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Buckley, C.

Publications and source records attributed to Buckley, C..

13 recordsLinked to original sources
bioRxiv2025

Extracellular Vesicles-Mediated Crosstalk in Bone: miR-150-5p as a Mechanosensitive Inhibitor of Osteoclastogenesis

Bone is a dynamic tissue that is constantly remodelling via a tightly controlled balance between formation and resorption. However, when osteoclast-mediated resorption exceeds formation, it can lead to net bone loss and the development of osteoporosis. Osteoclastogenesis is regulated by local environmental cues, including paracrine factors released by resident cell populations. Extracellular vesicles (EVs) have recently emerged as key mediators of paracrine communication, though their role in osteoclastogenesis remains underexplored. Therefore, this study investigated how resident mesenchymal-derived bone cells and their secreted EVs modulate osteoclastogenesis, and delineate the signalling factors mediating this anti-catabolic communication. We demonstrate that the secretome of mesenchymal-derived bone cells inhibit osteoclast differentiation to differing degrees depending on the stage of lineage commitment and on the mechanical environment, and demonstrate that this inhibition is mediated via the release of EVs. In addition, we identified the terminally differentiated osteocyte as the optimal parent cell for the production of anti-catabolic EVs and demonstrated the importance EV dosage. Finally, we show that mechanosensitive miR-150-5p is packaged within osteocyte-derived EVs and inhibits osteoclast differentiation. Taken together, this study identifies mechanically-activated osteocyte derived EVs and miR-150-5p as key regulators of osteoclastogenesis and novel molecular therapies for skeletal pathologies.

bioengineering↗
bioRxiv2025

VasoTracker 2: An Open-source Platform for Quantitative Analysis of Vascular Reactivity and Function

VasoTracker 2 is an open-source platform for studying blood vessel dynamics, featuring versatile diameter-tracking software and complementary low-cost hardware components. This system surpasses existing tools through accessible, high-resolution analysis across multiple imaging modalities, enabling comprehensive assessment of vascular dynamics in both real-time and pre-recorded experiments. Advanced algorithms enable multi-point diameter tracking in branched vessels, automated pressure-response protocols, and reliable edge detection. The software can assess vessels imaged by brightfield microscopy, fluorescence imaging, and in ultrasound recordings, supporting diverse applications from isolated vessel studies to in vivo assessment. For ex vivo applications, VasoTracker 2 includes modular open-source hardware components that can be used to create a low-cost pressure myograph system: a confocal-compatible vessel chamber and a programmable pressure controller, VasoMoto. By combining powerful analytical capabilities with an open-access approach, VasoTracker 2 provides free software and low-cost hardware alternatives to commercial systems, democratizing access to advanced vascular research tools for scientists worldwide.

physiology↗
bioRxiv2025

Klebsiella pneumoniae disrupts vasodilation by targeting eNOS post translational modifications via the type VI secretion system and the capsule polysaccharide

Vasodilation is a crucial protective response to inflammation and infection. Endothelial cells control vasodilation through the bioavailability of eNOS-produced nitric oxide (NO), and the generation of endothelium-dependent hyperpolarization (EDH). Here, we demonstrate that Klebsiella pneumoniae, one of the most prevalent blood stream infection pathogens, inhibits agonist-induced vasodilation by blunting the NO-dependent pathway and attenuating the EDH pathway. The type VI secretion system (T6SS) effector VgrG4 licences the kinase PKC{beta} in an NLRX1-controlled mitochondria reactive oxygen species (mtROS)-dependent manner to phosphorylate the eNOS inhibitory site Thr495, effectively dampening eNOS activity. The capsule polysaccharide, on the other hand, limits the phosphorylation of the eNOS activation site Ser1177 by inducing the phosphatase PP2Ac upon activation of an EGF receptor-dependent pathway. VgrG4-induced mtROS attenuates the EDH pathway. Overall, this work reveals a new anti-host activity of the T6SS and illustrates how pathogens can control vascular biology by targeting eNOS post translational modifications.

