Biology subjects

Thompson, C. M.

Publications and source records attributed to Thompson, C. M..

6 recordsLinked to original sources

bioRxiv2026

Development of a high throughput continuous fluorescence anisotropy-based assay to analyse peptidoglycan-digesting enzymes

Bacterial cell wall processing enzymes have a central role in the control of the construction, remodelling and breakdown of the peptidoglycan wall that protects bacteria from osmotic rupture. Many of these enzymes have biologically essential functions and exert mechanistically diverse activities on a complex polymer substrate. Despite this biological importance they are relatively understudied and there are significant technical challenges in measuring the activity of individual enzymes against the complex chemical structure of the bacterial cell wall peptidoglycan. To generate a suitable substrate, bacterial cells are grown with a fluorescent D-alanine analogue, allowing the isolation of natively fluorescently labelled PG. This material is then used to measure interactions with PG-targeting enzymes and regulatory proteins. The resulting assay enables analysis of multiple enzyme-substrate combinations within minutes while retaining molecular specificity. This approach provides a robust, cost-effective alternative to conventional PG activity assays and could accelerate the identification and characterisation of novel antimicrobial compounds targeting bacterial cell wall metabolism. Our results indicate this assay has the potential to be employed in high-throughput inhibitor discovery, making it effective for a broad-spectrum assay for bacterial peptidoglycan research applications.

biochemistry↗

bioRxiv2025

A microfluidic gradient and parallel-track system uncovers spatial control of endocytosis and adhesion formation in breast cancer cell migration

Cell migration through confined spaces is a critical step in cancer metastasis, yet the spatial regulation of endocytosis and adhesion dynamics during this process remains poorly understood. To address this, we developed a microfluidic platform that generates stable, spatially linear biochemical gradients across 5 m-tall migration channels while limiting confounding flow-induced shear stress (<0.05 dyn/cm2). COMSOL simulations and optical calibration using FITC-dextran confirmed that gradients form reliably within 5 minutes. The microdevice also supports long-term live imaging and is compatible with both spinning disk confocal and total internal reflection fluorescence structured illumination microscopy modalities, enabling high-resolution visualization of adhesion and endocytic structures. Localized application of the endocytic inhibitor Dyngo-4a to the front or rear of migrating cells revealed that front-targeted inhibition significantly increased the enrichment of paxillin and the clathrin adaptor AP-2 at the leading edge, whereas rear-targeted inhibition completely abolished their front-rear asymmetry. These changes were accompanied by enhanced migration speed and persistence, particularly under front-targeted inhibition. Together, these findings highlight the critical role of spatially coordinated endocytosis in sustaining polarized adhesion and persistent cell movement. Our platform offers a powerful tool for dissecting subcellular mechanisms of migration under confinement and provides a broadly applicable framework for probing spatially localized signaling in engineered microenvironments.

cell biology↗

bioRxiv2022

Targeted multi-omic analysis of human skin tissue identifies alterations of conventional and unconventional T cells associated with burn injury

Burn injuries are a leading cause of unintentional injury, associated with a dysfunctional immune response and an increased risk of infections. Despite this, little is known about the role of T cells in human burn injury. In this study, we compared the activation and function of conventional T cells and unconventional T cell subsets in skin tissue from acute burn (within 7 days from initial injury) burn, late phase burn (beyond 7 days from initial injury), and non-burn patients. We compared T cell functionality by a combination of flow cytometry and a multi-omic single-cell approach with targeted transcriptomics and protein expression. We found a significantly lower proportion of CD8+ T cells in burn skin compared to non-burn skin, with CD4+ T cells making up the bulk of the T cell population. Both conventional and unconventional burn tissue T cells show significantly higher IFN-{gamma} and TNF- levels after stimulation than non-burn skin T cells. In sorted T cells, clustering showed that burn tissue had significantly higher expression of homing receptors CCR7, S1PR1, and SELL compared to non-burn skin. In unconventional T cells, including mucosal-associated invariant T (MAIT) and {gamma}{delta} T cells, we see significantly higher expression of cytotoxic molecules GZMB, PRF1, and GZMK. Multi-omics analysis of conventional T cells suggests a shift from tissue-resident T cells in non-burn tissue to a circulating T cell phenotype in burn tissue. In conclusion, by examining skin tissue from burn patients, our results suggest that T cells in burn tissue have a pro-inflammatory rather than a homeostatic tissue-resident phenotype, and that unconventional T cells have a higher cytotoxic capacity. Our findings have the potential to inform the development of novel treatment strategies for burns.

immunology↗

bioRxiv2021

ROS-Degradable Polythioketal Urethane Foam Dressings to Promote Porcine Skin Wound Repair

