Cell Stress and Chaperones

Brown adipose tissue (BAT) thermogenesis is essential for cold defense, but its contribution to fever and emotionally induced hyperthermia (psychogenic fever) remains disputed. Here we address this issue using genetic, surgical, physiological, and molecular approaches in rats. We generated UCP1 knockout rats, in which classical BAT thermogenesis is abolished, and examined body temperature responses to systemic inflammation induced by lipopolysaccharide and to emotional stressors such as restraint and cage exchange. Despite profound impairment of cold-induced and {beta}3-adrenergic-induced thermogenesis, UCP1 deletion did not affect LPS- or stress-evoked elevations in core or interscapular temperature. Surgical removal of interscapular BAT in wild-type rats likewise failed to alter these hyperthermic responses. Consistent with these findings, LPS and emotional stress induced only small, strain-dependent changes in the expression of thermogenic genes in BAT and minimally affected BAT mass, in marked contrast to the robust BAT activation elicited by {beta}3-adrenergic stimulation. Notably, emotional stress induced UCP3 expression in neck muscles, suggesting a potential contribution of skeletal muscle metabolic processes to stress-induced hyperthermia. Together, these findings demonstrate that both immune-induced and psychogenic fever occur independently of BAT thermogenesis and point to non-BAT tissues - likely including skeletal muscle - as candidate peripheral effectors supporting fever and emotional hyperthermia.

The ubiquitin conjugating enzyme UBE2J1/Ubc6e localizes to the endoplasmic reticulum where it mediates the ubiquitination and proteasomal degradation of terminally misfolded proteins. Although the protein is known to undergo phosphorylation at serine S184, we have considered modification at an additional site and used a bespoke anti-phospho antibody to confirm phosphorylation also at serine residue S266. Despite the well-described role of UBE2J1 in ER associated degradation (ERAD), we found no evidence for regulation at S266 during Unfolded Protein Response (UPR) induction by thapsigargin. Instead, our studies suggest that phosphorylation occurs independently at the S184 and S266 sites, with mutation at one site failing to disrupt basal phosphorylation at the second. We identified several contexts in which these two phosphorylations were differentially regulated. For example, ER localization, which is important for phosphorylation at S184, was not required for modification at S266, and sensitivity to proteasome inhibitors, which is regarded as a distinguishing feature of the S184 phospho-variant, was unaltered by the S266A mutation. Regarding regulation at S266 on the other hand, we found that pharmacological activation of protein kinase A resulted in rapid phosphorylation, with differential use of phospho-specific antibodies confirming that phosphorylation at S184 was unchanged by this treatment. Hormonal stimulation by glucagon resulted in a similar pattern of UBE2J1 phosphorylation, which occurred exclusively at S266 and could be inhibited by H89. The differential regulation demonstrated in these studies extends our understanding of the UBE2J1 enzyme, and may indicate a role in the integration of energy metabolism with environmental stress conditions.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the cytoplasmic aggregation and nuclear depletion of the TDP-43 protein. The latter impairs TDP-43 function as an RNA-binding protein and compromises the repression of cryptic splicing events, affecting both glutamatergic upper motor neurons and cholinergic lower motor neurons. This study systematically investigated the molecular and functional consequences of TDP-43 knockdown in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons (iGNs) and cholinergic motor neurons (iMNs) using antisense oligonucleotides. The results demonstrated that TDP-43 loss elicits widespread, cell-type-specific changes in gene expression and alternative splicing. Notably, a shared subset of ALS-associated targets, including STMN2 and UNC13A, were consistently downregulated and mis-spliced across both neuronal subtypes. Functionally, Microelectrode Array (MEA) electrophysiology recordings revealed that TDP-43 knockdown induces a hyperexcitable phenotype in both neuronal populations, though they exhibited distinct network patterns: iGNs displayed synchronized bursting and significant shifts in overall electrophysiological profiles, while iMNs showed asynchronous firing. Furthermore, the inclusion of astrocytes in co-culture models expanded the repertoire of detectable cryptic splicing, including an event in HDGFL2 previously identified in patient cerebrospinal fluid. Despite these profound molecular and functional deficits, TDP-43 depletion did not impact neuronal viability or increase susceptibility to glutamate-induced excitotoxicity. These findings validate hiPSC-derived iGNs and iMNs as relevant models for ALS and highlight the critical necessity of considering cell-type specificity when elucidating pathogenesis and developing targeted therapies.

