Cell Stress and Chaperones

Heatstroke is a life-threatening condition in which heat-shock and unfolded-protein responses are strongly activated but fail to prevent proteostasis disruption and severe cellular injury. Whether post-transcriptional regulation contributes to this mismatch remains unknown. We integrated small RNA sequencing with mRNA profiling in peripheral blood mononuclear cells from patients with classical heatstroke and matched heat-exposed controls recruited during the Hajj pilgrimage. mRNA profiling was performed in 19 cases and 19 controls, and miRNA sequencing in 17 cases and 16 controls from the same cohort. Differentially expressed miRNAs were integrated with 4,462 differentially expressed mRNAs using high-confidence inverse-expression miRNA-mRNA pairs. Twenty-six miRNAs mapped to 376 mRNA targets, forming 414 regulatory pairs and two opposing programmes. Programme A, comprising 16 downregulated miRNAs, was associated with activation of PI3K-mTOR, NRF2 oxidative stress and HIF-1 signalling, consistent with stress-survival signalling. Programme B, comprising 10 upregulated miRNAs, was associated with suppression of stress-granule components and fatty-acid {beta}-oxidation genes, consistent with impaired protein quality control and reduced metabolic flexibility. miR-92a-3p emerged as a central regulatory node, and its target PIK3R3 connected 9 of the 10 enriched pathways. These findings suggest a post-transcriptional regulatory layer that could contribute to the limited protection afforded by activated stress defences in human heatstroke.

Redox balance is central to aerobic metabolism, yet acute heat stress can destabilize this balance by increasing metabolic rates and shifting the balance of critical electron carriers such as NADH. In early Drosophila melanogaster embryos, maintaining redox balance is particularly critical as embryos undergo a developmental redox shift and rely on oxidative phosphorylation to power nuclear divisions. Here, we assayed six isofemale D. melanogaster lines from temperate (Vermont, USA; France; Japan) and tropical (St. Kitts; Ghana; India) climates to assess metabolic responses to heat in heat-sensitive versus heat-tolerant embryos. We used untargeted LC--MS to measure 33 metabolites and the major redox couples (NADH/NAD+, NADPH/NADP+, and GSH/GSSG) at 25{degrees}C and after a 32{degrees}C heat shock. In all embryos, heat shock induced shared shifts in metabolic profiles, with increases in nucleotide monophosphates (e.g., AMP, CMP, and GMP) and amino acids (e.g., alanine, glutamic acid, serine). In contrast, redox metabolites diverged by region: heat-sensitive temperate embryos shifted toward a more oxidized state (46.6% decrease in NADH/NAD+ ratio and 4-fold increase in oxidized glutathione), while heat-tolerant tropical embryos maintained glutathione balance and increased the NADH/NAD+ ratio by 52.9%, indicating a more reduced state. These patterns are consistent with higher NADH oxidation and greater oxidative stress (inferred from oxidized glutathione) in the temperate embryos, versus better maintenance of redox balance in tropical embryos. Together, our results suggest that maintaining redox balance is a key determinant of acute heat tolerance, and healthy development overall, during early embryogenesis.

Many insects enter diapause, a programmed state of developmental arrest that enables survival under adverse environmental conditions. In Drosophila melanogaster Meigen, 1830, diapause is characterized by reproductive arrest and reduced intestinal growth, accompanied by suppressed intestinal stem cell (ISC) activity. Juvenile Hormone (JH) promotes ISC proliferation under favorable conditions, but its capacity to modulate stem cell dynamics during cold-induced diapause remains unclear. Here, we investigated whether JH signaling can reactivate midgut remodeling in adult females maintained at 11. At this temperature, flies exhibited pronounced gut atrophy and elevated Phospho-histone H3 (PH3+) cell abundance, consistent with temperature-dependent G2/M phase arrest JH treatment significantly increased the proportion of Delta-positive progenitor cells in the anterior (R2) and posterior (R5) midgut regions at both 11 and 25, demonstrating that JH acts as a conserved mitogen for the ISC pool irrespective of thermal environment. A trend toward reduced PH3+ accumulation in the posterior midgut following JH treatment (p = 0.061) suggests possible facilitation of mitotic exit, though this effect did not reach statistical significance. Despite cellular-level changes, JH treatment did not restore overall gut size, indicating that the 72-84 hour exposure window was insufficient for subsequent tissue hypertrophy. Additionally, we identified a recurrent cold-induced pathology of gut distension, provisionally termed Lumen Obstruction Syndrome (LOS), which was independent of JH signaling. These findings reveal an uncoupling of JH-driven stem cell expansion from gross organ growth under diapause conditions, highlighting the selective sensitivity of the ISC compartment to endocrine signaling during environmental stress.

