Journal of Hazardous Materials

Depleted uranium (DU) is an environmental contaminant with a 30 g/L (ppb; parts per billion) EPA maximum contaminant level (MCL) for drinking water. The mining of uranium and use of DU in modern weapons underly human exposure that disproportionally impacts military and tribal communities in the United States. Uranium's radiotoxic characteristics are understood, but its chemical hazards much less so. In zebrafish (Danio rerio) and human cell cultures we test the hypothesis that exposure to DU negatively impacts cellular function and development through disruption of mitochondrial metabolism. Using a novel shrapnel model with TEM/SEM+EDS, we showed uranium microparticles caused proximity-dependent mitochondrial disruption. In waterborne exposure paradigms, larval movement was reduced and hatching delayed as a result of reduced movement and not enzyme deficiencies in response to 18 ppb DU, below the MCL. Increased DNA damage accumulation was detected in exposed larva and cells. DNA-damage quantitative PCR of DU-exposed larvae showed increased damage in the ahr1 locus (nuclear gene) and decreased mitochondrial DNA (mtDNA) copy number, but mtDNA damage levels varied across experiments. Mitochondrial function was assessed using a resazurin-based assay in the presence and absence of antioxidants and showed diminished cytoplasmic reductive capacity. DU exposure alone did not enrich antioxidant gene expression, contrasting with arsenic exposure, a known ROS-inducer and Nrf2-activator. Sulforaphane (SFN), a potent Nrf2-activator, did not blunt the effects of DU exposure, despite activation of antioxidant response element (ARE) genes (gstp and gss), but did blunt the effects of arsenic exposure. The most enriched transcript in DU-exposed larvae coded for slingshot protein phosphatase (ssh), further exploration revealed ssh1b as the zebrafish-specific ortholog activated in response to DU, and inhibition using an identified SSH1 inhibitor, Sennoside A, partially rescued the metabolic and hatching defects observed. Our data points to a cytotoxic mechanism in which DU disrupts mitochondrial function through ssh1b enrichment that impairs normal mitophagy, leading to decreased cellular reductive potential independent of either ROS production or ARE-activation. Our results suggest that health impacts from DU exposure may be directly linked to impaired mitochondrial functions.

Chemically complex pharmaceutical mixtures in wastewater and sludge can affect microbial adaptation; however, the responses to different co-occurring compounds have not been elucidated well. In this study, the adaptation of a strain derived from hospital sludge, Klebsiella pneumoniae SS02, to 17-ethinylestradiol (EE2), warfarin sodium, and their combination has been studied. The organism grows under all three conditions, and pre-exposure experiments show induction and cross-induction to substrates. UHPLC MS/MS analyses demonstrated that there is conditional depletion of the parent compound EE2 by [~]15% at 36 h post-treatment compared to initial concentrations, but not for the abiotic and non-adapted controls. The rate of warfarin sodium depletion was approximately [~]30% within 36 h and was in accordance with first order kinetics (k = 0.0102 /h; t{square}/{square}= 67.9 h). Under the combined treatment regime, there was a delay in warfarin sodium depletion, suggesting staged substrate consumption. Growth inhibition with efflux inhibitors confirmed transport-driven tolerance. A genome-based study revealed the coordinated response strategy that involved a proposed flavin-dependent monooxygenase (RutA), an oxidative entry into the pathway; redox processing linked to Hpa; aromatic metabolism through {beta}-ketoadipate pathway; and RND efflux system. The structural study additionally supported ligand-mediated decrease in DNA binding affinity of RutR, which is in agreement with de-repression of the substrate-activated regulatory mechanism. All these findings lead to the development of a dual-strategy for adaptation model in which oxidative modification and efflux-mediated protection work together under the influence of a mixture of pharmaceuticals.

