O-Acetyl Migration within the Sialic Acid Side Chain: A Mechanistic Study Using the Ab Initio Nanoreactor.

Many disease-causing viruses target sialic acids on the surface of host cells. Some viruses bind preferentially to sialic acids with O-acetyl modification at the hydroxyl group of C7, C8, or C9 on the glycerol-like side chain. Studies of proteins binding to sialosides containing O-acetylated sialic acids are crucial in understanding the related diseases but experimentally difficult due to the lability of the ester group. We recently showed that O-acetyl migration among hydroxyl groups of C7, C8, and C9 in sialic acids occurs in all directions in a pH-dependent manner. In the current study, we elucidate a full mechanistic pathway for the migration of O-acetyl among C7, C8, and C9. We used an ab initio nanoreactor to explore potential reaction pathways and density functional theory, pKa calculations, and umbrella sampling to investigate elementary steps of interest. We found that when a base is present, migration is easy in any direction and involves three key steps: deprotonation of the hydroxyl group, cyclization between the two carbons, and the migration of the O-acetyl group. This dynamic equilibrium may play a defensive role against pathogens that evolve to gain entry to the cell by binding selectively to one acetylation state.

SARS-CoV-2 and MERS-CoV Spike Protein Binding Studies Support Stable Mimic of Bound 9-O-Acetylated Sialic Acids.

Many disease-causing viruses target sialic acids (Sias), a class of nine-carbon sugars known to coat the surface of many cells, including those in the lungs. Human beta coronaviridae, known for causing respiratory tract diseases, often bind Sias, and some preferentially bind to those with 9-O-Ac-modification. Currently, co-binding of SARS-CoV-2, a beta coronavirus responsible for the COVID-19 pandemic, to human Sias has been reported and its preference towards α2-3-linked Neu5Ac has been shown. Nevertheless, O-acetylated Sias-protein binding studies are difficult to perform, due to the ester lability. We studied the binding free energy differences between Neu5,9Ac2α2-3GalßpNP and its more stable 9-NAc mimic binding to SARS-CoV-2 spike protein using molecular dynamics and alchemical free energy simulations. We identified multiple Sia-binding pockets, including two novel sites, with similar binding affinities to those of MERS-CoV, a known co-binder of sialic acid. In our binding poses, 9-NAc and 9-OAc Sias bind similarly, suggesting an experimentally reasonable mimic to probe viral mechanisms.

An Audio Personal Health Library of Clinic Visit Recordings for Patients and Their Caregivers (HealthPAL): User-Centered Design Approach.

BACKGROUND: Providing digital recordings of clinic visits to patients has emerged as a strategy to promote patient and family engagement in care. With advances in natural language processing, an opportunity exists to maximize the value of visit recordings for patients by automatically tagging key visit information (eg, medications, tests, and imaging) and linkages to trustworthy web-based resources curated in an audio-based personal health library. OBJECTIVE: This study aims to report on the user-centered development of HealthPAL, an audio personal health library. METHODS: Our user-centered design and usability evaluation approach incorporated iterative rounds of video-recorded sessions from 2016 to 2019. We recruited participants from a range of community settings to represent older patient and caregiver perspectives. In the first round, we used paper prototypes and focused on feature envisionment. We moved to low-fidelity and high-fidelity versions of the HealthPAL in later rounds, which focused on functionality and use; all sessions included a debriefing interview. Participants listened to a deidentified, standardized primary care visit recording before completing a series of tasks (eg, finding where a medication was discussed in the recording). In the final round, we recorded the patients' primary care clinic visits for use in the session. Findings from each round informed the agile software development process. Task completion and critical incidents were recorded in each round, and the System Usability Scale was completed by participants using the digital prototype in later rounds. RESULTS: We completed 5 rounds of usability sessions with 40 participants, of whom 25 (63%) were women with a median age of 68 years (range 23-89). Feedback from sessions resulted in color-coding and highlighting of information tags, a more prominent play button, clearer structure to move between one's own recordings and others' recordings, the ability to filter recording content by the topic discussed and descriptions, 10-second forward and rewind controls, and a help link and search bar. Perceived usability increased over the rounds, with a median System Usability Scale of 78.2 (range 20-100) in the final round. Participants were overwhelmingly positive about the concept of accessing a curated audio recording of a clinic visit. Some participants reported concerns about privacy and the computer-based skills necessary to access recordings. CONCLUSIONS: To our knowledge, HealthPAL is the first patient-centered app designed to allow patients and their caregivers to access easy-to-navigate recordings of clinic visits, with key concepts tagged and hyperlinks to further information provided. The HealthPAL user interface has been rigorously co-designed with older adult patients and their caregivers and is now ready for further field testing. The successful development and use of HealthPAL may help improve the ability of patients to manage their own care, especially older adult patients who have to navigate complex treatment plans.