Escherichia coli is highly sensitive to acid and osmotic stress but adapts by modulating the expression of stress responsive genes. Nucleoid-associated proteins (NAPs) play key roles in DNA organization and sensing environmental changes. The histone-like nucleoid structuring protein H-NS is an NAP acting as a global regulator of stress genes. H-NS may alter local chromatin structure to modulate the expression of such genes in response to environmental stress. The H-NS homolog StpA co-regulates several target genes, but its precise role is poorly defined. To investigate the regulatory interplay between these two proteins, we examined transcription, DNA binding and chromatin structure at two regulated operons, hdeAB and proVWX, in E. coli following exposure to acid and salt shock. Our results show that H-NS senses pH and osmotic cues to remodel chromatin and relieve repression, while StpA compensates for H-NS loss, particularly at proVWX, highlighting a coordinated regulatory network.

Following our laboratorys earlier observations on systemic damage inflicted by sev-Gal4 driven activated Ras (sev>RasV12) over-expression in Drosophila larval eye discs, we now show that sev>RasV12 expressing males suffer enhanced eye roughening and pupal death than female sibs because the former have significantly greater Ras levels in ommatidial cells than in female counterpart. In normally developing ommatidial cells, TBPH/TDP-43 was more abundant in cytoplasm in male than in female eye discs. The sev>RasV12 expression reduced nuclear TBPH in female eye discs but caused no apparent change in males. Caz/Fus, an interacting partner of TBPH, was significantly downregulated in sev>RasV12 eye discs, more so in males. Significant reduction in the microtubule binding protein Futsch in eye discs of sev>RasV12 larvae of either sexes but female-specific elevation of Fas2 appears to be due to the above normal developmental differences in TBPH and Caz in female and male ommatidial cells and because Sxl, the master regulator of sex-determination, is present only in females. In view of known auto-regulatory loop between Fas2 and Ras, we suggest that elevated levels of Fas2 cause levels of Ras to be much less elevated in sev>RasV12 female eye discs than in male sibs. This results in greater local and systemic damage in males. These findings have general and clinical relevance since perturbed Ras signaling is a major factor in several diseases, including cancer.

Short-term heat acclimation (HA) induces cardiovascular and fluid-regulatory adaptations, but its impact on markers of renal tubular injury and acute kidney injury risk (AKI) during exercise-heat stress remains unclear. Fourteen healthy endurance athletes were randomised to five days of isothermic HA (HOT; n = 7; 32 {degrees}C, 70% relative humidity; target core temperature [&ge;]38.5 {degrees}C), or matched exercise in thermoneutral conditions (TEMP, n = 7). Heat stress tests (HST; 45 min cycling at 32 {degrees}C, 70% RH) were performed pre- and post-intervention. Blood biomarkers of kidney tubular stress (NGAL, KIM-1), fluid-regulation (copeptin, serum osmolality) and sympathetic activity (plasma normetanephrine) were measured at rest and immediately post-HST. HA reduced resting heart rate (-8 {+/-} 5 bpm, p = 0.007, d = 1.0), increased plasma volume (+7.3 {+/-} 5.1%, p = 0.022) and sweat loss (+500 {+/-} 539 mL, p = 0.018, d = 1.1). Copeptin rose during the pre-intervention HST in both groups (HOT: +11 {+/-} 6; TEMP: +12 {+/-} 13 pmol{middle dot}L-1, p < 0.05), but not post-intervention. NGAL increased only in TEMP during HST1 (+45 {+/-} 29 g{middle dot}L-1, p = 0.030), while KIM-1 remained unchanged. No group x time interactions were observed for any biomarkers (p > 0.05). Five days of HA improved cardiovascular and thermoregulatory responses but did not alter renal stress markers or fluid-regulatory responses during exercise in the heat. These findings suggest short-term HA enhances heat tolerance without reducing acute renal biomarker responses under hot, humid conditions. New & NoteworthyFive days of isothermic heat acclimation improved cardiovascular and thermoregulatory responses, related to a lower resting heart rate, plasma volume expansion, and greater sweat loss. However, these benefits did not reduce renal tubular stress markers (NGAL, KIM-1), fluid-regulatory strain (copeptin), or sympathetic activity (normetanephrine) during exercise in the heat. Short-term heat acclimation lowers cardiovascular strain but does not mitigate renal biomarker responses, suggesting kidney stress risk remains unchanged in hot, humid conditions.

