Search

Surveillance of SARS-CoV-2 infection is critical for controlling the current pandemic. Antigen rapid tests (ARTs) provide a means for surveillance. Available lateral flow assay format ARTs rely heavily on nitrocellulose paper, raising challenges in supply shortage. Vertical flow assay (VFA) with cellulose paper as test material attracts much attention as a complementary test approach. However, current reported VFAs are facing challenges in reading the test signal from the bottom face of the test cassette, complicating the test workflow and hindering translation into rapid test application. Here, we address this gap with an enhanced VFA against SARS-CoV-2 N protein that adapts a cellulose pull-down test format allowing (1) one-step sample application at the top of the test cassette and (2) readout of the test signal from the top. We also demonstrate the feasibility of translating the enhanced VFA into a point-of-care application that can help in SARS-CoV-2 surveillance.

Neutralizing antibodies (NAbs) prevent pathogens from infecting host cells. Detection of SARS-CoV-2 NAbs is critical to evaluate herd immunity and monitor vaccine efficacy against SARS-CoV-2, the virus that causes COVID-19. All currently available NAb tests are lab-based and time-intensive. We develop a 10 min cellulose pull-down test to detect NAbs against SARS-CoV-2 from human plasma. The test evaluates the ability of antibodies to disrupt ACE2 receptor—RBD complex formation. The simple, portable, and rapid testing process relies on two key technologies: (i) the vertical-flow paper-based assay format and (ii) the rapid interaction of cellulose binding domain to cellulose paper. Here we show the construction of a cellulose-based vertical-flow test. The developed test gives above 80% sensitivity and specificity and up to 93% accuracy as compared to two current lab-based methods using COVID-19 convalescent plasma. A rapid 10 min cellulose based test has been developed for detection of NAb against SARS-CoV-2. The test demonstrates comparable performance to the lab-based tests and can be used at Point-of-Care. Importantly, the approach used for this test can be easily extended to test RBD variants or to evaluate NAbs against other pathogens. In response to infections, the human body produces proteins called antibodies. Neutralizing antibodies (NAbs) are one type of such proteins that are capable of inactivating the target, such as the SARS-CoV-2 virus that causes COVID-19. Monitoring levels of NAb allows us to understand levels of protective immunity. However, current methods to measure NAb are laboratory-based and are not necessarily suitable for large scale NAb monitoring in a large population. We develop a rapid test to detect SARS-CoV-2 NAb in 10 min that can be operated outside a laboratory. Our test provides results that are comparable to lab-based tests, which require between 1 h and up to 2 days to get a result. Our test may be useful for large-scale monitoring of immunity, for example in populations that do not have routine access to a lab. Kongsuphol et al. develop a paper-based, vertical flow assay to detect SARS-CoV-2 neutralizing antibodies. The point-of-care assay has comparable performance to lab-based tests and provides results in 10 min.

There is clinical need for a quantifiable point-of-care (PoC) SARS-CoV-2 neutralizing antibody (nAb) test that is adaptable with the pandemic's changing landscape. Here, we present a rapid and semi-quantitative nAb test that uses finger stick or venous blood to assess the nAb response of vaccinated population against wild-type (WT), alpha, beta, gamma, and delta variant RBDs. It captures a clinically relevant range of nAb levels, and effectively differentiates prevaccination, post first dose, and post second dose vaccination samples within 10 min. The data observed against alpha, beta, gamma, and delta variants agrees with published results evaluated in established serology tests. Finally, our test revealed a substantial reduction in nAb level for beta, gamma, and delta variants between early BNT162b2 vaccination group (within 3 months) and later vaccination group (post 3 months). This test is highly suited for PoC settings and provides an insightful nAb response in a postvaccinated population. Ministry of Health (MOH) National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This study is supported by National Health Innovation Singapore (NHIC) grant # NHIC-COVID19-2005004, National Research Foundation via CREATE Share grant #R571-002-021-592 and the Anti-microbial Resistance Interdisciplinary Research Group (AMR-IRG) of Singapore-MIT Alliance in Research and Technology (SMART). All samples acquired from National Centre for Infectious Diseases (NCID) were supported by Singapore Ministry of Health's National Medical Research Council COVID-19 Research Fund: COVID19RF-0008.

