Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca ²⁺ release-activated Ca ²⁺ (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orai1 and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orai1 tandem multimers and a fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orai1 molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orai1 subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca ²⁺ stores were depleted, the channels clustered in aggregates containing ≈1,300 channels, amplifying the local Ca ²⁺ entry.

The rapid spread of coronavirus SARS-CoV-2 greatly threatens global public health but no prophylactic vaccine is available. Here, we report the generation of a replication-incompetent recombinant serotype 5 adenovirus, Ad5-S-nb2, carrying a codon-optimized gene encoding Spike protein (S). In mice and rhesus macaques, intramuscular injection with Ad5-S-nb2 elicits systemic S-specific antibody and cell-mediated immune (CMI) responses. Intranasal inoculation elicits both systemic and pulmonary antibody responses but weaker CMI response. At 30 days after a single vaccination with Ad5-S-nb2 either intramuscularly or intranasally, macaques are protected against SARS-CoV-2 challenge. A subsequent challenge reveals that macaques vaccinated with a 10-fold lower vaccine dosage (1 × 10¹⁰ viral particles) are also protected, demonstrating the effectiveness of Ad5-S-nb2 and the possibility of offering more vaccine dosages within a shorter timeframe. Thus, Ad5-S-nb2 is a promising candidate vaccine and warrants further clinical evaluation.

Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.

Neuropeptides play a variety of roles in many physiological processes and serve as potential therapeutic targets for the treatment of some nervous-system disorders. In recent years, there has been a tremendous increase in the number of identified neuropeptides. Therefore, we have developed NeuroPep, a comprehensive resource of neuropeptides, which holds 5949 non-redundant neuropeptide entries originating from 493 organisms belonging to 65 neuropeptide families. In NeuroPep, the number of neuropeptides in invertebrates and vertebrates is 3455 and 2406, respectively. It is currently the most complete neuropeptide database. We extracted entries deposited in UniProt, the database ( www.neuropeptides.nl ) and NeuroPedia, and used text mining methods to retrieve entries from the MEDLINE abstracts and full text articles. All the entries in NeuroPep have been manually checked. 2069 of the 5949 (35%) neuropeptide sequences were collected from the scientific literature. Moreover, NeuroPep contains detailed annotations for each entry, including source organisms, tissue specificity, families, names, post-translational modifications, 3D structures (if available) and literature references. Information derived from these peptide sequences such as amino acid compositions, isoelectric points, molecular weight and other physicochemical properties of peptides are also provided. A quick search feature allows users to search the database with keywords such as sequence, name, family, etc., and an advanced search page helps users to combine queries with logical operators like AND/OR. In addition, user-friendly web tools like browsing, sequence alignment and mapping are also integrated into the NeuroPep database. Database URL : http://isyslab.info/NeuroPep