The deepest ocean is one of the most extreme environments for life on our planet, combining near-freezing temperatures, low oxygen levels, and hydrostatic pressures reaching 111 MPa (1100 atm). Extreme pressures are predicted to alter many aspects of biology, including the physical properties of biological hydrogels, protein structure, and the solubility of gases in water. How organisms have adapted to live in these conditions is poorly understood. Studying these organisms in situ is difficult and requires specialized deep-sea equipment capable of withstanding the extreme pressure; raising these organisms in captivity is also challenging due to their extreme habitat requirements. Given these difficulties in studying deep-sea organisms, we set out to identify the problems shallow-dwelling organisms face due to increased pressure. These can provide insights into how organisms tolerate life in the deepest parts of the ocean. This project aims to take embryos of the shallow-dwelling aquatic organism Xenopus laevis, determine how surface-dwelling organisms fail under high hydrostatic pressure, and identify a means to survive this deadly pressure. We have designed a system to expose different embryonic stages of X. laevis to high pressures and observe its effects. After identifying the limits of survivability, we sought to understand how these embryos can acclimate to changing pressures. Comparative RNA-seq and cross-species analyses revealed a conserved, pressure-induced transcriptional response across phyla, with the heat shock pathway among the most strongly activated. Pre-activation of this pathway via prior pressure or other stressors enhances survival under otherwise lethal hydrostatic conditions.

Cellular redox homeostasis plays a critical role in regulating protein function, including chaperone activity, through reversible oxidation of cysteine and methionine residues. Previously, we found that budding yeast cells experiencing redox imbalance due to inactivated thioredoxin reductase (trr1{Delta}) activate the heat shock response and induce hyperaccumulation of the small heat shock protein/sequestrase Hsp42 with misfolded proteins. Building on that finding, this study identified cysteine 127 (C127) within Hsp42 as a redox-active residue that becomes oxidized in trr1{Delta} cells, upon treatment with the powerful oxidant hydrogen peroxide, or by exposure to the cysteine crosslinker divinyl sulfone (DVSF). In trr1{Delta} cells, C127 oxidation promoted intermolecular disulfide bond formation and contributed to Hsp42 homo-oligomerization. We show that stable oligomerization requires both the prion-like domain (PrLD) and C127 oxidation. While Hsp42-GFP formed prominent persistent foci in trr1{Delta} cells, replacement of C127 with non-thiol reactive serine decreased foci formation. Furthermore, the C127S mutation diminished Hsp42 oligomerization and sedimentability. Immunoprecipitation coupled with mass spectrometry analysis revealed that Hsp42 in trr1{Delta} cells preferentially associated with mitochondrial precursor proteins accumulated in the cytoplasm, as well as oxidation-reduction enzymes. The observed client selectivity was altered by the C127S mutation that diversified the spectrum of Hsp42-associated proteins. Collectively, these findings identify Cys127 as a redox-active switch that regulates Hsp42 assembly, foci formation, stability, and client specificity in response to oxidative stress.

Cyclooxygenase-1 (COX-1) is classically regarded as a constitutive enzyme that produces eicosanoids with housekeeping functions, but recent evidence indicates that it may also be involved in the acute phase of severe systemic inflammation. There is evidence indicating that COX-1 is selectively activated in the spleen via post-translational mechanisms early the course of LPS-induced systemic inflammation. However, the mechanistic link between COX-1 and the spleen has not yet been demonstrated in direct experiments. The present study was conducted to fill this gap. The effects of the COX-1 inhibitor SC-560 on the LPS-induced severity triad (hypotension, hypothermia and acidosis) were evaluated in rats subjected to splenectomy or in sham-operated controls. In the sham-operated group, SC-560 significantly attenuated the severity triad independently of changes in plasma cytokines (TNF and IL-1{beta}). In the splenectomized rats, SC-560 completely lost its ability to attenuate the hypotension and the acidosis induced by LPS. The effect of SC-560 on LPS-induced hypothermia was also impaired by splenectomy, though not completely. We then conducted a lipidomic screening to identify which COX-1-derived eicosanoids might be responsible for mediating the severity triad. Based on spleen-blood correlations, the screening identified PGE2 and PGD2 as putative candidates. In conclusion, the present study provides direct evidence for a mechanistic link between the spleen and COX-1 in the mediation of severity in systemic inflammation, and identifies PGE2 and PGD2 as putative candidates involved.

