular, E1 significantly reduces virus titers and muscle damage. E1 might represent a potential adjunct to EV-A71 treatment., (© 2022. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

CaCO 3 , which occurs in three crystalline anhydrous polymorphs named calcite, aragonite, and vaterite, is always found in mineralized skeletons or growing shells of many marine organisms. However, understanding how these organisms achieve this control has been a significant challenge in biomineralization. In this work, we proposed a novel vaterite stabilizer acidic matrix protein PNU7 that existed in both prism and nacre of Pinctada fucata, and identified its functional domain DDDDDDHDDVEETED. Our experiments reveal that PNU7 triggers a stable large vaterite formation with Mg 2+ deficiency even under low Ca 2+ . Increasing PNU7 in the calcium carbonate crystallization system with Mg 2+ leads to a significant shrinking in crystal size and rising in nucleation quantity. Moreover, it converts an atomically rough dome-like shape to a smooth sphere on unsiliconized glass. These effects rescind after removing the asp-rich region at the C-terminus. We also find that decreasing pnu7 mRNA in vivo leads to nacreous inner surface growth substantially lessened. Thus, PNU7 may not only supply vaterite in shell formation but also involve the nacreous regulation via surface energy minimization., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Adrenoleukodystrophy protein (ALDP) is responsible for the transport of very-long-chain fatty acids (VLCFAs) and corresponding CoA-esters across the peroxisomal membrane. Dysfunction of ALDP leads to peroxisomal metabolic disorder exemplified by X-linked adrenoleukodystrophy (ALD). Hundreds of ALD-causing mutations have been identified on ALDP. However, the pathogenic mechanisms of these mutations are restricted to clinical description due to limited structural and biochemical characterization. Here we report the cryo-electron microscopy structure of human ALDP with nominal resolution at 3.4 Å. ALDP exhibits a cytosolic-facing conformation. Compared to other lipid ATP-binding cassette transporters, ALDP has two substrate binding cavities formed by the transmembrane domains. Such structural organization may be suitable for the coordination of VLCFAs. Based on the structure, we performed integrative analysis of the cellular trafficking, protein thermostability, ATP hydrolysis, and the transport activity of representative mutations. These results provide a framework for understanding the working mechanism of ALDP and pathogenic roles of disease-associated mutations., Competing Interests: YJ, YZ, WW, JL, ZY, GY No competing interests declared, (© 2022, Jia, Zhang et al.)

A major hurdle for single particle cryo-EM in structural determination lies in the specimen preparation impaired by the air-water interface (AWI) and preferential particle-orientation problems. In this work, we develop functionalized graphene grids with various charges via a dediazoniation reaction for cryo-EM specimen preparation. The graphene grids are paraffin-assistant fabricated, which appear with less contaminations compared with those produced by polymer transfer method. By applying onto three different types of macromolecules, we demonstrate that the high-yield charged graphene grids bring macromolecules away from the AWI and enable adjustable particle-orientation distribution for more robust single particle cryo-EM structural determination., (© 2022. The Author(s).)

Understanding the molecular and cellular mechanisms of human primordial germ cells (hPGCs) is essential in studying infertility and germ cell tumorigenesis. Many RNA-binding proteins (RBPs) and non-coding RNAs are specifically expressed and functional during hPGC developments. However, the roles and regulatory mechanisms of these RBPs and non-coding RNAs, such as microRNAs (miRNAs), in hPGCs remain elusive. In this study, we reported a new regulatory function of DAZL, a germ cell-specific RBP, in miRNA biogenesis and cell proliferation. First, DAZL co-localized with miRNA let-7a in human PGCs and up-regulated the levels of &gt;100 mature miRNAs, including eight out of nine let-7 family, miR21, miR22, miR125, miR10 and miR199. Purified DAZL directly bound to the loops of precursor miRNAs with sequence specificity of GUU. The binding of DAZL to the precursor miRNA increased the maturation of miRNA by enhancing the cleavage activity of DICER. Furthermore, cell proliferation assay and cell cycle analysis confirmed that DAZL inhibited the proliferation of in vitro PGCs by promoting the maturation of these miRNAs. Evidently, the mature miRNAs up-regulated by DAZL silenced cell proliferation regulators including TRIM71. Moreover, DAZL inhibited germline tumor cell proliferation and teratoma formation. These results demonstrate that DAZL regulates hPGC proliferation by enhancing miRNA processing., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sensors with high-sensitivity for resistive methanol gas detection are highly desirable. Herein, we report newly designed ultrathin anionic metal-organic framework (MOF) nanosheets (NSs), with an average thickness of 10 nm and an electrical conductivity of 3.77 × 10 -4 S cm -1 . The ultrathin MOF NSs can be used as the active material in an electronic methanol gas sensor, which exhibits high sensitivity toward methanol gas at room temperature, i.e. , high R air / R gas (363.2 at 100 ppm), fast gas response/recovery speed (6 s/2 s at 20 ppm), long-term stability, and superior cross-selectivity against other interfering gases.