Reversible O-Acetyl Migration within the Sialic Acid Side Chain and Its Influence on Protein Recognition.

O-Acetylation is a common naturally occurring modification of carbohydrates and is especially widespread in sialic acids, a family of nine-carbon acidic monosaccharides. O-Acetyl migration within the exocyclic glycerol-like side chain of mono-O-acetylated sialic acid reported previously was from the C7- to C9-hydroxyl group with or without an 8-O-acetyl intermediate, which resulted in an equilibrium that favors the formation of the 9-O-acetyl sialic acid. Herein, we provide direct experimental evidence demonstrating that O-acetyl migration is bidirectional, and the rate of equilibration is influenced predominantly by the pH of the sample. While the O-acetyl group on sialic acids and sialoglycans is stable under mildly acidic conditions (pH < 5, the rate of O-acetyl migration is extremely low), reversible O-acetyl migration is observed readily at neutral pH and becomes more significant when the pH increases to slightly basic. Sialoglycan microarray studies showed that esterase-inactivated porcine torovirus hemagglutinin-esterase bound strongly to sialoglycans containing a more stable 9-N-acetylated sialic acid analog, but these compounds were less resistant to periodate oxidation treatment compared to their 9-O-acetyl counterparts. Together with prior studies, the results support the possible influence of sialic acid O-acetylation and O-acetyl migration to host-microbe interactions and potential application of the more stable synthetic N-acetyl mimics.

A combined NMR, MD and DFT conformational analysis of 9-O-acetyl sialic acid-containing GM3 ganglioside glycan and its 9-N-acetyl mimic.

O-Acetylation of carbohydrates such as sialic acids is common in nature, but its role is not clearly understood due to the lability of O-acetyl groups. We demonstrated previously that 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc) is a chemically and biologically stable mimic of the 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) of the corresponding sialoglycans. Here, a systematic nuclear magnetic resonance (NMR) spectroscopic and molecular dynamics (MD) simulation study was undertaken for Neu5,9Ac2-containing GM3 ganglioside glycan (GM3-glycan) and its Neu5Ac9NAc analog. GM3-glycan with Neu5Ac as the non-O-acetyl form of Neu5,9Ac2 was used as a control. Complete 1H and 13C NMR chemical shift assignments, three-bond 1H-13C trans-glycosidic coupling constants (3JCH), accurate 1H-1H coupling constants (3JHH), nuclear Overhauser effects and hydrogen bonding detection were carried out. Results show that structural modification (O- or N-acetylation) on the C-9 of Neu5Ac in GM3 glycan does not cause significant conformational changes on either its glycosidic dihedral angles or its secondary structure. All structural differences are confined to the Neu5Ac glycerol chain, and minor temperature-dependent changes are seen in the aglycone portion. We also used Density Functional Theory (DFT) quantum mechanical calculations to improve currently used 3JHH Karplus relations. Furthermore, OH chemical shifts were assigned at -10°C and no evidence of an intramolecular hydrogen bond was observed. The results provide additional evidence regarding structural similarities between sialosides containing 9-N-acetylated and 9-O-acetylated Neu5Ac and support the opportunity of using 9-N-acetylated Neu5Ac as a stable mimic to study the biochemical role of 9-O-acetylated Neu5Ac.