Drug metabolism has traditionally been interpreted within enzyme-centric and extended clearance frameworks, in which intrinsic clearance is determined primarily by catalytic activity, enzyme abundance, and plasma membrane transport. Although these paradigms have been highly successful for cytochrome P450 (CYP)-mediated pathways, they do not fully explain persistent discrepancies in non-CYP metabolism, particularly glucuronidation. A key unresolved issue is that many drug-metabolizing enzymes, including UDP-glucuronosyltransferases (UGTs) and carboxylesterases, have catalytic domains oriented toward the endoplasmic reticulum (ER) lumen. Thus, substrates, cofactors, and metabolites must traverse intracellular membranes before and after catalysis, indicating that metabolic flux may be constrained by membrane-delimited transport processes. Here, we propose the multilayered clearance concept, a membrane-resolved flux framework in which effective metabolic clearance emerges from sequential transport and enzymatic processes across plasma and intracellular membranes. In this framework, clearance is governed by the rate-determining step among substrate delivery, ER access, cofactor supply, catalytic turnover, metabolite removal, and cellular export, rather than by enzyme activity alone. This perspective provides a mechanistic basis for interpreting microsome-hepatocyte discrepancies, underprediction of glucuronidation clearance in in vitro-in vivo extrapolation, limitations of current physiologically based pharmacokinetic models, and UGT-mediated drug-drug interactions (DDIs) that cannot be explained by direct enzyme inhibition alone. We further propose classification of UGT-mediated DDIs into enzyme-limited, system-level flux-limited, and intracellular transport-limited types. Although not yet based on a fully parameterized predictive model, the multilayered clearance concept identifies hidden flux-controlling processes that may improve mechanistic interpretation of non-CYP clearance and DDI prediction.
Since the discovery of cytotoxic agents in the mid-20th century, acute leukemia has consistently served as a model for oncology research. As the Human Genome Project and subsequent genomic profiling elucidated the landscape of somatic mutations and cytogenetic aberrations driving leukemogenesis, the development of molecularly targeted therapies has dramatically accelerated, yielding significant improvements in patient outcomes.In acute myeloid leukemia (AML), the emergence of selective inhibitors targeting high-frequency alterations such as FLT3, NPM1, and IDH1/2 has redefined the standard of care, demonstrating superior efficacy when combined with conventional intensive chemotherapy or hypomethylating agents. Simultaneously, for acute lymphoblastic leukemia (ALL), in addition to the significant improvements achieved by tyrosine kinase inhibitors (TKIs) for BCR-ABL-positive ALL, the advent of CD19- or CD22-targeted monoclonal antibodies and CAR-T cell therapies has marked an epoch-making milestone, representing a major paradigm shift in the management of relapsed or refractory cases. Bridging these two distinct lineages, menin inhibitors have emerged as a novel class of agents targeting a common pathogenic mechanism in KMT2A-rearranged AML/ALL and NPM1-mutated AML, exhibiting promising antileukemic activity across these subtypes. In this review, we describe the evolution of leukemia therapy—highlighting historical trajectory across AML, APL, and ALL from uniform cytotoxic chemotherapy to molecularly targeted agents, antibody-based therapies, and chemo-free paradigms, while outlining future perspectives for precision hematology.
ATP-site resistance mutations, exemplified by T315I in BCR::ABL1, limit the durability of kinase inhibitor therapy in hematological malignancies. Allosteric sites outside the catalytic cleft offer an alternative: ligands that bind regulatory pockets can stabilize inactive conformations and retain activity against mutations that defeat ATP-site drugs. Several reviews have addressed this principle across the kinome, but none has applied a hematology-focused druggability appraisal anchored in the BCR::ABL1/asciminib precedent. This review fills that gap with two contributions: mechanistic evidence that crizotinib engages BCR::ABL1 through a putative dual ATP-site/myristoyl-pocket mechanism, supported by indirect evidence and pending direct structural confirmation; and a hypothesis linking recurrent synonymous mutations in non-receptor tyrosine kinases to transiently structured regulatory regions, as a strategy for identifying latent allosteric sites Asciminib is the proof of concept. It binds the MBP of ABL1, locking the kinase in an autoinhibited-like state without competing for ATP. In the ASCEMBL trial, it achieved a major molecular response rate of 25.5% at 24 weeks versus 13.2% for bosutinib in heavily pretreated CML, with better tolerability-the first regulatory-site inhibitor approved for a hematological malignancy. The question is whether this can extends further. Dual-site strategies may raise the barrier to resistance, but the structural and biochemical validation remains incomplete for FLT3, JAK2, and BTK. Asciminib resistance is already real: A337V and P465S mutations reduce binding, and bypass signaling adds another layer. Each approved allosteric agent-asciminib, trametinib, and ivosidenib-required extensive structural and functional validation before reaching the clinic; structural prediction alone is not enough.
