Biology subjects

Hoyland, J. A.

Publications and source records attributed to Hoyland, J. A..

7 recordsLinked to original sources

bioRxiv2026

Circadian transcriptome in cultured cells depends on synchronisation method and differs from in vivo condition

In vitro synchronisation is widely used to study circadian clocks in cells, but whether cultured cells recapitulate tissue-level rhythmic outputs remains unclear. Articular cartilage provides a useful model to address this because chondrocytes are the only resident cell type. Here, we compared circadian time series transcriptomes between primary mouse chondrocytes synchronised by heat shock, dexamethasone, or osmotic stress and in vivo cartilage tissue. All three stimuli robustly synchronised core clock gene rhythms but produced distinct circadian phases and markedly different rhythmic transcriptomes, depending on the synchronizer. Heat shock, dexamethasone, and osmotic stress yielded 5255, 2008, and 879 transcripts classified as rhythmic, respectively, in primary chondrocytes, yet only 64 genes were shared across the three in vitro datasets, and only 15 were shared when in vivo cartilage transcriptome was included. Pairwise comparisons between synchronizers revealed some statistically enriched overlaps, but only marginally above chance, and shared genes showed limited conservation of circadian phase. Functional enrichment analysis also revealed stimulus-dependent rhythmic programmes with modest pathway-level overlap. These findings indicate that circadian output in cultured cells is shaped by the synchronising cue and cellular microenvironment. We also present BodyClocks.org, an interactive resource implementing this comparative framework across a curated collection of circadian transcriptomic datasets.

genomics↗

bioRxiv2025

Multimodal X-ray imaging reveals hierarchical fibre mechanics

Fibrous materials--ranging from connective tissues to engineered composites--are vital to many biological and man-made systems, optimised to withstand complex in-operando or in-vivo loading. The spines intervertebral discs (IVD) load-bearing capacity depends on a hierarchical extracellular matrix, where plywood-like lamellae of collagen fibres in the annulus fibrosus contain nanometre-scale fibrils built from staggered triple-helical monomers. How intact IVDs couple fibril-scale mechanics to fibre-scale organisation under load remains unresolved. Here we introduce TomoSAXS, a full-field 3D small-angle X-ray scattering tomography that maps fibril-to-fibre mechanics across an intact tissue. We show that intrafibrillar molecular pre-strain (D-period stagger) is lamellar textured and tightly correlated with microscale fibre strain. Pre-strain is inversely related to fibril strain and its variability, consistent with load-sharing through molecular unwinding. Radial strain bridges and high-curvature zones at the annulus fibrosus-nucleus pulposus interfaces emerge as critical regulators of local mechanics. These findings reveal concerted fibril-fibre interactions that sustain mechanical equilibrium in the IVD, preserving elasticity and shape. More broadly, TomoSAXS establishes a platform to visualise nano- to micro-scale matrix mechanics across biological and synthetic fibrous materials, with applications in ageing and disease, therapeutic evaluation, and the design of bio-based and bioinspired materials.

bioengineering↗

bioRxiv2024

Modulation of circadian rhythms in articular cartilage by heat pulses

ObjectivePrior studies have shown that disruption of the circadian clock leads to cartilage degeneration in mice while shift work is associated with higher risk of osteoarthritis (OA) in humans. In this study we investigated the potential of heat pulses to restore dampened circadian rhythms in articular cartilage. MethodsFemoral head cartilage explants and primary chondrocytes were isolated from PER2::LUC mice. Human femoral condyle cartilage was obtained from osteoarthritic patients undergoing total knee replacement. Tissues and cells were exposed to heat shock at various temperatures (37-43 {degrees}C) and incubation lengths. Bioluminescence from explants and cells was recorded in real-time. RNA sequencing and qPCR were used to assess gene expression changes in response to heat. ResultsWe established that a 60-min pulse at 43 {degrees}C was sufficient to restore dampened PER2::LUC rhythms in mouse cartilage explants or in primary chondrocytes. Transcriptome analysis in mouse articular cartilage showed an up-regulation of genes encoding heat shock proteins and collagens, and a transient down-regulation of Sox9, Runx2, Per1, Clock and Cry2. Heat induced the expression of circadian clock genes in human osteoarthritic knee cartilage. Mechanistically, inhibition of HSP90 activity or perturbation of F-actin polymerisation blocked the heat-induced resynchronisation of circadian rhythms. ConclusionTogether, these data have contributed to a greater understanding of the multifaceted nature of the connections between circadian timekeeping, heat stress responses and homeostasis in articular cartilage. These findings also suggest that time-prescribed temperature increases could be developed into a non-invasive intervention to slow down tissue ageing and restore homeostasis in osteoarthritic joints by improving circadian oscillations of cartilage rhythmic pathways.

