Snoring-related vibrations have been proposed as a pathogenic factor contributing to upper airway muscle dysfunction in patients with obstructive sleep apnea (OSA). To investigate whether exposure to snoring vibration is linked to muscle weakness, we used an in vitro vibration model to examine its effects on mitochondrial homeostasis in L6 muscle cells at 8, 12, 24, and 48 h. The findings were then compared with mitochondrial alterations in the upper airway muscles from snorers and patients with OSA. Proteomic analysis of L6 myoblasts revealed extensive remodeling of the mitochondrial proteome at 8 h, affecting pathways involved in oxidative phosphorylation, protein import, ribosome biogenesis, and RNA processing. Respiratory chain remodeling was subunit-specific, with increased abundance of selected components of Complexes I, IV, and V, including NDUFS4, COX5A, and ATP5PD. However, reductions in spliceosome-associated factors, such as SRSF2 and DDX46, along with alterations in mitochondrial ribosomal proteins, indicated impaired RNA processing and protein synthesis. Furthermore, both proteomic and transcriptomic analyses revealed activation of a mechanosensing-mechanotransduction axis, with early upregulation of integrin subunits and mechanosensitive ion channels, followed by transient activation of focal adhesion signaling. Despite transcriptional upregulation of selected Complex IV subunits Cox5a and Cox6a2, this response was accompanied by accumulation of unspliced pre-mRNA, indicating impaired RNA processing efficiency and a decoupling between transcript and protein levels. Real-time Seahorse assay revealed a collapse of mitochondrial respiration and glycolytic reserve at 8 h. Although mitochondrial oxygen consumption recovered after 48 h, the ability to dynamically upregulate glycolysis remained impaired. In patients, muscle capillarization was impaired, COX activity was reduced, and mitochondrial organization was disrupted. Moreover, transcription of Complex IV subunits COX5A and COX6A2 was, as in vibrated L6 cells, upregulated, suggesting a mismatch between transcript levels and protein expression. We conclude that snoring-induced vibrations are an unrecognized stressor that disrupts mitochondrial homeostasis in muscle by impairing RNA processing, protein synthesis, and mechanotransduction-driven mitochondrial remodeling, leading to transcript-protein uncoupling and likely muscle dysfunction.

Amyotrophic lateral sclerosis (ALS) caused by mutation in superoxide dismutase 1 ( SOD1 ) accounts for 15-30% of familial ALS and is typically autosomal dominant. How single base pair/amino acid changes in this small protein cause neurodegeneration is unknown. In North America, SOD1A4V is the most common familial ALS SOD1 mutation and results in an aggressive form of ALS. Here, we present a novel genomically humanised mouse model of SOD1A4V , in which the mouse Sod1 locus has been replaced by the human SOD1 gene, with intact genomic architecture of exons and introns, but bearing an A4V mutation. In agreement with previously reported human genomic knock-in mice, the phenotype is mild; however, transcriptomic and metabolomic profiling reveal significant dysregulation of glycolysis, the tricarboxylic acid (TCA) cycle, and lipid metabolism. These changes suggest an early bioenergetic imbalance that precedes neuromuscular impairment. Our findings support metabolic dysfunction as an early event in ALS pathogenesis. This freely available SOD1A4V model provides a valuable tool for studying ALS progression and identifying therapeutic targets for pre-symptomatic treatment. SUMMARY STATEMENT This study describes the generation and analysis of novel genomically humanised SOD1A4V mice, revealing metabolic dysfunction through integrated multi-omic analyses, characterising a freely available potential pre-symptomatic ALS model for future research. ### Competing Interest Statement The authors have declared no competing interest.