Irreversible cisplatin-induced hearing loss (CIHL) is a refractory chemotherapy-related adverse effect with limited clinical treatments. Schisantherin B (STB), a lignan isolated from Schisandra chinensis, is widely recognized for its neuroprotective properties, while its role in auditory injury remains unclear. Herein, we found that STB alleviated cisplatin-induced ototoxicity in House Ear Institute Organ of Corti 1 (HEI-OC1) cells and guinea pig models, protecting cochlear hair cells, synaptic ribbons and spiral ganglion neurons, and partially restoring auditory brainstem response (ABR) thresholds. Furthermore, combined drug affinity responsive target stability (DARTS) assay, the cellular thermal shift assay (CETSA), and the surface plasmon resonance (SPR) assay, we confirmed STB directly binds to the canopy FGF signaling regulator 2 (CNPY2), a key initiator of endoplasmic reticulum (ER) stress. Notably, consistent dual in vitro and in vivo validation confirmed that STB exerts no regulatory effect on CNPY2 protein abundance, yet suppressed the downstream Protein kinase R-like endoplasmic reticulum kinase / C/EBP homologous protein (PERK/CHOP) signaling cascade and ER stress-mediated apoptosis. Moreover, molecular docking and co-immunoprecipitation (co-IP) validated the physical binding of STB to CNPY2 and the endogenous interaction between CNPY2 and PERK. Additionally, CNPY2 overexpression and shRNA knockdown further verified this functional relationship. Integrated proteomic and transcriptomic analyses showed STB partially reversed cisplatin-triggered inflammation and excessive ER stress. Collectively, our results suggest STB may serve as a potential otoprotective agent. The CNPY2-PERK/CHOP axis is closely linked to cisplatin-induced cochlear damage and offers a feasible target for intervention against CIHL. Abbreviations: CIHL, cisplatin-induced hearing loss; STB, Schisantherin B; HEI-OC1, house ear institute organ of corti 1; ABR, auditory brainstem response; DARTS, drug affinity responsive target stability; CETSA, cellular thermal shift assay; SPR, surface plasmon resonance; CNPY2, canopy FGF signaling regulator 2; ER, endoplasmic reticulum; PERK, protein kinase R-like endoplasmic reticulum kinase; CHOP, C/EBP homologous protein; co-IP, co-immunoprecipitation; STA, Schisantherin A; STC, Schisantherin C; dB SPL, decibels sound pressure level; EDTA, ethylenediaminetetraacetic acid; dB SPL, decibels sound pressure level; SGN, spiral ganglion neuron; IHCs, inner hair cells; OHCs, outer hair cells; CCK-8, Cell Counting Kit-8; OD, optical density; ODb, blank sample, ODc, control sample; NC, negative control; PVDF, polyvinylidene difluoride; RT, room temperature; LC-MS/MS, liquid chromatography tandem mass spectrometry; MS, mass spectrometry; DMSO, dimethyl sulfoxide; KDs, equilibrium dissociation constants; SP, standard precision; SEM, standard error of the mean; HSD, honestly significant difference; Ctrl, control group; CV, cell viability; Kd, dissociation rate constant; Ka, association rate constant; STS, sodium thiosulfate; UPR, unfolded protein response; BLB, blood-labyrinth barrier.
