Biology subjects

Allbritton, N. L.

Publications and source records attributed to Allbritton, N. L..

5 recordsLinked to original sources

bioRxiv2024

Clostridioides difficile-mucus interactions encompass shifts in gene expression, metabolism, and biofilm formation

In a healthy colon, the stratified mucus layer serves as a crucial innate immune barrier to protect the epithelium from microbes. Mucins are complex glycoproteins that serve as a nutrient source for resident microflora and can be exploited by pathogens. We aimed to understand how the intestinal pathogen, Clostridioides diffiicile, independently uses or manipulates mucus to its benefit, without contributions from members of the microbiota. Using a 2-D primary human intestinal epithelial cell model to generate physiologic mucus, we assessed C. difficile-mucus interactions through growth assays, RNA-Seq, biophysical characterization of mucus, and contextualized metabolic modeling. We found that host-derived mucus promotes C. difficile growth both in vitro and in an infection model. RNA-Seq revealed significant upregulation of genes related to central metabolism in response to mucus, including genes involved in sugar uptake, the Wood-Ljungdahl pathway, and the glycine cleavage system. In addition, we identified differential expression of genes related to sensing and transcriptional control. Analysis of mutants with deletions in highly upregulated genes reflected the complexity of C. difficile-mucus interactions, with potential interplay between sensing and growth. Mucus also stimulated biofilm formation in vitro, which may in turn alter viscoelastic properties of mucus. Context-specific metabolic modeling confirmed differential metabolism and predicted importance of enzymes related to serine and glycine catabolism with mucus. Subsequent growth experiments supported these findings, indicating mucus is an important source of serine. Our results better define responses of C. difficile to human gastrointestinal mucus and highlight a flexibility in metabolism that may influence pathogenesis. IMPORTANCEClostridioides difficile results in upwards of 250,000 infections and 12,000 deaths annually in the United States. Community-acquired infections continue to rise and recurrent disease is common, emphasizing a vital need to understand C. difficile pathogenesis. C. difficile undoubtedly interacts with colonic mucus, but the extent to which the pathogen can independently respond to and take advantage of this niche has not been explored extensively. Moreover, the metabolic complexity of C. difficile remains poorly understood, but likely impacts its capacity to grow and persist in the host. Here, we demonstrate that C. difficile uses native colonic mucus for growth, indicating C. difficile possesses mechanisms to exploit the mucosal niche. Furthermore, mucus induces metabolic shifts and biofilm formation in C. difficile, which has potential ramifications for intestinal colonization. Overall, our work is crucial to better understand dynamics of C. difficile-mucus interactions in the context of the human gut.

microbiology↗

bioRxiv2022

Single-cell dynamics of core pluripotency factors in human pluripotent stem cells

The human transcription factors OCT4, SOX2, and NANOG form a core signaling network critical for maintaining stem cell pluripotency and self-renewal potential. The spatiotemporal expression dynamics of these pluripotency factors throughout differentiation is unclear, limiting our understanding of stem cell fate decisions. Here, we combined CRISPR/Cas9-mediated gene editing with microraft array technology to generate human embryonic stem cell lines with endogenously tagged fluorophores for OCT4, SOX2, and NANOG. Fluorescence time-lapse imaging revealed that pluripotent stem cells show gastrulation-like patterning without direct chemical induction. Directed differentiation to the three primary germ layers--endoderm, mesoderm, and ectoderm--revealed distinct spatiotemporal patterns of SOX2 and NANOG expression in single cells. Finally, we captured dynamic changes in cell morphology during ectoderm differentiation corresponding to the formation of neural rosettes. This study provides a robust method for generating live-cell reporters in human stem cells and describes the single-cell dynamics of human pluripotency factors during differentiation.

developmental biology↗

bioRxiv2021

Magnetically-Propelled Fecal Surrogates for Modeling the Impact of Solid-Induced Shear Forces on Primary Colonic Epithelial Cells

