Metallomics

Purpose. Neuroblastoma is the most common malignancy of infancy, and cancer cells strongly depend on iron metabolism for proliferation. Whether sub-cytotoxic iron load reprograms mitochondrial and redox metabolism, and how this relates to the malignant behavior of neuroblastoma cells, is poorly defined. We hypothesized that exposure of human neuroblastoma cells to a non-toxic iron overload perturbs mitochondrial and redox metabolism, thereby affecting cancer cell properties. Methods. SH-SY5Y neuroblastoma cells were exposed to 100 M iron sulfate for 24 h. The resulting changes were monitored using complementary methods, including cell viability, cytotoxicity, proliferation and migration assays, intracellular iron quantification by ICP-MS, confocal imaging of mitochondrial morphology and membrane potential ({Delta}{Psi}m), high-resolution respirometry with flux control ratios, probe-based oxidant and H2O2 measurements, and label-free quantitative proteomics with Ingenuity Pathway Analysis. Results. Non-toxic iron loading raised intracellular iron about four-fold, promoted cell proliferation and reduced migration. Iron induced mitochondrial fragmentation and {Delta}{Psi}m depolarization while preserving mitochondrial content and OXPHOS coupling efficiency. Among respiratory states, only proton-leak respiration increased, paralleled by a higher leak/ETS coupling ratio. Total cellular oxidant levels and H2O2 output were strongly reduced. The proteome showed downregulation of ribosomal proteins and translation/RNA-processing components and upregulation of histones and ferritin. Conclusion. Mild iron exposure reprograms mitochondrial and redox metabolism in neuroblastoma cells. The observed selective increase in proton leak lowers {Delta}{Psi}m to a range that biophysically limits oxidant production while OXPHOS coupling is preserved. Interfering with this adaptive, proton leak-dependent and preventive antioxidant program may offer a way to alter neuroblastoma progression.

Palladium (Pd), a technology critical platinum group metal, is increasingly lost to the environment through anthropogenic activity, yet the molecular basis of Pd uptake and tolerance in plants remains poorly understood. Here, we examine the role of the Arabidopsis thaliana (Arabidopsis) COPT2 copper transporter in Pd transport and plant stress responses. Expression of COPT2 in Saccharomyces cerevisiae conferred Pd sensitivity, indicating a capability for Pd transport. Arabidopsis copt2 loss-of-function mutants accumulated less Pd in shoots and exhibited reduced reactive oxygen species (ROS) production, supporting a direct role for COPT2 in Pd distribution and associated oxidative stress. Our subsequent transcriptome profiling revealed extensive changes under Pd exposure, including downregulation of HMA2 and aquaporins, and upregulation of HMA7, glutathione transferases, and glutamine synthetase GLN1;1, consistent with the activation of metal detoxification and redox homeostasis pathways. Together, these findings identify COPT2 as a key transport protein mediating the uptake of Pd in Arabidopsis and suggest that Pd induces similar detoxification mechanisms as zinc (Zn), copper (Cu) and cadmium (Cd), providing a foundation for engineering plants for precious metal recovery.

Dehydroascorbic acid (DHA), the oxidized form of vitamin C, exhibits specific toxicity against KRAS-mutant cancer cells; however, the early metabolic reprogramming mechanisms leading to cell death remain incompletely understood. In this study, we reveal the non-linear, dose-dependent response of the intracellular metabolome during the ultra-early phase of 1-hour DHA exposure. Under 1 mM DHA exposure, an early significant accumulation of the primary catabolite erythrulose (p = 0.030) and moderate consumption of glutamate (p = 0.0086) were observed, while core TCA cycle homeostasis and cell viability were preserved. In contrast, under 10 mM DHA exposure, an acute and profound alteration of the metabolic profile was observed. Along with the significant accumulation of glycerate, indicative of a glycolytic blockade, and the terminal peroxidation product oxalate, core TCA cycle intermediates such as -ketoglutarate and malate showed rapid depletion (Log2FC < -2.0, p < 10-7). Furthermore, coupled with this acute energy depletion, a statistically significant accumulation of free methionine (Log2FC = 1.14, p = 0.011), which was below the limit of detection in the control, was identified alongside profound depletions of glutamate (p < 0.001) and cysteine (p = 0.0027). These results strongly suggest that the initial response to high-dose DHA is not merely a linear accumulation of oxidative stress, but rather an "abrupt metabolic switch" characterized by rapid TCA cycle dysfunction followed by the early signature of a methionine trap.