Birds exhibit longer lifespans than similarly sized mammals, despite having higher mass-adjusted blood glucose levels. This makes them a valuable model for the comparative study of the metabolic and physiological aspects of aging. Circulating glucose contributes to multiple pathological processes, primarily through glycation reactions and the formation of advanced glycation end-products (AGEs), as well as by promoting oxidative stress. These mechanisms are interconnected by feedback loops and play a key role in the development of age-related pathologies. To explore the causal role of glycaemia in avian ageing, we conducted a one-year dietary supplementation experiment in captive zebra finches. Birds received either glucose- or methylglyoxal-enriched water. Previously, we observed that chronic glucose supplementation in zebra finches increased mortality, an effect that did not appear to be mediated by the associated increase in plasma protein glycation or AGE levels. Therefore, the mechanisms underlying increased mortality in the glucose group remained unclear. In the present study, we investigated how glucose and methylglyoxal supplementation affect blood oxidative status and red blood cell telomere dynamics and apoptosis. We found that methylglyoxal supplementation decreased the non-enzymatic antioxidant capacity (OXY) of plasma and increased DNA damage, while glucose supplementation had no significant effect on oxidative stress, although circulating glucose levels influenced oxidative status in a sex-dependent manner. Males exhibited a positive correlation between glucose levels and organic hydroperoxides and protein carbonyls. Additionally, we report, for the first time in birds, a seasonal variation in telomere length, which was more pronounced in glucose-supplemented individuals, yet seemed independent of oxidative status. Apoptosis probability increased with both treatments, particularly with the methylglyoxal supplementation. These results highlight that glucose and methylglyoxal trigger different glucotoxicity-related pathways, with distinct effects on bird health and aging. However, the relationship between glucose supplementation and mortality remains still unclear and warrants further investigation.

CHI3L1 (YKL-40) is a chitinase-like glycoprotein involved in immune regulation, tissue remodeling, and cancer, yet the molecular principles governing its glycan interactions remain incompletely defined. Previous reports suggested that CHI3L1 can bind to chitin oligosaccharides (COS) and glycosaminoglycan (GAG) ligands, however, the molecular basis and binding sites underlying these interactions remain controversial. Here, a combination of biophysical and computational methods is employed to shed light on carbohydrate interactions of the protein and delineate a potential crosstalk between its glycan-binding interfaces. Our results demonstrate that COS and GAGs bind to distinct, non-overlapping sites on CHI3L1. Both ligand classes exhibit a strong dependence of binding affinity on the degree of polymerization. Molecular dynamics simulations, supported by mutational analysis, identify a GAG-binding site centered on residues R144, R145, and K147 and reveal an additional distal interaction site for longer GAG ligands. Biophysical and biochemical assays fail to confirm a previously proposed allo- or orthosteric interaction between both binding sites. However, physiologically relevant protein-protein interactions mediated by the chitin binding site of CHI3L1 are differentially regulated by GAG and COS ligands. COS inhibit binding of galectin-3 to CHI3L1, whereas GAG ligands enhance the affinity between the proteins by ca. 14-fold. Together, these findings establish CHI3L1 as a dual carbohydrate-binding protein with distinct recognition interfaces and reveal a previously unrecognized role for GAGs in modulating CHI3L1-mediated signaling interactions.

It has been previously shown that chronic social defeat stress caused by paired agonistic interactions between male mice is accompanied by the development of depression-like state and immune deficiency. The aim of this study was to investigate changes in the expression of C1qtnf superfamily genes (encoding the complement component related with tumor necrosis factor) in the hypothalamus, thymus and lungs against the background of the Lewis lung adenocarcinoma growth. In the experiments, on the 5th day of social stress, male mice were injected with tumor cells into the tail vein. Chronic social stress continued for the next two weeks. The transcriptomes of the hypothalamus, thymus and lungs of mice were sequenced at the Genoanalytica Collective Center (http://genoanalytica.ru/, Moscow). Changes in the expression of the C1qtnf genes in the tissues of stressed mice were studied compared with the control and mice that were additionally injected with tumor cells. Overall, significant correlations were found between expression of most genes in each tissue of the experimental groups. In the hypothalamus of stressed animals, when tumor cells were introduced, an increase in the expression of the genes C1qtnf1, C1qtnf2, C1qtnf3, C1qtnf6 and C1qtnf7 was observed compared to controls. In the thymus of these animals, tumor cell injection increased expression of the C1qtnf1, C1qtnf5, and C1qtnf6 genes. In the lung of tumor-injected stressed mice, expression of the C1qtnf1, C1qtnf2, C1qtnf7, and C1qtnf9 genes was decreased relative to controls and non-tumor-injected depressed mice, reaching near-zero levels in some mice. Analysis of C1qtnf superfamily gene expression in the all tissues revealed negative correlations between the expression of the C1qtnf1, C1qtnf2, and C1qtnf7 genes in the hypothalamus and lungs indicating synchronization of processes against the background of social stress and Levis lung adenocarcinoma.

