Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant utilized as a preservative in many products from foods to cosmetics. Population-based studies have detected BHT in the vast majority (>90%) of human specimens (serum, urine, fingernails) as well as home dust samples. Thus, BHT constitutes a ubiquitous environmental contaminant without obvious adverse health effects to humans. In contrast, exposure of mice to a single intraperitoneal dose of BHT triggers distal epithelial damage including loss of gas-exchanging alveolar type 1 epithelial cells, thus providing an invaluable tool to study alveolar repair and transient fibrosis. Presently, the molecular basis of BHT lung toxicity remains unknown. To address this, mouse lung single cell transcriptomic data were used to identify cytochrome P450 2B10 (CYP2B10) in AT1 cells as a BHT-activating enzyme. In cell culture systems, expression of CYP2B10 leads to marked BHT sensitization consistent with bioactivation of BHT into a toxic quinone methide. Targeted and proteome-wide experiments identify BHT-induced protein alkylation, DNA damage response, stress kinase activation and intrinsic apoptosis in a CYP2B10-dependent manner. Structure-activity relationship studies reveal the necessity of para -methyl and ortho - t -butyl groups necessary for BHT bioactivation. Our findings elucidate the molecular basis by which BHT is converted from an innocuous antioxidant into a highly toxic quinone methide and identify the first mammalian enzyme known to catalyze BHT bioactivation. Further, CYP2B10-catalyzed BHT bioactivation may provide a strategy for future targeted cell ablation technologies or environmental remediation of BHT.

Genetic variants throughout the multi-domain protein leucine-rich repeat kinase 2 ( LRRK2) gene are the most common cause of autosomal dominant Parkinson's disease, and the most prevalent Parkinson's-associated LRRK2 variants enhance kinase activity. Hence, the kinase domain has been extensively targeted for therapeutic development. However, clinical progression of LRRK2 kinase inhibitors has been limited by on-target peripheral toxicities linked to strong kinase suppression, raising the question of whether targeting LRRK2 kinase activity through other means could offer a more tunable therapeutic window. LRRK2 is one of two proteins in the human proteome that possess a Roc-COR GTPase domain in tandem with a kinase domain, and the GTPase domain has been shown to intramolecularly regulate kinase output. Here, we ask whether the Roc-COR GTPase can be targeted as an alternative approach to downregulate kinase activity. We utilize a chemical genetic approach to sensitize LRRK2 to existing GTPase inhibitors and show that pharmacologically engaging the Roc-COR GTPase domain in cells decreases but does not fully inhibit LRRK2-mediated Rab10 T73 phosphorylation. This study lays the groundwork for future efforts directed at the development of pharmacological inhibitors for the LRRK2 GTPase, providing an alternative approach for therapies targeting LRRK2-driven Parkinson's disease.

Dietary proteins induce antigen-specific immune responses, leading to oral tolerance or food allergies. While mouse models of allergic sensitization rely on antigen context and adjuvant signaling, exposure to purified proteins is usually sufficient for tolerance development in studies with soluble model antigens. In previous work, we discovered that the maize protein zein induces robust antigen-specific intestinal Tregs following normal chow exposure, but unexpectedly does not elicit a T cell response when delivered as a purified protein. Here, we investigated the mechanisms underlying these differential T cell responses, considering both biochemical properties of zein and its food matrix, as well as intestinal antigen processing. We focused on three zein preparations that induced different frequencies of antigen-specific intestinal T cells. While in vitro studies showed comparable presentation by dendritic cells, in vivo studies revealed stark differences in intestinal uptake. We identified differences in protein solubility and active sampling mechanisms as cooperative drivers of intestinal zein uptake. Different zein preparations could be sampled by either goblet cells, a route previously described for the model antigen OVA, or M cells, a route hypothesized for dietary proteins and established for transport of pathogenic bacteria. These findings also extend to peanuts, where the allergen Ara h 1 was selectively transported by goblet or M cells depending on food preparation, establishing examples of naturally occurring dietary M cell ligands. Finally, solubilizing zein increased intestinal uptake, and correspondingly feeding a diet with solubilized zein led to more intestinal zein-specific T cells compared to feeding the insoluble form. Overall, these findings suggest that intestinal uptake of dietary antigens is a regulated and context-dependent determinant of antigen-specific T cell induction, with implications for development of tolerance-restoring immunotherapies.