Cholestatic liver injury (CLI) arises from hepatic bile acid overload due to impaired bile flow, often progressing to liver fibrosis and even failure. While endoplasmic reticulum (ER) stress activation is a key driver for CLI pathogenesis, its regulatory mechanisms remain incompletely understood. In this study, we identify that mesencephalic astrocyte-derived neurotrophic factor (MANF), as an ER-resident protein, was markedly upregulated within the livers of both CLI patients and murine models. In rodents with bile duct ligation (BDL)-triggered cholestasis, hepatic-specific MANF knockout aggravated bile acid overload, liver injury, and inflammation by hyperactivating ER stress. Furthermore, hepatic-specific MANF deletion exacerbated tunicamycin-induced ER stress and hepatocyte damage, whereas hepatic MANF overexpression conferred protection against ER stress and liver injury. Mechanistically, MANF directly interacts with GRP78, altering the association between GRP78 and PERK and thereby inhibiting PERK-eIF2α-ATF4 pathway activation. Disruption of the GRP78-PERK axis abolished MANF-mediated ER stress attenuation. Hepatic MANF overexpression mitigated BDL-induced liver damage via deactivating PERK-ATF4 signaling. Our studies unveil MANF serving as a pivotal regulator of ER stress in CLI, functioning through the GRP78-PERK-ATF4 axis. These results not only provide novel mechanistic insights into cholestatic liver injury but also position MANF as a potential candidate therapeutic target for CLI.
Small cell lung cancer is one of the most aggressive malignancies, characterized by rapid tumor growth, early metastatic spread and extremely poor survival. Although most patients initially respond to platinum-based chemotherapy, relapse is almost inevitable and treatment options at recurrence remain limited. The recent introduction of immune checkpoint inhibitors has provided only modest clinical benefit, largely due to the fact that these tumors are immunologically cold. These limitations highlight the urgent need to better understand the molecular features of small cell lung cancer in order to identify more effective therapeutic strategies. In this review, we summarize current knowledge of the molecular landscape of small cell lung cancer, with particular emphasis on transcriptome-based classifications that have identified four major molecular subtypes defined by distinct transcriptional regulators and gene expression programs. We discuss how these classifications have improved the biological understanding of the disease and stimulated efforts to develop subtype-specific therapeutic strategies. At the same time, we highlight important limitations of this framework, including the remarkable transcriptional plasticity of tumor cells, which allows dynamic transitions between subtypes and may contribute to therapeutic resistance. To address these challenges, we examine additional molecular features that may represent more stable vulnerabilities, including recurrent genomic alterations, such as the widespread loss of tumor suppressor genes or oncogene amplifications through extrachromosomal DNA. We also discuss emerging approaches aimed at identifying novel context-specific cancer dependencies, including genome-scale functional screens in vitro and in vivo and genetic restraint analyses. Finally, we consider the growing potential of liquid biopsy strategies, which exploit the high level of circulating tumor DNA in patients with this disease to detect clinically relevant genomic alterations and monitor tumor evolution. Overall, this review highlights both the opportunities and challenges associated with molecular stratification in small cell lung cancer. The integration of transcriptional classifications with genomic and functional approaches may help identify more robust therapeutic vulnerabilities and guide the development of more effective treatments for this highly aggressive disease.
The global spread of multidrug-resistance (MDR) bacteria is devastating health systems and economies worldwide, and this critical trend has revitalized antibiotic adjuvant research. The extensive dissemination of plasmid- or chromosome-mediated resistance genes emphasize the importance of reshaping colistin sensitivity. Here, we report the discovery that Melissa officinalis essential oil isolated from Traditional Chinese medicine, effectively potentiates bactericidal activities of colistin against mobile colistin resistance (MCR-1) positive Escherichia coli (E. coli). Mechanistic analysis demonstrated that Melissa officinalis essential oil interacts with the MCR-1 protein to disrupt LPS modification, while concurrently inducing microbial iron homeostasis disruption and oxidative damage. To enhance its essential oil-mediated therapeutic potential in clinical management, Melissa officinalis essential oil-loaded nanostructured lipid carrier (MOEO-NLC) was designed with improved biocompatibility and solubility. Encouragingly, the combination of MOEO-NLC and colistin achieves bacterial burden reduction and inflammatory response suppression in a peritonitis-sepsis model. Collectively, this study advances antibacterial tactics and underscores the transformative potential of MOEO-NLC in paving the way for enduring countermeasures against antibiotic-resistant threats.