Ferroptosis has attracted significant attention for its potential to treat drug resistant tumors, along with its role in neurodegeneration and ischemia. Several ferroptosis targets were identified in the past decade, yet it is also highly affected by cellular metabolism and epithelial or mesenchymal phenotypes which limits the utility of these targets across diverse cell and tissue types. To address this, we utilized chemo-resistant cancer cell lines with different metastatic potential and tissue origins and discovered a common role of cytoplasmic ion changes during ferroptosis. We observed significant changes in intracellular potassium, sodium and chloride levels upon induction of ferroptosis. Therefore, we screened several ion channel inhibitors and activators (i.e. modulators) and identified Kv7.2 and CFTR modulators influence ferroptosis execution despite the fact that these cell lines do not express these proteins. We demonstrated that clinically approved modulators of CFTR and Kv7.2 can inhibit ferroptosis which indicates that these inhibitors could be repurposed as treatments in ferroptosis-associated diseases such as neurodegeneration and kidney diseases independent of ion channel expression. These modulators also influenced potassium and chloride levels in different contexts indicating off-target effects may also lead to changes in ion levels and may serve as a ferroptosis target. Further, we demonstrated that sustained imbalance of sodium, potassium and chloride ions with ferroptosis inducer salinomycin and digoxin, a Na+/K+ ATPase inhibitor, enhanced ferroptosis. The combination of salinomycin and digoxin successfully enhanced ferroptotic cell death across both cell lines with different metabolic signatures in vitro, offering a novel ferroptosis target. This combination significantly reduced tumor growth in vivo compared with the single agent alone, offering a new therapeutic modality against drug resistant tumors.

Microplastics are pervasive environmental pollutants that pose a growing concern for human health. Oral ingestion is a common route of human microplastic exposure, yet the proteomic response of the gut epithelium to microplastics remains unclear. This study aimed to investigate the cellular effects of pristine and artificially digested microplastic exposure in primary rat duodenal epithelial cells using untargeted proteomics. Cells were exposed to pristine or digested 0.5 um polystyrene microplastics at 10 or 100 ug/mL for 72 hours and were then analyzed by tandem liquid chromatography and mass spectrometry (LC-MS). Proteins that were both significantly different in intensity compared to controls, with a threshold change of 1.3 or greater, were considered to be differentially expressed. This criterion identified 41 differentially expressed proteins after 100 ug/mL pristine MP exposure, with 19 downregulated and 21 upregulated. Following exposure to 100 ug/mL digested MP, only 3 differentially expressed proteins were upregulated and 7 were down regulated, demonstrating the impact of microplastic physicochemistry. FGSEA pathway analysis revealed that 270 Reactome pathways were significantly altered following microplastic exposure in either condition at both concentrations. These pathways contributed to functional domains including protein synthesis, DNA replication, cell cycle control and aerobic respiration. Overall, microplastic exposure was associated with upregulated mitochondrial respiration, and downregulation of nuclear-related processes including DNA synthesis, transcription and cell proliferation. This study provides targets for future investigation (mitochondria and nucleus) and emphasizes the need to consider biological and environmental conditions for in vitro models of microplastic exposure.