Fibrillarin (FBL), a core component of the C/D box small nucleolar ribonucleoprotein (snoRNP) complex, catalyzes the 2'-O-methylation (Nm) of the ribose 2'-hydroxyl moiety in ribosomal RNA (rRNA). Distinct Nm patterns contribute to ribosome heterogeneity, which is linked to selective translation of oncogenes. FBL dysregulation generates an aberrant Nm signature in triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype. This study investigated the role of FBL in TNBC via translation-driven mechanisms. Our findings show that FBL knockdown impairs oncogenic traits, triggers metabolic stress, and reduces the translation efficiency of oncogenes, such as metastasis-associated protein 1 (MTA1), interleukin-1 receptor-associated kinase 1 (IRAK1), and thymosin beta 10 (TMSB10). RiboMethSeq confirmed that the rRNA Nm sites exhibited differential sensitivity to FBL depletion. Additionally, FBL knockdown led to alterations in 18S ribosome structure confirmed by SHAPE and specifically reduced RPS28 incorporation into ribosomes. Notably, silencing RPS28 also disrupted both the oncogenic phenotype and downregulated MTA1, IRAK1, and TMSB10 expression. These findings reveal a complex interplay between FBL, rRNA Nm modifications, and RPS28 in shaping oncogenic protein pools and ribosomal composition in TNBC, offering promising insights into therapeutic approaches targeting this aggressive cancer subtype.

Fibrillarin (FBL), a core component of the C/D box snoRNP complex, catalyzes 2'-O-methylation (Nm) of ribosomal RNA (rRNA), influencing ribosome heterogeneity and oncogene translation. In triple-negative breast cancer (TNBC), FBL dysregulation creates an aberrant Nm signature. This study explores role of FBL in TNBC progression via translation-driven mechanisms. FBL knockdown impaired tumorigenic traits, induced metabolic stress, and reduced the translation efficiency of oncogenes, including metastasis-associated protein 1 (MTA1), interleukin-1 receptor-associated kinase 1 (IRAK1), and thymosin beta 10 (TMSB10). RiboMethSeq analysis revealed differential rRNA Nm site sensitivity to FBL depletion. Additionally, FBL knockdown lowered RPS28 protein levels, suggesting its misincorporation into ribosomes. Notably, silencing RPS28 also suppressed oncogenic traits and downregulated MTA1, IRAK1, and TMSB10, highlighting its role in FBL-mediated translation. These findings uncover a complex interplay between FBL, rRNA Nm modifications, and RPS28 in shaping oncogenic protein pools and ribosomal composition in TNBC. Targeting this pathway could offer novel therapeutic strategies for this aggressive cancer subtype. ### Competing Interest Statement The authors have declared no competing interest.

The Shaker/Kv1 subfamily of voltage-gated potassium (K+) channels is essential for modulating membrane excitability. Their loss results in prolonged depolarization and excessive calcium influx. These channels have also been implicated in a variety of other cellular processes, but the underlying mechanisms remain poorly understood. Through comprehensive screening of K+ channel mutants in C. elegans, we discovered that shk-1 mutants are highly susceptible to bacterial pathogen infection and oxidative stress. This vulnerability is associated with reduced glycogen levels and substantial mitochondrial dysfunction, including decreased ATP production and dysregulated mitochondrial membrane potential under stress conditions. SHK-1 is predominantly expressed and functions in body wall muscle to maintain glycogen storage and mitochondrial homeostasis. RNA-sequencing data reveal that shk-1 mutants have decreased expression of a set of cation-transporting ATPases (CATP), which are crucial for maintaining electrochemical gradients. Intriguingly, overexpressing catp-3, but not other catp genes, restores the depolarization of mitochondrial membrane potential under stress and enhances stress tolerance in shk-1 mutants. This finding suggests that increased catp-3 levels may help restore electrochemical gradients disrupted by shk-1 deficiency, thereby rescuing the phenotypes observed in shk-1 mutants. Overall, our findings highlight a critical role for SHK-1 in maintaining stress tolerance by regulating glycogen storage, mitochondrial homeostasis, and gene expression. They also provide insights into how Shaker/Kv1 channels participate in a broad range of cellular processes.