Climate-driven warming is challenging the physiological limits of aquatic ectotherms, with cardiac performance emerging as one of the key determinants of thermal tolerance. Cardiac function relies on mitochondrial ATP production, and mitochondrial dysfunction has been linked to cardiac failure at critical temperatures. However, mitochondria are plastic and may represent a target for interventions aimed at improving thermal tolerance in fish. Exercise training improves whole-animal performance in fish, including cardiac thermal performance, and improves mitochondrial function in other taxa. However, its effects on the thermal sensitivity of cardiac mitochondria remain unknown. This study investigated whether exercise-training alters cardiac mitochondrial performance at optimal and critical temperatures in rainbow trout Oncorhynchus mykiss. Farmed rainbow trout were subjected to a four-week exercise training regime, while control fish remained under standard rearing conditions. Cardiac mitochondrial respiration was assessed in permeabilised heart fibres at 16{degrees}C (optimal growth temperature) and 26{degrees}C (temperature associated with cardiac arrhythmia) and several biochemical and nuclear indicators were measured. No significant differences were detected between treatments for any measured variable. However, trained fish generally exhibited higher maximal respiratory capacities and respiratory control ratios, particularly at the elevated temperature, suggesting subtle improvements in mitochondrial function despite considerable inter-individual variation. Temperature influenced mitochondrial performance, increasing proton leak and reducing coupling efficiency. These findings demonstrate that cardiac mitochondrial function is thermally sensitive and represents a potential targeted for improving thermal resilience in aquaculture species.

Heat-related illness is an increasing global health threat, with heatstroke representing its most severe form and frequently causing cerebellar injury characterised by selective Purkinje neuron loss. The molecular basis of this regional vulnerability remains unclear. Here, we integrated transcriptomic data from human heat exposure experiments, patients with heatstroke, primary human neurons, and cortical organoids to define conserved responses to heat stress and investigate determinants of cerebellar susceptibility. Across datasets, heat stress induced a highly conserved transcriptional programme dominated by suppression of ribosome biogenesis, RNA processing, translation, and metabolic pathways, consistent with reduced biosynthetic and energy-demanding activity. Mapping these signatures to human brain atlases revealed that the cerebellum showed the lowest enrichment of downregulated genes, while heatstroke exhibited a distinct regional transcriptional pattern compared with experimental heat exposure. At the cellular level, granule and Purkinje neurons showed the strongest association with heat-responsive gene suppression. Purkinje-enriched genes were linked to synaptic organisation, neurotransmission, membrane excitability, and ion transport. Connectivity Map analysis identified compounds predicted to reverse the heatstroke transcriptional signature, including the mTOR inhibitor KU-0063794. These findings identify a conserved molecular response to heat stress and suggest that selective Purkinje vulnerability reflects intrinsic metabolic and electrophysiological properties rather than preferential activation of canonical heat-shock pathways.

Thymosin {beta}4 (T{beta}4) is a conserved acidic polypeptide with 43-amino acids participating in multiple pathophysiological processes. In this study in vivo effects of T{beta}4 on liver regeneration are investigated in carbon-tetrachloride (CCL4) induced rodent animal liver jury models. Results illustrate that exogenous T{beta}4 treatment significantly reduced CCL4-rendered liver necrosis around central vein. At 48 hours after CCL4 insults hepatocytes proliferation occur mainly around the periportal area, while hepatocytes proliferation around the necrosis area is prominently increased by exogenous T{beta}4 treatment. The holistic proliferation level of liver tissues are also enhanced by exogenous T{beta}4. Hepatocyte proliferation activities negatively correlate with the necrosis extent of the liver tissue. These results suggested firstly exogenous T{beta}4 treatment could enhance liver regeneration and exhibit prosperous potential for application in clinical conditions such as liver transplantation.