Riluzole is the most commonly prescribed among the limited approved therapies for amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by progressive motoneuron loss and paralysis. It is thought to act by suppressing motoneuron excitability and glutamate release, but its clinical benefits are modest and often diminish over time. We previously showed that homeostatic mechanisms in the SOD1G93A (mSOD1) mouse model of ALS are hyperactive and prone to overcompensation. Here, we tested whether such dysregulated homeostasis antagonizes the effects of riluzole. Wild-type (WT) and presymptomatic mSOD1 mice received therapeutic doses of riluzole in drinking water for 10 days, with untreated littermates of both genotypes serving as controls. Motoneuron excitability and synaptic inputs were then examined using intracellular recordings from the isolated sacral spinal cord. The data showed that chronic riluzole treatment increased motoneuron excitability and polysynaptic inputs in mSOD1 mice but produced no detectable changes in WT motoneurons. These results suggest that hyperactive homeostatic mechanisms in ALS counteract the suppressive effects of riluzole. Notably, mSOD1 motoneurons exhibited larger membrane capacitance than WT, consistent with their increased cell size at this disease stage. Riluzole treatment reduced motoneuron membrane capacitance in mSOD1 mice to the range observed in WT animals, indicating normalization of cell size and potentially reduction in metabolic demand. Together, these findings help explain the limited clinical efficacy of riluzole while revealing a previously unrecognized neuroprotective mechanism of the drug in ALS.

BackgroundHumidity ramp protocols are widely used to assess human heat tolerance limits, but the impact of ramp temporal structure (e.g., step duration) on estimated critical environmental limits (CELs) remains unclear. This study integrated theoretical modeling and empirical testing to assess these effects on apparent core temperature (Tcr) inflection points. MethodsA first-order thermal model described Tcr dynamics during stepwise humidity changes at fixed dry-bulb temperature (Tdb), with analytical solutions for increments of duration {Delta}t and sensitivity analyses across relevant time constants ({tau}). Twenty-six healthy young adults (14 males, 12 females) completed randomized trials at Tdb=42 {degrees}C: (1) slow-ramp (4-hour equilibration at 40% RH, then +6% RH/hour for 2 hours followed by +3% RH/hour; RH range: 40-61%) and (2) aggressive-ramp (30 min equilibration, then +2% RH every 5 min; RH range: 28-88%). Rectal and skin temperatures, heart rate, and perceptual ratings were monitored continuously. ResultsWhen {Delta}t/{tau} <<1, thermal disequilibrium accelerated Tcr rises, yielding prematurely low CELs; dwell times [&ge;] 60 min/step permitted near-equilibrium and higher thresholds. Aggressive-ramp CELs were significantly lower than slow-ramp (males: 29.9{+/-}1.6 {degrees}C vs. 33.4{+/-}0.5 {degrees}C; females: 30.3{+/-}0.9 {degrees}C vs. 33.8{+/-}0.5 {degrees}C), with downward shifts of 3.4{+/-}1.9 {degrees}C and 3.5{+/-}0.9 {degrees}C, respectively. ConclusionRapid humidity increments systematically underestimate heat tolerance due to thermal lag. Accurate CEL determination requires prolonged stable exposures (gold standard) or slow ramps ensuring sufficient equilibration ({Delta}t [&ge;] 60 min/step). Our findings reveal a core limitation of aggressive-ramp protocols and offer a framework for improved assessment of human environmental compensability. NEW & NOTEWORTHYThis study reveals how ramp temporal structure affects heat tolerance assessment. Rapid humidity increments in aggressive-ramp protocols cause premature underestimation of critical environmental limits (CELs) due to thermal disequilibrium. In contrast, prolonged dwell times ([&ge;] 60 min/step) in slow ramps allow near-equilibrium conditions, resulting in higher and more accurate CELs. These findings emphasize the importance of equilibration time in defining heat tolerance and provide a more reliable approach for assessing heat stress in extreme environments.