Impaired skin healing and progression into chronic wounds is a prevalent and growing medical problem. Porous, resorbable biomaterials can be used as temporary substrates placed into skin defects to support cell infiltration, neo-tissue formation, and remodeling of nonhealing wounds. Naturally-derived biomaterials have promising healing benefits, but their low mechanical properties and exuberant costs limit their performance and use. Synthetic materials can be affordably manufactured and tuned across a broader range of physiochemical properties, but opportunities remain for tailoring them for ideal host immune and regenerative responses. Polyesters are the most clinically-tested class of synthetic biomaterials, but their hydrolysis releases acidic degradation products that can cause autocatalytic degradation processes that are poorly controlled and are not tied to cellular or other biologic activities. Here, we systemically explored a series of ROS-degradable polythioketal (PTK) urethane (UR) foams with varied hydrophilicity as an alternative class of synthetic biomaterials for wound healing. It was found that the most hydrophilic PTK- UR variant, which had 7 ethylene glycol (EG7) repeats flanking each side of each thioketal bond, had the highest ROS reactivity of the PTK-URs tested. In an in vivo porcine excisional skin wound healing model, hydrophilic EG7 PTK-UR foams more effectively promoted tissue integration, ECM deposition, and re- epithelialization of full-thickness skin wound compared to more hydrophobic PTK-UR variants. Resolution of type 1 inflammation and lower foreign body response to scaffold remnants was also observed for EG7 versus more hydrophobic PTK-UR scaffolds. Finally, porcine wound healing studies showed that EG7 PTK-UR foams had similar wound healing response to a collagen-based clinical gold standard product, Integra Bilayer Wound Matrix (BWM), while outperforming polyester UR foam-based NovoSorb Biodegradable Temporizing Matrix (BTM) with respect to increased ECM production, vascularization, and biomaterial-associated immune phenotype. In sum, PTK-UR foams warrant further development toward a new class of synthetic biomaterial foams for skin wound healing applications.

bioengineering↗

bioRxiv2018

Global emergence and population dynamics of divergent serotype 3 CC180 pneumococci

Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the United States, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3-31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identify a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939-1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development.\n\nAuthor SummaryStreptococcus pneumoniae is a leading cause of bacterial pneumoniae, meningitis, and otitis media. Despite inclusion in the most recent pneumococcal conjugate vaccine, PCV13, serotype 3 remains epidemiologically important globally. We investigated the persistence of serotype 3 using whole-genome sequencing data form 301 isolates collected among 24 countries from 1993-2014. Through phylogenetic analysis, we identified three distinct lineages within a single clonal complex, CC180, and found one has recently emerged and grown in prevalence. We then compared genomic difference among lineages as well as variations in pneumococcal vaccine use among sampled countries. We found that the recently emerged lineage, termed Clade II, has a higher prevalence of antibiotic resistance compared to other lineages, diverse surface protein antigens, and a higher rate of recombination, a process by which bacteria can uptake and incorporate genetic material from its surroundings. Differences in vaccine use among sampled countries did not appear to be associated with the emergence of Clade II. We highlight the need to routine, representative sampling of bacterial isolates from diverse geographic areas and show the utility of genomic data in resolving epidemiological differences within a pathogen population.

evolutionary biology↗

bioRxiv2017

Population genomics of pneumococcal carriage in Massachusetts children following PCV-13 introduction

BackgroundThe 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced in the United States in 2010. Using a large pediatric carriage sample collected from shortly after the introduction of PCV-7 to several years after the introduction of PCV-13, we investigate alterations in the composition of the pneumococcal population following the introduction of PCV-13, evaluating the extent to which the post-vaccination non-vaccine type (NVT) population mirrors that from prior to vaccine introduction and the effect of PCV-13 on vaccine type lineages.\n\nMethods and FindingsDraft genome assemblies from 736 newly sequenced and 616 previously published pneumococcal carriages isolates from children in Massachusetts between 2001 and 2014 were analyzed. Isolates were classified into one of 22 sequence clusters (SCs) on the basis of their core genome sequence. We calculated the SC diversity for each sampling period as the probability that any two randomly drawn isolates from that period belong to different SCs. The sampling period immediately after the introduction of PCV-13 (2011) was found to have higher diversity than preceding (2007) or subsequent (2014) sampling periods (Simpsons D 2007: 0.915 95% CI [0.901, 0.929]; 2011: 0.935 [0.927, 0.942]; 2014: 0.912 [0.901, 0.923]). Amongst NVT isolates, we found the distribution of SCs in 2011 to be significantly different from that in 2007 or 2014 (Fishers Exact Test p=0.018, 0.0078), but did not find a difference comparing 2007 to 2014 (Fishers Exact Test p=0.24), indicating greater similarity between samples separated by a longer time period than between samples from closer time periods. We also found changes in the accessory gene content of the NVT population between 2007 and 2011 to have been reduced by 2014. Amongst the new serotypes targeted by PCV-13, four were present in our sample. The proportion of our sample composed of PCV-13-only vaccine serotypes 19A, 6C, and 7F decreased between 2007 and 2014, but no such reduction was seen for serotype 3. We did, however, observe differences in the genetic composition of the pre- and post-PCV-13 serotype 3 population. Our isolates were collected during discrete sampling periods from a small geographic area, which may limit the generalizability our findings.\n\nConclusionPneumococcal diversity increased immediately following the introduction of PCV-13, but subsequently returned to pre-vaccination levels. This is reflected in the distribution of NVT lineages, and, to a lesser extent, their accessory gene frequencies. As such, there may be a period during which the population is particularly disrupted by vaccination before returning to a more stable distribution. The persistence and shifting genetic composition of serotype 3 is a concern and warrants further investigation.

evolutionary biology↗