Biology subjects

Duncan, G. A.

Publications and source records attributed to Duncan, G. A..

5 recordsLinked to original sources

bioRxiv2021

Shaping nanoparticle diffusion through biological barriers to drug delivery

Nanoparticle drug delivery systems encounter many biological barriers, such as the extracellular matrix and mucus gels, that they must bypass to gain access to target cells. A design parameter that has recently gained attention is nanoparticle shape, as it has been shown elongated rod-shaped nanoparticles achieve higher diffusion rates through biological gels. However, the optimal dimensions of rod-shaped nanoparticles to enhance this effect has yet to be established. To systematically approach this, rod-shaped nanoparticles were synthesized by mechanically stretching 100 nm, 200 nm, and 500 nm spherical nanoparticles. Transmission electron microscopy confirmed this procedure yields a significant fraction of elongated rods and remaining spheres could be removed by centrifugation. Fluorescent microscopy and multiple particle tracking analysis was then used to characterize rod-shaped and spherical nanoparticle diffusion in MaxGel(R), a model extracellular matrix hydrogel. When dispersed in MaxGel, we found rod-shaped nanoparticles exhibited the greatest enhancement in diffusion rate when their length far exceeds the average hydrogel network size. These results further establish the importance of shape as a design criterion to improve nanoparticle-based drug delivery systems.

bioengineering↗

bioRxiv2021

Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium

The clinical impact of rhinovirus C (RV-C) is well-documented; yet the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24 hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection and correlating with the formation of large intracellular vesicles. To further define RV-C15 replication sites, we analyzed the expression of giantin, phosphatidylinositol-4- phosphate, and calnexin, as well as the colocalization of these markers with dsRNA. Fluorescence levels of all three cellular markers were elevated during infection and altered giantin distribution further indicated Golgi fragmentation. However, unlike previously characterized RVs, the high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression, facilitating replication, and the induction of incomplete autophagy, a mechanism used by other RVs to promote non-lytic release of progeny virions. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality - aspects of infection that may contribute to pathogenesis in vivo. Author summaryRhinovirus C has a global distribution and significant clinical impact - especially in those with underlying lung disease. Although RV-C is genetically, structurally, and biologically distinct from RV-A and -B viruses, our understanding of the RV-C life cycle has been largely inferred from these and other related viruses. Here, we performed a detailed analysis of RV-C15 replication in a physiologically-relevant model of human airway epithelium. Our single-cell, microscopy-based approach revealed that - unlike other RVs - the endoplasmic reticulum is the primary site for RV- C15 replication. RV-C15 replication also stimulated STING expression, which was proviral, and triggered dramatic changes in cellular organization, including altered virus receptor distribution, fragmented Golgi stacks, and the induction of incomplete autophagy. Additionally, we observed a loss of epithelial barrier function and a decrease in mucociliary clearance, a major defense mechanism in the lung, during RV-C15 infection. Together, these data reveal novel insight into RV-C15 replication dynamics and resulting cytopathic effects in the primary target cells for infection, thereby furthering our understanding of the pathogenesis of RV-C. Our work highlights similar, as well as unique, aspects of RV-C15 replication compared to related pathogens, which will help guide future studies on the molecular mechanisms of RV-C infection.

microbiology↗

bioRxiv2021

Membrane-Tethered Mucin 1 is Stimulated by Interferon in Multiple Cell Types and Antagonizes Influenza A Virus Infection in Human Airway Epithelium