The migratory endoparasitic pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, causing significant economic and ecological losses in conifer forest ecosystems in Europe and Asia. Understanding the molecular mechanisms regulating PWN parasitism-related genes may lead to new sustainable solutions for control. Based on previous PWN transcriptomic datasets from the pre-parasitic and parasitic stages and from the pharyngeal gland cells (GC), an in silico analysis was performed to identify transcription factors (TF) highly expressed in the GC. Seven candidates TF genes were selected, and their spatial expression validated by in situ hybridisation. From those, two GC-expressed TFs, BXY_079 and BXY_022, each encoding zinc finger domains, were successfully knocked down by RNA interference. Transcriptomic data from silenced BXY_079 and BXY_022 TFs, analysed with existing life cycle specific transcriptomic data, showed that both TFs control genes expressed at similar times, by repressing male-related genes while activating genes expressed during the J3 and D3 stages, yet each represents the extreme of the others minor function. In addition to these common roles, BXY_079 also activates parasitism-related genes in the J2 stage. These BXY_079-activated parasitism-related genes predominantly encode proteins with lytic functions, including secreted peptidases and glycoside hydrolases. Consistent with their proposed role in parasitism, these genes are highly expressed during the parasitic juvenile stages and are likely involved in nematode feeding, tissue penetration, and migration within the host. In contrast, BXY_022 also represses the expression of several genes related to the reproduction system, such as major sperm proteins and cytosolic motility proteins, particularly in the adult male stage. Taken together, both dual-functional TFs work together, non-redundantly, to regulate gene expression across the life cycle, while each is additionally specialised to regulate diverse and distinct gene sets: ranging from genes implicated in lytic parasitic functions to sexual dimorphism.

Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome with incompletely understood molecular mechanisms. Histone serotonylation is a recently identified epigenetic modification in which serotonin is covalently conjugated to glutamine 5 of histone H3 in H3K4me3-marked nucleosomes. Here, we investigated the role of transglutaminase 2 (TGM2)-mediated histone serotonylation in HFpEF. In a mouse model of HFpEF induced by salty drinking water, unilateral nephrectomy and aldosterone infusion (SAUNA), cardiac H3K4me3Q5ser and nuclear TGM2 levels were increased. Cardiomyocyte-specific TGM2-deficient mice developed aggravated HFpEF phenotypes after SAUNA exposure, including worsened diastolic dysfunction, reduced exercise capacity, pulmonary congestion and delayed cardiomyocyte relaxation. CUT&RUN sequencing identified H3K4me3Q5ser-enriched regions predominantly around transcription start sites after SAUNA exposure, with notable enrichment at genes associated with G2/M checkpoint-related stress-response signaling. RNA sequencing further showed that activation of this pathway was impaired in SAUNA-exposed TGM2-deficient hearts. In cardiac myocytes, calcium-binding sites and nuclear localization of TGM2 support checkpoint-related stress-response gene activation in cardiac myocytes. Pharmacological WEE1 inhibition, which activates downstream CDK1-associated checkpoint signaling, partially rescued the aggravated HFpEF phenotype in TGM2-deficient mice. Finally, in patients with HFpEF, lower circulating serotonin levels were associated with adverse cardiac outcomes, and cardiomyocyte H3K4me3Q5ser levels correlated with serum serotonin concentrations. These findings suggest that cardiomyocyte TGM2-mediated histone serotonylation represents a stress-adaptive, cardioprotective epigenetic mechanism in HFpEF.

The ability to adapt to changing environmental conditions is essential for cellular survival. A central feature of the eukaryotic stress response is the inhibition of bulk mRNA nuclear export, while stress-induced transcripts are specifically exported. However, the molecular mechanisms that simultaneously inhibit bulk mRNA export while mediating selective export of specific transcripts remain poorly understood. Here, we performed comparative phosphoproteomic analyses of S. cerevisiae under different stress conditions. We identified a heat shock-induced increase in phosphorylation within the N-terminal domain of the mRNA export adaptor Yra1. Preventing this phosphorylation significantly reduces nuclear accumulation of poly(A)+ RNA during heat stress and concomitantly enhances the export of heat-induced transcripts. Mechanistically, Yra1 phosphorylation appears to weaken its interaction with the export receptor Mex67, thereby contributing to nuclear accumulation of bulk poly(A)+ RNA under heat stress. Together, our findings establish Yra1 phosphorylation as a previously unrecognized regulatory mechanism that promotes nuclear mRNA accumulation during heat stress and contributes to selective mRNA export.