microbiology↗
bioRxiv2024

CD4+ tissue resident memory Th17 cells drive IL-17A-mediated joint pathology in Spondyloarthritis

ObjectivesInterleukin (IL)-17A is a key driver of Spondyloarthritis (SpA) joint pathology. We aimed to identify its cellular source in synovial tissue from patients with two forms of SpA namely Axial SpA (AxSpA) and Psoriatic arthritis (PsA). MethodsSynovial tissue from patients with SpA was profiled using single-cell RNA sequencing (scRNA-seq: AxSpA, n=5 and PsA, n=6) or spatial RNA profiling (PsA, n=4). CellPhoneDB was used to infer cell-cell communication. Tissue resident memory Th17 (TRM17)-like cells were generated in vitro using blood memory CD4+ T cells from SpA patients. An epigenetic inhibitor library, siRNA and clustered regularly interspaced short palindromic repeats (CRISPR) were used to identify epigenetic regulator(s) for TRM17. ResultsscRNA-seq showed that CD4+CXCR6+ TRM17 cells are the predominant spontaneous IL17A producers in SpA synovium. Cell-cell communication and single-cell spatial analysis support the interaction between TRM17 and CLEC10A+ dendritic cells, which were activated in SpA. Both sublining and lining fibroblasts in SpA synovium showed evidence of IL-17A activation. In vitro-generated CD4+ TRM17-like cells phenocopied joint tissue TRM17, producing IL-17A/F upon T cell receptor (TCR) stimulation, which was further enhanced by cytokines. Perturbation of BRD1 inhibited the generation of TRM17-like cells. ConclusionsCD4+ TRM17 cells are the predominant source of IL-17A in SpA synovial tissue. TCR stimulation is essential for the secretion of IL-17A by CD4+TRM17-like cells. The epigenetic regulator BRD1 contributes to the generation of CD4+TRM17. Depleting CD4+TRM17 cells in SpA is thus a therapeutic strategy with potential to induce long-term remission. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSInterleukin (IL)-17A plays a key role in the immunopathogenesis of Spondyloarthritis (SpA), but its cellular source in joint tissue has not been determined previously. The induction and accumulation of CD4+ tissue resident memory Th17 (TRM17) cells following the clearance of pathogens has been described in skin, lung and kidney. Whether CD4+ TRM17 cells also accumulate in the joint and contribute to the pathology of SpA is not clear. What this study addsO_LICD4+ TRM17 cells are present in SpA synovial tissue and are the predominant source of IL17A C_LIO_LICD4+ TRM17 cells in SpA joints express IL17A without any in vitro exogenous stimulation C_LIO_LIT cell receptor (TCR) rather than cytokine stimulation is essential for IL-17A production by CD4+ TRM17-like cells C_LIO_LIThe epigenetic regulator BRD1 contributes to the generation of CD4+ TRM17-like cells. C_LI How this study might affect research, practice or policyOur findings identify CD4+ TRM17 cells as the primary source of IL-17A in SpA synovium, a previously unrecognized role for these cells. Key questions remain: How do CD4+ TRM17 cells relate to IL-17A producers in synovial fluid? What mechanisms induce and maintain them in the joint? How do they interact with other cells to promote arthritis? These questions warrant further investigation. In addition, our data suggest that targeting CD4+ TRM17 cells, the "factory" of IL-17A in SpA synovial tissue, has the potential to induce long-term remission, encouraging future efforts to develop new therapies to deplete CD4+ TRM17 cells in SpA.

immunology↗
bioRxiv2024

Increased TRPV4 channel expression enhances and impairs blood vessel function in hypertension