Nanoplastics (NPs) are emerging environmental contaminants able to cross biological barriers, disrupt cellular and organelle homeostasis, and alter the brain microenvironment. This study investigated whether NPs affect microglia to neuron communication, a key mechanism underlying neuronal resilience, via the nuclear factor erythroid 2 like 2 (NFE2L2) pathway. Using an in vitro model, we evaluated the effects of polystyrene nanoplastics on microglial metabolic fitness and microglia-mediated neuronal stress responses. Increasing NP concentrations induced a dose dependent biphasic effect. Low to intermediate concentrations increased intracellular adenosine triphosphate (ATP) levels in microglia and enhanced microglia-mediated activation of neuronal NFE2L2. In contrast, high NP concentration impaired microglial metabolism, reduced ATP availability, and decreased microglia to neuron communication. These findings indicate that NPs alter microglial energetic status and modulate neuroprotective signalling, potentially contributing to impaired neuron to microglia interactions and increased susceptibility to neurotoxicity.

Microplastics are pervasive environmental pollutants increasingly implicated in adverse human health effects, with emerging evidence linking MPLs exposure to elevated cardiovascular risk, including atherosclerosis. However, their specific mechanisms of action remain unknown. Human aortic endothelial cells (HAECs), located in the innermost layer of blood vessels, play a crucial role in maintaining vascular homeostasis and the development of atherosclerosis. This study demonstrates that polystyrene microplastics (80 nm MPLs) can enter HAECs through multiple pathways, including macropinocytosis, clathrin-mediated endocytosis, and caveolin-mediated endocytosis, and co-localize with mitochondria and lysosomes. MPLs exposure resulted in coordinated transcriptional, epitranscriptomic, and metabolomic reprogramming in HAECs, characterized by disruption of mitochondrial genes and an inflammatory response with activation of TNF-a; and NF-kB signaling. Integrative analysis revealed remodeling of the epitranscriptomic profile, demonstrated by an increase in 1-methyladenosine (m1A) modification along with reciprocal regulation (TRMT61A upregulation and ALKBH3 suppression) of its transcriptomic machinery, alongside other enzymes associated with 3-methylcytidine (m3C), pseudouridine (Y), 5-methylcytidine (m5C), and 7-methylguanosine (m7G) pathways. By comparing transcriptomic data from MPLs-treated HAECs with those of human atherosclerotic plaques, several common dysregulated pathways were identified, particularly those related to vascular physiological regulation and cell signaling. Metabolomic profiling further revealed significant remodeling of lipid metabolic networks associated with oxidative stress and inflammatory signaling. In summary, this study reveals that HAECs can internalize MPLs, leading to multiple disturbances in the transcriptome, epigenome, and metabolic networks, suggesting that MPLs exposure may pose a potential hazard to human cardiovascular health.

Per- and polyfluoroalkyl substances (PFAS) are persistent toxicants with immunological, metabolic and epithelial effects, but their relevance to inflammatory skin disease remains unclear. We developed a computational toxicology framework to test whether perfluoroalkyl sulfonate programs, especially perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS), converge with psoriasis-associated keratinocyte inflammation. Exposure transcriptomes were derived from GSE236956, in which human embryonic stem cell-derived epithelial-lineage models were exposed to 10 M PFAS for 8-16 days. Six PFAS were prioritized using descriptors, Tanimoto similarity, toxicology evidence, adverse outcome pathway (AOP)-like key events, exposure differentially expressed gene burden and read-across support. PFAS signatures were integrated with psoriasis bulk transcriptomes, single-cell RNA sequencing, keratinocyte-state mapping, regulator and communication inference, spatial transcriptomics and token-aware Geneformer-compatible virtual perturbation. PFOS ranked highest in integrated prioritization, followed by PFHxS and perfluorooctanoic acid. PFHxS produced a smaller but directionally informative signature within a PFOS-dominant perfluoroalkyl sulfonate footprint. The shared PFOS and PFHxS program converged with psoriasis through inflammatory keratinocyte, epidermal-stress, cytoskeletal and lipid-related modules. Single-cell and spatial analyses localized the program to activated keratinocytes and inflammatory epidermal niches, with strong spatial co-localization with inflammatory keratinocyte and epidermal stress scores. Virtual perturbation prioritized S100A9, S100A8, KRT16, IL36G, CCL20, CXCL8, FABP5, KRT17, FOS, JUN and NFKBIZ as candidate effectors. These findings support an exposure-informed, experimentally testable hypothesis linking persistent perfluoroalkyl sulfonate programs to keratinocyte inflammatory niches in psoriasis.