Brown adipose tissue (BAT) is a unique tissue with mitochondria specialized for thermogenesis via the BAT-specific uncoupling protein 1 (UCP1). Ucp1-/- mice cannot tolerate acute exposure to cold, illustrating the necessity of UCP1 for efficient mitochondrial thermogenesis. However, these mice adapt to low temperatures through a gradual acclimation process, suggesting a high degree of mitochondrial plasticity in brown and beige fat cells. This phenomenon, which remains to be fully elucidated, indicates the potential for these mitochondria to implement effective thermogenic mechanisms in the absence of uncoupling protein 1 (UCP1). Here, we investigated mitochondrial remodeling in beige and brown fat of Ucp1-/- mice to determine how they fulfill their thermogenic role. Upon gradual acclimation to a cold environment, Ucp1-/- mice exhibited body metabolic parameters and temperatures in the interscapular region similar to those of wild-type mice of BAT, highlighting effective thermogenesis. Interestingly, mitochondrial patch-clamp analysis and a mitochondrial Ca2+ swelling assay revealed a dramatic increase in Ca2+ uptake depending on the mitochondrial calcium uniporter (MCU) in BAT mitochondria from Ucp1-/- mice when robust thermogenesis was required. Mitochondrial remodeling was accompanied by markedly increased tethering between mitochondria and the endoplasmic reticulum (ER) in Ucp1-/- mice, confirming a significant restructuring of the contact sites between the ER and mitochondria, likely to adapt to a new Ca2+ homeostasis. Respiratory complexes also underwent significant reorganization, which partly led to a reduction in their assembly. Levels of ATP synthase and its F1 subcomplex increased, suggesting a major source of ATP consumption and energy expenditure. We propose a new role for MCU as a key regulator of mitochondrial plasticity, enabling efficient thermogenesis in beige and brown adipose tissues in the absence of UCP1.

Alzheimers disease (AD) is a devastating neurodegenerative disorder characterized by memory loss and a decline in cognitive function. Hallmarks of AD include an age-dependent accumulation of toxic amyloid beta (A{beta}) 42 in the brain, energy dyshomeostasis caused by mitochondrial dysfunction, and iron overload. However, the role of iron overload and mitochondrial dysfunction in AD pathology is unknown and their precise relationship with A{beta} 42 toxicity in AD pathology is unclear. C. elegans provide a powerful model system to untangle and clarify these relationships. In this study, we quantify the temperature-dependence of iron toxicity (16, 20 and 25C) in neurons and muscle of C. elegans that overexpress A{beta} 42. We found that A{beta} 42, regardless of the cell-type expression, caused accelerated paralysis compared to age-matched WT worms with the greatest degree of paralysis observed at an elevated temperature (25C). Moreover, the combination of iron toxicity and A{beta} 42 results in an enhanced paralytic phenotype at 16C. Thus, iron exposure potentiates A{beta} toxicity observed at low temperatures. Iron toxicity stimulated both maximum (State 3) and leak (State 4) respiration in WT and A{beta} 42 worms. A{beta} 42 worms also exhibited increased leak respiration at baseline that was further exacerbated by iron toxicity. Iron burden and sensitivity increased A{beta} 42 peptide toxicity. A{beta} 42 worms exhibited reduced levels of Ca, Zn, Mn, and K. Overall, our results suggest that iron potentiates A{beta} toxicity at low temperature and enhances A{beta} peptide mediated mitochondrial bioenergetic dysfunction in C. elegans. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/714217v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@9eaf46org.highwire.dtl.DTLVardef@542eforg.highwire.dtl.DTLVardef@16d9678org.highwire.dtl.DTLVardef@1b1b16d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITemperature stress modulates the synergetic interactions of iron toxicity and A{beta} 42 pathology C_LIO_LIIron sensitivity drives increased cell-type specific A{beta} 42 pathology C_LIO_LIEnergy dyshomeostasis via impaired mitochondrial function and increased proton leak contributes to iron- and A{beta}-induced pathology C_LI

