Protein–protein interactions at organelle membranes bridge organelles in close proximity, facilitating regulated metabolite exchange and maintaining cellular homeostasis. Enzyme-catalyzed proximity labeling (PL) has been widely used to uncover the molecular composition of these interactions, but excessive labeling of irrelevant cytosolic proteins complicates data analysis. To address this, we developed a streamlined protocol that combines the TurboID system with digitonin-permeabilization to efficiently map protein interactions at organelle membranes in live mammalian cells. Digitonin selectively permeabilizes the plasma membrane, removing cytosolic proteins while preserving the integrity of inner membranes like the ER and mitochondria. This approach enhances spatial resolution in proteo-mic analysis, enabling a more precise map for protein interactome. Using this method, we successfully achieved proximal labeling of ER-localized proteins REEP1 and REEP6 to decipher their interaction networks, demonstrating its applicability for studying membrane-associated interactions with greater clarity and reduced contamination.

Abnormal amyloid fibrils are characteristic features and common pathological mechanisms of various neurodegenerative diseases, often found in disease-related brain regions, leading to neuroinflammation and neuronal apoptosis. Many disease-associated amyloid fibrils consist of a rigid fibril core primarily composed of cross-β sheets, surrounded by a fuzzy coat formed by intrinsically disordered regions (IDR). Over the past two decades, substantial structural knowledge of the rigid fibril core has been accumulated through cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) based on cross-polarization. In contrast, the highly disordered conformations of the fuzzy coats have hindered their structural characterization. Here, we describe the application of two-dimensional (2D) heteronuclear single quantum coherence (HSQC) and three-dimensional (3D) HNCO, HNCA, and HN(CO)CA spectra, utilizing the scalar coupling-based ¹H detection magic angle spinning (MAS) ssNMR techniques for backbone assignment of the IDR in amyloid fibrils, with the aim of further elucidating the conformational changes of the IDR during ligand binding processes.

Identifying immunoglobulin (Ig) genes from antigen-specific B cells is crucial for understanding immune responses and generating monoclonal antibodies for diagnostic and therapeutic purposes. Despite single B cell PCR-based mouse antibody development is well established, several practical challenges remain. Here, we present an optimized protocol for the sequencing and cloning of variable regions of antibodies from single antigen-specific mouse B cells, along with high-throughput antibody expression and characterization. This method builds upon existing techniques, incorporating laboratory refinements and detailed troubleshooting insights. By integrating fluorescence-activated cell sorting (FACS) with reverse transcription polymerase chain reaction (RT-PCR) to amplify immunoglobulin heavy and light chain genes, along with a 12-well format for antibody expression, our refined approach enables efficient monoclonal antibody production and functional screening, thereby accelerating the antibody discovery workflow across a range of experimental applications.

Algae, especially microalgae, are versatile in terms of nutrition, feed, biofertilizers, biofuel, and so on. In the realm of oncotherapy, algal extracts have been extensively used as anti-cancer active ingredients; however, what has been ignored is the anti-cancer value induced by themselves. Thanks to their unparalleled advantages, for example, intrinsic tumor homing, immunogenicity, and in situ production of anti-cancer agents, algae pave a new way in anti-cancer research. Algae have been reported about selective cytotoxicity to cancer cells and could work for oxygen-dependent strategies such as photodynamic therapy, owing to their natural photosynthetic abilities. Interestingly, integrating with customized nanomaterials (NMs), algae have been demonstrated to have unprecedented potential in overcoming barriers to existing treatment methods. Thus, in this review, starting from the classification of algae, the diverse effects of algae are thoroughly introduced, followed by the current engineering strategies of algae; lastly, the emerging development of algae-based therapeutics is timely summarized with an emphasis on the intelligent creation of biohybrid systems by choosing algae and tailored NMs. This review presents a comprehensive exploration of engineered algae-involved innovative cancer therapy, with a discussion of the future challenges and outlook, which will help design creative therapy paradigms and facilitate their clinical applications.

In bacterial DNA replication, helicase DnaB and primase DnaG form the primosome. Helicase DnaB unwinds double-stranded DNA (dsDNA) to provide templates for DNA polymerase, whereas primase DnaG supplies RNA primers to DNA polymerase for the synthesis of Okazaki fragments. How primase DnaG coordinates with helicase DnaB at the DNA replication fork remains unclear. In this study, the interactions between the helicase-binding domain of DnaG (DnaG (HBD)) and DnaB hexamer were studied. A stable ternary complex of DnaB₆/dT₁₆/DnaG(HBD) from Bacillus stearothermophilus was prepared and the homogeneity of the DnaB₆/dT₁₆/DnaG(HBD) complex was verified by dynamic light scattering. The stoichiometry of DnaG(HBD) to process DnaB₆ was investigated by isothermal titration calorimetry. The results show that a single primase DnaG binds to DnaB₆ in the presence of single-stranded DNA. Based on these results, a model is proposed to explain how the primase DnaG couples with the processing DnaB₆ helicase during the Okazaki fragment synthesis cycle. These findings provide valuable insights into the coupling between dsDNA unwinding and RNA primer synthesis in DNA replication.