Biology subjects

Stewart, C. E.

Publications and source records attributed to Stewart, C. E..

13 recordsLinked to original sources

bioRxiv2025

Inflammasome Activation in Cutaneous Squamous Cell Carcinoma

Epithelia maintain their barrier function by exploiting the proliferative and plastic properties of stem cells to promote continual tissue regeneration. Consequently, however, these features make epithelia prone to tumorigenesis. Skin is the largest epithelial barrier and the source of cutaneous cancers, the deadliest of which are cutaneous squamous cell carcinomas (cSCCs). Despite the pervasiveness of these cancers, however, the molecular mechanisms employed by stem cells and their microenvironment to promote skin tumor development remain poorly defined. Our previous work revealed that genotoxic damage to normal skin activates inflammasome signaling crosstalk between epidermal epithelia and fibroblasts to promote epithelial stem cell hyperproliferation and fate misspecification. We hypothesized that these phenomena would also be featured during skin cancer development. Using mouse and human skin disease specimens, we determined that in vivo stem cell misspecification is a generalizable feature across diverse pathophysiological skin conditions, including cutaneous cancers, but is not present in normal high proliferative contexts. Strikingly, in vivo inflammasome activation was observed in both the epithelial and dermal compartments of cSCC tumors but was not evident in other skin pathologies. Probing further into the mechanism, we revealed that inflammasome pathway activation in cSCC is epithelial autonomous but non-cell autonomous. Furthermore, fibroblasts juxtaposed to the cSCC epithelial tumor interface exhibited population expansion as well as IL-1 signaling activation, which was absent in overlying epithelia. Based on these findings, we propose a model whereby epithelial-to-fibroblast inflammasome crosstalk initiates a fibroblast feed-forward IL-1 signaling loop that augments the tumor-promoting cSCC milieu.

cell biology↗

bioRxiv2025

Temperature signals drive grass secondary cell wall thickening

In grasses, stem elongation is driven by intercalary meristems at node-internode junctions, where cells divide, elongate, and in some cell types secondary wall maturation. Cellulose is the predominant polymer in plant cells and the most abundant biopolymer on Earth. It is synthesized at the plasma membrane by multi-protein complexes that include CELLULOSE SYNTHASE A (CESA) proteins. To investigate the spatiotemporal regulation of cellulose deposition during development, we developed a CESA8 luciferase gene expression reporter system in Brachypodium distachyon. High bioluminescence was observed in stem nodes, a specific region of elongating internodes, and the inflorescence, indicating sites of active secondary wall deposition. Within internodes, luminescence followed a distinct pattern, with a "dark zone" directly above the node with minimal signal, followed by a "bright zone" approximately 5 mm above the node where bioluminescence peaked. Histological, biophysical, and transcript analysis confirmed that luminescence intensity correlates with thickened secondary cell walls, increased cellulose crystallinity, and elevated CESA8 transcript levels. Time-lapse imaging revealed that CESA8 expression follows a robust diurnal rhythm governed by thermocycles alone, with peak expression occurring in the early morning. Temperature pulse experiments revealed an immediate but transient response of CESA8 to temperature shifts, which we modeled as an incoherent feed-forward loop. Finally, we found a strong correlation between CESA8 expression and stem elongation, highlighting the role of secondary cell wall thickening in supporting upright growth. These findings provide new insights into the regulation of secondary wall formation and its integration with environmental cues, advancing our understanding of grass stem development. SIGNIFICANCEUnderstanding how grasses build strong stems is essential for improving biomass production and crop resilience. In grasses, stem elongation and secondary cell wall thickening occur in distinct zones, yet the precise timing and regulation of this process remain unclear. To investigate this phenomenon, we developed a real-time imaging system to track the expression of CESA8, a key gene involved in cellulose synthesis. Our findings reveal that secondary wall thickening follows a daily rhythm controlled by temperature rather than light. These insights provide a foundation for optimizing plant architecture in bioenergy crops, improving their efficiency and sustainability.