Circulating stem and progenitor cells (SPCs), including mesenchymal stromal cells (MSCs) and hematopoietic stem/progenitor cells (HSPCs), are mobilised after tissue injury but their temporal behaviour after hemorrhagic shock (HS) and relationship to cytokine milieus and outcome remain unclear. In a prospective observational cohort at JPN Apex Trauma Centre, AIIMS, New Delhi we studied 100 participants: 50 trauma patients with hemorrhagic shock and traumatic brain injury (HS index group), 25 trauma patients without HS, and 25 minor-injury controls. Peripheral blood was collected at admission (day 0) for all groups and additionally at days 3, 7 and 14 for the HS group. PBMCs were phenotyped by flow cytometry (HSPC markers: CD45, CD123, CD38, CD34; MSC markers: CD105, CD73, CD90) and serum SDF-1, VEGF-A, EGF, GRO- and GRO-{beta}, GM-CSF and G-CSF were measured by ELISA; group and time effects were evaluated with mixed-effects models and correlations by Spearman tests (two-tailed p<0.05). At admission, trauma patients without HS had significantly higher MSC and HSPC-like populations versus controls (p<0.0001). In the HS cohort SPC percentages rose modestly at day 0-3 then declined sharply by days 7-14 (time effect p<0.0001); non-survivors exhibited significantly higher early SPC and cytokine levels that persisted until death while survivors showed an early rise followed by decline (outcome and time interaction p<0.0001). All cytokines were up-regulated in trauma groups, peaked at day 0-3 in HS patients, and correlated positively with SPC counts (notably SDF-1, VEGF-A, G-CSF, Gro- and GM-CSF; Spearman p<0.05); higher early SPC and cytokine signatures associated with greater organ dysfunction (higher SOFA) and with timing of sepsis. These findings indicate that trauma provokes an early SPC and cytokine response that in HS is followed by later decline, and that persistent early elevation predicts worse outcomes, suggesting serial SPC and cytokine profiling may have prognostic value and identify an early therapeutic window for regenerative or immunomodulatory interventions.

Disturbances of 24-hour or circadian rhythms imposed by everyday irregular work and/or social schedules have been linked to vascular disease, including ischemic stroke. Using an established shift work-like paradigm and preclinical model for ischemic stroke, we have shown that environment-induced circadian dysregulation exacerbates stroke outcomes differentially to a greater extent in male than female rats. Because more severe stroke outcomes and circadian rhythm disturbances have been linked to gut pathophysiology, present study examined the effects of chronic LD cycle shifting on gut cytoarchitecture, microbiota composition, metabolites, and gut-derived inflammatory mediators. Adult (5-7mo) rats were divided into 2 groups and exposed for 50d to a fixed or shifted (lights-on advanced by 12hr/5d) LD 12:12 cycle. Circadian entrainment of activity rhythms was stable in all rats on the fixed LD 12:12 cycle but was severely disrupted during exposure to shifted LD cycles. Significant changes in the composition of the gut microbiome including reduced alpha diversity, shifts in beta diversity and correlations between the abundance of beneficial gut bacteria and stroke survival were observed in male but not female rats exposed to shifted LD cycles relative to fixed LD controls. This effect of circadian dysregulation on gut microbiota was accompanied by evidence of pathologic gut morphology (i.e., shorter and blunted villi, crypt hyperplasia disruption of tight junction proteins and gut barrier integrity), elevated serum endotoxin concentrations, decreased levels of the short-chain fatty acid (SCFA) butyrate, and increased circulating levels of the inflammatory cytokine IL-17A in shifted LD male rats. These results suggest that alterations in gut morphology, microbiota and metabolites may contribute to sex differences in the effects of shift work-related circadian dysregulation on ischemic stroke outcomes.

In yeast, transcriptional adaptor 2 (ADA2; SAGA complex subunit ADA2), a member of histone acetyltransferase (HAT) complex, regulates transcription through cell signalling, but its precise role in cellular metabolism remains unclear. In this study, genetic loss of ADA2 (ada2{Delta}) induces squalene (SQ) accumulation, indicating aberrant triterpene metabolism, coupled with endoplasmic reticulum (ER)/nuclear ER (nER) expansion. Lipid analyses of ada2{Delta} revealed elevated phosphatidic acid (PA) and phosphatidylcholine (PC) levels, indicating disrupted phospholipid metabolism. The expanded ER causes basal autophagy elevation, cellular recycling, and nER phagy, suggesting a regulatory role for ADA2 in autophagy. Downregulation of phosphatidate cytidylyltransferase (CDS1) and inositol-3-phosphate synthase (INO1), coupled with elevated PA and PC in ada2{Delta}, points to a significant disruption in cytidine-diphosphate-diacylglycerol and phosphatidylinositol pathway. Overexpression of CDS1 or INO1, or the inositol supplementation, in ada2{Delta} restores SQ, basal autophagy and ER phagy. The observed target of rapamycin Ser/Thr kinase complex (TORC1) activity in ada2{Delta} is due to the high PA content. Rapamycin-mediated inhibition of TORC1 reduced SQ, PA and ER expansion while increasing lipid droplets. In contrast, a rapamycin-treated ada2{Delta}pah1{Delta} strain retained high PA, SQ and ER expansion, underscoring the functional role of TORC1-nuclear envelope morphology protein 1 (Nem1)/sporulation-specific protein SPO7 (Spo7)-Pah1 axis. Notably, SQ levels remained unchanged in a rapamycin-treated ada2{Delta}atg39{Delta} strain, suggesting that loss of nER-phagy receptor, Atg39, impairs the effectiveness of TORC1 inhibition. In conclusion, our data unveiled a critical role for Ada2 in maintaining the intricate relationship between lipid and triterpene/sterol metabolism and connecting autophagy and ER homeostasis.

