Biology subjects

Fleming, A.

Publications and source records attributed to Fleming, A..

12 recordsLinked to original sources

bioRxiv2025

Molecular Mechanisms of Priming Innate Immunity by Small Extracellular Vesicles Released during Infection with Gram-negative Bacteria

Much still remains to understand about the underlying molecular mechanisms by which the trafficking of small extracellular vesicles (sEVs) modulates innate immune responses during infection with pathogenic Gram-negative bacteria. To address this significant gap in knowledge, we used two infection models to investigate innate immune regulation by the sEVs released from cells infected with either Yersinia pestis (Yp) or Burkholderia thailandensis (Bt), designated as EXi-Yp and EXi-Bt respectively. The EXi induced differentiation of naive human monocytes to macrophages and triggered robust pro-inflammatory cytokine release, including release of IL-6, mirroring direct bacterial infection effects. Comprehensive cell signaling analyses revealed that the EXi modulate a small set of host signaling proteins, with p38 activation being primarily responsible for the observed protective effects. EXi-induced p38 activation leads to increased IL-6 release, which in turn is responsible for decreased bacterial survival within recipient immune cells that are subsequently infected. Consistent with the in vitro results, mice administered with EXi-Yp exhibited elevated serum IL-6 levels and were protected from Yp infection. Furthermore, using our microfluidic chip platform that allows functional interrogation of EV effects under physiologically relevant conditions, we have demonstrated that EXi exchange between Yp-infected cells and naive recipient monocytes leads to differentiation of the recipient cells to macrophages. Together, our findings reveal a largely unexplored aspect of innate immunity and provide a mechanistic model in which EXi prime local and distant naive monocytes via p38-induced differentiation and IL-6 production to protect against infection with Gram-negative bacteria.

immunology↗

bioRxiv2025

Divergent roles of DNA methylation, TRIM28, and p53 surveillance in human embryonic and trophoblast stem cells

Transcriptional regulation of transposons and genes by TRIM28 and 5mC is critical for proper mammalian embryonic development, but the specific roles for these mediators in human embryonic and placental lineage remain unclear. We find that loss of TRIM28 has a limited effect on global transposon expression and instead results in upregulation of genes proximal to TRIM28-bound Long Terminal Repeats (LTRs) in both human trophoblast stem cells (hTSCs) and human embryonic stem cells (hESCs). MER11A elements show especially strong regulatory importance in hTSCs: these elements are bound by both TRIM28 and placental transcription factors and show both heterochromatic and euchromatic features. Some genes are positively regulated by MER11A elements in hTSC basal state, while other MER11A-proximal genes show upregulation only upon TRIM28 deletion. By contrast, loss of DNA methylation in hESCs or hTSCs leads to a global increase in transposon expression. While many genic 5mC targets are shared in hESCs and hTSCs, we also observe evidence that a handful of genes important for somatic development are repressed by 5mC in trophoblast, while a small parallel set of placental genes are repressed by methylation in embryonic tissue. Interestingly, loss of DNMT1 causes hESCs to be rapidly lost from culture in a TP53 and mitotic surveillance checkpoint-dependent manner, while hTSCs show little p53 response to DNMT1 loss or DNA damage generally, instead showing gradual mitotic defects and aneuploidy and slow loss from culture. This discrepancy may explain the higher frequency of karyotypic abnormality found in human placental cells. Together, this study charts the role of TRIM28 and DNA methylation in regulating embryonic and placental transcription and demonstrates divergent p53-dependent responses to genomic instability.

molecular biology↗

bioRxiv2025

Intestinal challenges shape the polarisation of protective dural memory CD4 T cells

The meninges house several innate and adaptive immune cell populations1-3. These predominantly localise within the dura mater and include gut-derived IgA-secreting plasma cells4. Whether T cell adaptive memory in the dura is similarly linked to the gut is currently unknown. Here we show that dural CD4 T cell polarisation to a T helper (Th) 1, Th2 and Th17 state is determined by the nature of the immunological challenge encountered in the gastrointestinal tract. We find that intestinally polarised CD4 T cells seed to the dura in a CXCR6-CXCL16-dependent manner, express tissue-residency markers and are long-lived. Functionally, these orally-primed dural CD4 T are capable of a rapid antigen-specific recall response that limits pathogen spread into the brain following intravenous re-challenge. Our work reveals how linked intestinal and dural immunity enables the central nervous system to accrue immunological memory of gut microbes, the most likely source of life-threatening bloodborne pathogens.

immunology↗

bioRxiv2025

Semi-Automated Seal Detection on the Western Antarctic Peninsula: An Unsupervised Machine Learning Approach for Detecting Ice Seals in Aerial Survey Data

