Mucin glycans drive oral microbial community composition and function.

Human microbiome composition is closely tied to health, but how the host manages its microbial inhabitants remains unclear. One important, but understudied, factor is the natural host environment: mucus, which contains gel-forming glycoproteins (mucins) that display hundreds of glycan structures with potential regulatory function. Leveraging a tractable culture-based system to study how mucins influence oral microbial communities, we found that mucin glycans enable the coexistence of diverse microbes, while resisting disease-associated compositional shifts. Mucins from tissues with unique glycosylation differentially tuned microbial composition, as did isolated mucin glycan libraries, uncovering the importance of specific glycan patterns in microbiome modulation. We found that mucins shape microbial communities in several ways: serving as nutrients to support metabolic diversity, organizing spatial structure through reduced aggregation, and possibly limiting antagonism between competing taxa. Overall, this work identifies mucin glycans as a natural host mechanism and potential therapeutic intervention to maintain healthy microbial communities.

Comparison of Physicochemical Properties of Native Mucus and Reconstituted Mucin Gels.

Simulating native mucus with model systems such as gels made from reconstituted mucin or commercially available polymers presents experimental advantages including greater sample availability and reduced inter- and intradonor heterogeneity. Understanding whether these gels reproduce the complex physical and biochemical properties of native mucus at multiple length scales is critical to building relevant experimental models, but few systematic comparisons have been reported. Here, we compared bulk mechanical properties, microstructure, and biochemical responses of mucus from different niches, reconstituted mucin gels (with similar pH and polymer concentrations as native tissues), and commonly used commercially available polymers. To evaluate gel properties across these length scales, we used small-amplitude oscillatory shear, single-particle tracking, and microaffinity chromatography with small analytes. With the exception of human saliva, the mechanical response of mucin gels was qualitatively similar to that of native mucus. The transport behavior of charged peptides through native mucus gels was qualitatively reproduced in gels composed of corresponding isolated mucins. Compared to native mucus, we observed substantial differences in the physicochemical properties of gels reconstituted from commercially available mucins and the substitute carboxymethylcellulose, which is currently used in artificial tear and saliva treatments. Our study highlights the importance of selecting a mucus model system guided by the length scale relevant to the scientific investigation or disease application.

Genome-wide association analysis of COVID-19 mortality risk in SARS-CoV-2 genomes identifies mutation in the SARS-CoV-2 spike protein that colocalizes with P.1 of the Brazilian strain.

SARS-CoV-2 mortality has been extensively studied in relation to host susceptibility. How sequence variations in the SARS-CoV-2 genome affect pathogenicity is poorly understood. Starting in October 2020, using the methodology of genome-wide association studies (GWAS), we looked at the association between whole-genome sequencing (WGS) data of the virus and COVID-19 mortality as a potential method of early identification of highly pathogenic strains to target for containment. Although continuously updating our analysis, in December 2020, we analyzed 7548 single-stranded SARS-CoV-2 genomes of COVID-19 patients in the GISAID database and associated variants with mortality using a logistic regression. In total, evaluating 29,891 sequenced loci of the viral genome for association with patient/host mortality, two loci, at 12,053 and 25,088 bp, achieved genome-wide significance (p values of 4.09e-09 and 4.41e-23, respectively), though only 25,088 bp remained significant in follow-up analyses. Our association findings were exclusively driven by the samples that were submitted from Brazil (p value of 4.90e-13 for 25,088 bp). The mutation frequency of 25,088 bp in the Brazilian samples on GISAID has rapidly increased from about 0.4 in October/December 2020 to 0.77 in March 2021. Although GWAS methodology is suitable for samples in which mutation frequencies varies between geographical regions, it cannot account for mutation frequencies that change rapidly overtime, rendering a GWAS follow-up analysis of the GISAID samples that have been submitted after December 2020 as invalid. The locus at 25,088 bp is located in the P.1 strain, which later (April 2021) became one of the distinguishing loci (precisely, substitution V1176F) of the Brazilian strain as defined by the Centers for Disease Control. Specifically, the mutations at 25,088 bp occur in the S2 subunit of the SARS-CoV-2 spike protein, which plays a key role in viral entry of target host cells. Since the mutations alter amino acid coding sequences, they potentially imposing structural changes that could enhance viral infectivity and symptom severity. Our analysis suggests that GWAS methodology can provide suitable analysis tools for the real-time detection of new more transmissible and pathogenic viral strains in databases such as GISAID, though new approaches are needed to accommodate rapidly changing mutation frequencies over time, in the presence of simultaneously changing case/control ratios. Improvements of the associated metadata/patient information in terms of quality and availability will also be important to fully utilize the potential of GWAS methodology in this field.