The colonic epithelium is continuously exposed to an array of biological and mechanical stimuli as its luminal contents are guided over the epithelial surface through regulated smooth muscle contraction. In this report, the propulsion of solid fecal contents over the colonic epithelium is recapitulated through noninvasive actuation of magnetic agarose hydrogels over primary intestinal epithelial cultures, in contrast to the vast majority of platforms that apply shear forces through liquid microflow. Software-controlled magnetic stepper motors enable experimental control over the frequency and velocity of these events to match in vivo propulsive contractions, while the integration of standardized well plate spacing facilitates rapid integration into existing assay pipelines. The application of these solid-induced shear forces did not deleteriously affect cell monolayer surface coverage, viability, or transepithelial electrical resistance unless the device parameters were raised to a 50x greater contraction frequency and 4x greater fecal velocity than those observed in healthy humans. At a frequency and velocity that is consistent with average human colonic motility, differentiation of the epithelial cells into absorptive and goblet cell phenotypes was not affected. Protein secretion was modulated with a two-fold increase in luminal mucin-2 secretion and a significant reduction in basal interleukin-8 secretion. F-actin, zonula occludens-1, and E-cadherin were each present in their proper basolateral locations, similar to those of static control cultures. While cellular height was unaffected by magnetic agarose propulsion, several alterations in lateral morphology were observed including decreased circularity and compactness, and an increase in major axis length, which align with surface epithelial cell morphologies observed in vivo and may represent early markers of luminal exfoliation. This platform will be of widespread utility for the investigation of fecal propulsive forces on intestinal physiology, shedding light on how the colonic epithelium responds to mechanical cues. HIGHLIGHTSO_LIMagnetic nanoparticle-embedded agarose hydrogels were developed as surrogates for human feces C_LIO_LISoftware-controlled magnetic motors enable programmable studies of colonic motility C_LIO_LIPropulsive shear forces were optimized for primary cell viability, surface coverage, and electrical resistance C_LIO_LIMucus production and cytokine secretion were modulated by magnetic agarose propulsion C_LIO_LIStructural and cytoskeletal proteins remain properly distributed with alterations in lateral cell morphology C_LI

bioengineering↗

bioRxiv2020

Decreased Colonic Activin Receptor Like Kinase 1 Disrupts Epithelial Barrier integrity and is associated with a poor clinical outcome in Crohns disease

ObjectiveIntestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohns disease (CD). However, the mechanism is understudied. We recently reported increased microRNA-31-5p (miR-31-5p) expression in colonic IECs of CD patients, but downstream targets are unknown. DesignMiR-31-5p target genes were identified by integrative analysis of RNA- and small RNA-sequencing data from colonic mucosa and confirmed by qPCR in colonic IECs. Functional characterization of Activin Receptor-Like Kinase 1 (ACVRL1 or ALK1) in IECs was performed ex vivo using 2 dimensional-cultured human primary colonic IECs. The impact of altered colonic ALK1 signaling in CD for the risk of surgery and endoscopic relapse was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator. ResultsALK1 was identified as a target of miR-31-5p in colonic IECs of CD patients and confirmed using a 3-UTR reporter assay. Activation of ALK1 restricted the proliferation of colonic IECs in an EdU proliferation assay and down-regulated the expression of stemness-related genes. Activated ALK1 signaling directed the fate of colonic IEC differentiation toward colonocytes. Down-regulated ALK1 signaling was associated with increased stemness and decreased colonocyte-specific marker expression in colonic IECs of CD patients compared to healthy controls. Activation of ALK1 enhanced epithelial barrier integrity in a trans-epithelial electrical resistance permeability assay. Lower colonic ALK1 expression was identified as an independent risk factor for surgery and associated with a higher risk of endoscopic relapse in CD patients. ConclusionDecreased colonic ALK1 disrupted colonic IEC barrier integrity and associated with deteriorated clinical outcomes in CD patients.

cell biology↗

bioRxiv2017

LITE microscopy: a technology for high numerical aperture, low photobleaching fluorescence imaging

Fluorescence microscopy is a powerful approach for studying sub-cellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel LSFM method: Lateral Interference Tilted Excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically-limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.

cell biology↗