Development of a cellulose-based 96-well plate vertical flow pull-down assay.

The abundance and low production cost of biomaterial cellulose paper have attracted attention for many applications. Point-of-care (PoC) diagnostic tests have been successfully developed using patterned cellulose paper. Although PoC diagnostic tests are rapid and simple to perform, their sample processing throughput is limited, allowing for only one sample to be evaluated at a time, which restricts potential applications. Thus, it was appealing to expand cellulose-based PoC tests to high-throughput versions to increase their applicability. Here, we present the development of a high-throughput cellulose-based 96-well plate vertical flow pull-down assay that can process 96 tests, is easy to prepare, and can be customized for different detection targets. The device has two key features: (i) patterned cellulose paper for 96 tests that do not require pre-immobilization of capturing reagents, and (ii) reusable sturdy housing. We believe that a variety of applications, including laboratory testing, population surveillance tests, and sizable clinical trials for diagnostic tests, can benefit from the adoption of this cellulose-based 96-well plate assay.

Rapid Evaluation of Vaccine Booster Effectiveness against SARS-CoV-2 Variants.

As the COVID-19 pandemic continues, countries around the world are switching toward vaccinations and boosters to combat the pandemic. However, waning immunity against SARS-CoV-2 wild-type (WT) and variants have been widely reported. Booster vaccinations have shown to be able to increase immunological protection against new variants; however, the protection observed appears to decrease quickly over time suggesting a second booster shot may be appropriate. Moreover, heterogeneity and waning of the immune response at the individual level was observed suggesting a more personalized vaccination approach should be considered. To evaluate such a personalized strategy, it is important to have the ability to rapidly evaluate the level of neutralizing antibody (nAbs) response against variants at the individual level and ideally at a point of care setting. Here, we applied the recently developed cellulose pulled-down virus neutralization test (cpVNT) to rapidly assess individual nAb levels to WT and variants of concerns in response to booster vaccination. Our findings confirmed significant heterogeneity of nAb responses against a panel of SARS-CoV-2 variants, and indicated a strong increase in nAb response against variants of concern (VOCs) upon booster vaccination. For instance, the nAb response against current predominant omicron variant was observed with medians of 88.1% (n = 6, 95% CI = 73.2% to 96.2%) within 1-month postbooster and 70.7% (n = 22, 95% CI = 66.4% to 81.8%) 3 months postbooster. Our data show a point of care (POC) test focusing on nAb response levels against VOCs can guide decisions on the potential need for booster vaccinations at individual level. Importantly, it also suggests the current booster vaccines only give a transient protective response against some VOC and new more targeted formulations of a booster vaccine against specific VOC may need to be developed in the future. IMPORTANCE Vaccination against SARS-CoV-2 induces protection through production of neutralization antibodies (nAb). The level of nAb is a major indicator of immunity against SARS-CoV-2 infection. We developed a rapid point-of-care test that can monitor the nAb level from a drop of finger stick blood. Here, we have implemented the test to monitor individual nAb level against wild-type and variants of SARS-CoV-2 at various time points of vaccination, including post-second-dose vaccination and postbooster vaccination. Huge diversity of nAb levels were observed among individuals as well as increment in nAb levels especially against Omicron variant after booster vaccination. This study evaluated the performance of this point-of-care test for personalized nAb response tracking. It verifies the potential of using a rapid nAb test to guide future vaccination regimens at both the individual and population level.

Finger stick blood test to assess postvaccination SARS-CoV-2 neutralizing antibody response against variants.

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.

Development and translation of a paper-based top readout vertical flow assay for SARS-CoV-2 surveillance.

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.

Structures of FHOD1-Nesprin1/2 complexes reveal alternate binding modes for the FH3 domain of formins.

The nuclear position in eukaryotes is controlled by a nucleo-cytoskeletal network, critical 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 directly as well as through FHOD1, a formin primarily involved in actin bundling. Here, we report the crystal structure of Nes2G bound to FHOD1 and show that the presumed G-binding domain of FHOD1 is rather a spectrin repeat (SR) binding enhancer for the neighboring FH3 domain. The structure reveals that SR binding by FHOD1 is likely not regulated by the diaphanous-autoregulatory domain helix of FHOD1. Finally, we establish that Nes1G also has one FHOD1 binding SR, indicating that these abundant, giant Nesprins have overlapping functions in actin-bundle recruitment for nuclear movement.

A rapid simple point-of-care assay for the detection of SARS-CoV-2 neutralizing antibodies.

Background: 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. Method: 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. Results: 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. Conclusions: 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.

The structure and catalytic mechanism of human sphingomyelin phosphodiesterase like 3a--an acid sphingomyelinase homologue with a novel nucleotide hydrolase activity.

UNLABELLED: 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 Zn(2+) ions and that excess Zn(2+) 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.

Structural and dynamic insights into substrate binding and catalysis of human lipocalin prostaglandin D synthase.

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.