Chronic exposure to inorganic arsenic affects more than 200 million people, and disease outcome varies widely among individuals with comparable intake. Explanations have centered on arsenic metabolism, which acts on arsenite that is already inside the cell. Uptake sits upstream of metabolism and sets how much arsenite reaches the genome, yet whether it acts as a gene-by-environment modifier of genotoxicity has been asserted far more often than it has been measured. We expressed human aquaporin-3 (AQP3), its point mutants and the paralogues AQP7, AQP9 and AQP10 in three human cell lines, and measured DNA damage as {gamma}H2AX by flow cytometry against a damage threshold re-derived from mock-transfected cells in every experiment. In lung fibroblasts, AQP3 raised the {gamma}H2AX-high fraction 2.36-fold at 5 M arsenite. Because a gene-by-environment interaction widens the genotype gap with dose while an independent genotoxin would not, the combination exceeds what the two factors would produce if they simply added by 6.36 percentage points, which is an interaction. The response required the AQP3 sequence, was suppressed by N-acetylcysteine in proportion to arsenite dose, was absent in RKO cells, and was shared with AQP9 and AQP10, which created an arsenite dose-response in a cell line that had none of its own. Low-input RNA sequencing of 135 ten-cell pools located the effect inside the cell: at an identical 1 M applied dose and within a single batch, AQP3 raised HMOX1 (induced when trivalent arsenic modifies KEAP1 thiols, and so a report of intracellular rather than applied exposure) 3.3-fold, a shift equivalent to 40% of the 1-to-10 M interval on the measured dose-response, while AQP3 without arsenite was indistinguishable from untreated. Duplex sequencing detected no consistent change in somatic mutation frequency from arsenite alone at any dose to 10 M, in an assay that resolved a 26-fold response to N-ethyl-N-nitrosourea; the arsenite-induced mutation frequency at the EGFR locus nonetheless differed 1.61-fold by AQP3 genotype. The interaction is therefore not unique to AQP3, and aquaglyceroporin complement belongs alongside metabolism among the host factors that set arsenic susceptibility.

Nanoplastics (NPs) are widespread environmental contaminants, yet most toxicity studies rely on short-term, single-dose exposures that poorly reflect daily human exposure. The kidney, particularly the proximal tubule, is a likely site of repeated NP exposure given its role in filtration and reabsorption, but chronic exposure effects are largely unexplored. Here, a long-term in vitro model was developed in which human proximal tubule (HK-2) cells were continuously exposed to 20 nm or 100 nm carboxylated polystyrene NPs (100 {micro}g/mL) over 12 days. Continuous 20 nm NP exposure caused progressive loss of viability whereas 100 nm NPs left viable cell numbers largely unaffected. RNA-sequencing showed 20 nm NPs drove extensive, time-dependent transcriptional dysregulation (1,548 differentially expressed genes at day 6, rising to 4,431 at day 9), far exceeding the minimal alterations in gene expression (35 genes) caused by 100 nm NPs. Cell-cycle analysis and pathway enrichment revealed KRAS pathway activation and S-phase arrest in 20 nm-treated cells, with ANGPTL4 the most upregulated gene. Validation with qRT-PCR confirmed increased ANGPTL4, HMOX1 and FN1 expression, genes implicated in renal fibrosis and chronic kidney disease. These findings show that continuous, size-dependent NP exposure elicits progressive cytotoxic and transcriptional responses in kidney proximal tubule cells and indicate ANGPTL4 as a candidate early biomarker of NP-induced renal stress.

Heavy metal contamination of aquatic environments poses persistent risks to ecosystems and human health. Aquatic macrophytes such as Pistia stratiotes are increasingly used for phytoremediation due to their capacity to accumulate contaminants without displaying symptoms of abiotic stress. However, the molecular mechanisms underlying their metal tolerance remain poorly understood. Metallothioneins (MTs) are cysteine-rich proteins involved in metal binding and detoxification in plants. In this study, genome-wide analysis was performed to identify and characterise candidate MT genes in P. stratiotes and to assess their transcriptional response to copper exposure. Six novel MT genes (PsMT2f, PsMT3a-d, and PsMT4) were identified and classified into MT2, MT3, and MT4 subfamilies based on conserved cysteine motifs and domain architecture. Quantitative real-time PCR showed differential and tissue-specific expression of MT genes in roots and leaves following exposure to copper for up to 4 h. These findings provide the first comprehensive characterisation of the MT gene family in P. stratiotes and demonstrate their potential role in copper tolerance, supporting the application of this species in the phytoremediation of metal-contaminated aquatic systems.