Hypothyroidism is associated with cardiovascular complications, including supraventricular arrhythmias. Thus, this study investigated functional and morphological alterations in the atrial of hypothyroid rats and evaluated the effects of acute ranolazine treatment on arrhythmia susceptibility. Hypothyroidism was induced in Wistar rats with methimazole (0.02 % in drinking water) for 28 days. The experimental approach included in vivo electrocardiography (ECG), ex vivo assessment of atrial contractility and arrhythmia induction by burst pacing, histological analyses, PCR, and immunofluorescence. Hypothyroid rats exhibited reduced heart rate, prolonged P-wave duration, and increased PR interval, indicating impaired atrial function. Ex vivo atrial preparations also showed increased susceptibility to arrhythmias, characterized by a higher number and longer duration of arrhythmic episodes following burst pacing. Acute ex vivo administration of the antiarrhythmic drug ranolazine significantly reduced arrhythmias susceptibility. Histological analysis revealed myocardial disorganization, and increased vacuolization in hypothyroid rat atria. In addition, Cx43 expression was markedly reduced, potentially contributing to impaired intercellular electrical coupling and increased arrhythmogenic susceptibility. Overall, these findings demonstrate that hypothyroidism induces significant electrophysiological, structural and contractile atrial remodeling, resulting in increased susceptibility to atrial arrhythmia. These findings highlight the importance of thyroid hormone signaling in maintaining atrial electrical stability and suggest that targeting electrical instability with ranolazine may have therapeutic potential in hypothyroid cardiac remodeling.
Triple-negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, associated with poor prognosis and frequent drug resistance. Ferroptosis refers to an iron-dependent form of non-apoptotic cell death triggered by lipid peroxidation, offering a promising strategy to overcome this resistance. Here, we describe a dual-pharmacophore fusion strategy that hybridizes the electrophilic warhead of a GPX4 inhibitor with the clinical PARP inhibitor niraparib, leading to the design and synthesis of (S)-2-(4-(1-(4-ethynylthiazole-2-carbonyl)piperidin-3-yl)phenyl)-2H-indazole-7-carboxamide (YHD-26), a novel ferroptosis inducer. YHD-26 inhibits PARP (IC50 = 92 nM) and binds GPX4 (KD = 23.4 μM). Mechanistically, YHD-26 exerts potent antiproliferative effects, induces ferroptosis, and impairs DNA damage repair, resulting in G2/M arrest in MDA-MB-468 and 4 T1 cells. In a 4 T1 xenograft mouse model, YHD-26 inhibited GPX4 and PARP, validating its in vivo antitumor efficacy and dual-target engagement. Collectively, YHD-26 represents a novel dual GPX4/PARP inhibitor with therapeutic potential for TNBC.
Ependymal cells are specialized multiciliated epithelial cells that line the brain ventricles. Their coordinated ciliary beating contributes to cerebrospinal fluid circulation and neural homeostasis. Hydrocephalus, a severe neurological condition affecting ∼1-3 per 1000 births worldwide, can arise from defects in ciliary motility, ependymal cell differentiation, planar cell polarity (PCP), and cytoskeletal organization. Mutations in genes that regulate these processes have been identified in patients with congenital hydrocephalus and related ciliopathies, establishing the clinical relevance of these pathways. This review summarizes the recent advances in the molecular mechanisms underlying ependymal cell biology. We describe the structural and functional organization of the ependymal cilia and discuss how ciliary defects range from severe developmental disruptions causing neonatal hydrocephalus to subtle maintenance defects underlying late-onset forms of the disease. We then examine the hierarchical transcriptional programs controlling ependymal differentiation, from master regulators such as GemC1 and Multicilin to downstream effectors, including FoxJ1 and Regulatory Factor X proteins. The core PCP pathway, comprising Vangl, Celsr, Frizzled, and Dishevelled proteins, coordinates tissue-wide ciliary orientation. Recent cryo-electron microscopy studies have provided structural insights into core components. Finally, we discuss the role of cytoskeletal networks in ependymal maintenance. Actin networks support structural integrity through mechanosensitive feedback loops and transduce mechanical forces into transcriptional activation of multiciliogenesis. In parallel, microtubule-based systems coordinate the planar polarized ciliary orientation via the Daple-dynein axis. Taken together, these molecular insights advance our understanding of the pathogenesis of hydrocephalus and may inform future diagnostic and therapeutic strategies.