Metastatic melanoma remains highly lethal despite advances in immunotherapy and MAPK-targeted therapy. The Rac1 P29S mutation, present in 4-9% of cutaneous melanomas, confers intrinsic resistance to BRAF and MEK inhibitors. Like other small GTPases, Rac1 lacks suitable pockets for small molecule inhibitor binding, motivating indirect approaches to suppressing its activity. We have previously shown that wild-type Rac1 is sensitive to inhibition of de novo GTP biosynthesis in cancer cells, particularly suppression of the rate-limiting inosine monophosphate dehydrogenase (IMPDH) enzymes. Therefore, we asked whether IMPDH inhibition could suppress Rac1 P29S and its associated phenotypes in melanoma. We found that IMPDH inhibition reduced Rac1 activity, impaired Rac1-dependent phenotypes, and induced S-phase arrest in Rac1 P29S -harboring cells. Moreover, IMPDH inhibition synergized with the BRAF inhibitor vemurafenib and the MEK inhibitor trametinib in Rac1 P29S melanoma cells. Dual BRAF and IMPDH inhibition also resulted in enhanced suppression of MEK and ERK phosphorylation in vitro. In syngeneic mouse models, the FDA-approved IMPDH inhibitor mycophenolate mofetil sensitized Rac1 P29S -expressing melanoma tumors to trametinib, including tumors made refractory by prior trametinib exposure. We further identified a feed-forward circuit in which Rac1 sustains IMPDH2 expression levels through JNK and c-Jun/AP1 signaling, potentially coupling the GTPase to its own nucleotide supply. These findings establish GTP biosynthesis as a targetable vulnerability in Rac1 P29S melanoma and support IMPDH inhibition as a rational partner for MAPK-targeted therapy.

Chronic alcohol consumption is associated with increased breast cancer risk; however, the long-term effects of alcohol exposure on DNA damage response (DDR) signaling and therapeutic vulnerability in breast epithelial cells remain incompletely understood. In the present study, we established a chronic alcohol exposure model using immortalized human breast epithelial MCF-10A cells to investigate persistent alterations in DNA damage signaling and sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition. MCF-10A cells that chronically exposed to 0.2% alcohol for 20 weeks and then withdrew alcohol treatment for 4 weeks still retained elevated γH2AX levels and sustained activation of ATM/ATR-associated DDR pathways. Chronic alcohol-exposed cells also exhibited increased sensitivity to the PARP inhibitors olaparib and veliparib, as demonstrated by reduced cell viability, decreased clonogenic survival, and enhanced apoptosis. Mechanistically, olaparib induced greater G2/M checkpoint activation and replication-associated DNA damage in chronic alcohol-exposed cells compared with control cells. In addition, chronic alcohol exposure increased PARP expression and enhanced activation of DDR signaling following PARP inhibition. These findings demonstrate that chronic alcohol exposure induces persistent DNA repair stress and sensitizes breast epithelial cells to PARP inhibition. Our study suggests that chronic alcohol-associated genomic stress may create potential vulnerability to PARP inhibition.

Bacteria employ diverse DNA-targeting systems, including restriction-modification (R-M) and CRISPR-Cas, to cleave invading bacteriophage genomes. In response, phages encode counter-defense strategies that block or mitigate DNA damage. Here, we screened a panel of Pseudomonas aeruginosa phages against native and heterologous DNA-targeting systems and identified the Phikmvvirus phage genus as broadly resistant to multiple CRISPR-Cas and R-M systems. Following CRISPR-Cas12a exposure, most protospacer sequences remained genetically unchanged. However, at an intergenic protospacer, mutations accumulated with high frequency at the Cas12a cleavage site rather than within PAM or seed sequences, resembling repair-associated indels observed after genome editing in eukaryotic cells. Genetic screens to isolate Cas12a- and EcoRI-sensitized phage mutants revealed perturbations to the phage DNA ligase. A Cas12a-sensitive mutant phage was rescued by DNA ligase expression in trans, which was also sufficient to reverse CRISPR targeting of an unrelated phage. Together, our results support a model in which ΦKMV-like phages tolerate certain nucleases through ligase-dependent repair of nuclease-induced double-stranded breaks.