Over the past 25 years, the Western Antarctic Peninsula (WAP) has experienced dramatic shifts in sea ice extent. This change has coincided with rapid alterations in ice-dependent ecosystems, including those supporting crabeater seals - the most abundant Antarctic seal and one of the largest mammalian consumers of krill. Despite their ecological importance, population estimates for ice seals remain scarce due to the difficulty of surveying large-scale, remote, ice-covered habitats. In 2023, during an abnormally low sea ice year, we conducted aerial surveys over Crystal Sound and Marguerite Bay during the end of the breeding season, flying over 1,000 km of transects. Seals were extremely sparse in the resulting imagery - occupying less than 1% of the surveyed area. This posed a significant challenge for both manual annotation and automated detection. Here we present a semi-automated, rules-based image analysis pipeline to substantially reduce human annotation time. Our method leverages hierarchical clustering with just two tunable parameters, avoiding the computational burden and opacity of deep learning models. Using this method, we identified 758 seals within a [~]350 km2 survey subset, achieving a test recall of 82%. In the absence of concurrent tagging data to estimate haul-out corrections, we refrain from extrapolating to a population estimate. However, the low observed densities highlight the urgent need for continued monitoring. Our improved data processing pipeline is a key step in facilitating the large-scale analysis required to inform conservation strategies for this key species.

ecology↗

bioRxiv2024

PROSER1 Modulates DNA Demethylation through Dual Mechanisms to Prevent Syndromic Developmental Malformations

The link between DNA methylation and neurodevelopmental disorders is well established. However, how DNA methylation is fine-tuned - ensuring precise gene expression and developmental fidelity - remains poorly understood. PROSER1, a known TET2 interactor, was recently linked to a severe neurodevelopmental disorder. Here, we demonstrate that PROSER1 interacts with all TET enzymes and stabilizes chromatin-bound TET-OGT-PROSER1-DBHS (TOPD) complexes, which regulate DNA demethylation and developmental gene expression. Surprisingly, we find that PROSER1 also sequesters TET enzymes, preventing widespread demethylation and transposable element de-repression. Our findings identify PROSER1 as a key factor which both positively and negatively regulates DNA demethylation essential for mammalian neurodevelopment.

developmental biology↗

bioRxiv2021

A video-based behavioural intervention associated with improved HPV knowledge and intention to vaccinate

AimsThe aim of this study was to design, develop and evaluate a theory and evidence-based intervention to improve human papillomavirus (HPV) and HPV vaccine knowledge, and intention to vaccinate, among parent-daughter dyads. MethodsA theory and evidence-based online behavioural intervention, "Is the HPV vaccine for me?" was developed to improve HPV and HPV vaccine knowledge, and intention to vaccinate. The impact and feasibility of the intervention was evaluated in a prospective randomised controlled feasibility trial. ResultsA total of 49 parent-daughter dyads completed baseline knowledge assessment (n=24 control, n=25 intervention), and 35 dyads completed knowledge assessment at week 2 (n=17 control, n=18 intervention). The intervention was associated with a statistically significant increase in HPV, and HPV vaccine knowledge and intention to vaccinate. All intervention participants found the video interesting, while 96% found it useful. ConclusionsThis intervention was found to be affordable, practicable, effective (cost-effective), acceptable, safe, and equitable, in this feasibility study.

scientific communication and education↗

bioRxiv2021

Cells of the human intestinal tract mapped across space and time

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used singlecell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprungs disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.

cell biology↗

bioRxiv2020

Radiation resistance in Placozoa: Trichoplax adhaerens upregulates DNA repair genes and extrudes cells after exposure.

Trichoplax adhaerens is the simplest multicellular animal with tissue differentiation and somatic cell turnover. Like all other multicellular organisms, it should be vulnerable to cancer, yet there have been no reports of cancer in T. adhaerens, or any other placozoan. We investigated the cancer resistance of T. adhaerens, discovering that they are able to tolerate high levels of radiation damage (218.6 Gy). To investigate how T. adhaerens survive levels of radiation that are lethal to other animals, we examined gene expression after the X-ray exposure, finding overexpression of genes involved in DNA repair and apoptosis including the MDM2 gene. We also discovered that T. adhaerens extrudes clusters of inviable cells after X-ray exposure. T. adhaerens is a valuable model organism for studying the molecular, genetic and tissue-level mechanisms underlying cancer suppression.

cancer biology↗

bioRxiv2020

Direct Continuous EMG control of a Powered Prosthetic Ankle for Improved Postural Control after Guided Physical Training: a Case Study

