Biology subjects

Day, N. J.

Publications and source records attributed to Day, N. J..

9 recordsLinked to original sources

bioRxiv2026

CaMKII Activation Enhances Antioxidant Defence and Mitochondrial Function in human articular chondrocytes

BackgroundThe articular cartilage has limited vascular supply and repair capacity, making it particularly susceptible to reactive oxygen species (ROS)-driven oxidative damage. Excess ROS contributes to extracellular matrix breakdown and is a major factor in osteoarthritis (OA) progression. We previously identified Calcium/Calmodulin-dependent protein kinase II (CaMKII) as a regulator of cartilage homeostasis, prompting us to investigate its role during oxidative stress. MethodsPrimary adult human articular chondrocytes were isolated from OA cartilage. CaMKII activity was modulated using adenoviral overexpression of a constitutively active form of the kinase or of Autocamtide-2-related inhibitory peptide, a CaMKII inhibitor. Redox status and mitochondrial function were assessed by molecular and metabolic assays. ResultsOxidative stress increased CaMKII phosphorylation. CaMKII inhibition elevated cellular and mitochondrial ROS, whereas CaMKII activation enhanced mitochondrial respiration capacity, improved mitochondrial morphology, and was associated to NRF2 nuclear translocation. ConclusionCaMKII supports redox homeostasis in chondrocytes and may represent a therapeutic strategy to preserve cartilage integrity and delay disease onset.

cell biology↗

bioRxiv2025

Autologous human iPSC-derived Alveolus-on-Chip reveals early pathological events of M. tuberculosis infection

Immunocompetent and experimentally accessible alveolar systems to study human respiratory diseases are lacking. Here, we developed a single donor human induced pluripotent stem cell (iPSC)-derived Lung-on-Chip (iLoC) containing Type II and I alveolar epithelial cells, vascular endothelial cells, and macrophages in a microfluidic device that mimic lung 3D mechanical stretching and air-liquid interface. Imaging and scRNA-seq analysis revealed that the iLoC recapitulated cellular profiles present in the human distal lung. Infection of the iLoC with the human pathogen Mycobacterium tuberculosis (Mtb) showed that both macrophages and epithelial cells were infected and showed limited bacterial replication. Stochastically, large macrophage clusters containing necrotic core-like structure and Mtb replication were observed. A genetically engineered autophagy deficient iLoC revealed that after Mtb infection, macrophage necrosis was higher upon ATG14 deficiency without bacterial replication. Altogether, we report an autologous, genetically tractable human alveolar model to study lung diseases and therapies.

bioengineering↗

bioRxiv2024

microRNA-544a as a new modulator of the Wnt-signalling network in the articular cartilage and osteoarthritis

ObjectiveDetermining the effect of microRNA-544a (miR-544a) in articular chondrocytes isolated from patients affected by osteoarthritis (OA) and its role in the modulation of the Wnt signalling. MethodsArticular chondrocytes were isolated from patients undergoing joint replacement because of OA. Expression levels of miR-544a were measured by PCR and by in situ hybridization. Putative targets of miR-544a were confirmed by reporter assay and by qPCR in cells stimulated with a miR-544a mimic. The effect of miR-544a on chondrocyte metabolism was monitored by qPCR for phenotypic markers, protein expression levels of aggrecan neoepitopes/MMP-13 and modulation of alcian blue content in micromass cultures, upon stimulation with a miR-544a mimic. The expression levels of MMP-13 and Aggrecan neoepitopes in response to miR-544a stimulation was also measured in co-stimulation with Xav-939 and KN93, which are respectively {beta}-catenin and CaMKII inhibitors. ResultsOur results suggest that miR-544a enhances the activation of the Wnt-signalling in the articular chondrocytes, by downregulating the expression of components of the Wnt/{beta}-catenin destruction complex. The expression of miR-544a is higher in chondrocytes isolated from damaged areas of the articular cartilage removed from OA patients, and can be upregulated by pro-inflammatory and pro-fibrotic cytokines. miR-544a exerts a pro-catabolic effect of articular chondrocytes, which is rescued both by the inhibition of the Wnt/{beta}-catenin and Wnt/CaMKII signalling pathways. Conclusionour results point to miR-544a as a new, important modulator of the Wnt signalling network within the articular cartilage suggesting a key role for microRNAs in regulating how the multiple branches of the network and their interaction modulate cartilage homeostasis.