Nuclear bodies are membrane-less compartments formed via liquid-liquid phase separation (LLPS) and serve as "dynamic information-processing hubs" for key molecular processes, including transcription and RNA metabolism. In the nervous system, these functional condensates provide a physical substrate for the formation of diverse splicing isoforms, which are essential for maintaining the complexity of the human brain. Recent findings suggest that the collapse of this "nuclear infrastructure" is the underlying cause of several psychiatric disorders. This collapse is due to an aberrant phase transition, where the nuclear environment shifts from a reversible, dynamic state to an irreversible, rigid state-a disruption of the homeostatic mechanism known as "Interstasis." This review discusses how this physical transition leads to systemic splicing failure and explores next-generation therapeutic strategies, such as antisense oligonucleotides (ASOs) and small-molecule modifiers, to restore nuclear integrity. This review is structured as follows to systematically explore the nexus between subnuclear architecture and neuropsychiatric pathogenesis. Section 1 establishes the biophysical foundations of nuclear bodies, highlighting LLPS and homeostatic mechanisms like interstasis. Section 2 addresses the advanced diversity and spatiotemporal regulation of alternative splicing in the nervous system, with a particular focus on the neuron-specific long non-coding RNA Gomafu. Section 3 comprehensively dissects the molecular mechanisms of nuclear infrastructure collapse-encompassing aberrant phase transitions, microexon/novel exon dysregulation, and 3D genome and transport deficits-in neurodevelopmental disorders and schizophrenia. Finally, Section 4 outlines next-generation drug discovery strategies, focusing on therapeutic modalities such as ASOs and small molecules capable of modulating network-level analog states.
The mechanistic role of microglial activation in Alzheimer's disease pathology is typically investigated using mouse models with aggressive amyloid accumulation, leaving the dynamics of microglial responses under the relatively slow deposition of amyloid-β (Aβ) characteristic of the early stages of the disease poorly understood. In this study, we examined microglial gene expression in the brains of AppNL-F knock-in mice, which gradually develop Aβ pathology in an aging-dependent manner without amyloid-β precursor protein overexpression. Quantitative PCR (qPCR) analysis revealed that microglial gene expression presents a stepwise activation pattern: early-induced genes increased at 12 months, whereas late-induced genes emerged at 18 months. Notably, neither group showed further induction at 24 months despite continued Aβ accumulation, indicating that microglial activation does not scale proportionally with the amyloid burden. Several canonical components of the disease-associated microglia program were not induced in AppNL-F mice, whereas a set of previously unrecognized microglial genes (Ly86, Snx20, and Pram1) was upregulated. The induction of these novel genes was preserved in the brains of Trem2 R47H knock-in mice, corroborating that the R47H variant exhibits only mild, if any, phenotype. Immunoblotting of selected proteins confirmed these qPCR-based findings. Together, these results reveal a stepwise mode of microglial gene activation under slow amyloid progression and identify novel genes that may be relevant to the early stages of Alzheimer's disease.
Drug-food interactions are highly diverse, sometimes leading to clinically significant outcomes. A classic example is the contraindicated coadministration of grapefruit juice with specific medications due to the inhibition of intestinal drug-metabolizing enzymes. This review delineates the diverse mechanisms by which fruit-derived small- and macromolecules modulate intestinal transporters. First, the key determinants of intestinal drug absorption and how dietary factors influence these processes are briefly described. Polyphenols in apple, grapefruit, and orange juices competitively inhibit intestinal uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1), thereby reducing the systemic exposure of its substrate drugs. Notably, apple polyphenols trigger the internalization of OATP2B1 from the plasma membrane via protein kinase C activation, a phenomenon experimentally characterized as a "pre-incubation effect." A pre-incubation effect is also induced by apple-derived extracellular vesicles (APEVs); microRNAs contained within APEVs bind to the SLCO2B1-3'UTR, leading to the down-regulation of OATP2B1 expression. Furthermore, both apple polyphenols and APEV-contained microRNAs suppress the expression of the apical sodium-dependent bile acid transporter (ASBT). Mechanistically, these apple-derived microRNAs down-regulate retinoic acid receptor alpha, a transcription factor for ASBT expression. The physiological and pharmacological relevance of these APEV effects has been demonstrated. Regarding OATP2B1, suppressing the intestinal accumulation of SN-38 significantly mitigates SN-38-induced gastrointestinal toxicity. Meanwhile, the reduction in ASBT activity aligns with the well-documented health benefits of apple consumption on functional constipation. In conclusion, fruit-derived small and macromolecules regulate intestinal uptake transporters through multifaceted mechanisms. Elucidating these pathways is essential not only for preventing adverse interactions but also for understanding and leveraging the health-promoting benefits of functional foods.