Genomic instability manifests in two distinct ways based on DNA alteration scale, differentially affecting the tumor-immune microenvironment. Mutational instability involves excessive accumulation of small-scale alterations, such as substitutions and indels, from defective DNA repair or aberrant mutagenic processes, generating neoantigens and increased immunogenicity. Chromosomal instability encompasses large-scale alterations in which cells gain, lose, or rearrange chromosomes, resulting in aneuploidy and immune evasion through chronic innate activation and myeloid remodeling. This review outlines differences between mutational and chromosomal instability and discuss how their interplay shapes tumor immunogenicity and response to immunotherapy. Understanding these opposing mechanisms could inform immunotherapeutic approaches in cancer.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with a median survival of less than two years despite surgery, radiotherapy, and temozolomide. Its poor outcome reflects diffuse invasion, cellular heterogeneity, and the activation of overlapping pathways (PI3K/AKT/mTOR, Wnt/β-catenin, Notch, and NF-κβ) that sustain growth, therapy resistance, and recurrence. This article reviews evidence that natural polyphenols, especially curcumin, resveratrol, quercetin, and Epigallocatechin Gallate (EGCG), can act as multitarget agents against GBM. Polyphenols promote apoptosis by disrupting mitochondrial potential and regulating caspases; restoring the autophagic balance via AMPK/ULK1 activation and mTOR inhibition; and dampening angiogenesis by reducing HIF-1α and VEGF. They moderate oxidative stress, inhibit NF-κB/STAT3, and remodel the tumor microenvironment by lowering IL-6/TNF-α and Matrix Metalloproteinases (MMP-2/MMP-9), thereby curbing invasion. Epigenetically, they inhibit DNMTs and HDACs and modulate noncoding RNAs, re-expressing tumor-suppressor programs. Importantly, several polyphenols impair glioblastoma stem-like cells by reducing self-renewal, downregulating the expression of stemness markers, and sensitizing cells to temozolomide and radiation. These pleiotropic actions suggest value as adjuncts to current care and in rational combinations with precision approaches. Although these beneficial effects are promising, the efficacy of natural polyphenolic compounds in GBM is limited by their bioavailability and blood‒brain barrier permeability.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype characterized by the absence of actionable molecular targets, high metastatic propensity, and limited responsiveness to current immunotherapies. Although immune checkpoint inhibitors show promise, their clinical efficacy is constrained by low tumor immunogenicity and a highly immunosuppressive tumor microenvironment (TME). This study reports a hyaluronic acid (HA)-functionalized TiO2@Cu2-XSe@HA nanosystem that promotes the coordinated induction of ferroptosis and cuproptosis, thereby enhancing sonodynamic immunotherapy against TNBC. It features a rationally designed Cu2-XSe/TiO2 heterostructured nanosonosensitizer and an integrated therapeutic strategy combining ultrasound-activated sonodynamic effects with the coordinated induction of ferroptosis and cuproptosis. Under ultrasound irradiation, the Cu2-XSe/TiO2 heterointerface promotes efficient electron transfer and reactive oxygen species (ROS) generation, markedly amplifying sonodynamic efficacy. Meanwhile, released Cu2+ depletes intracellular glutathione, disrupts redox homeostasis, thereby promoting copper-dependent proteotoxic stress. In parallel, Cu2+ induces autophagic degradation of glutathione peroxidase 4, further enhancing ferroptotic signaling. This coordinated induction effectively suppresses tumor growth, remodels the immunosuppressive TME, and potentiates antitumor immunity, as further corroborated by single-cell RNA sequencing. Collectively, this study provides a rational strategy for integrating sonodynamic therapy with dual non-apoptotic cell death pathways, offering a promising strategy to sensitize TNBC to immunotherapy.

Previous adversity increases sensitivity to subsequent stressful events1-8, but the causal underlying changes in brain circuitry are poorly understood. Here we harnessed unbiased whole-brain activity mapping to identify circuits that are functionally remodelled by previous adversity to promote heightened stress sensitivity. The anterior hypothalamic nucleus (AHN)-a region that has received little attention until now in the context of stress-displayed heightened stress reactivity in previously stressed mice. This was accompanied by increased correlational strength between the AHN and a threat-related brain network. Using in vivo Miniscope imaging, we then found that neuronal activity in the AHN scales with negative valence. Moreover, previous stress amplified the proportion of valence-sensitive AHN neurons, indicating inflated processing of negative valence in the AHN might drive heightened stress sensitivity. Providing causal support for the role of AHN in negative valence and stress sensitivity, inhibiting AHN neurons blunted, and exciting their activity promoted, stress responses. Finally, amygdala neurons that project to the AHN were found to track negative valence, and silencing amygdala inputs to the AHN abolished sensitized stress responses. These findings define a key role of the AHN in regulating negative valence signals from the amygdala and highlight a new pathway that heightens sensitivity to stressful events.