Tenuazonic acid (TeA) is an emerging Alternaria mycotoxin frequently detected in food and feed commodities, raising concerns about its toxicological relevance. Chronic oral exposure to TeA has been reported to induce dysplastic alterations in the esophageal mucosa of mice, while human biomonitoring data indicate an association between TeA exposure and esophageal cancer, although a causal relationship has not yet been established. At a mechanistic level, the effects of TeA in esophageal cells remain poorly characterized. Therefore, this study investigated the impact of TeA on cytotoxicity, oxidative stress, DNA damage, mitochondrial homeostasis, cell-cycle distribution and transcriptomic stress responses in human esophageal KYSE-510 cells. TeA induced a concentration-dependent reduction in metabolic activity and total protein content after 24 h exposure to 0.1-100 M. Significant cytotoxicity was measured starting from 20 M. At sub-cytotoxic concentrations, TeA triggered rapid ROS formation within 5-30 min exposure and induced formamidopyrimidine-DNA glycosylase (FPG) sensitive DNA damage after 1 h exposure (5-7.5 M), indicating oxidative DNA lesions. In addition, TeA altered mitochondrial morphology after 4 h exposure at 7.5 M, manifested by shrinkage of the mitochondrial network area and perinuclear redistribution, while mitochondrial respiration showed only a non-significant tendency towards reduced respiratory capacity. RNA sequencing after 6 h exposure to 10 M TeA revealed oxidative stress-associated transcriptional changes, impaired antioxidant and stress-adaptive responses, and p53-associated stress signaling. Furthermore, TeA induced significant G2/M phase accumulation after 24 h exposure to 1-10 M.

The use of nitrogen (N) fertilizers to meet global food demands is expected to continue rising. However, up to 70% of N applied to agricultural soils is lost through microbially mediated processes such as nitrification. Inhibiting nitrification is thus a key strategy to reduce N losses and improve fertilizer N use efficiency. Various plant-derived compounds, termed biological nitrification inhibitors (BNIs), have been shown to reduce accumulation of nitrification products, intermediates, and byproducts (nitrite, nitrate, nitric and nitrous oxides). However, the mechanisms by which BNIs affect nitrifiers, along with their specificity and persistence in soil are not well understood. Here, we evaluated the effects of three BNIs: methyl 3-(4-hydroxyphenyl) acrylate (MHPA), 6-methoxy-2(3H)-benzoxazolone (MBOA), and limonene, on ammonia-oxidizing, total microbial, and fungal communities in two soils with contrasting pH. Their persistence in each soil was also evaluated. Although ammonia-oxidizing archaea initially dominated nitrifier communities in both soils, their bacterial counterparts significantly increased after mineral N addition but also were more sensitive to BNI application. Limonene and the synthetic inhibitor DMPP stimulated ammonium immobilization, as total soil mineral N was significantly reduced. Limonene and MHPA had the strongest off-target effects, increasing the relative abundance of hydrocarbon-degrading bacteria and potential fungal pathogens, respectively. In contrast, MBOA inhibited nitrification with minimal off-target effects. Among the tested BNIs, MBOA was also the most persistent in the high-pH, high-nitrification-rate soil. Our results show that MBOA is a promising biological inhibitor and highlight the importance of understanding BNIs ecological effects to develop targeted and sustainable N management strategies.