Groundwater contamination by lead and nitrate exceeding the permissible limits of the World Health Organization (WHO) and the Bureau of Indian Standards (BIS) of 10 ppb lead and 45 to 50 ppm nitrate poses a severe health risk of systemic toxicity in humans and animals. Microbial nitrate reduction coupled with microbiologically induced calcite precipitation (MICP) proved to be a novel bioremediation strategy to transform soluble lead nitrate into insoluble lead biominerals. This study presents statistical optimization of operational parameters for nitrate reduction and co-precipitation of biogenic lead carbonate-calcium carbonate by nitrate-reducing bacterial consortia. The SEM, TEM with SEAD, and XRD analysis of the biomineral confirmed lead carbonate co-precipitation with CaCO3. A two-level factorial design was applied to screen significant variables and interactive factors that influence nitrate reduction and biomineral precipitation. The highest nitrate reduction, maximum ammonia production, and biogenic Pb-mineralization efficiency of > 90% were obtained at the optimum conditions (initial nitrate requirement of 600 ppm, temperature 37 oC, pH 8, and carbon content of 5.5 g/L) compared to those obtained under the common process parameters. ANOVA with a high correlation coefficient (R2 > 0.99) corroborated the linear model for the biogenic mineralization process. This study on the biogenic removal of Pb by nitrate-reducing bacterial consortia revealed possible opportunities for large-scale treatment of Pb-contaminated surface and subsurface water bodies.

Although therapeutic advancements have dramatically improved outcomes for cancer patients in recent decades, advanced cancer remains a significant challenge. There is still an urgent need for new and more effective strategies that minimize toxicity and improve survival outcomes in cancer patients. Natural products from microorganisms are a rich and promising source of bioactive compounds with substantial therapeutic potential in cancer treatment. Recently, B. proteolyticus strain OSUB18, isolated from switchgrass, has been demonstrated to promote plant growth and regulate reactive oxygen species (ROS) production. Here, we investigated the anticancer potential of this plant-associated microbe and demonstrated that its crude extracts exhibited broad-spectrum cytostatic activity across diverse solid tumor cell lines. Importantly, OSUB18 extracts displayed limited cytotoxicity toward normal human mammary epithelial cells and mouse fibroblasts. This potentially selective cytostatic activity was retained within a water soluble, heat-labile, and protease-resistant fraction. The extracts primarily suppressed cell proliferation, accompanied by increased lipid peroxidation, with preliminary evidence suggesting the potential involvement of ferroptosis. These findings suggest that the bioactive component may be a selective cytostatic agent with multifaceted mechanisms of action.

Carbaryl, a widely used carbamate pesticide, has been epidemiologically associated with genotoxic and carcinogenic outcomes in exposed human populations, including elevated cancer risk at several tissue sites and, in some agricultural communities, chronic tubulointerstitial kidney disease of unknown etiology (CKDu). However, the cellular mechanisms underlying these associations, and whether carbaryl acts uniformly across tissues, remain poorly defined. Screening a panel of cell lines of distinct species and lineage, we found that carbaryl elicits pronounced, dose-dependent cytotoxicity and cell cycle arrest selectively in a renal tubular epithelial line (IMCD3) and, to a lesser extent, a cervical epithelial line (HeLa), while a fibroblast line (NIH3T3) was unaffected. In IMCD3 cells, carbaryl induces S- and G2/M-phase arrest through direct binding to DNA and consequent DNA damage, triggers Bcl-2-mediated late apoptosis independently of reactive oxygen species, and drives irreversible mitotic arrest. Molecular dynamics simulations, cellular thermal shift assays, and kinase measurements converge on CDK1 as a direct target of carbaryl: carbaryl destabilises the CDK1 active site and inhibits its catalytic activity, whereas CDK2 is engaged through an opposing, rigidifying mode with no functional consequence. This asymmetry provides a coherent mechanistic basis for the combined S-phase and G2/M arrest observed. Together, these findings establish carbaryl as a genotoxic, cell-cycle-disrupting agent with tissue-selective potency acting directly on CDK1, and provide a cell-based mechanistic framework for interpreting its reported associations with renal and other tissue pathologies in exposed human populations.

Environmental exposures are major but understudied determinants of chronic disease. Cadmium is a pervasive toxic metal encountered through diet and tobacco smoke that has been associated with cognitive impairment and Alzheimer's disease mortality, yet strategies to mitigate its neurotoxicity remain limited. S-equol is a gut microbial metabolite of soy daidzein that crosses the blood brain barrier and preferentially activates estrogen receptor beta. Here, we show that cadmium disrupts an axis linking the equol producing gut bacterium Adlercreutzia equolifaciens to circulating S-equol and hippocampal estrogen receptor expression in mice, that S-equol attenuates cadmium-induced cytotoxicity and restores neuronal, glutamatergic and synaptic transcriptional programs in mouse neural progenitor cells, and that circulating S-equol in older humans is lower in apolipoprotein E4 carriers and covaries with body mass index, identifying S-equol as a microbiome-derived, host-modulated candidate countermeasure against cadmium neurotoxicity.