BackgroundEndothelial cell TRPV4 channels provide a control point that is pivotal in regulating blood vessel diameter by mediating the Ca2+-dependent release of endothelial-derived vasoactive factors. In hypertension, TRPV4-mediated control of vascular function is disrupted but the underlying mechanisms, and precise physiological consequences remain controversial. MethodsHere, using a comprehensive array of methodologies, endothelial TRPV4 channel function was examined in intact arteries from normotensive WKY and hypertensive SHR rats. ResultsOur results show there is a notable shift in vascular reactivity in hypertension, characterized by enhanced endothelium-dependent vasodilation at low levels of TRPV4 channel activation. However, at higher levels of TRPV4 activity, this vasodilatory response is reversed, contributing to the aberrant vascular tone observed in hypertension. The change in response, from dilation to constriction, was accompanied by a shift in intracellular Ca2+ signaling modalities arising from TRPV4 activity. Oscillatory TRPV4-evoked IP3-mediated Ca2+ release, which underlies dilation, decreased, while the contraction inducing sustained Ca2+ rise, arising from TRPV4-mediated Ca2+ influx, increased. Our findings also reveal that while the sensitivity of endothelial cell TRPV4 to activation was unchanged, expression of the channel is upregulated and IP3 receptors are downregulated in hypertension. ConclusionsThese data highlight the intricate interplay between endothelial TRPV4 channel expression, intracellular Ca2+ signaling dynamics, and vascular reactivity. Moreover, the data support a new unifying hypothesis for the vascular impairment that accompanies hypertension. Specifically, that endothelial cell TRPV4 channels play a dual role in modulating blood vessel function in hypertension.

physiology↗
bioRxiv2023

A longitudinal single-cell therapeutic atlas of anti-tumour necrosis factor treatment in inflammatory bowel disease

Precision medicine in immune-mediated inflammatory diseases (IMIDs) requires an understanding of how cellular networks change following therapy. We describe a therapeutic atlas for Crohns disease (CD) and ulcerative colitis (UC) following anti-tumour necrosis factor (TNF) therapy. We generated ~1 million single-cell transcriptomes, organised into 109 cell states, from 216 gut biopsies from 38 patients and three controls, revealing disease- and therapy-specific differences. A systems-biology analysis identified distinct spatially-resolved cellular microenvironments: granuloma signatures in CD and interferon (IFN)-response signatures localising to T-cell aggregates and epithelial damage in CD and UC. Longitudinal comparisons demonstrated that disease progression in non-responders associated with myeloid and stromal cell perturbations in CD and increased multi-cellular IFN signalling in UC. IFN signalling was also observed in rheumatoid arthritis (RA) synovium with a lymphoid pathotype. Our therapeutic atlas informs drug positioning across IMIDs, and suggests a rationale for the use of janus kinase (JAK) inhibition following anti-TNF resistance.

immunology↗
bioRxiv2022

An optimised method for bacterial nucleic acid extraction from positive blood culture broths for whole genome sequencing, resistance phenotype prediction and downstream molecular applications