Emerin (EMD), an inner nuclear membrane protein essential for nuclear architecture integrity, gene expression, cellular signaling, and chromatin stability, interacts with the LINC complex and participates in cytoskeleton-nucleoskeleton communication by binding to nuclear actin filaments. EMD is implicated in migration, invasion, and metastasis in some tumors, but its role in glioblastoma (GBM) remains unclear. This study evaluated the effects of EMD knockdown and overexpression in GBM cell lines following genotoxic treatment with cisplatin. In both wild-type p53 (U87-MG) and mutant p53 (U138-MG) GBM cells, EMD expression is high, and cisplatin treatment did not affect these protein levels. EMD knockdown in U87-MG cells significantly increased cisplatin IC50, viability, and proliferation. Conversely, stable overexpression of EMD in U87-MG cells led to reduced cisplatin IC50, viability, proliferation, and migration. EMD knockdown or overexpression did not affect any U138-MG phenotypes, with or without cisplatin treatment. Modulation of EMD levels causes morphological changes in stress fiber cytoskeleton, whereas overexpression of EMD in U87-MG cells promotes an increase and a decrease in nuclear and cytoplasmic actin levels, respectively. These biological responses of U87-MG cells overexpressing EMD were coincidentally associated with alterations in the levels of pH2AX(Ser139), p-p53(Ser15), p53, and p21Kip1 proteins after cisplatin exposure. In sum, modulation of EMD levels affects the viability, migration, and proliferation of wild-type p53 GBM cells treated with cisplatin, suggesting unknown roles in the DNA damage response and repair. This work highlights EMD as a potential regulator of GBM chemoresistance and a target for therapeutic intervention.

White, beige and brown adipocytes store energy as lipids, secrete hormones and produce heat, playing an important role in the regulation of energy balance through not completely defined mechanisms. We investigate herein the impact of the almost complete absence of mature adipocytes (severe lipoatrophy) in the determination of energy balance (energy intake and expenditure) and homeothermy in mice. For this, mice with severe lipoatrophy induced by adipocyte deletion of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) (PPAR{gamma} flox adiponectin-Cre) and littermate controls (PPAR{gamma} flox) were evaluated for energy balance, thermoneutral zone, core body temperature, locomotor activity, and gene expression profiles at different ambient temperatures. Severely lipoatrophic mice are heavier, hypermetabolic and hyperphagic and feature a widened thermoneutral zone, lower ambulatory activity, and metabolic inflexibility at both 23 and 17{degrees}C, along with unstable thermal behavior characterized by hyperthermia at 30{degrees}C, normothermia at 23{degrees}C, and bouts of hypothermia at 17{degrees}C. Noteworthy, lipoatrophic mice hypermetabolism at 30{degrees}C is not due to thyroid hormones, impaired insulation or increased body and lean masses and is not altered by pharmacological blockade of either {beta}-adrenergic receptor signaling with propranolol or skeletal muscle sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) and sarcolipin (SLN)-mediated calcium cycling with dantrolene, but is partially attenuated by pharmacological inhibition of acetyl-CoA carboxylase (ACC) and de novo lipogenesis with ND-630. In conclusion, severe lipoatrophy causes hypermetabolism and hyperthermia at 30{degrees}C partly through the activation of liver de novo fatty acid synthesis.