The nuclear position in eukaryotic cells is controlled by a nucleo-cytoskeletal network, with important roles in cell differentiation, division and movement. Forces are transmitted through conserved linker of nucleoskeleton and cytoskeleton (LINC) complexes that traverse the nuclear envelope and engage on either side of the membrane with diverse binding partners. Nesprin-2 giant (Nes2G), a LINC element in the outer nuclear membrane, connects to the actin network directly as well as through FHOD1, a formin whose major activity is bundling actin. Much of the molecular details of this process remain poorly understood. Here, we report the crystal structure of Nes2G bound to FHOD1. We show that the G-binding domain of FHOD1 is rather a spectrin repeat binding enhancer for the neighboring FH3 domain, possibly establishing a common binding mode among this subclass of formins. The FHOD1-Nes2G complex structure suggests that spectrin repeat binding by FHOD1 is likely not regulated by the DAD helix of FHOD1. Finally, we establish that Nes1G also has one FHOD1 binding spectrin repeat, indicating that these abundant, giant Nesprins have overlapping functions in actin-bundle recruitment for nuclear movement.

Human sphingomyelinase phosphodiesterase like 3a (SMPDL3a) is a secreted enzyme that shares a conserved catalytic domain with human acid sphingomyelinase (aSMase), the enzyme carrying mutations causative of Niemann–Pick disease. We have solved the structure of SMPDL3a revealing a calcineurin-like fold. A dimetal site, glycosylation pattern and a disulfide bond network are likely to be conserved also in human aSMase. We show that the binuclear site of SMPDL3a is occupied by two Zn2+ ions and that excess Zn2+ leads to inhibition of enzyme activity through binding to additional sites. As an extension of recent biochemical work we uncovered that SMPDL3a catalyses the hydrolysis of several modified nucleotides that include cytidine 5′-diphosphocholine, cytidine diphosphate ethanolamine and ADP-ribose, but not the aSMase substrate, sphingomyelin. We subsequently determined the structure of SMPDL3a in complex with the product 5′-cytidine monophosphate (CMP), a structure that is consistent with several distinct coordination modes of the substrate/product in the active site during the reaction cycle. Based on the structure of CMP complexes, we propose a phosphoryl transfer mechanism for SMPDL3a. Finally, a homology model of human aSMase was constructed to allow for the mapping of selected Niemann–Pick disease mutations on a three-dimensional framework to guide further characterization of their effects on aSMase function. Database Structural data are available in the PDB database under the accession numbers 5EBB and 5EBE.

Lipocalin prostaglandin D synthase (L-PGDS) regulates synthesis of an important inflammatory and signaling mediator, prostaglandin D2 (PGD2). Here, we used structural, biophysical, and biochemical approaches to address the mechanistic aspects of substrate entry, catalysis, and product exit of this enzyme. Structure of human L-PGDS was solved in a complex with a substrate analog (SA) and in ligand-free form. Its catalytic Cys 65 thiol group was found in two different conformations, each making a distinct hydrogen bond network to neighboring residues. These help in elucidating the mechanism of the cysteine nucleophile activation. Electron density for ligand observed in the active site defined the substrate binding regions, but did not allow unambiguous fitting of the SA. To further understand ligand binding, we used NMR spectroscopy to map the binding sites and to show the dynamics of protein-substrate and protein-product interactions. A model for ligand binding at the catalytic site is proposed, showing a second binding site involved in ligand exit and entry. NMR chemical shift perturbations and NMR resonance line-width alterations (observed as changes of intensity in two-dimensional cross-peaks in [1H,15N]-transfer relaxation optimization spectroscopy) for residues at the Ω loop (A-B loop), E-F loop, and G-H loop besides the catalytic sites indicate involvement of these residues in ligand entry/egress.