BackgroundA prerequisite to rapid molecular detection of pathogens causing bloodstream infections is an efficient, cost effective and robust DNA extraction solution. We describe methods for microbial DNA extraction direct from positive blood culture broths, suitable for metagenomic sequencing and the application of machine-learning based tools to predict antimicrobial susceptibility. MethodsProspectively collected culture-positive blood culture broths with Gram-negative bacteria, were directly extracted using various commercially available kits. We compared methods for efficient inhibitor removal, avoidance of DNA shearing or degradation, to achieve DNA of high quality and purity. Bacterial species identified via whole-genome metagenomic sequencing (Illumina, MiniSeq) from blood culture extracts were compared to conventional methods from cultured isolates (Vitek MS). A machine-learning algorithm (AREScloud) was used to predict susceptibility against commercially available antibiotics, compared to susceptibility testing (Vitek 2) and other commercially available rapid diagnostic instruments (Accelerate Pheno and BCID). ResultsA two-kit method using a modified MolYsis Basic kit (for host DNA depletion) and extraction using Qiagen DNeasy UltraClean microbial kits resulted in optimal extractions appropriate for multiple molecular applications, including PCR, short-read and long-read sequencing. DNA extracts from 40 blood culture broths were included. Taxonomic profiling by direct metagenomic sequencing matched species identification by conventional methods in 38/40 (95%) of samples, with two showing agreement to genus level. In two polymicrobial samples, a second organism was missed by sequencing. Whole genome sequencing antimicrobial susceptibility testing (WGS-AST) models were able to accurately infer profiles for 6 common pathogens against 17 antibiotics. Overall categorical agreement (CA) was 95%, with 11% very major errors (VME) and 3.9% major errors (ME). CA for WGS-AST was >95% for 5/6 of the most common pathogens (E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and C. jejuni) while it was lower for K. oxytoca (66.7%), likely due to the presence of inducible cephalosporinases. Performance of WGS-AST was sub-optimal for uncommon pathogens (e.g. Elizabethkingia) and some combination antibiotic compounds (e.g. ticarcillin-clavulanate). Time to pathogen identification and resistance gene detection was fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a rapid MIC result in approximately 8 h. While Illumina based direct metagenomic sequencing did not result in faster turn-around times compared conventional methods, use of real-time nanopore sequencing may allow faster data acquisition. ConclusionsThe application of direct metagenomic sequencing from positive blood culture broths is a feasible approach and solves some of the challenges of sequencing from low-bacterial load samples. Machine-learning based algorithms are also accurate for common pathogen / drug combinations, although additional work is required to optimise algorithms for uncommon species and more complex resistance genotypes, as well as streamlining methods to provide more rapid sequencing results.

microbiology↗
bioRxiv2021

Mitochondrial ATP production is required for endothelial cell control of vascular tone

Arteries and veins are lined by non-proliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis to fuel each of these energy-demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells in angiogenesis, and little is known about energy production in endothelial cells within the fully-formed vascular wall. Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial oxidative phosphorylation disrupts endothelial control of vascular tone. The role for endothelial cell energy production is independent of species, sex, or vascular bed. Basal, mechanically-activated, and agonist-evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. This effect is mimicked by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. These data show that mitochondrial ATP is necessary for calcium-dependent, nitric oxide mediated endothelial control of vascular tone, and identifies the critical role of endothelial mitochondrial energy production in fueling perfused blood vessel function.

physiology↗
bioRxiv2020

Disrupted Endothelial Cell Heterogeneity and Network Organization Impairs Vascular Function in Prediabetic Obesity

RationaleObesity is a major risk factor for diabetes and cardiovascular diseases such as hypertension, heart failure, and stroke. Impaired endothelial function occurs in the earliest stages of obesity and underlies vascular alterations giving rise to cardiovascular disease. However, the mechanisms that link weight gain to endothelial dysfunction are ill-defined. Increasing evidence suggests that, rather than being a population of uniformly responding cells, neighboring endothelial cells are highly heterogeneous and are organized as a communicating multicellular network that controls vascular function. ObjectiveTo investigate the hypothesis that disrupted endothelial heterogeneity and network-level organization contributes to impaired vascular reactivity in obesity. Methods and ResultsTo study obesity-related vascular function without the complications associated with diabetes, we induced a state of prediabetic obesity in rats. Small artery diameter recordings confirmed nitric-oxide mediated vasodilator responses were dependent on increases in endothelial calcium levels and were impaired in obese animals. Single-photon imaging revealed a linear relationship between blood vessel relaxation and network-level calcium responses. Obesity did not alter the slope of this relationship, but impaired network-level endothelial calcium responses. The network itself was comprised of structural and functional components. The structural component, a hexagonal lattice network of endothelial cells, was unchanged in obesity. The functional network contained sub-populations of clustered agonist-sensing cells from which signals were communicate through the network. In obesity there were fewer but larger clusters of agonist-sensing cells and communication path lengths between clusters was increased. Communication between neighboring cells was unaltered in obesity. Altered network organization resulted in impaired, population-level calcium signaling and deficient endothelial control of vascular tone. Specialized subpopulations of endothelial cells had increased agonist sensitivity. These agonist-responsive cells were spatially clustered in a non-random manner and drove network level calcium responses. Communication between adjacent cells was unaltered in obesity, but there was a decrease in the size of the agonist-sensitive cell population and an increase in the clustering of agonist-responsive cells ConclusionsThe distribution of cells in the endothelial network is critical in determining overall vascular function. Altered cell heterogeneity and arrangement in obesity decrease endothelial function and provide a novel framework for understanding compromised endothelial function in cardiovascular disease.

