Snoring-generated fluid droplets as a potential mechanistic link between sleep-disordered breathing and pneumonia.

The soft palate and back of the throat represent vulnerable early infection sites for SARS-CoV-2, influenza, streptococci, and many other pathogens. We demonstrate that snoring causes aerosolization of pharyngeal fluid that covers these surfaces, which previously has escaped detection because the inspired airstream carries the micron-sized droplets into the lung, inaccessible to traditional aerosol detectors. While many of these droplets will settle in the lower respiratory tract, a fraction of the respirable smallest droplets remains airborne and can be detected in exhaled breath. We distinguished these exhaled droplets from those generated by the underlying breathing activity by using a chemical tracer, thereby proving their existence. The direct transfer of pharyngeal fluids and their pathogens into the deep lung by snoring represents a plausible mechanistic link between the previously recognized association between sleep-disordered breathing and pneumonia incidence.

Targeting the Main Protease (Mpro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies.

The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.

Snoring may transmit infectious aerosols from the upper to the lower respiratory tract.

Migration to the lungs of an initial upper airway infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or other respiratory pathogens can lead to pneumonia, associated with progression from mild to severe symptoms. Chemical pneumonitis or bacterial pneumonia may be caused by the 'macroaspiration' of large volumes of oropharyngeal or gastroesophageal secretions into the lower respiratory tract. 'Microaspiration', i.e., a similar mechanism but involving much smaller amounts of oropharyngeal secretions, is considered the pathogenetic mechanism for most pneumonias, including that associated with COVID-19. Here, we hypothesize an alternative mechanism: Rather than by microaspiration, these fluids enter the lungs as microdroplets that are generated by snoring and then carried by the inspired airstream. Laboratory measurements indicate that snoring generates (a) comparable numbers and sizes of oral fluid droplets as loud speaking and (b) total fluid quantities that are similar to those reported for microaspiration. Snoring propensity is strongly correlated to known risk factors for severe COVID-19, including male gender, age, obesity, diabetes, obstructive sleep apnea, and pregnancy. Therefore, more research is urgently needed to determine if various methods that decrease snoring can prevent progression to pneumonia after initial infection of the upper airways.

Hybrid measurement of respiratory aerosol reveals a dominant coarse fraction resulting from speech that remains airborne for minutes.

Accurate measurements of the size and quantity of aerosols generated by various human activities in different environments are required for efficacious mitigation strategies and accurate modeling of respiratory disease transmission. Previous studies of speech droplets, using standard aerosol instrumentation, reported very few particles larger than 5 µm. This starkly contrasts with the abundance of such particles seen in both historical slide deposition measurements and more recent light scattering observations. We have reconciled this discrepancy by developing an alternative experimental approach that addresses complications arising from nucleated condensation. Measurements reveal that a large volume fraction of speech-generated aerosol has diameters in the 5- to 20-µm range, making them sufficiently small to remain airborne for minutes, not hours. This coarse aerosol is too large to penetrate the lower respiratory tract directly, and its relevance to disease transmission is consistent with the vast majority of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiating in the upper respiratory tract. Our measurements suggest that in the absence of symptoms such as coughing or sneezing, the importance of speech-generated aerosol in the transmission of respiratory diseases is far greater than generally recognized.

Quantitative detection of hydrogen peroxide in rain, air, exhaled breath, and biological fluids by NMR spectroscopy.

Hydrogen peroxide (H2O2) plays a key role in environmental chemistry, biology, and medicine. H2O2 concentrations typically are 6 to 10 orders of magnitude lower than that of water, making its quantitative detection challenging. We demonstrate that optimized NMR spectroscopy allows direct, interference-free, quantitative measurements of H2O2 down to submicromolar levels in a wide range of fluids, ranging from exhaled breath and air condensate to rain, blood, urine, and saliva. NMR measurements confirm the previously reported spontaneous generation of H2O2 in microdroplets that form when condensing water vapor on a hydrophobic surface, which can interfere with atmospheric H2O2 measurements. Its antimicrobial activity and strong seasonal variation speculatively could be linked to the seasonality of respiratory viral diseases.

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

The airborne lifetime of small speech droplets and their potential importance in SARS-CoV-2 transmission.

Speech droplets generated by asymptomatic carriers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are increasingly considered to be a likely mode of disease transmission. Highly sensitive laser light scattering observations have revealed that loud speech can emit thousands of oral fluid droplets per second. In a closed, stagnant air environment, they disappear from the window of view with time constants in the range of 8 to 14 min, which corresponds to droplet nuclei of ca. 4 µm diameter, or 12- to 21-µm droplets prior to dehydration. These observations confirm that there is a substantial probability that normal speaking causes airborne virus transmission in confined environments.

Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell.

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-µs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 µM, oligomeric off-pathway intermediates compete with folding of the native state.

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Dissociation of the trimeric gp41 ectodomain at the lipid-water interface suggests an active role in HIV-1 Env-mediated membrane fusion.

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. The actual fusion process involves a switch from a homotrimeric prehairpin intermediate conformation, consisting of parallel coiled-coil helices, to a postfusion state where the ectodomains are arranged as a trimer of helical hairpins, adopting a six-helix bundle (6HB) state. Here, we show by solution NMR spectroscopy that a water-soluble 6HB gp41 ectodomain binds to zwitterionic detergents that contain phosphocholine or phosphatidylcholine head groups and phospholipid vesicles that mimic T-cell membrane composition. Binding results in the dissociation of the 6HB and the formation of a monomeric state, where its two α-helices, N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), become embedded in the lipid-water interface of the virus and host cell. The atomic structure of the gp41 ectodomain monomer, based on NOE distance restraints and residual dipolar couplings, shows that the NHR and CHR helices remain mostly intact, but they completely lose interhelical contacts. The high affinity of the ectodomain helices for phospholipid surfaces suggests that unzippering of the prehairpin intermediate leads to a state where the NHR and CHR helices become embedded in the host cell and viral membranes, respectively, thereby providing a physical force for bringing these membranes into close juxtaposition before actual fusion.

pH-triggered, activated-state conformations of the influenza hemagglutinin fusion peptide revealed by NMR.

The highly conserved first 23 residues of the influenza hemagglutinin HA2 subunit constitute the fusion domain, which plays a pivotal role in fusing viral and host-cell membranes. At neutral pH, this peptide adopts a tight helical hairpin wedge structure, stabilized by aliphatic hydrogen bonding and charge-dipole interactions. We demonstrate that at low pH, where the fusion process is triggered, the native peptide transiently visits activated states that are very similar to those sampled by a G8A mutant. This mutant retains a small fraction of helical hairpin conformation, in rapid equilibrium with at least two open structures. The exchange rate between the closed and open conformations of the wild-type fusion peptide is ~40 kHz, with a total open-state population of ~20%. Transitions to these activated states are likely to play a crucial role in formation of the fusion pore, an essential structure required in the final stage of membrane fusion.