Influenza A virus (IAV) causes seasonal epidemics and periodic pandemics, resulting in significant morbidity and mortality in the human population. Tethered mucin 1 (MUC1) is highly expressed in airway epithelium, the primary site of IAV replication, and also by other cell types that influence IAV infection, including macrophages. MUC1 has the potential to influence infection dynamics through physical interactions and/or signaling activity, and recent work suggests MUC1 acts as a releasable decoy receptor and anti-inflammatory molecule during IAV infection. Still, the modulation of MUC1 and its impact during viral pathogenesis remains unclear. Thus, we sought to further investigate the interplay between MUC1 and IAV in an in vitro model of primary human airway epithelium (HAE). Our data indicate that a recombinant IAV hemagglutinin (H3) and H3N2 virus can bind endogenous HAE MUC1. We find that infection of HAE cultures with H1N1 or H3N2 IAV strains does not trigger enhanced MUC1 shedding, but instead stimulates an increase in cell-associated MUC1 protein. We observed a similar increase after stimulation with either type I or type III interferon (IFN); however, inhibition of IFN signaling during H1N1 infection only partially abrogated this increase, indicating multiple soluble factors contribute to MUC1 upregulation during the antiviral response. We expanded these findings and demonstrate that in addition to HAE, primary human monocyte-derived macrophages also upregulate MUC1 protein in response to both IFN treatment and conditioned media from IAV-infected HAE cultures. We then developed HAE genetically depleted for MUC1 to determine its impact on IAV pathogenesis, finding that MUC1 knock-out cultures exhibited enhanced viral growth compared to control cultures for several IAV strains. Together, our data support a model whereby MUC1 antagonizes productive uptake of IAV in HAE. Infection then stimulates MUC1 expression on multiple cell types through IFN-dependent and -independent mechanisms that may further impact infection dynamics. Author SummaryThe mucosal surface of the respiratory epithelium is an important site of first contact for viral respiratory pathogens. Large and heavily glycosylated molecules known as tethered mucins extend from the cell surface and may physically restrict access to underlying cells. Recently, one of these tethered mucins, MUC1, has also been shown to influence cell signaling and inflammation. Still, despite its abundance in the airway and multifunctional capability, the role of MUC1 during influenza virus infection in the human respiratory tract remains unclear. Here, we demonstrate that influenza virus directly interacts with MUC1 in a physiologically-relevant model of human airway epithelium and find that MUC1 protein expression is elevated throughout the epithelium and in primary human monocyte-derived macrophages in response to important antiviral signals produced during infection. Using genetically-modified human airway cultures lacking MUC1, we then provide evidence of more efficient influenza virus infection in the absence of this mucin. Our data suggest that MUC1 not only physically restricts influenza virus uptake, but also represents a dynamic component of the host response that acts to further stem viral spread.

microbiology↗

bioRxiv2020

Modeling airway dysfunction in asthma using synthetic mucus biomaterials

As asthma worsens, occlusion of airways with mucus significantly contributes to airflow obstruction and reduced lung function. Recent evidence from clinical studies has shown mucus obtained from adults and children with asthma possesses altered mucin composition. However, how these changes alter the functional properties of the mucus gel is not yet fully understood. To study this, we have engineered a synthetic mucus biomaterial to closely mimic the properties of native mucus in health and disease. We demonstrate this model possesses comparable biophysical and transport properties to native mucus ex vivo collected from human subjects and in vitro isolated from human airway epithelial (HAE) tissue cultures. We found by systematically varying mucin composition that mucus gel viscoelasticity is enhanced when predominantly composed of mucin 5AC (MUC5AC), as is observed in asthma. As a result, asthma-like synthetic mucus gels are more slowly transported on the surface of HAE tissue cultures and at a similar rate to native mucus produced by HAE cultures stimulated with the type 2 cytokine IL-13, known to contribute to airway inflammation and MUC5AC hypersecretion in asthma. We also discovered the barrier function of asthma-like synthetic mucus towards influenza A virus was impaired as evidenced by the increased frequency of infection in MUC5AC-rich hydrogel coated HAE cultures. Together, this work establishes a biomaterial-based approach to understand airway dysfunction in asthma and related muco-obstructive lung diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/342766v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@bed446org.highwire.dtl.DTLVardef@626c28org.highwire.dtl.DTLVardef@1aabb6borg.highwire.dtl.DTLVardef@1f69081_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Diversity of tRNA Clusters in the Chloroviruses

Viruses rely on their host's translation machinery for the synthesis of their own proteins. Problems belie viral translation when the host has a codon usage bias (CUB) that is different from an infecting virus with differences in the GC content between the host/virus genome. Here, we evaluate the hypothesis that chloroviruses adapted to host CUB by acquisition and selection of tRNAs that at least partially favor their own CUB. The genomes of 41 chloroviruses comprising three clades of three different algal hosts have been sequenced, assembled and annotated. All 41 viruses not only encode tRNAs, but their tRNA genes are located in clusters. One tRNA gene was common to all three clades of chloroviruses, while differences were observed between clades and even within clades. By comparing the codon usage of one chlorovirus algal host, whose genome has been sequenced and annotated (67% GC content), to that of two of its viruses (40% GC content), we found that the viruses were able to at least partially overcome the host's CUB by encoding tRNAs that recognize AU-rich codons. In addition, 39/41 chloroviruses encode a putative lysidine synthase, which alters the anticodon of tRNAmet that normally recognizes AUG to recognize the codon AUA, a codon for isoleucine. This is advantageous to the viruses because the AU-rich codon AUA is 12-13 times more common in the chloroviruses than their host. Evidence is presented that supports the concept that chlorovirus tRNA clusters were acquired prior to events that separated them into the three clades.View Full Text

genomics↗