cell biology↗
bioRxiv2020

Coordinated assembly and release of adhesions builds apical junctional belts during de novo polarisation of an epithelial tube

Using the zebrafish neural tube as a model, we uncover the in vivo mechanisms allowing the generation of two opposing apical epithelial surfaces within the centre of an initially unpolarised, solid organ. We show that NOK/Pals1/Mpp5a and Rab11a play a dual role in coordinating the generation of ipsilateral junctional belts whilst simultaneously releasing contralateral adhesions across the centre of the tissue. We show that Nok and Rab11a mediated resolution of cell-cell adhesions are both necessary for midline lumen opening and contribute to later maintenance of epithelial organisation. We propose these roles for both Nok and Rab11a operate through the transmembrane protein Crumbs. In light of a recent conflicting publication, we also clarify that the junction remodelling role of Nok is not specific to dividing cells.

neuroscience↗
bioRxiv2019

Hybrid optical gating for long-term 3D time-lapse imaging of the beating embryonic zebrafish heart

Three-dimensional fluorescence time-lapse imaging of structural, cellular and sub-cellular processes in the beating heart is an increasingly achievable goal using the latest imaging and computational techniques. However, previous approaches have had significant limitations. Temporarily arresting the heart using drugs disrupts the hearts physiological state, and the use of ultra-high frame-rates for fluorescence image acquisition causes phototoxic cell damage. Real-time triggered imaging, synchronized to a specific phase in the cardiac-cycle, can computationally "freeze" the heart to acquire the minimal number of fluorescence images required for 3D time-lapse imaging. However, until now no solution has been able to maintain phase-lock to the same point in the cardiac cycle for more than about one hour. Our new hybrid optical gating system maintains phase-lock for up to 24 h, acquiring synchronized 3D+time video stacks of the unperturbed heart in vivo. This approach has enabled us to observe detailed developmental, structural, cellular and subcellular processes, including live cell division and cell fate tracking, in the embryonic zebrafish heart using transgenic fish lines expressing cell-specific fluorophores. We show that our approach not only provides high spatial and temporal resolution 3D-imaging, but also avoids phototoxic injury, where alternative approaches induce measurable harm. This provides superb cellular and subcellular imaging of the heart while it is beating in its normal physiological state, and opens up new and exciting opportunities for further study in the heart and other moving cellular and subcellular structures in vivo.

bioinformatics↗
bioRxiv2017

Use of whole genome sequencing to investigate an outbreak of gonorrhoea among females in urban New South Wales, Australia, 2012 to 2014.

Increasing rates of gonorrhoea have been observed among urban heterosexuals within the Australian state of New South Wales (NSW). Here, we applied whole genome sequencing (WGS) to better understand transmission dynamics. Ninety-four isolates of a particular N. gonorrhoeae genotype (G122) associated with female patients (years 2012 to 2014) underwent phylogenetic analysis using core single nucleotide polymorphisms (SNPs). Context for genetic variation was provided by including an unbiased selection of 1,870 N. gonorrhoeae genomes from a recent United Kingdom (UK) study. NSW genomes formed a single clade, with the majority of isolates belonging to one of five clusters, and comprised patients of varying age groups. Intra-patient variability was less than 7 core SNPs. Several patients had indistinguishable core SNPs, suggesting a common infection source. These data have provided an enhanced understanding of transmission of N. gonorrhoeae among urban heterosexuals in NSW, Australia, and highlight the value of using WGS in N. gonorrhoeae outbreak investigations.

epidemiology↗