Microbial biological control agents (mBCAs) offer a promising alternative to conventional disease management, but identifying effective strains requires screening large microbial collections against many pathogens. Here, we developed a scalable screening approach to characterize the antagonistic activity of potato-associated bacteria against multiple pathogens and investigated how bacterial origin and phylogeny relate to antagonistic phenotypes. A collection of 600 bacterial strains isolated from two potato cultivars, three plant compartments using four cultivation media was screened against six major potato pathogens: the bacteria Dickeya solani and Pectobacterium carotovorum, the fungi Alternaria solani and Rhizoctonia solani, and the oomycetes Phytophthora infestans and Pythium ultimum. A scalable confrontation method was developed for non-filamentous bacteria, while complementary lower-throughput assays were used for filamentous strains. This approach enabled successful assessment of 92% of non-filamentous and 97% of filamentous strain-pathogen combinations. Isolation cultivar, plant compartment and cultivation medium had only limited effects on the proportion of antagonistic strains. In contrast, pathogen sensitivity varied markedly: P. infestans was the most susceptible pathogen, whereas D. solani and P. carotovorum were generally resistant to bacterial antagonism. Antagonistic activity clustered phylogenetically, with Bacillus and Streptomyces containing many broad-spectrum inhibitors. In addition, several taxa showed preferential activity against the oomycetes, including P. infestans-specific antagonists from the Pseudomonas genus and P. ultimum-specific antagonists from the Frigoribacterium, Curtobacterium, Pedobacter and Phyllobacterium genera. Overall, multi-pathogen screening revealed distinct generalist and specialist antagonistic profiles and identified candidate strains and taxa for further evaluation as mBCAs.

The sulfur mobilization (SUF) system is the only Fe-S cluster biosynthesis pathway in Mycobacterium tuberculosis (M. tb) and Mycolicibacterium smegmatis (M. smegmatis). In this pathway, SufC protein acts as an ATPase in the SufBC2D scaffold for Fe-S cluster biogenesis. This crucial role positions SufC as a promising target for the development of antimycobacterial therapeutics. However, the SufC ATPases from Mycobacterial species are not yet characterized. This study includes cloning, expression, purification, and characterization of SufC homologs from M. tb (Rv1463) and M. smegmatis (MSMEG_3124) using biochemical, biophysical, and computational approaches. Both SufC proteins were purified using affinity and size-exclusion chromatography and observed to exist as monomers in solution. Although both proteins have the conserved domains, they exhibit different ATP-dependent kinetic efficiencies, demonstrating differences in their local environment and catalytic behavior. Furthermore, CRISPR interference targeting MSMEG_3124 results led to diminished M. smegmatis growth, highlighting the critical role of SufC in cellular viability. Modeling and virtual screening led to the identification of three candidate compounds, of which Z2911048515 and Z66741142 were selected for experimental evaluation. Circular dichroism spectroscopy analysis revealed slight differences in the spectral features of Rv1463 in the presence of Z2911048515 and Z66741142, while significant spectral variations were observed for MSMEG_3124. Importantly, Z66741142 was able to cause changes in the fluorescence spectra of both SufC protein homologs. Surprisingly, both compounds showed only limited inhibition of SufC ATPase activity, exhibited distinct modes of action, and may serve as preliminary chemical scaffolds for optimization and testing as effective inhibitors. This study presents functional diversity of mycobacterial SufC proteins across pathogenic and non-pathogenic species and provides a basis for developing a framework for further mechanistic and structure-guided investigation within the indispensable SUF pathway.