The voltage-gated potassium channel AKT1 is responsible for primary K + uptake in Arabidopsis roots. AKT1 is functionally activated through phosphorylation and negatively regulated by a potassium channel α-subunit AtKC1. However, the molecular basis for the modulation mechanism remains unclear. Here we report the structures of AKT1, phosphorylated-AKT1, a constitutively-active variant, and AKT1-AtKC1 complex. AKT1 is assembled in 2-fold symmetry at the cytoplasmic domain. Such organization appears to sterically hinder the reorientation of C-linkers during ion permeation. Phosphorylated-AKT1 adopts an alternate 4-fold symmetric conformation at cytoplasmic domain, which indicates conformational changes associated with symmetry switch during channel activation. To corroborate this finding, we perform structure-guided mutagenesis to disrupt the dimeric interface and identify a constitutively-active variant Asp379Ala mediates K + permeation independently of phosphorylation. This variant predominantly adopts a 4-fold symmetric conformation. Furthermore, the AKT1-AtKC1 complex assembles in 2-fold symmetry. Together, our work reveals structural insight into the regulatory mechanism for AKT1., (© 2022. The Author(s).)

MicroRNAs (miRNAs) are key players in human hepatocellular carcinoma (HCC) tumorigenesis. Therefore, small molecules targeting components of miRNA biogenesis may provide new therapeutic means for HCC treatment. By a high-throughput screening and structural simplification, we identified a small molecule, CIB-3b, which suppresses the growth and metastasis of HCC in vitro and in vivo by modulating expression profiles of miRNAome and proteome in HCC cells. Mechanistically, CIB-3b physically binds to transactivation response (TAR) RNA-binding protein 2 (TRBP) and disrupts the TRBP-Dicer interaction, thereby altering the activity of Dicer and mature miRNA production. Structure-activity relationship study via the synthesis of 45 CIB-3b derivatives showed that some compounds exhibited a similar inhibitory effect on miRNA biogenesis to CIB-3b. These results support TRBP as a potential therapeutic target in HCC and warrant further development of CIB-3b along with its analogues as a novel therapeutic strategy for the treatment of HCC.

DNA hydrogels have attracted increasing attention owing to their excellent permeability and high mechanical strength, together with thixotropy, versatile programmability and good biocompatibility. However, the moderate biostability and immune stimulation of DNA have arisen as big concerns for future potential clinical applications. Herein, we report the self-assembly of a novel l-DNA hydrogel, which inherited the extraordinary physical properties of a d-DNA hydrogel. With the mirror-isomer deoxyribose, this hydrogel exhibited improved biostability, withstanding fetal bovine serum (FBS) for at least 1 month without evident decay of its mechanical properties. The low inflammatory response of the l-DNA hydrogel has been verified both in vitro and in vivo. Hence, this l-DNA hydrogel with outstanding biostability and biocompatibility can be anticipated to serve as an ideal 3D cell-culture matrix and implanted bio-scaffold for long-term biomedical applications., (© 2022 Wiley-VCH GmbH.)

Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes 1,2 . In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs) 3,4 . ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes 5,6 . Here we report the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5'-phosphate-binding pocket. The overall conformation of Dcr-2-Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2-Loqs-PD., (© 2022. The Author(s).)

Biomolecular condensation driven by liquid-liquid phase separation (LLPS) is key to assembly of membraneless organelles in numerous crucial pathways. It is largely unknown how cellular structures or components spatiotemporally regulate LLPS and condensate formation. Here we reveal that cytoskeletal dynamics can control the condensation of p62 bodies comprising the autophagic adaptor p62/SQSTM1 and poly-ubiquitinated cargos. Branched actin networks are associated with p62 bodies and are required for their condensation. Myosin 1D, a branched actin-associated motor protein, drives coalescence of small nanoscale p62 bodies into large micron-scale condensates along the branched actin network. Impairment of actin cytoskeletal networks compromises the condensation of p62 bodies and retards substrate degradation by autophagy in both cellular models and Myosin 1D knockout mice. Coupling of LLPS scaffold to cytoskeleton systems may represent a general mechanism by which cells exert spatiotemporal control over phase condensation processes., (© 2022. The Author(s) under exclusive licence to Center for Excellence in Molecular Cell Science, CAS.)