plant biology↗

bioRxiv2024

Wavelet transform of single-trial vestibular short-latency evoked potential reveals temporary reduction in signal detectability and temporal precision following noise exposure

The vestibular short-latency evoked potential (VsEP) reflects the activity of irregular vestibular afferents and their target neurons in the brain stem. Attenuation of trial-averaged VsEP waveforms is widely accepted as an indicator of vestibular dysfunction, however, more quantitative analyses of VsEP waveforms could reveal underlying neural properties of VsEP waveforms. Here, we present a time-frequency analysis of the VsEP with a wavelet transform on a single-trial basis, which allows us to examine trial-by-trial variability in the strength of VsEP waves as well as their temporal coherence across trials. Using this method, we examined changes in the VsEP following 110 dB SPL noise exposure in rats. We found detectability of head jerks based on the power of wavelet transform coefficients was significantly reduced 1 day after noise exposure but recovered nearly to pre-exposure level in 3 - 7 days and completely by 28 days after exposure. Temporal coherence of VsEP waves across trials was also significantly reduced on 1 day after exposure but recovered with a similar time course. Additionally, we found a significant reduction in the number of calretinin-positive calyces in the sacculi collected 28 days after noise exposure. Furthermore, the number of calretinin-positive calyces was significantly correlated with the degree of reduction in temporal coherence and/or signal detectability of the smallest-amplitude jerks. This new analysis of the VsEP provides more quantitative descriptions of noise-induced changes as well as new insights into potential mechanisms underlying noise-induced vestibular dysfunction. Significance StatementOur study presents a new method of VsEP quantification using wavelet transform on a single-trial basis. It also describes a novel approach to determine the stimulus threshold of the VsEP based on signal-detection theory and Rayleigh statistics. The present analysis could also be applied to analysis of auditory brain stem response (ABR). Thus, it has the potential to provide new insights into the physiological properties that underlie peripheral vestibular and auditory dysfunction.

neuroscience↗

bioRxiv2023

Noise-induced changes in rat head stability and orientation correlate with dysfunction of otolith irregular afferents

Exposure to intense noise damages both the cochlea and vestibular end organs. Our group previously reported attenuated vestibular short-latency evoked potentials (VsEP) and reduced numbers of calretinin-positive (CR+) calyces in the saccule following noise exposure. Here, we examined rats resting head orientation with respect to gravity as well as head stability following a 4-hour exposure to 120 dB SPL noise. We also assessed how behavioral changes are related to changes in VsEP waveforms and calretinin expression in the utricle and saccule to elucidate potential underlying mechanisms. We found significant reductions in the P2N2 and N2P3 amplitudes following noise exposure. The number of CR+ calyces in both saccule and utricle were also significantly reduced. The size of the reduction in N2P3 amplitude was significantly correlated to the number of CR+ calyces. Animals with a greater loss of CR+ calyces in the utricle following noise showed significant decreases in the average speed of y-axis rotational head motion, while those with a fewer loss of CR+ calyces showed significant increases. In addition, animals with larger noise-induced changes in VsEP and CR+ calyces held their heads motionless longer following noise exposure. We hypothesize that noise exposure is inherently destructive to an animals head stability and thereby manifests as an increase in average head speed in mildly to moderately affected animals. But when the damage was large enough, animals exhibited reduced duration and head motion speed as a behavioral adaptation. The noise exposure also significantly altered the pitch angle of head orientation in animals who had the largest reduction in CR+ calyces in the saccule, suggesting that saccular irregular afferents, including those that are CR+, are critical in control of head and body posture.

neuroscience↗

bioRxiv2023

Ataxia-telangiectasia mutated (Atm) disruption sensitizes spatially-directed H3.3K27M/TP53 diffuse midline gliomas to radiation therapy