Lipid peroxidation (LPO) is widely used as a biomarker of oxidative stress in coral bleaching research, yet its measurement remains poorly standardized across the field. A systematic review of the coral LPO literature reveals substantial variation in methodological approaches, including tissue fraction analysis, lysis protocols, assay choice, and normalization metrics, confounding cross-study comparison and obscuring the biological interpretation of results. We experimentally investigate two key sources of variation: the use of bulk holobiont vs separated host and algal symbiont fractions, and the choice of normalization metric. To do so, we used Montastraea cavernosa (n = 6 colonies) exposed to ambient (28C), heat stress (30.5C), and heat stress + artificial upwelling (AU; heat stress intermitted by daily pulses of cooler water, 30.5/27.5C) conditions in a controlled mesocosm experiment. Using a TBARS-based MDA assay with a lysis buffer optimized for coral tissue, we measured LPO separately in coral host and algal symbiont fractions across four time points throughout the day. Host MDA remained stable across all treatments and time points, consistent with either sufficient antioxidant buffering capacity or thermal acclimation over the experimental period. Algal symbiont MDA, in contrast, exhibited pronounced diel and treatment-specific dynamics, and the two fractions responses were decoupled from one another. Normalizing MDA to coral surface area instead of total protein content produced largely consistent diel and treatment patterns, but the two metrics diverged at specific time points, indicating that normalization choice is not interchangeable and can itself affect interpretation. Together, our literature review and empirical results demonstrate that host and algal symbiont LPO dynamics are not comparable when aggregated and argue for host-symbiont fraction separation and consistent, explicitly reported normalization as minimum standards for interpretable and cross-comparable coral LPO measurement.

Some key inflammatory genes controlled by the RELA transcription factor are thought to be highly expressed in fibroblasts induced into senescence by ionizing radiation (IR) as part of the Senescent-Associated Secretory Phenotype (SASP). However, this view is based largely on studies of a limited number of fibroblast cell lines derived from fetal lung or neonatal foreskin. Here, we show that more than half of the primary adult fibroblast strains examined exhibit only weak induction of RELA-dependent inflammatory genes following IR-induced senescence. We define these fibroblasts as "low-responding" to distinguish them from fibroblasts that express high levels of inflammatory gene expression in response to IR. RNA-seq analysis indicated particularly weak IL1A and IL1B expression in low-responding fibroblasts. IL1-alpha and IL1-beta participate in a positive amplification loop for inflammatory gene expression in senescence. Addition of recombinant IL1-alpha or IL1-beta to these fibroblasts sufficed to induce high expression of inflammatory genes. Low-responding fibroblasts thus exhibit cell-autonomous defects in IL1A and IL1B gene activation in response to IR that explains their overall low expression of RELA-targeted inflammatory genes. This defect was correlated with reduced chromatin accessibility and H3-K27-acetylation at 2 putative enhancers in the intergenic region separating IL1A and IL1B, and deletion of either of these enhancers inhibited inflammatory gene expression in IR-induced senescence. Fibroblasts express distinct transcriptomes and we found that differential expression of the FOXF1 transcription factor gene in high-responding WI38 fetal lung fibroblasts contributes to inflammatory gene expression after IR. Our observations indicate that fibroblasts can be distinguished by their ability to manifest cell-autonomous induction of inflammatory genes under conditions of IR-induced senescence.

Objectives; The role of nuclear receptor subfamily 4, group A, member 3 (NR4A3) in hepatic steatosis, inflammation, and insulin resistance (IR) within the context of metabolic dysfunction-associated steatotic liver disease (MASLD) remains largely underexplored. Consequently, this study aimed to examine NR4A3's impact on MASLD and the potential underlying mechanisms. Methods; We aimed to elucidate the functional role of NR4A3 in MASLD through its knockdown in cell culture and animal models. To establish the cell culture model of MASLD, LO2 cells were treated with free fatty acids (FFAs), while male C57BL/6 mice were fed a high-fat diet (HFD) to create the animal model. NR4A3 knockdown was achieved using specific short hairpin RNA (NR4A3-shRNA) in the mice model and three small interfering RNAs (NR4A3-siRNAs) in the cell culture model. The lipids content, fatty acid synthesis, inflammatory factors, and IR were then assessed with and without NR4A3 knockdown. Furthermore, the underlying mechanism through which NR4A3 exerts its influence was explored by analyzing the interaction between NR4A3 and activating transcription factor 3 (ATF3). Results: In the cell culture experiments, the knockdown of NR4A3 significantly decreased the lipids content, fatty acid synthesis, and inflammatory factors in the LO2 cells treated with FFAs in the NR4A3-shRNA group compared with those in the NC-shRNA control group. In the animal model experiments, NR4A3 knockdown in the HFD male C57BL/6 mice significantly ameliorated HFD-induced hepatic steatosis, inflammation, and IR. Mechanistically, the knockdown of NR4A3 downregulated the expression and transcriptional activity of ATF3, resulting in an impaired ATF3 function. ATF3 overexpression significantly reversed lipid accumulation decline and reduced inflammation after NR4A3 knockdown. Conclusion: The downregulation of NR4A3 alleviates MASLD by modulating ATF3, suggesting this may be a promising therapeutic target.