cell biology↗

bioRxiv2023

The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc

The circadian clock in mammals temporally coordinates physiological and behavioural processes to anticipate daily rhythmic changes in their environment. Chronic disruption to circadian rhythms (e.g., through ageing or shift work) is thought to contribute to a multitude of diseases, including degeneration of the musculoskeletal system. The intervertebral disc (IVD) in the spine contains circadian clocks which control [~]6% of the transcriptome in a rhythmic manner, including key genes involved in extracellular matrix (ECM) homeostasis. However, it remains largely unknown to what extent the local IVD molecular clock is required to drive rhythmic gene transcription and IVD physiology. In this work, we identified profound age-related changes of ECM microarchitecture and an endochondral ossification-like phenotype in the annulus fibrosus (AF) region of the IVD in the Col2a1-Bmal1 knockout mice. Circadian time series RNA-Seq of the whole IVD in Bmal1 knockout revealed loss of circadian patterns in gene expression, with an unexpected emergence of 12-hour ultradian rhythms, including FOXO transcription factors. Further RNA sequencing of the AF tissue identified region-specific changes in gene expression, evidencing a loss of AF phenotype markers and a dysregulation of ECM and FOXO pathways in Bmal1 knockout mice. Consistent with an up-regulation of FOXO1 mRNA and protein levels in Bmal1 knockout IVDs, inhibition of FOXO1 in AF cells suppressed their osteogenic differentiation. Collectively, these data highlight the importance of the local molecular clock mechanism in the maintenance of the cell fate and ECM homeostasis of the IVD. Further studies may identify potential new molecular targets for alleviating IVD degeneration.

physiology↗

bioRxiv2022

Peptide location fingerprinting identifies species- and tissue-conserved structural remodelling of proteins as a consequence of ageing and disease

Extracellular matrix (ECM) in the intervertebral disc (IVD), lung and artery are thought to undergo the age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins. Here we applied PLF to publicly available ageing human IVD (outer annulus fibrosus), ageing mouse lung and human arterial atherosclerosis datasets and identified novel target proteins alongside common age-associated differences within protein structures which were conserved between tissue regions, organs, sexes and species and in age-related disease. We identify peptide yield differences across protein structures which coincide with biological regions, potentially reflecting the functional consequences of ageing or atherosclerosis for macromolecular assemblies (collagen VI and fibrin), enzyme/inhibitor activity (cathepsin B and alpha-2 macroglobulin), activation states (complement C3 and thrombin) and interaction states (laminins, perlecan, fibronectin, filamin-A, collagen XIV and apolipoprotein-B). Furthermore, we show that alpha-2 macroglobulin, prothrombin, collagen XIV and apolipoprotein-B all exhibit possible shared structural consequences in IVD ageing and arterial atherosclerosis, providing novel links between an age-related disease and intrinsic ageing. Crucially, we also demonstrate that fibronectin, laminin beta chains and filamin-A all exhibit conserved age-associated structural differences between mouse lung and human IVD, providing evidence that ECM, and their associating proteins, may be subjected to potentially similar mechanisms or consequences of ageing across species, irrespective of differences in lifespan and tissue function.

molecular biology↗

bioRxiv2021

Mechanical loading and hyperosmolarity as a daily resetting cue for skeletal circadian clocks

Daily rhythms in mammalian behaviour and physiology are generated by a multi-oscillator circadian system entrained through environmental cues (e.g. light). Presence of niche-dependent physiological time cues has been proposed, allowing local tissues flexibility of phase adjustment. However, to date, such stimuli have remained elusive. Here we show that cycles of mechanical loading and osmotic stimuli within physiological range drive rhythmic expression of clock genes and reset clock phase and amplitude in cartilage and intervertebral disc tissues. Hyperosmolarity (not hypo-osmolarity) resets clocks in young and ageing skeletal tissues through mTORC2-AKT-GSK3{beta} pathway, leading to genome-wide induction of rhythmic genes. These results advocate diurnal patterns of mechanical loading and consequent daily surges in osmolarity as a bona fide tissue niche-specific time cue to maintain skeletal circadian rhythms in sync. One-Sentence SummaryCircadian clocks in aneural skeletal tissues sense the passage of time through rhythmic patterns of loading and osmolarity

physiology↗

bioRxiv2019

Circadian time series proteomics reveals daily dynamics in cartilage physiology

ObjectivesArticular cartilage undergoes cyclical heavy loading and low load recovery during the 24-hour day/night cycle. We investigated the daily changes of protein abundance in mouse femoral head articular cartilage by performing 24-hour time-series proteomics study.\n\nMethodsTandem mass spectrometry analysis was used to quantify proteins extracted from mouse cartilage. Bioinformatics analysis was performed to quantify rhythmic changes in protein abundance. Primary chondrocytes were isolated and cultured for independent validation of selected rhythmic proteins.\n\nResults145 rhythmic proteins were detected. Among these were key cartilage molecules including CCN2, MATN1, PAI-1 and PLOD1 & 2. Pathway analysis revealed that proteins related to protein synthesis, cytoskeleton and glucose metabolism exhibited time-of-day dependent peaks in their abundance. Meta-analysis of published proteomics datasets from articular cartilage revealed that numerous rhythmic proteins were dysregulated in osteoarthritis and/or ageing.\n\nConclusionsOur circadian proteomics study revealed that articular cartilage is a much more dynamic tissue than previously thought. Chondrocytes exhibit circadian rhythms not only in gene expression but also in protein abundance. Our results clearly call for the consideration of circadian timing in understanding cartilage biology, osteoarthritis pathogenesis, treatment strategies and biomarker detection.

physiology↗