Increasing evidence suggests that the calcium-binding and proinflammatory protein S100A9 is an important player in neuroinflammation-mediated Alzheimer’s disease (AD). The amyloid co-aggregation of S100A9 with amyloid-β (Aβ) is an important hallmark of this pathology. Apolipoprotein E (ApoE) is also known to be one of the important genetic risk factors of AD. ApoE primarily exists in three isoforms, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). Even though the difference lies in just two amino acid residues, ApoE isoforms produce differential effects on the neuroinflammation and activation of the microglial state in AD. Here, we aim to understand the effect of the ApoE isoforms on the amyloid aggregation of S100A9. We found that both ApoE3 and ApoE4 suppress the aggregation of S100A9 in a concentration-dependent manner, even at sub-stoichiometric ratios compared to S100A9. These interactions lead to a reduction in the quantity and length of S100A9 fibrils. The inhibitory effect is more pronounced if ApoE isoforms are added in the lipid-free state versus lipidated ApoE. We found that, upon prolonged incubation, S100A9 and ApoE form low molecular weight complexes with stochiometric ratios of 1:1 and 2:1, which remain stable under SDS-gel conditions. These complexes self-assemble also under the native conditions; however, their interactions are transient, as revealed by glutaraldehyde cross-linking experiments and molecular dynamics (MD) simulation. MD simulation demonstrated that the lipid-binding C-terminal domain of ApoE and the second EF-hand calcium-binding motif of S100A9 are involved in these interactions. We found that amyloids of S100A9 are cytotoxic to neuroblastoma cells, and the presence of either ApoE isoforms does not change the level of their cytotoxicity. A significant inhibitory effect produced by both ApoE isoforms on S100A9 amyloid aggregation can modulate the amyloid-neuroinflammatory cascade in AD.

Skeletal muscle adaptation to exercise involves various phenotypic changes that enhance the metabolic and contractile functions. One key regulator of these adaptive responses is the activation of AMPK, which is influenced by exercise intensity. However, the mechanistic understanding of AMPK activation during exercise remains incomplete. In this study, we utilized an in vitro model to investigate the effects of mechanical loading on AMPK activation and its interaction with the mTOR signaling pathway. Proteomic analysis of muscle cells subjected to static loading (SL) revealed distinct quantitative protein alterations associated with RNA metabolism, with 10% SL inducing the most pronounced response compared to lower intensities of 5% and 2% as well as the control. Additionally, 10% SL suppressed RNA and protein synthesis while activating AMPK and inhibiting the mTOR pathway. We also found that SRSF2, necessary for pre-mRNA splicing, is regulated by AMPK and mTOR signaling, which, in turn, is regulated in an intensity-dependent manner by SL with the highest expression in 2% SL. Further examination showed that the ADP/ATP ratio was increased after 10% SL compared to the control and that SL induced changes in mitochondrial biogenesis. Furthermore, Seahorse assay results indicate that 10% SL enhances mitochondrial respiration. These findings provide novel insights into the cellular responses to mechanical loading and shed light on the intricate AMPK-mTOR regulatory network in muscle cells.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mech-anisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain un-clear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degra-dation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demon-strate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

BACKGROUND:Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized. METHODS:The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model. RESULTS:We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model. CONCLUSIONS:Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.

G protein-coupled receptors (GPCRs) mediate responses to various extracellular and intracellular cues. However, the large number of GPCR genes and their substantial functional redundancy make it challenging to systematically dissect GPCR functions in vivo. Here, we employ a CRISPR/Cas9-based approach, disrupting 1654 GPCR-encoding genes in 284 strains and mutating 152 neuropeptide-encoding genes in 38 strains in C. elegans . These two mutant libraries enable effective deorphanization of chemoreceptors, and characterization of receptors for neuropeptides in various cellular processes. Mutating a set of closely related GPCRs in a single strain permits the assignment of functions to GPCRs with functional redundancy. Our analyses identify a neuropeptide that interacts with three receptors in hypoxia-evoked locomotory responses, unveil a collection of regulators in pathogen-induced immune responses, and define receptors for the volatile food-related odorants. These results establish our GPCR and neuropeptide mutant libraries as valuable resources for the C. elegans community to expedite studies of GPCR signaling in multiple contexts.