BackgroundDespite the promise of powered lower limb prostheses, the existing control of these modern devices is insufficient to assist many daily activities, such as postural control while lifting weight, that require continuous control of prosthetic joints according to human states and environments. The objective of this case study was to investigate the feasibility and potential of direct, continuous electromyographic (dEMG) control of a powered ankle prosthesis, combined with physical therapist (PT)-guided training, for improved standing postural control in an individual with transtibial amputation. MethodsA powered prosthetic ankle was directly controlled by EMG signals of the residual lateral gastrocnemius and tibialis anterior muscles. The participant with transtibial amputation received 4-week PT-guided training on posture while using the dEMG control of powered ankle. A subset of activities in the mini-BESTest (a clinical balance assessment tool) were used in the training and evaluation protocol. We quantified EMG signals in the bilateral shank muscles, biomechanics that captures postural control and stability, and score for the clinical balance evaluation. ResultsCompared to the participants daily passive prosthesis, the dEMG-controlled ankle, combined with the training, yielded improved clinical balance score and reduced compensation from the intact joints. In addition, cross correlation coefficient of bilateral CoP excursions, a metric for quantifying standing postural control, increased to 0.83({+/-}0.07) when using dEMG ankle control, compared with 0.39({+/-}0.29) when using the passive device. Between-limb coordination was also observed as synchronized activation of homologous muscles in the shank. We witnessed rapid improvement in performance on the first day of the training for load transfer tasks, where bilateral CoP synchronization improvement was significantly related to repetition order (R=0.459, p = 0.045). Finally, the participant further improved this performance significantly across training days. ConclusionThis case study showed the feasibility of dEMG control of powered prosthetic ankle by a transtibial amputee after a PT-guided training to assist postural control. This studys training protocol and dEMG control method that lays the foundation for future study to extend these results through the inclusion of more participants and activities.

bioengineering↗

bioRxiv2019

Alpha synuclein aggresomes inhibit ciliogenesis and multiple functions of the centrosome

Protein aggregates are the pathogenic hallmarks of many different neurodegenerative diseases and include the Lewy bodies found in Parkinsons disease. Aggresomes are closely-related cellular accumulations of misfolded proteins. They develop in a juxtanuclear position, adjacent to the centrosome, the microtubule organizing centre of the cell, and share some protein components. Despite the long-standing observation that aggresomes/Lewy bodies and the centrosome sit side-by-side in the cell, no studies have been done to see whether these protein accumulations impede the organelle function. We investigated whether the formation of aggresomes affected key centrosome functions: its ability to organize the microtubule network and to promote cilia formation. We find that when aggresomes are present, neuronal cells are unable to organise their microtubule network. New microtubules are not nucleated and extended, and the cells fail to respond to polarity cues. Since dopaminergic neurons are polarised, ensuring correct localisation of organelles and the effective intracellular transport of neurotransmitter vesicles, loss of centrosome activity could contribute to loss of dopaminergic function and neuronal cell death in Parkinsons disease. In addition, we provide evidence that many cell types, including dopaminergic neurons, cannot form cilia when aggresomes are present, which would affect their ability to receive extracellular signals.

neuroscience↗

bioRxiv2019

The autophagic response to Staphylococcus aureus provides an intracellular niche in neutrophils

Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus, multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutrophil subversion by intracellular S. aureus remains unknown. Targeting of intracellular pathogens by autophagy is recognised as an important component of host innate immunity, but whether autophagy is beneficial or detrimental to S. aureus-infected hosts remains controversial. Here, using larval zebrafish we show that S. aureus is rapidly decorated by the autophagy marker Lc3 following engulfment by macrophages and neutrophils. Upon phagocytosis by neutrophils, Lc3-positive, non-acidified spacious phagosomes are formed. This response is dependent on phagocyte NADPH oxidase as both cyba knockdown and diphenyleneiodonium (DPI) treatment inhibited Lc3 decoration of phagosomes. Importantly, NADPH oxidase inhibition diverted neutrophil S. aureus processing into tight acidified vesicles, which resulted in increased host resistance to the infection. Some intracellular bacteria within neutrophils were also tagged by p62-GFP fusion protein and loss of p62 impaired host defence. Taken together, we have shown that intracellular handling of S. aureus by neutrophils is best explained by Lc3-associated phagocytosis (LAP), which appears to provide an intracellular niche for bacterial pathogenesis, while the selective autophagy receptor p62 is host-protective. The antagonistic roles of LAP and p62-mediated pathways in S. aureus-infected neutrophils may explain the conflicting reports relating to anti-staphylococcal autophagy and provide new insights for therapeutic strategies against antimicrobial resistant staphylococci.

immunology↗

bioRxiv2018

Social licence through citizen science: A tool for marine conservation

1. Introduction 1. Introduction 2. Methods 3. Results Discussion 4. Conclusion References Meaningful public engagement with science and research is essential to improving knowledge about the environment and supporting sustainable use of ecosystems and natural resources. Transparent and culturally-appropriate natural resource management is imperative (Christie et al. 2017) and the publics role in decision-making and data collection is increasingly recognised (Hecker et al. 2018), supported by a rise in the number of citizen science projects in recent years (Chandler et al. 2017;Pocock et al. 2017), including marine-based projects (Cigliano and Ballard 2017). In parallel, social licence has become an important theme for development, pa ...

scientific communication and education↗