Oxidative phosphorylation (OxPhos) and substrate-level phosphorylation (SLP) are the primary biochemical mechanisms for ATP generation in mammalian cells. Increased dependency on SLP can arise from either transient (e.g., Crabtree effect) or chronic (e.g., Warburg effect) suppression of OxPhos. We evaluated the steady-state bioenergetics of quiescent and proliferating mouse cells matched in genetic background and cell biology, including primary thioglycollate-elicited peritoneal macrophages, malignant VM-M3 glioma with macrophage characteristics, and malignant CT-2A astrocytoma. Data are presented showing: 1) OxPhos insufficiency was the primary bioenergetic hallmark of proliferating malignant cells. 2) Dysregulated proliferation was associated with lower metabolic flexibility under nutrient stress than was growth arrest or regulated proliferation. 3) The Crabtree effect was present in macrophages and involved a glucose- and time-dependent suppression of OxPhos coupled to ATP production through SLP and resistance to mitochondrial inhibitors; lowering glucose concentration or substituting glucose with respiratory fuels increased OxPhos and restored sensitivity to mitochondrial inhibitors. 4) The Warburg effect in tumor cells involved a persistent SLP dependency and resistance to mitochondrial inhibitors under equivalent glucose-replete conditions. 5) OxPhos could sustain the regulated proliferation of non-tumor cells in the absence of a Warburg effect. Collectively, our findings emphasize that comparisons between normal and cancer cell metabolism require long-term experimental endpoints that ensure steady-state ATP sufficiency.

Pyrogenic black carbon, such as biochar and wildfire char, possesses a reversible electron storage capacity (ESC), allowing it to serve as an electron donor or acceptor for microorganisms. However, not all the ESC is bioavailable, and the proportion available varies by culture. We hypothesized microbes capable of producing soluble redox mediators could access the ESC in biochar interior and thus utilize a higher percentage of the ESC than organisms without a mediator. To test this hypothesis, we grew wild-type Shewanella oneidensis (MR-1) and {Delta}bfe MR-1, a mutant incapable of exporting flavins, on air-oxidized wood biochar. Cell counts and redox titration show that both strains of MR-1 grew to similar cell densities and accessed comparable amounts of biochar ESC (~19%). The addition of exogenous flavin mononucleotide (FMN) yielded no measurable increase in final cell density, due to the strong adsorption of FMN to biochar. SEM images show attachment of MR-1 and {Delta}bfe to biochar surfaces and the presence of nanowires. This study demonstrates MR-1 can utilize biochar ESC for growth, through direct physical contact without the involvement of flavins. Understanding how microbes metabolize biochar provides insight into the impacts and biogeochemical roles of pyrogenic carbon.

Chlorella sorokiniana is an industrially important microalga, but its genetic manipulation remains limited by poor transformation efficiency. Although several barriers have been proposed, the role of intracellular nuclease activity has received little attention. Here, we compared the degradation of exogenous double-stranded DNA in whole-cell lysates from transformation-recalcitrant (Chlorella sorokiniana and Tetradesmus obliquus) and transformation-proficient (Phaeodactylum tricornutum, Microchloropsis gaditana, and Nannochloropsis oceanica) microalgae. C. sorokiniana rapidly degraded both linear and plasmid DNA, while T. obliquus exhibited intermediate activity. In contrast, no detectable degradation was observed in the transformation-proficient species. DNA degradation in C. sorokiniana occurred within minutes, and nuclease activity in both recalcitrant species was inhibited by EDTA and Zn2+, consistent with a metal ion-dependent endonuclease. These findings suggest that intracellular nuclease activity may represent an underexplored barrier to exogenous DNA stability during transformation and provide a potential target for improving genetic transformation of recalcitrant microalgae.