In numerous neurodegenerative diseases known collectively as tauopathies, the microtubule-associated protein tau forms fibrillar aggregates that are hallmarks of disease pathology. Tauopathies represent a substantial fraction of diseases associated with protein misfolding. Cellular chaperones known as small heat shock proteins (sHSPs) play a critical role in maintaining protein homeostasis by delaying the onset of protein aggregation. Two sHSPs, HSPB1 (Hsp27) and HSPB5 (B-crystallin), are constitutively expressed in brain and neurons. Here, we show that HSPB1 and HSPB5 delay tau aggregation in vitro through distinct mechanisms dictated by their disordered N-terminal regions (NTRs). HSPB1 inhibits tau aggregation under normal cellular conditions, whereas HSPB5 displays activity towards tau when activated by stress conditions such as pH acidosis. Using chimeric HSPB1/HSPB5 constructs in which small NTR subregions are swapped, we identify functional regions within the NTRs that modulate chaperone function for tau. The functional regions contain known sites of phosphorylation, suggesting that they are also control points that respond to cellular stress conditions. Our findings support an emerging model in which specific functional motifs within disordered regions of sHSPs govern activity and client engagement under normal and stress conditions. Broader AudienceIn many neurodegenerative diseases, the microtubule-associated protein tau forms fibrillar aggregates in the brain. Small heat shock proteins (sHSP) help prevent such aggregation, but their mechanisms of action remain enigmatic. We show HSPB1 and HSBP5, two sHSPs that are abundant and co-localize with tau, delay the onset of tau aggregation through distinct mechanisms. Each relies on specific small regions within their disordered N- terminal domains whose accessibility can be regulated by stress conditions and post- translational modifications.

Stress Granules (SGs) are cytoplasmic biomolecular condensates that form in response to a variety of stress conditions, though their function remains unclear. "Canonical" SGs - caused by stressors like sodium arsenite - are dynamic and cytoprotective, allowing cells to evade cell death during periods of stress. Ultraviolet (UV) irradiation is known to elicit a "non-canonical" SG subtype, lacking canonical SG components such as eukaryotic initiation factor 3 and polyadenylated mRNAs. The exact function of UV SGs, and the mechanisms driving their formation, remain unknown. Here we report the findings of a comparative analysis of UVA, UVB and UVC exposures on SG formation in three cell types: osteosarcoma (U2OS), keratinocytes (HaCaT), and mouse embryonic fibroblasts (MEF). We observed that SG formation in response to UV is highly cell type dependent. UVB and UVC induce robust SG formation in U2OS cells. However, only UVC exposure induced modest SG formation in MEFs, and none of the wavelengths caused SGs in HaCaT. While UVC-induced SGs in U2OS cells appear to be cell cycle dependent and specific to G1, UVB induced SG formation regardless of cell cycle stage. We tested the hypothesis that oxidative stress triggered by UV may be driving UV SG formation, and that keratin may buffer this effect, by overexpressing keratin in U2OS. Interestingly, we found that keratin and antioxidant treatment efficiently suppressed arsenite-induced SGs but had no effect on UV SGs. Our work confirms that UV SG formation is cell type specific and is not driven by oxidative stress.