Focal impaired consciousness seizures (FICS) are among the most common seizure types in epilepsy and are a major cause of reduced QOL, occurring predominantly in temporal lobe epilepsy. Although seizure activity often remains confined to a focal temporal lobe network, patients develop widespread impairment of consciousness, indicating dysfunction of distributed brain systems rather than local seizure activity alone. Over the past two decades, converging evidence from human electrophysiological recordings, neuroimaging, and experimental animal models has substantially advanced our understanding of the neural mechanisms underlying this phenomenon. This review summarizes current knowledge of the clinical features and pathophysiology of FICS in temporal lobe epilepsy. We discuss evidence supporting the network inhibition hypothesis, in which seizure activity propagates from the hippocampus to subcortical relay structures that suppress the ascending arousal system, leading to impaired consciousness. We further examine emerging evidence implicating thalamocortical oscillations as a potential mechanism of cortical disconnection and review findings on the bilateral network engagement observed during FICS. By reviewing the substantial progress in defining the circuitry underlying impaired consciousness, we highlight the key questions that remain to be addressed in future research.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening conditions characterized by severe pulmonary dysfunction, with persistently high global incidence and mortality. Although glucocorticoids, the standard pharmacotherapy for ALI/ARDS, have been reported to reduce short-term mortality and improve outcomes, their clinical benefit is limited by dose- and duration-dependent adverse effects, thereby driving the exploration of complementary or alternative approaches. Natural saponins, a structurally diverse group of glycosides widely distributed in medicinal plants, have gained considerable interest owing to their multitarget anti-inflammatory, antioxidant, and antiapoptotic activities demonstrated in various in vitro and in vivo ALI models. Here, we systematically review the protective mechanisms of natural saponins from the perspectives of cellular regulation and signaling pathways, highlighting their modulatory effects on macrophages, neutrophils, and other effector cells, as well as their actions through key pathways related to inflammation, oxidative stress, and apoptosis. We further discuss current strategies to overcome the major translational barriers of saponins, particularly structural modifications and formulation approaches aimed at improving oral bioavailability and reducing hemolytic activity. This review consolidates the pharmacological basis for saponin-based ALI therapy and provides a forward-looking perspective on addressing their limitations to facilitate clinical translation.
Retinitis pigmentosa (RP) is an inherited retinal disorder characterized by the progressive degeneration of photoreceptor cells, representing a leading cause of blindness. A substantial proportion of patients with RP report photopsia-the perception of spontaneous flashes of light in the visual field. Photopsia can act as visual "noise" and may interfere with visual processing, even compromising restored visual signals following emerging therapeutic interventions. In retinal degeneration (rd) animal models, retinal ganglion cells (RGCs) exhibit pathological periodic spontaneous firing, or oscillations, which have been implicated as a retinal circuit substrate for photopsia. In this review, we summarize current findings on pathological retinal oscillations in rd models and incorporate recent evidence from congenital stationary night blindness (CSNB) models. Together, these studies suggest that RGC oscillations are not simply a consequence of photoreceptor degeneration, but instead arise from a pathological state of the ON bipolar cell-AII amacrine cell network associated with TRPM1-dependent signaling. We propose that altered input balance within this network, including reduced rod bipolar cell-to-AII amacrine cell signaling and altered ON cone bipolar cell activity, may represent a shared mechanism underlying pathological oscillations across distinct retinal diseases.
Neuronal Intranuclear Inclusion Disease (NIID) is caused by GGC repeat expansions in the 5' untranslated region of the notch 2 N-terminal like C (NOTCH2NLC) gene. An upstream open reading frame within the mutant transcript produces the NOTCH2NLC upstream open reading frame-derived polyglycine protein (uN2CpolyG) containing expanded polyglycine (polyG), which forms intranuclear inclusions. Although uN2CpolyG is thought to play a critical role in disease pathogenesis, the mechanisms underlying its toxicity and inclusion formation remain incompletely understood. In this study, we first expressed a pure GGC repeat encoding polyG in Neuro2a cells and identified aggregate-associated proteins by mass spectrometry. We then confirmed the formation of intracellular aggregates using both transient expression and drug-inducible expression systems for uN2CpolyG. Among the proteins identified by mass spectrometry, Calpain small subunit 1 (Capns1), the regulatory subunit of calpain, was found to be sequestered into uN2CpolyG aggregates. Notably, the N-terminus of Capns1 contains a glycine-rich sequence, which mediated its co-aggregation with uN2CpolyG. Furthermore, knockdown of Capns1 appeared to reduce the accumulation of uN2CpolyG aggregates. Collectively, these findings identify Capns1 as a potential modifier of NIID pathology.
Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, promotes cardiac hypertrophy, yet the molecular mechanisms linking microbial metabolism to cardiac gene expression remain incompletely defined. Ketohexokinase (KHK), the rate-limiting enzyme of fructose catabolism, is implicated in cardiac pathology, but its role in TMAO-induced hypertrophy is unknown. Here, we show that dietary choline supplementation elevates plasma TMAO and induces pathological cardiac hypertrophy in mice, effects significantly attenuated by the microbial inhibitor 3,3-dimethyl-1-butanol (DMB). RNA sequencing identified Khk as a top upregulated transcript in TMAO-exposed cardiomyocytes, confirmed at the protein level both in vitro and in vivo. Functionally, lentiviral-mediated Khk silencing mitigated hypertrophic growth, mitochondrial dysfunction, and oxidative stress, while pharmacological KHK inhibition with osthole ameliorated cardiac hypertrophy and preserved cardiac function. Mechanistically, TMAO selectively enriched histone H3 lysine 27 acetylation (H3K27ac) at a downstream enhancer of Khk, as revealed by CUT&Tag sequencing and qPCR. Pharmacological blockade of histone acetyltransferase activity with C646 abolished this enhancer activation and KHK upregulation. Targeted motif discovery within the Khk enhancer further identified conserved binding motifs for GATA family transcription factors, which are known to cooperate with p300 to modulate H3K27ac deposition and regulate cardiac hypertrophy genes, suggesting a potential mechanism by which this enhancer may be regulated. Collectively, these findings define a novel TMAO-H3K27ac-KHK axis linking gut microbial metabolism to cardiac remodeling, and identify KHK as a promising druggable target for therapeutic intervention in microbiota-associated heart disease.
Glioblastoma (GBM) is among the most aggressive primary brain tumors, marked by rapid proliferation, therapeutic resistance, and profound intratumoral heterogeneity. Epigenetic regulators such as lysine-specific demethylase 1A (KDM1A) and histone deacetylase 2 (HDAC2) are aberrantly expressed in resistant GBM subpopulations and strongly correlate with poor clinical outcomes. Here, we assessed the therapeutic potential of MPT0G521, a dual KDM1A/class I HDAC inhibitor, in disrupting epigenetic regulation and cell cycle progression. Bioinformatic analyses of resistance-associated gene profiles (temozolomide and 2 Gy radiation) and single-cell transcriptomic datasets from distinct tumor regions revealed enrichment of KDM1A and HDAC2 in high-cycling GBM clusters, particularly at invasive margins prone to recurrence. Functional assays demonstrated that MPT0G521 potently inhibited proliferation of both parental and temozolomide-resistant GBM cells, inducing G2/M arrest and apoptosis. Transcriptomic profiling further identified significant downregulation of centrosome integrity genes (FSD1, KIFC1), spindle regulators (TUBB, STMN1, KIF2C, KIF15), kinetochore components (AURKB, CDCA8, SPAG5), and G2/M checkpoint mediators (CENPF, MYBL2, CCNF, MYT1, CDC25A), resulting in disrupted mitotic progression. Mechanistically, MPT0G521 increased histone H3 methylation and acetylation, validating its dual inhibitory activity against KDM1A and class I HDACs. Collectively, these findings indicate that MPT0G521 disrupts the G2/M activation and mitotic machinery, thereby suppressing proliferative and resistant GBM subpopulations. This dual epigenetic strategy holds strong promise for overcoming GBM heterogeneity and reducing recurrence.
Ferroptosis is a distinct, iron-dependent form of regulated cell death characterized by lipid peroxidation. Despite its growing significance in physiology and disease, the molecular networks that govern ferroptosis are not yet fully understood. Genome-wide CRISPR screens have broadened the regulatory landscape of ferroptosis by revealing both conserved and context-dependent mechanisms. In this review, we summarize recent advances in CRISPR-based ferroptosis screens, highlighting a transition from in vitro CRISPR screens to in vivo platforms and single-cell CRISPR screens. We also discuss the potential translation of key targets, focusing on their structural druggability and therapeutic potential. By outlining objective-driven screening strategies, this review seeks to provide options for exploring the distinct mechanisms of ferroptosis and to accelerate its translation into therapeutic opportunities for various diseases.