Diffuse midline gliomas (DMGs) are lethal brain tumors characterized by p53-inactivating mutations and oncohistone H3.3K27M mutations that rewire the cellular response to genotoxic stress, which presents therapeutic opportunities. We used RCAS/tv-a retroviruses and Cre recombinase to inactivate p53 and induce K27M in the native H3f3a allele in a lineage- and spatially-directed manner, yielding primary mouse DMGs. Genetic or pharmacologic disruption of the DNA damage response kinase Ataxia-telangiectasia mutated (ATM) enhanced the efficacy of focal brain irradiation, extending mouse survival. This finding suggests that targeting ATM will enhance the efficacy of radiation therapy for p53-mutant DMG but not p53-wildtype DMG. We used spatial in situ transcriptomics and an allelic series of primary murine DMG models with different p53 mutations to identify transactivation-independent p53 activity as a key mediator of such radiosensitivity. These studies deeply profile a genetically faithful and versatile model of a lethal brain tumor to identify resistance mechanisms for a therapeutic strategy currently in clinical trials.

cancer biology↗

bioRxiv2023

Exercise on cytokine responses in males and females: Effect of leucine, HMB and BCAA.

PurposeTo identify the effects of leucine, {beta}-hydroxy {beta}-methylbutyrate (HMB) and branched chain amino acid (BCAA) on post-exercise cytokine responses in females and males. MethodsMales (n=53) and females (n=37) completed 100 drop jumps and consumed either no supplement, leucine (3g/d), HMB (3g/d) or BCAA (4.5g/d) from 1d pre to 14d post-exercise. Muscle soreness, squat jumps, chair rises and creatine kinase (CK) were measured at pre, post, 24h, 48h, 7 and 14d. Blood lactate (pre, post), 10 cytokines (pre, 24h, 48h, 7d) and oestradiol (pre, 7d) were also measured. ResultsWithout supplementation post-exercise, soreness was induced in both males (6-fold) and females (5-fold). With supplementation, there were no increases in CK or oestradiol in females and no impact on muscle soreness, performance, or function in both sexes. In males, CK was elevated in untreated (48%) and leucine (69%) conditions vs baseline, but these were suppressed with HMB and BCAA. IL-7 was elevated in females vs males at baseline (6.3-fold), leucine increased IL-7 concentrations in females at 24h (17.0-fold), 48h (5.1-fold) vs males. With HMB, TNFr1- increased in females at 24h (2.2-fold), 48h (2.3-fold) and 7d (2.3-fold) vs males. In males with BCAA, TNFr1- decreased (P=0.06) from pre to 24h (6.8-fold), then increased (P<0.05) from 24 to 48h (8.0-fold). ConclusionAlthough supplements were without effect on soreness following exercise, the cytokine response was evoked by exercise and impacted significantly by leucine, HMB and BCAA in females vs males. This improved cytokine response in females could lead to improved resistance to damage.

physiology↗

bioRxiv2023

Evidence of Disrupted-in Schizophrenia 1 (DISC1) as an arsenic binding protein and implications regarding its role as a translational activator

Disrupted-in-schizophrenia-1 (DISC1) is a scaffold protein that plays a pivotal role in orchestrating signaling pathways involved in neurodevelopment, neural migration, and synaptogenesis. Among those, it has recently been reported that the role DISC1 in the Akt/mTOR pathway can shift from a global translational repressor to a translational activator in response to oxidative stress induced by arsenic. In this study we are providing evidence that DISC1 can directly bind arsenic via a C-terminal cysteine motif (C-X-C-X-C). A series of fluorescence-based binding assays were conducted with a truncated C-terminal domain construct of DISC1 and a of series of single, double, and triple cysteine mutants. We found that arsenous acid, a trivalent arsenic derivative, specifically binds to the C-terminal cysteine motif of DISC1 with low micromolar affinity. All three cysteines of the motif are required for high-affinity binding. Electron microscopy experiments combined with in silico structural predictions revealed that that the C-terminal of DISC1 forms an elongated tetrameric complex. The cysteine motif is consistently predicted to be located within a loop, fully exposed to solvent, providing a simple molecular framework to explain the high-affinity of DISC1 toward arsenous acid. This study sheds light on a novel functional facet of DISC1 as an arsenic binding protein and highlights its potential role as both a sensor and translational modulator within the Akt/mTOR pathway.