Rare earth elements (REEs) are critical components of green technologies, but current mining and purification methods remain environmentally unsustainable due to their high energy consumption and intensive chemical requirements. Bio-hydrometallurgical processes have the potential to concentrate and recover REEs at a circumneutral pH. Work presented here uses bacteria at neutral pH to concentrate REEs from solution and subsequently recover those REEs using sodium citrate. Shewanella oneidensis MR-1 was incubated anaerobically in a culture media solution spiked with 14 REEs and yttrium for one to six days. REE concentrations remaining in solution were then compared to REE concentrations on cell pellets. For these same timepoints, the loosely bound extracellular polymeric substance (LB-EPS) was removed from cells prior to quantifying REEs on pellets to narrow down the location of REE binding. Moreover, cell pellets collected after 5 days in REE spiked solution were subjected to a time series desorption assay using sodium citrate. Shewanella oneidensis at a starting OD600 of 0.6 adsorbed 1.18mg/g of REE after 3 days. 80% of these REEs were located in the LB-EPS. In 10 minutes, 0.5 M sodium citrate desorbed about 75% of REEs from cells and over 95% after 24 hours. This method was also applied to Alaskan coal and showed that 68-86% of REEs were desorbed form S. oneidensis. This study elucidates the REE binding location and capacity of S. oneidensi, REE removal efficiency of sodium citrate overtime, and the application of this sustainable biotechnology for REE recovery at a circumneutral pH from Alaskan coal.

The wide variety of physical and chemical properties in materials makes the study of unknown substances challenging. We have previously proposed a theoretical framework for agnostic material characterization based on using nucleic acid imprints of the materials and then analyzing material-specific patterns of derived sequences. Here we demonstrate an experimental and computational pipeline that can agnostically identify and distinguish varied materials based on DNA k-mer imprints and validate the ability of these imprints to distinguish closely related materials. This work lays the foundation for expansion of purely agnostic sensing technologies for the unbiased characterization and categorization of a much wider variety of biotic and abiotic materials.

The widespread use of synthetic food dyes, such as Acid Yellow 23 (AY 23), in the food, cosmetics, and pharmaceutical industries raises questions about their potential effects on biological systems and public health. The concentration-dependent interaction between AY 23 and bovine serum albumin (BSA), a crucial model protein for understanding pharmacokinetics and protein-ligand behaviour, was examined in this study. We demonstrate that, under physiological conditions, increasing dye concentrations from 50 M to 200 M results in notable conformational changes, increased surface hydrophobicity, and protein aggregation using a multimodal biophysical approach that includes fluorescence spectroscopy. Direct visualisation verified these structural changes and aggregate formation, whereas hemolytic assay confirmed the high hemolytic nature of AY 23-induced fibrils. Additionally, this study provides a mechanistic basis for the toxicological effects of AY 23, underscoring the implications of food dyes for public health.

Evidence of negative health and environmental effects of glyphosate-based herbicides (GBHs) has led to marketing of glyphosate-free formulations. A frequent glyphosate replacement is pelargonic acid, which is rapidly degraded, leading to claims of greater safety and less environmentally damaging than GBHs. However, toxicity of commercial pelargonic acid formulations containing several co-formulants have not been determined. Using Roundup NL, a representative pelargonic acid-based herbicide, we undertook tissue culture cell assays measuring viability, plasma membrane integrity, DNA damage, and activation of stress-response pathways. In human hepatoma HepG2 cells, Roundup NL was more cytotoxic than pelargonic acid, and more toxic than the GBH Roundup ProBio and glyphosate as shown by reduced viability underpinned by plasma membrane damage. Pelargonic acid and Roundup NL did not induce oxidative stress. However, comet assays revealed that pelargonic acid but not Roundup NL caused a modest but significant increase in DNA damage at sub-cytotoxic concentrations. The murine embryonic stem cell-based ToxTracker system confirmed Roundup NL as not directly genotoxic but triggered oxidative stress and protein damage (ER stress, impaired proteostasis) indicating cell and assay dependency of oxidative stress pathway activation. Our results suggest that exposure to pelargonic acid-based herbicides constitutes a health hazard and that co-formulants present in Roundup NL contribute substantially to its overall toxicity.