pharmacology and toxicology↗

bioRxiv2024

CaMKII induces an autophagy-dependent anabolic response in Articular Chondrocytes

ObjectiveThe objective of this study was to elucidate the role of Calcium calmodulin-dependent Kinase II (CaMKII) in articular chondrocytes and its involvement in osteoarthritis (OA) pathogenesis. By performing gain and loss of function experiments, the research aimed to determine how CaMKII modulates chondrocyte metabolism, anabolic and catabolic processes, hypertrophic differentiation, and autophagy within the articular cartilage. DesignArticular cartilage was harvested from patients undergoing joint replacement surgery for OA, and adult human articular chondrocytes (AHACs) were isolated and cultured. Recombinant adenoviruses were used to overexpress a constitutively active form of CaMKII{gamma} (AdCaMKII) or inhibit CaMKII activity (AdAIP). Various assays, including RT-PCR analysis, alcian blue staining of Micromass cultures, immunofluorescence, and Western blotting, were performed to assess the effects of CaMKII modulation on chondrocyte function. ResultsOverexpression of activated CaMKII{gamma} promoted anabolism, evidenced by increased expression of SOX9, COL2A1, and ACAN, and decreased MMP-13 levels. It also enhanced proteoglycan content in AHAC micromass cultures. Furthermore, CaMKII counteracted the catabolic effects of IL-1{beta} and preserved proteoglycan content. We also observed decreased chondrocyte proliferation and increased synthesis of hypertrophic marker Type X Collagen. CaMKII activation was found to induce autophagy, as indicated by increased phosphorylation of Beclin1 and decreased p62 expression. The anabolic effects of CaMKII were dependent on autophagy, as inhibition of autophagy with Bafilomycin prevented the CaMKII-induced increase in glycosaminoglycan content. ConclusionsCaMKII plays a significant role in modulating chondrocyte metabolism and maintaining cartilage homeostasis. It promotes anabolic processes, counteracts catabolic stimuli, and induces autophagy in articular chondrocytes. However, it also promotes hypertrophic differentiation, highlighting the complexity of CaMKII-mediated signalling in cartilage. Understanding these pathways could lead to new therapeutic strategies that leverage CaMKIIs anabolic potential while mitigating its pro-degenerative effects.

cell biology↗

bioRxiv2022

A Deep Redox Proteome Profiling Workflow and Its Application to Skeletal Muscle of a Duchene Muscular Dystrophy Model

Perturbation to the redox state accompanies many diseases and its effects are viewed through oxidation of biomolecules, including proteins, lipids, and nucleic acids. The thiol groups of protein cysteine residues undergo an array of redox post-translational modifications (PTMs) that are important for regulation of protein and pathway function. To better understand what proteins are redox regulated following a perturbation, it is important to be able to comprehensively profile protein thiol oxidation at the proteome level. Herein, we report a deep redox proteome profiling workflow and demonstrate its application in measuring the changes in thiol oxidation along with global protein expression in skeletal muscle from mdx mice, a model of Duchenne Muscular Dystrophy (DMD). In depth coverage of the thiol proteome was achieved with >18,000 Cys sites from 5608 proteins in muscle being quantified. Compared to the control group, mdx mice exhibit markedly increased thiol oxidation, where ~2% shift in the median oxidation occupancy was observed. Pathway analysis for the redox data revealed that coagulation system and immune-related pathways were among the most susceptible to increased thiol oxidation in mdx mice, whereas protein abundance changes were more enriched in pathways associated with bioenergetics. This study illustrates the importance of deep redox profiling in gaining a greater insight into oxidative stress regulation and pathways/processes being perturbed in an oxidizing environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/504013v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@1917f1org.highwire.dtl.DTLVardef@1730cd0org.highwire.dtl.DTLVardef@4e4820org.highwire.dtl.DTLVardef@1615f4c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeep redox profiling workflow results in stoichiometric quantification of thiol oxidation for > 18,000 Cys sites in muscle C_LIO_LIThiol redox changes were much more pronounced than protein abundance changes for the overlapping set of proteins C_LIO_LIRedox changes are most significant in coagulation and immune response pathways while abundance changes on bioenergetics pathways C_LI

biochemistry↗

bioRxiv2022

Hydroquinone, a cigarette smoke compound, affects cartilage homeostasis through activation of the aryl hydrocarbon receptor pathway.