biophysics↗

bioRxiv2022

Atm loss does not radiosensitize a primary mouse model of Pten-deleted brainstem glioma

Diffuse midline gliomas arise in the brainstem and other midline brain structures and cause a large proportion of childhood brain tumor deaths. Radiation therapy is the most effective treatment option, but these tumors ultimately progress. Inhibition of the phosphoinositide-3-kinase (PI3K)-like kinase ataxia telangiectasia mutated (ATM), which orchestrates the cellular response to radiation-induced DNA damage, may enhance the efficacy of radiation therapy. Diffuse midline gliomas in the brainstem contain loss-of-function mutations in the tumor suppressor PTEN, or functionally similar alterations in the phosphoinositide-3-kinase (PI3K) pathway, at moderate frequency. Here, we sought to determine if Atm inactivation could radiosensitize a primary mouse model of brainstem glioma driven by Pten loss. Using Cre/loxP recombinase technology and the RCAS/TVA retroviral gene delivery system, we established a mouse model of brainstem glioma driven by Pten deletion. We find that Pten-null brainstem gliomas are relatively radiosensitive at baseline. In addition, we show that deletion of Atm in the tumor cells does not extend survival of mice bearing Pten-null brainstem gliomas after focal brain irradiation. These results characterize a novel primary mouse model of PTEN-mutated brainstem glioma and provide insights into the mechanism of radiosensitization by Atm deletion, which may guide the design of future clinical trials. Brief SummaryWe develop a mouse model of PTEN-mutated brainstem glioma and find that perturbation of the ATM does not enhance radiation efficacy in this model.

cancer biology↗

bioRxiv2022

Replicative ageing perturbs the metabolic signature of murine C2C12 skeletal myotubes

IntroductionChronological ageing is associated with mitochondrial dysfunction and increased reactive oxygen species (ROS) production in skeletal muscle. However, the effects of replicative ageing on skeletal muscle cellular metabolism are not well known. Using an established myoblast model of cellular (replicative) ageing, we investigated the impact of ageing on energy metabolism in murine C2C12 myotubes. MethodsControl (P7-11) and replicatively aged (P48-51) C2C12 myoblasts were differentiated over 72-120 h. Mitochondrial bioenergetics were investigated by respirometry and mitochondrial superoxide and cellular ROS were measured in the absence and presence of antimycin A (AA). Genes related to mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Intracellular metabolites were quantified using an untargeted 1H-NMR metabolomics pipeline. ResultsMitochondrial coupling efficiency (Control: 79.5 vs. Aged: 70.3%, P=0.006) and relative oxidative ATP synthesis (Control: 48.6 vs. Aged: 31.7%, P=0.022) were higher in control vs. aged myotubes, but rates of mitochondrial superoxide production were lower (Control: 2.4x10-5 {+/-} 0.4 x 10-5 vs. Aged: 9.7x10-5 {+/-} 1.6x10-5 RFU/sec/cell; P=0.035). Replicatively aged myotubes had greater mRNA expression of mfn2 and Tfam compared to control. Yet, Nrf2 and PGC-1 expression were 2.8-fold and 3.0-fold higher in control versus aged myotubes over 24 h and 48 h (P<0.05), respectively. Branched chain amino acids L-leucine, L-isoleucine and L-valine, and L-carnitine were less abundant in aged versus control myotubes. Conclusion(s)Replicative ageing is associated with bioenergetic uncoupling, increased ROS production and impaired amino acid metabolism. Our findings suggest that cellular mitochondrial dysfunction and altered energy metabolism may exacerbate the age-related decline in skeletal muscle mass and function.

cell biology↗

bioRxiv2021

Degradation of ribosome and chaperone proteins is attenuated during the Differentiation of Replicatively Aged C2C12 Myoblasts

