Targeted protein S-nitrosylation of ACE2 inhibits SARS-CoV-2 infection.

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.

Breaking Down the Bottlebrush: Atomically Detailed Structural Dynamics of Mucins.

Mucins, the biomolecular components of mucus, are glycoproteins that form a thick physical barrier at all tissue-air interfaces, forming a first line of defense against pathogens. Structural features of mucins and their interactions with other biomolecules remain largely unexplored due to the challenges associated with their high-resolution characterization. Combining limited mass spectrometry glycomics and protein sequencing data, we present all-atom, explicitly solvated molecular dynamics simulations of a major respiratory mucin, MUC5B. We detail key forces and degrees of freedom imposed by the extensive O-glycosylation, which imbue the canonically observed bottlebrush-like structures to these otherwise intrinsically disordered protein backbones. We compare our simulation results to static structures observed in recent scanning tunneling microscopy experiments as well as other published experimental efforts. Our work represents the demonstration of a workflow applied to a mucin example, which we hope will be employed by other groups to investigate the dynamics and interactions of other mucins, which can inform on structural details currently inaccessible to experimental techniques.

Simulation-driven design of stabilized SARS-CoV-2 spike S2 immunogens.

The full-length prefusion-stabilized SARS-CoV-2 spike (S) is the principal antigen of COVID-19 vaccines. Vaccine efficacy has been impacted by emerging variants of concern that accumulate most of the sequence modifications in the immunodominant S1 subunit. S2, in contrast, is the most evolutionarily conserved region of the spike and can elicit broadly neutralizing and protective antibodies. Yet, S2's usage as an alternative vaccine strategy is hampered by its general instability. Here, we use a simulation-driven approach to design S2-only immunogens stabilized in a closed prefusion conformation. Molecular simulations provide a mechanistic characterization of the S2 trimer's opening, informing the design of tryptophan substitutions that impart kinetic and thermodynamic stabilization. Structural characterization via cryo-EM shows the molecular basis of S2 stabilization in the closed prefusion conformation. Informed by molecular simulations and corroborated by experiments, we report an engineered S2 immunogen that exhibits increased protein expression, superior thermostability, and preserved immunogenicity against sarbecoviruses.

SARS-CoV-2 evolved variants optimize binding to cellular glycocalyx.

Viral variants of concern continue to arise for SARS-CoV-2, potentially impacting both methods for detection and mechanisms of action. Here, we investigate the effect of an evolving spike positive charge in SARS-CoV-2 variants and subsequent interactions with heparan sulfate and the angiotensin converting enzyme 2 (ACE2) in the glycocalyx. We show that the positively charged Omicron variant evolved enhanced binding rates to the negatively charged glycocalyx. Moreover, we discover that while the Omicron spike-ACE2 affinity is comparable to that of the Delta variant, the Omicron spike interactions with heparan sulfate are significantly enhanced, giving rise to a ternary complex of spike-heparan sulfate-ACE2 with a large proportion of double-bound and triple-bound ACE2. Our findings suggest that SARS-CoV-2 variants evolve to be more dependent on heparan sulfate in viral attachment and infection. This discovery enables us to engineer a second-generation lateral-flow test strip that harnesses both heparin and ACE2 to reliably detect all variants of concern, including Omicron.

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE.

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. In particular, the T cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, -3, -4) decorate immune cells and act as key checkpoint inhibitors in cancer. However, their dense O-glycosylation remains enigmatic both in terms of glycoproteomic landscape and structural dynamics, primarily due to the challenges associated with studying mucin domains. Here, we present a mucinase (SmE) and demonstrate its ability to selectively cleave along the mucin glycoprotein backbone, similar to others of its kind. Unlike other mucinases, though, SmE harbors the unique ability to cleave at residues bearing extremely complex glycans which enabled improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we performed molecular dynamics (MD) simulations of TIM-3 and -4 to demonstrate how glycosylation affects structural features of these proteins. Overall, we present a powerful workflow to better understand the detailed molecular structures of the mucinome.

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE.

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. In particular, the T cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, -3, -4) decorate immune cells and act as key regulators in cellular immunity. However, their dense O-glycosylation remains enigmatic, primarily due to the challenges associated with studying mucin domains. Here, we demonstrate that the mucinase SmE has a unique ability to cleave at residues bearing very complex glycans. SmE enables improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we perform molecular dynamics (MD) simulations of TIM-3 and -4 to understand how glycosylation affects structural features of these proteins. Finally, we use these models to investigate the functional relevance of glycosylation for TIM-3 function and ligand binding. Overall, we present a powerful workflow to better understand the detailed molecular structures and functions of the mucinome.

Spike-heparan sulfate interactions in SARS-CoV-2 infection.

