Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc fusion proteins.

Coronavirus disease 2019 (COVID-19) is a newly emerged human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In a global pandemic, development of a cheap, rapid, accurate, and easy-to-use diagnostic test is necessary if we are to mount an immediate response to this emerging threat. Here, we report the development of a specific lateral flow immunoassay (LFIA)-based biosensor for COVID-19. We used phage display technology to generate four SARS-CoV-2 nucleocapsid protein (NP)-specific single-chain variable fragment-crystallizable fragment (scFv-Fc) fusion antibodies. The scFv-Fc antibodies bind specifically and with high affinity to the SARS-CoV-2 NP antigen, but not to NPs of other coronaviruses. Using these scFv-Fc antibodies, we screened three diagnostic antibody pairs for use on a cellulose nanobead (CNB)-based LFIA platform. The detection limits of the best scFv-Fc antibody pair, 12H1 as the capture probe and 12H8 as the CNB-conjugated detection probe, were 2 ng antigen protein and 2.5 × 104 pfu cultured virus. This LFIA platform detected only SARS-CoV-2 NP, not NPs from MERS-CoV, SARS-CoV, or influenza H1N1. Thus, we have successfully developed a SARS-CoV-2 NP-specific rapid diagnostic test, which is expected to be a simple and rapid diagnostic test for COVID-19.

NMR spectroscopy uncovers direct interaction between BAF60A and p53.

The BRG1-associated factor 60A (BAF60A), an SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1, has been known to be important for transcriptional activation and inhibition through the alteration of the DNA nucleosome. Although the association between BAF60A and p53 plays a critical role in tumor suppression, the interaction mode is still unclear. Here, we report the detailed interactions between BAF60A and p53 by both NMR spectroscopy and pull-down analysis. Both N-terminal region (BAF60ANR) and the SWIB domain (BAF60ASWIB) of BAF60A directly interact with the tetramerization domain of p53 (p53TET). NMR data show that Ile315, Met366, Ala388, and Tyr390 of BAF60ASWIB are mostly involved in p53TET binding. The calculated dissociation constant (KD) value between BAF60ASWIB and p53TET revealed relatively weak binding affinity, at approximately 0.3 ± 0.065 mM. Our data will enhance detailed interaction mechanism to elucidate the molecular basis of p53-mediated integration via BAF60A interaction.

Pumping mechanism of NM-R3, a light-driven bacterial chloride importer in the rhodopsin family.

A newly identified microbial rhodopsin, NM-R3, from the marine flavobacterium Nonlabens marinus, was recently shown to drive chloride ion uptake, extending our understanding of the diversity of mechanisms for biological energy conversion. To clarify the mechanism underlying its function, we characterized the crystal structures of NM-R3 in both the dark state and early intermediate photoexcited states produced by laser pulses of different intensities and temperatures. The displacement of chloride ions at five different locations in the model reflected the detailed anion-conduction pathway, and the activity-related key residues-Cys105, Ser60, Gln224, and Phe90-were identified by mutation assays and spectroscopy. Comparisons with other proteins, including a closely related outward sodium ion pump, revealed key motifs and provided structural insights into light-driven ion transport across membranes by the NQ subfamily of rhodopsins. Unexpectedly, the response of the retinal in NM-R3 to photostimulation appears to be substantially different from that seen in bacteriorhodopsin.

Non-cryogenic structure of a chloride pump provides crucial clues to temperature-dependent channel transport efficiency.

Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.

Side branching and luminal lineage commitment by ID2 in developing mammary glands.

Mammary glands develop through primary ductal elongation and side branching to maximize the spatial area. Although primary ducts are generated by bifurcation of terminal end buds, the mechanism through which side branching occurs is still largely unclear. Here, we show that inhibitor of DNA-binding 2 (ID2) drives side branch formation through the differentiation of K6+ bipotent progenitor cells (BPs) into CD61+ luminal progenitor cells (LPs). Id2-null mice had side-branching defects, along with developmental blockage of the differentiation of K6+ BPs into CD61+ LPs. Notably, CD61+ LPs were found in budding and side branches, but not in terminal end buds. Hormone reconstitution studies using ovariectomized MMTV-hemagglutinin-nuclear localized sequence-tagged Id2 transgenic mice revealed that ID2 is a key mediator of progesterone, which drives luminal lineage differentiation and side branching. Our results suggest that CD61 is a marker of side branches and that ID2 regulates side branch formation by inducing luminal lineage commitment from K6+ BPs to CD61+ LPs.