Home, sweet home: how mucus accommodates our microbiota.

As a natural environment for human-microbiota interactions, healthy mucus houses a remarkably stable and diverse microbial community. Maintaining this microbiota is essential to human health, both to support the commensal bacteria that perform a wide array of beneficial functions and to prevent the outgrowth of pathogens. However, how the host selects and maintains a specialized microbiota remains largely unknown. In this viewpoint, we propose several strategies by which mucus may regulate the composition and function of the human microbiota and discuss how compromised mucus barriers in disease can give rise to microbial dysbiosis.

Controlling Macroscopic Phase Separation of Aqueous Two-Phase Polymer Systems in Porous Media.

In previous work, our group discovered a phenomenon in which a mixed polymer-salt or mixed micellar aqueous two-phase system (ATPS) separates into its two constituent phases as it flows within paper. While these ATPSs worked well in their respective studies to concentrate the target biomarker and improve the sensitivity of the lateral-flow immunoassay, different ATPSs can be advantageous for new applications based on factors such as biomarker partitioning or biochemical compatibility between ATPS and sample components. However, since the mechanism of phase separation in porous media is not completely understood, introducing other ATPSs to paper is an unpredictable process that relies on trial and error experiments. This is especially true for polymer-polymer ATPSs in which the characteristics of the two phases appear quite similar. Therefore, our group aimed to develop semiquantitative guidelines for choosing ATPSs that can phase separate in paper. In this work, we evaluated the Washburn equation and its parameters as a potential mathematical framework to describe the flow behavior of polymer-salt and micellar ATPSs in fiberglass paper. We compared bulk phase fluid characteristics and identified the viscosity difference between the phases as a key determinant of the potential for phase separation in paper. We then used this parameter to predict the phase separation capabilities of polyethylene glycol (PEG)-dextran ATPSs in paper and control the composition of the leading and lagging phases. We also, for the first time, successfully demonstrated the phase separation phenomenon in hydrogels, thereby extending its application and potential benefits to an alternative porous medium.

An Aqueous Two-Phase System for the Concentration and Extraction of Proteins from the Interface for Detection Using the Lateral-Flow Immunoassay.

The paper-based immunoassay for point-of-care diagnostics is widely used due to its low cost and portability over traditional lab-based assays. Lateral-flow immunoassay (LFA) is the most well-established paper-based assay since it is rapid and easy to use. However, the disadvantage of LFA is its lack of sensitivity in some cases where a large sample volume is required, limiting its use as a diagnostic tool. To improve the sensitivity of LFA, we previously reported on the concentration of analytes into one of the two bulk phases of an aqueous two-phase system (ATPS) prior to detection. In this study, we preserved the advantages of LFA while significantly improving upon our previous proof-of-concept studies by employing a novel approach of concentrating gold nanoparticles, a common LFA colorimetric indicator. By conjugating specific antibodies and polymers to the surfaces of the particles, these gold nanoprobes (GNPs) were able to capture target proteins in the sample and subsequently be concentrated within 10 min at the interface of an ATPS solution comprised of polyethylene glycol, potassium phosphate, and phosphate-buffered saline. These GNPs were then extracted and applied directly to LFA. By combining this prior ATPS interface extraction with LFA, the detection limit of LFA for a model protein was improved by 100-fold from 1 ng/µL to 0.01 ng/µL. Additionally, we examined the behavior of the ATPS system in fetal bovine serum and synthetic urine to more closely approach real-world applications. Despite using more complex matrices, ATPS interface extraction still improved the detection limit by 100-fold within 15 to 25 min, demonstrating the system's potential to be applied to patient samples.