Recent biochemical, biophysical, and genetic studies have shown that heparan sulfate, a major component of the cellular glycocalyx, participates in infection of SARS-CoV-2 by facilitating the so-called open conformation of the spike protein, which is required for binding to ACE2. This review highlights the involvement of heparan sulfate in the SARS-CoV-2 infection cycle and argues that there is a high degree of coordination between host cell heparan sulfate and asparagine-linked glycans on the spike in enabling ACE2 binding and subsequent infection. The discovery that spike protein binding and infection depends on both viral and host glycans provides insights into the evolution, spread and potential therapies for SARS-CoV-2 and its variants.

GlycoGrip: Cell Surface-Inspired Universal Sensor for Betacoronaviruses.

Inspired by the role of cell-surface glycoproteins as coreceptors for pathogens, we report the development of GlycoGrip: a glycopolymer-based lateral flow assay for detecting SARS-CoV-2 and its variants. GlycoGrip utilizes glycopolymers for primary capture and antispike antibodies labeled with gold nanoparticles for signal-generating detection. A lock-step integration between experiment and computation has enabled efficient optimization of GlycoGrip test strips which can selectively, sensitively, and rapidly detect SARS-CoV-2 and its variants in biofluids. Employing the power of the glycocalyx in a diagnostic assay has distinct advantages over conventional immunoassays as glycopolymers can bind to antigens in a multivalent capacity and are highly adaptable for mutated strains. As new variants of SARS-CoV-2 are identified, GlycoGrip will serve as a highly reconfigurable biosensor for their detection. Additionally, via extensive ensemble-based docking simulations which incorporate protein and glycan motion, we have elucidated important clues as to how heparan sulfate and other glycocalyx components may bind the spike glycoprotein during SARS-CoV-2 host-cell infection. GlycoGrip is a promising and generalizable alternative to costly, labor-intensive RT-PCR, and we envision it will be broadly useful, including for rural or low-income populations that are historically undertested and under-reported in infection statistics.

Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection.

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

NIDCD/VA hearing aid clinical trial and follow-up: background.

This report provides background regarding the Long Term Follow-Up of Patients in the NIDCD/VA Hearing Aid Clinical Trial study and serves as an introduction to the detailed reports that follow in this issue of Journal of the American Academy of Audiology. The authors investigated five- to seven-year benefit/satisfaction in participants from the original NIDCDNA Hearing Aid Clinical Trial. The new study was designed to investigate current use of the original study hearing aids, to compare changes in selected audiological measures, and to assess possible predictors of long-term hearing aid use. The outcome measures included estimates of speech intelligibility in quiet and noise, self-reported patterns of hearing aid usage, self-reported estimates of activity limitations and quality-of-life issues, estimates of hearing aid satisfaction, and self-reported hearing aid benefit. Overall, the short-term benefits of hearing aid use observed during the original trial were noted to persist in the long term.

NIDCD/VA hearing aid clinical trial and follow-up: coupler and real-ear measurement.

A total of 190 individuals participated in a clinical visit during the Cooperative Studies Program (CSP) 418-A Long Term Follow-Up Study. Of this cohort, 158 participants were considered current hearing aid users, and 32 were non-hearing aid users. Of the current hearing aid users, 81 were still using their original 418 study devices, and 77 had acquired new hearing aids. Coupler and real ear measurements were completed on all available hearing aids. Results showed that study aids had remained relatively stable over the six years between CSP 418 and CSP 418-A. On average, these hearing aid wearers preferred use gain settings that were 6-9 dB less than current NAL-RP insertion gain targets. Mean real ear insertion gain (REIG) was comparable to the mean real ear insertion gain of the same participants in the original study, and users did not tend to increase gain as hearing decreased. Real ear saturation responses (RESR) remained unchanged. Loudness discomfort levels (LDL) obtained during 418-A were significantly lower than LDLs obtained on those same participants at both the initial and final visits in the previous study.

Coupler and real-ear measurement of hearing aid gain and output in the NIDCD/VA Hearing Aid Clinical Trial.

OBJECTIVE: Because the NIDCD/VA Hearing Aid Clinical Trial was conducted across eight clinical sites, rigorous control of the electroacoustic characteristics of the experimental devices was required. DESIGN: The parameters monitored included the gain and output of the approximately 720 hearing aids in the trial, measured both in the 2 cm3 coupler and in situ. Each measurement was repeated six times on each hearing aid across the 9-mo duration of the study to insure both the stability and the accuracy of the circuits under investigation. RESULTS: The gain data obtained in the coupler and in situ adequately demonstrated the stability of the instrument and the repeated measurements over time and across study sites. The output values produced by the experimental device also maintained acceptable constancy, both within and across treatment periods. CONCLUSIONS: These measurements reflected satisfactory stability and sufficient accuracy within the circuits to achieve the intended goals of the study.