Hibernators cycle between torpor, a state of profound metabolic and thermoregulatory suppression, and brief arousals during which metabolic rate and body temperature rapidly return to euthermic levels. These repeated physiological pressures require robust mechanisms to preserve brain integrity. Because the cerebral cortex is not thought to control hibernation directly yet must remain viable throughout torpor and recover rapidly during arousal, it provides a useful model for studying neural adaptation to hibernation. We therefore performed RNA sequencing of cerebral cortex from garden dormice (Eliomys quercinus) sampled during summer euthermia (SE), early torpor (TE), late torpor (TL), early arousal (AE), and late arousal (AL). Differential expression analysis revealed strongly stage-specific transcriptional remodeling across the hibernation cycle. Entry into torpor (SE-TE) and the transition from early to late arousal (AE-AL) showed minimal change, with 16 and 2 differentially expressed genes (DEGs), respectively. In contrast, extensive regulation was observed during torpor progression (TE-TL; 576 DEGs) and especially during the transition from late torpor to early arousal (TL-AE; 697 DEGs). Intermediate numbers of DEGs were detected in AL-TE (260) and AL-SE (50). Principal component and enrichment analyses indicated that the dominant axes of variation were associated with RNA processing and proteostatic control, metabolic and redox-related adaptation, and changes in intracellular trafficking and protein handling. In addition, comparison of adjacent contrasts revealed a marked opposite-direction transcriptional reversal between TE-TL and TL-AE, consistent with coordinated reactivation of torpor-associated programs during arousal. Together, these findings support a model in which cortex adaptation to hibernation involves transcriptional reprogramming consistent with metabolic suppression during torpor progression, especially in pathways related to carbohydrate and central carbon metabolism, redox homeostasis, and cellular signalling, followed by rapid reversal of these programs during early arousal.

Adult survivors of pediatric cancers are at elevated risk for neurocognitive late effects, but how these effects relate to metabolic perturbations in the brain remains unclear. To address this knowledge gap, the present study explored associations between neurometabolite levels and neurocognitive function in adult survivors of Hodgkin lymphoma (HL) and acute lymphocytic leukemia (ALL). Data were collected from a single-center observational study conducted at St. Jude Childrens Research Hospital (SJCRH) between October 2022 and November 2024. Adult survivors of HL (N=11 [5 females]; [&ge;]5 years post-diagnosis; mean [SD] current age 34 [9.5] years) and ALL (N=24 [16 females]; [&ge;]5 years post-diagnosis; current age 40 [12.6] years) and community controls (N=35 [17 females]; current age 40 [11] years) completed standardized neurocognitive tests of memory, attention, executive function, and processing speed. Participants also underwent proton magnetic resonance spectroscopy (1H MRS) to quantify neurometabolite levels in the left dorsolateral prefrontal cortex (dlPFC), left hippocampus, and left cerebellum. Analyses used regression models to examine differences in the slope of the relationship between neurometabolite and neurocognitive function or between neurometabolite and age. When comparing HL survivors vs controls, significant interactions were identified for group x age on the ratio of myo-inositol to N-Acetyl aspartic acid (mI/NAA; p=0.007) and group x Gamma-Aminobutyric Acid (GABA) on processing speed (p=0.04) in the left dlPFC. When comparing ALL survivors vs controls, significant interactions were identified for group x myo-inositol on verbal fluency in the left hippocampus (p=0.01) and group x GABA on cognitive flexibility in the left cerebellum (p=0.01). These preliminary findings suggest that neuroinflammation may be a mechanistic underpinning of age-associated neurocognitive impairment in pediatric cancer survivors.

Exercise provides broad health benefits, including improved emotional well-being and cognitive function. Emerging evidence suggests that exercising at different times during the day can have differential effects. However, how circadian phase and sex influence behavioral and physiological responses to exercise remains unclear. To address this question, we examined male and female wild-type mice maintained in either regular (REG, lights on/off at 7AM/7PM) or inverted (INV, lights off/on at 10AM/10PM) light cycles. Mice were then subjected to daily 20-min group swimming exercise sessions at ZT2-3 for 3 weeks. Exercised and sedentary controls mice were then subjected to an open field test (OFT) and blood corticosterone (CORT) measurements 24 hours post-exercise. We quantified several behaviors during swimming: escape attempts, floating, climbing and collisions. We also identified a novel swimming behavior: floating with only nostrils-above-water events (NAWEs). We found that expression of these behaviors was differentially modulated by sex, light-cycle and their interaction. Notably, behavioral differences were more pronounced in REG mice (rest phase). REG mice also lost weight after exercise and had elevated CORT levels compared to mice kept in INV conditions (active phase). Interestingly, OFT behaviors showed significant differences primarily in INV mice, particularly females, when comparing exercised vs sedentary groups. Our novel findings reveal that circadian rhythms and sex significantly interact to shape swimming exercise and stereotyped behaviors in mice. This emphasizes the need to consider the animals circadian phase when designing preclinical studies to match intended behavioral and physiological outcomes. HIGHLIGHTSCircadian phase and sex jointly shape swimming behavior patterns. Newly identified swimming behavior is more prevalent during rest-phase Restphase exercise produced stronger behavioral and physiological effects. Rest-phase exercise resulted in weight loss and elevated stress markers. Active-phase exercised females showed the strongest open field behavioral differences.