Vector-borne pathogens must adapt to sharply distinct host environments, yet the regulatory logic that coordinates transitions between opposing host-specific programs remains poorly defined. Here, we identify a minimal regulatory module in the flea-borne pathogen Yersinia pestis that integrates vector transmission with resistance to mammalian innate immunity. Screening of flea-induced genes uncovered the regulator HdfR, which acts primarily through activation of maoP, encoding a nucleoid-associated protein. This HdfR-MaoP module promotes biofilm-dependent foregut blockage in the flea while coordinating baseline envelope adaptations that limit complement recognition and inducible responses that confer resistance to antimicrobial peptides under mammalian host-like conditions. Functional interchangeability of HdfR and MaoP homologs reveals evolutionary conservation of this regulatory logic, and pharmacological perturbation of the module sensitizes Y. pestis to antimicrobial peptides. Together, these findings define a parsimonious and evolutionarily conserved regulatory hub that orchestrates bacterial success across abrupt environmental transitions and exposes a tractable point of intervention.

Glyoxalase I (GLO I) detoxifies the reactive glycolytic byproduct methylglyoxal (MG) and is frequently overexpressed in cancer cells. High GLO I expression is closely associated with resistance to anticancer drugs, including doxorubicin (DOX). However, the functional relationship between GLO I activity and DOX-induced cell death remains unclear. In this study, we investigated whether inhibition of GLO I enhances cellular sensitivity to DOX. Using non-small cell lung cancer cell lines with low (NCI-H460 cells) or high (NCI-H522 cells) GLO I expression, we found that NCI-H522 cells, which we previously reported to be highly sensitive to the GLO I inhibitor TLSC702 and to accumulate MG following GLO I inhibition, were more resistant to DOX than NCI-H460 cells. Cotreatment with exogenous MG significantly potentiated DOX-induced cytotoxicity through multiple cell death pathways, supporting a role for MG-related processes in modulating DOX responses. While simultaneous treatment with TLSC702 and DOX showed limited enhancement, pretreatment with TLSC702 followed by DOX markedly increased cell death in GLO I-high cells, suggesting that prior disruption of MG detoxification may sensitize cells to subsequent DOX exposure. This effect was accompanied by enhanced poly(ADP-ribose) polymerase (PARP) cleavage and was partially suppressed by caspase and autophagy inhibitors, indicating enhanced apoptosis with contributions from additional cell death pathways. Collectively, these results demonstrate that GLO I inhibition primes cancer cells for enhanced DOX-induced apoptosis, providing mechanistic insights into GLO I-associated chemoresistance.

Transitional pain describes the clinically important phase in which acute pain resolves or progresses to chronic pain. This phase is particularly relevant after surgery and trauma, where the onset of tissue injury can be identified. Evidence suggests that chronic postsurgical or post-traumatic pain may arise through the interaction of biological, psychological, and social mechanisms, including peripheral and central sensitization, neuroimmune activation, psychological distress, pain catastrophizing, functional avoidance, and prolonged opioid exposure. Transitional Pain Services address this risk period through early identification, multidisciplinary intervention, opioid stewardship, rehabilitation, and psychological support. Current evidence suggests benefits for opioid tapering, pain interference, mood, and function, although stronger trials are needed to confirm whether such services reduce the incidence of chronic pain. Therefore, transitional pain should be understood as a modifiable risk state and a promising target for preventive perioperative pain care.

Escherichia coli YmdA protein modulates biofilm formation and susceptibility to the aminoglycoside antibiotic apramycin (APR). Here, we renamed this protein DbaM (Dsb-associated Biofilm and Antibiotic Susceptibility Modulatory protein) to reflect its physiological role. We show that DbaM function requires translocation to the periplasm via its N-terminal signal sequence and subsequent Dsb-dependent maturation. The formation of an intramolecular disulfide bond between conserved cysteine residues (C41 and C54) is required for activity; mutations at these sites negate biofilm inhibition and APR sensitization. Rather than forming a stable complex, DbaM modulates the catalytic turnover of the disulfide bond (Dsb) network, likely through transient and catalytic mixed disulfide intermediates. Interestingly, we demonstrated that periplasmic redox modulation by disulfide bond formation protein A (DsbA)-associated action is dependent on DbaM activity. This process involves both the DsbA oxidative and disulfide bond isomerase C (DsbC) pathways and alters the periplasmic thiol-disulfide balance toward net oxidation. Additionally, oxidized DbaM structurally mimics major fimbrial subunits, suggesting that it inhibits biofilm development by engaging the chaperone-usher machinery to disrupt polymer elongation. Functional DsbA is required for the selective packaging of DbaM into extracellular vesicles (EVs). Exogenously applied DbaM-EVs disrupted biofilms and altered APR resistance in the recipient cells. Collectively, these results define the Dsb-fimbriae-DbaM axis, linking periplasmic redox homeostasis, EV dynamics, and antibiotic susceptibility, which may offer potential targets for antimicrobial strategies.