Age-related impairments in myoblast differentiation may contribute to reductions in muscle function in older adults, however, the underlying proteostasis processes are not well understood. Young (P6-10) and replicatively aged (P48-50) C2C12 myoblast cultures were investigated during early (0h-24h) and late (72h-96h) stages of differentiation using deuterium oxide (D2O) labelling and mass spectrometry. The absolute dynamic profiling technique for proteomics (Proteo-ADPT) was applied to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins. Proteo-ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P<0.05, FDR <5 %) different changes in abundance between young and aged cells at early and later periods of differentiation. Young cells exhibited a steady pattern of growth, protein accretion and fusion, whereas aged cells failed to gain protein mass or undergo fusion during later differentiation. Maturation of the proteome was retarded in aged myoblasts at the onset of differentiation, but their proteome appeared to catch up with the young cells during the early phase of the differentiation period. However, this catch up process in aged cells was not accomplished by higher levels of protein synthesis. Instead, a lower level of protein degradation in aged cells was responsible for the elevated gains in protein abundance. Our novel data point to a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts and highlights dysregulation of protein degradation, particularly of ribosomal and chaperone proteins, as a key mechanism that may contribute to age-related declines in the capacity of myoblasts to undergo differentiation.

cell biology↗

bioRxiv2020

UBR5 knockdown in human myotubes in-vitro and mouse skeletal muscle tissue in-vivo determines its pertinent role in anabolism and hypertrophy

UBR5 is an E3-ubiquitin-ligase positively associated with anabolism, hypertrophy and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5s role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in-vitro and in-vivo. The siRNA-induced reduction (-77%) in UBR5 gene expression in human myotubes was prevented by mechanical loading, suggesting that UBR5 gene expression may be regulated via mechano-transduction signalling. Therefore, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle in-vivo to investigate the impact of reduced UBR5 on mechano-transduction signalling MEK/ERK/p90RSK and Akt/p70S6K/4E-BP1/rpS6 pathways. Seven days post UBR5 RNAi electroporation, while reductions in overall muscle mass were not detected, mean CSA of GFP-positive fibers was reduced (-9.5%) and the number of large fibers was lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2 and Akt phosphorylation. Whilst p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early signalling events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (-4.6%) and larger reductions in fiber CSA (-18.5%) after 30 days. This was associated with increased levels of the phosphatase, PP2Ac, and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, and corresponding reductions in eIF4e. This study demonstrates UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.

physiology↗

bioRxiv2019

DNA methylation across the genome in aged human skeletal muscle tissue and muscle stem cells: The role of HOX genes and physical activity

Skeletal muscle tissue demonstrates global hypermethylation with aging. However, methylome changes across the time-course of differentiation in aged human muscle derived stem cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 {+/-} 4.4 years) and aged (83 {+/-} 4 years) human skeletal muscle and that of young/aged muscle stem cells over several time points of differentiation (0, 72 hours, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged muscle stem cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alteration in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in the focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and isolated muscle stem cells, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle and tissue and stem cells (on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P). Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6-8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and muscle stem cells and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

physiology↗

bioRxiv2019

Neuromuscular fatigue and recovery after strenuous exercise depends on skeletal muscle size and stem cell characteristics

Hamstring muscle injury is highly prevalent in sports involving repeated maximal sprinting. Although neuromuscular fatigue is thought to be a risk factor, the mechanisms underlying the fatigue response to repeated maximal sprints are unclear. Here, we show that repeated maximal sprints induce neuromuscular fatigue accompanied with a prolonged strength loss in hamstring muscles. The immediate hamstring strength loss was linked to both central and peripheral fatigue, while prolonged strength loss was associated with indicators of muscle damage. The kinematic changes immediately after sprinting likely protected fatigued hamstrings from excess elongation stress, while larger hamstring muscle physiological cross-sectional area and lower myoblast:fibroblast ratio appeared to protect against fatigue/damage and improve muscle recovery within the first 48 h after sprinting. We have therefore identified novel mechanisms that likely regulate the fatigue/damage response and initial recovery following repeated maximal sprinting in humans.

physiology↗