The RUNX1 transcription factor is a critical regulator of hematopoiesis and frequently mutated in myeloid malignancies. In the myeloproliferative neoplasm, chronic myeloid leukemia (CML), secondary somatic RUNX1 mutations and RUNX1::MECOM/EVI1, are associated with tyrosine kinase inhibitor (TKI) resistance and progression to the blast-phase (BP-CML). Research has predominantly focussed on transcriptional dysregulation mediated by RUNX1 mutations in myeloid malignancies, whilst post-transcriptional dysregulation remains comparatively unexplored. To address this, we used orthogonal organic phase separation (OOPS), to characterise the RNA-binding proteome of RUNX1 deficient BP-CML cells. RUNX1 depleted BP-CML cells exhibited significant alterations to RBP abundance involved in stress response pathways and translation/ribosome-biogenesis (RiBi). Furthermore, RUNX1 depletion or expression of RUNX1::EVI1 in BP-CML cells induced expression and RNA binding activity of SPATS2L, a component of stress granules (SG); membraneless cytoplasmic condensates protecting mRNAs from degradation, promoting survival under stress. Whilst RUNX1 depletion increased SG-assembly, SPATS2L depletion reduced SG-assembly in BP-CML cells and inhibited the growth and survival of multiple BP-CML cell lines. The translation inhibitor homoharringtonine (HHT), used historically in TKI-resistant CML, ablated SG-assembly in BP-CML cells with RUNX1 depletion, and, primary BP-CML cells with LOF/hypomorphic RUNX1 mutations (characterised by defective DNA-binding/CBF{beta}-interaction) were preferentially sensitised to HHT. Finally, suppressing SPATS2L expression induced by RUNX1 depletion, increased the HHT-sensitivity of RUNX1 depleted BP-CML cells, suggesting SPATS2L contributes to therapeutic resistance in CML with RUNX1 mutations. This study suggests that SPATS2L and SG induction could be critical to RUNX1-mutant leukemias, and, provides preliminary evidence for a mutationally-targeted approach in CML with RUNX1 aberrations.

Medulloblastoma is the most common pediatric brain cancer, but current treatments are largely non-specific, often causing developmental side effects. Genomic sequencing identified the RNA helicase DDX3X as one of the most frequently mutated genes in this cancer and a potential treatment target, yet its role in tumor progression remains elusive. Prior studies have indicated that the mutations cause specific defects in translation; however, both DDX3X and its yeast ortholog Ded1 have also been associated with cellular stress responses, suggesting that the contribution of the DDX3X mutations to medulloblastoma might result from defects in the translational response to stress. Building on our prior study that replicated the DDX3X mutations in yeast DED1 (ded1-mam), we examined the mutants effects following TOR pathway inactivation. First, we demonstrated that ded1-mam displayed substantial rapamycin-resistant growth compared to wild-type cells. In addition, similar to other ded1 mutants, the ded1-mam had decreased degradation of Ded1 and the translation factor eIF4G1 under TOR inactivation. Notably, these differences did not result in increased bulk translation following rapamycin; rather, the growth phenotypes appeared to be driven by translation of specific mRNAs. Reporter assays demonstrated enhanced translation of mRNAs with unstructured 5' UTRs in ded1-mam following TOR inhibition and a decrease in structured reporters. Furthermore, known Ded1 target genes with relatively unstructured 5 UTRs showed upregulated protein levels in rapamycin. We thus hypothesize that mutant DDX3X selectively upregulates translation of unstructured, pro-growth transcripts while downregulating other structured transcripts, allowing tumor cells to bypass stress-induced growth controls and promoting medulloblastoma progression.