We describe a novel superoxide dismutase (SOD1) mutation-associated clinical phenotype of cerebellar ataxia and motor neuron disease with a variant in the ceruloplasmin (Cp) gene, which may have possibly contributed to a multi-factorial phenotype, supported by genetic and protein structure analyses.

Molecular chaperones are essential to maintain proteostasis. While the functions of intracellular molecular chaperones that oversee protein synthesis, folding and aggregation, are established, those specialized to work in the extracellular environment are less understood. Extracellular proteins reside in a considerably more oxidizing milieu than cytoplasmic proteins and are stabilized by abundant disulfide bonds. Hence, extracellular proteins are potentially destabilized and sensitive to aggregation under reducing conditions. We combine biochemical and mass spectrometry experiments and elucidate that the molecular chaperone functions of the extracellular protein domain Bri2 BRICHOS only appear under reducing conditions, through the assembly of monomers into large polydisperse oligomers by an intra- to intermolecular disulfide bond relay mechanism. Chaperone-active assemblies of the Bri2 BRICHOS domain are efficiently generated by physiological thiol-containing compounds and proteins, and appear in parallel with reduction-induced aggregation of extracellular proteins. Our results give insights into how potent chaperone activity can be generated from inactive precursors under conditions that are destabilizing to most extracellular proteins and thereby support protein stability/folding in the extracellular space. SIGNIFICANCE: Chaperones are essential to cells as they counteract toxic consequences of protein misfolding particularly under stress conditions. Our work describes a novel activation mechanism of an extracellular molecular chaperone domain, called Bri2 BRICHOS. This mechanism is based on reducing conditions that initiate small subunits to assemble into large oligomers via a disulfide relay mechanism. Activated Bri2 BRICHOS inhibits reduction-induced aggregation of extracellular proteins and could be a means to boost proteostasis in the extracellular environment upon reductive stress.

Little is known about the early pathogenic events by which mutant superoxide dismutase 1 (SOD1) causes amyotrophic lateral sclerosis (ALS). This lack of mechanistic understanding is a major barrier to the development and evaluation of efficient therapies. Although protein aggregation is known to be involved, it is not understood how mutant SOD1 causes degeneration of motoneurons (MNs). Previous research has relied heavily on the overexpression of mutant SOD1, but the clinical relevance of SOD1 overexpression models remains questionable. We used a human induced pluripotent stem cell (iPSC) model of spinal MNs and three different endogenous ALS-associated SOD1 mutations (D90Ahom, R115Ghet or A4Vhet) to investigate early cellular disturbances in MNs. Although enhanced misfolding and aggregation of SOD1 was induced by proteasome inhibition, it was not affected by activation of the stress granule pathway. Interestingly, we identified loss of mitochondrial, but not lysosomal, integrity as the earliest common pathological phenotype, which preceded elevated levels of insoluble, aggregated SOD1. A super-elongated mitochondrial morphology with impaired inner mitochondrial membrane potential was a unifying feature in mutant SOD1 iPSC-derived MNs. Impaired mitochondrial integrity was most prominent in mutant D90Ahom MNs, whereas both soluble disordered and detergent-resistant misfolded SOD1 was more prominent in R115Ghet and A4Vhet mutant lines. Taking advantage of patient-specific models of SOD1-ALS in vitro, our data suggest that mitochondrial dysfunction is one of the first crucial steps in the pathogenic cascade that leads to SOD1-ALS and also highlights the need for individualized medical approaches for SOD1-ALS.

Recent data have demonstrated multiple benefits of intra- and postoperative fluid restriction in major abdominal surgery; however, data regarding the outcomes of fluid restriction in cytoreductive surgery and hyperthermic intraperitoneal chemoperfusion (CRS/HIPEC) are limited. This study evaluates the safety and short-term clinical outcomes of restricted intraoperative fluid therapy in CRS/HIPEC.