The modulation of iron metabolism is increasingly explored as a therapeutic strategy in cancer, particularly through the induction of ferroptosis. Given the central role of ferritin as a major intracellular iron buffer, targeting ferritin represents an attractive strategy to disrupt iron homeostasis; however, the lack of pharmacological approaches capable of selectively targeting ferritin currently limits the therapeutic exploitation of this vulnerability. MMRi62, initially developed to target the MDM2-MDM4 axis, has been proposed to induce ferroptosis via ferritin degradation. Here, we revisited this hypothesis in the context of medulloblastoma (MB). Contrary to this proposed mechanism, we found that MMRi62 does not trigger ferroptosis but instead induces robust apoptotic cell death. This effect is observed in both p53-mutant DAOY and p53 wild-type HD-MB03 cells, as well as in c-Myc/OTX2-driven medulloblastoma-like tumours genetically induced in brain organoids. Although ferritin degradation occurs upon treatment, genetic dissection demonstrates that neither ferritin itself nor ferritinophagy are required for MMRi62-induced cytotoxicity. Together, these findings rule out ferritin-dependent mechanisms as primary drivers of cytotoxicity. Instead, we uncovered that MMi62 induces a profound rewiring of iron metabolism consistent with a canonical iron starvation response, accompanied by a marked reduction in intracellular iron levels. Consistently, the UV-visible spectroscopic data were in agreement with the proposed iron-chelating properties of MMRi62. Taken together, our findings identify iron depletion-driven apoptosis, rather than ferroptosis, independently of ferritin degradation, as the primary mechanism of MMRi62 cytotoxicity in MB, refining its mode of action and highlighting iron homeostasis as a therapeutic vulnerability.

Characterizing the enzymatic accommodation of circular agricultural fertilizers is critical for informing strategies to remediate widespread soil sulfur hunger. Here, we present an exploratory in-silico investigation evaluating the active-site cleft of sulfate thiohydrolase (SoxB) across representative soil Proteobacteria. Following the crystallographic precedent of uncomplexed thiosulfate in PDB 2WDE, site-directed molecular docking indicated that the free thiosulfate polyanion binds favorably within the catalytic pocket (predicted affinity: -3.506 kcal/mol), yielding a substantially lower empirical energy barrier than hydrophobic elemental sulfur (S8, -1.409 kcal/mol). Comparative evaluation against the alkaline-adapted Thiobacillus denitrificans homolog revealed an elevated predicted binding affinity of -4.610 kcal/mol, suggesting a potential structural accommodation in high-pH calcareous soils (pH > 8.0). Unrestrained 5.0 ns all-atom molecular dynamics in explicit TIP3P solvent demonstrated initial structural stability of the unliganded host backbone (RMSD = 1.10 +/- 0.15 Angstrom). Energetic decomposition indicated that ligand association is governed predominantly by electrostatic interactions (delta-E_elec ~ -40 kcal/mol) with basic residues (His146, His269, Trp147; RMSF < 0.60 Angstrom), with a single-trajectory unbinding event observed at 3.2 ns. These computational observations provide an exploratory baseline characterizing the active-site electrostatic landscape of SoxB, generating working hypotheses for downstream empirical soil microcosm and in-planta trials.

Ultraviolet-C (UV-C) radiation is a potent environmental stressor that induces DNA damage, accumulation of reactive oxygen species (ROS), and cellular dysfunction. This also initiates coordinated molecular responses to regulate cellular adaptation and recovery. Small extracellular vesicles (sEVs) have emerged as key mediators of intercellular communication through the selective incorporation of microRNAs (miRNAs), proteins, and lipids. However, the temporal changes of sEV-associated cargo during the progression from UV-C-induced damage to recovery remain uncharacterized. We hypothesised that UV-C exposure induces stage-specific changes in sEV cargo (miRNA and proteins) that promote the transition of cells from damage to the response and recovery phases. To examine this, a time-resolved UV-C exposure model was established using Caenorhabditis elegans (C. elegans). In this paper, we studied 2.5 hours of UV-C exposure, including cellular damage, intracellular ROS levels, and DNA integrity, to define the damage and repair state. Based on these temporal variations, samples were collected: i) immediately after exposure, ii) 6 hours post-exposure, and iii) 16 hours post-exposure were designated as damage, response, and recovery conditions, respectively. sEVs isolated from whole-worm preparations were characterised using biophysical and molecular approaches, followed by miRNA sequencing and proteomic analyses. Integrative analysis further identified miRNA-protein associations and associated them with pathways involved in stress adaptation, metabolism, signal transduction, and cellular homeostasis. Collectively, these findings provide a temporal molecular framework of sEV cargo associated with UV-C-induced damage, response, and recovery in C. elegans. Overall, this study identified candidate molecular signatures and miRNA-protein associations that underlie the temporally selective packaging of sEV-associated cargo following UV-C-induced damage, providing a basis for future tissue-specific and mechanistic investigations.