Exposure to cigarette smoke has a proven detrimental impact on different aspects of human health. Increasing evidences link smoking to degeneration of joint tissues. However, the toxic mechanisms elicited by the different components of cigarette smoke have not been fully elucidated yet. We have previously shown that exposure to hydroquinone (HQ), a pro-oxidant chemical present in cigarette smoke, can promote joint tissue degradation in murine models of rheumatoid arthritis through the activation of the aryl hydrocarbon receptor (AhR) pathway. Osteoarthritis (OA) is a chronic debilitating articular disease characterized by progressive degradation of the articular cartilage, whose onset and progression have also been associated with smoking. In this work we aimed to investigate the effect of HQ exposure on articular chondrocytes and how it affects cartilage homeostasis. Cell viability, gene expression, oxidative stress and inflammatory parameters were quantified in primary articular chondrocytes exposed to HQ in presence or absence of IL-1{beta} pre-stimulation. HQ stimulation downregulated phenotypic markers genes such as SOX-9 and Col2a1, whereas upregulated the expression of the catabolic enzymes MMP-3 and ADAMTS5. HQ also promoted oxidative stress and reduced proteoglycan content. HQ exacerbated the pro-inflammatory effects mediated by the IL-1{beta} co-stimulation. Finally, we showed that HQ-degenerative effects were mediated by the activation of AhR. Together, our findings address the harmful effects of HQ in the articular cartilage health, providing novel evidence surrounding the toxic mechanisms of environmental pollutants underlying the onset of articular diseases.

pharmacology and toxicology↗

bioRxiv2021

LOTVS: a global collection of permanent vegetation plots

Analysing temporal patterns in plant communities is extremely important to quantify the extent and the consequences of ecological changes, especially considering the current biodiversity crisis. Long-term data collected through the regular sampling of permanent plots represent the most accurate resource to study ecological succession, analyse the stability of a community over time and understand the mechanisms driving vegetation change. We hereby present the LOng-Term Vegetation Sampling (LOTVS) initiative, a global collection of vegetation time-series derived from the regular monitoring of vascular plants in permanent plots. With 79 datasets from five continents and 7789 vegetation time-series monitored for at least six years and mostly on an annual basis, LOTVS possibly represents the largest collection of temporally fine-grained vegetation time-series derived from permanent plots and made accessible to the research community. As such, it has an outstanding potential to support innovative research in the fields of vegetation science, plant ecology and temporal ecology.

ecology↗

bioRxiv2019

PUF family proteins FBF-1 and FBF-2 regulate germline stem and progenitor cell proliferation and differentiation in C. elegans

Stem cells support tissue maintenance, but the mechanisms that balance the rate of stem cell self-renewal with differentiation at a population level remain uncharacterized. Through investigating the regulation of germline stem cells by two PUF family RNA-binding proteins FBF-1 and FBF-2 in C. elegans, we find that FBF-1 restricts differentiation, while FBF-2 promotes both proliferation and differentiation. FBFs act on a shared set of target mRNAs; however, FBF-1 destabilizes target transcripts, while FBF-2 promotes their accumulation. These regulatory differences result in complementary effects of FBFs on stem cells. We identify a mitotic cyclin as one of the targets affecting stem cell homeostasis. FBF-1-mediated translational control requires the activity of CCR4-NOT deadenylase. Distinct abilities of FBFs to cooperate with CCR4-NOT depend on protein sequences outside of the conserved PUF family RNA-binding domain. We propose that the combination of FBF activities regulates the dynamics of germline stem cell proliferation and differentiation.

developmental biology↗

bioRxiv2018

Multivariate methods for testing hypotheses of temporal community dynamics

For ecological research to make important contributions towards understanding and managing temporally-variable global change processes, such as responses to land-use and climatic change, we must have effective and comparable ways to quantify and analyse compositional change over time in biological communities. These changes are the sum of local colonisation and extinction events, or changes in the biomass and relative abundance of taxa within and among samples. We conducted a quantitative review of currently available methods for the analysis of multivariate datasets collected at temporal intervals. This review identified the need for the application of quantitative, hypothesis-based approaches to understand temporal change in community composition, particularly for small datasets with less than 15 temporal replicates. To address this gap, we: (1) conceptually present how temporal patterns in community dynamics can be framed as specific, testable hypotheses; (2) provide three fully-worked case-studies, complete with R code, demonstrating multivariate analysis methods for temporal hypothesis testing and pattern visualisation; and (3) present a road map for testing specific, quantitative hypotheses relating to the underlying mechanisms of temporal community dynamics.

ecology↗