Plant growth-promoting bacteria are gaining significant attention as promising biofertilizers. However, the inconsistency between in vitro plant growth-promoting traits and actual field performance remains a challenge, driven partly by a limited understanding of in situ colonization. This study characterized the colonization patterns of Citrobacter sedlakii CESi7, a novel plant growth-promoting bacterium, isolated from oil palm waste compost, during Brassica rapa cultivation. The in situ behavior of CESi7 was observed in both sterilized medium and non-sterilized soil using fluorescence in situ hybridization with a strain-targeting probe. The results revealed that CESi7 can establish both epiphytic and endophytic populations that transiently colonize roots. In a sterilized medium, CESi7 was widely distributed throughout the root tissues. Conversely, in non-sterilized soil, the bacterium formed dense aggregates specifically at the root tips. This study provides direct microscopic evidence of the colonization strategy of CESi7, offering crucial insights for its development as an effective biofertilizer.

Flavins are organic redox cofactors central to metabolism and uniquely capable of acting as extracellular electron shuttles. For life to have emerged, it must have disengaged itself from its stationary geochemical environment, a step requiring mobile redox-active components. The role of flavins at life's origin has been debated for decades, centered on their capacity for both one- and two-electron chemistry, distinguishing them from nicotinamides and iron-sulfur clusters. Here we chart the abiotic reduction of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin under hydrothermal conditions (40 {degrees}C, 1 bar N2 or 5 bar H2, pH 6, 8, and 10) by nickel (Ni) and iron (Fe). Flavins show greater environmental versatility than hydride carriers such as NAD and can harvest electrons from metals that would otherwise reduce water's protons to H2. Reduction is favoured under acidic conditions, while increasing molecular charge at higher pH impedes electron transfer. Ni acts as a hydrogenation catalyst, reducing deprotonated flavins via hydride transfer, suggesting mineral composition could have influenced geochemical selection of early electron carriers. Reduced FMNH2 and FADH2 were tested as electron shuttles toward Fe3+-containing minerals, revealing that FMNH2 enables faster mineral dissolution than FADH2. We further demonstrate complete redox cycling of FMN through Ni-assisted H2 reduction and subsequent oxidation by magnetite (Fe3O4) under inert atmosphere, releasing Fe2+. This study highlights the versatility, stability and redox chemical capabilities of flavins in prebiotic context.

Manual intervention for concrete repair and replacement comes at high environmental and economic costs. Bioconcrete, which can be formed by bacteria via microbially-induced carbonate precipitation (MICP), is a sustainable method for concrete repair. Bioconcrete-forming bacteria can be incorporated into the concrete at mixing and then heal cracks where and when they occur. Bioconcrete is not intentionally made by bacteria; rather, it is a byproduct of alterations to the local environment that occur during their normal metabolic activities. Bacteria thus make bioconcrete by different metabolic mechanisms, and the environment plays a substantial role in the yield and physical properties of the bioconcrete produced by a given bacterium. The ureolytic bacterium Sporosarcina pasteurii is the most commonly used model organism for MICP, but it requires urea supplementation, which is not feasible for all applications because of nitrogenous waste. In particular, the marine environment is understudied for bioconcrete applications, yet there is a need for self-healing structures in this environment, wherein urea and nitrogenous waste would be detrimental to native biota. Here, we assessed the ability of S. pasteurii to form bioconcrete under marine-like media conditions with urea and calcium supplementation. We found that S. pasteurii generated higher bioconcrete yields in these media conditions compared to standard growth media. We then designed an enrichment protocol to isolate and characterize non-urea-requiring bioconcrete-forming bacteria from Atlantic seawater. We identified three isolates, from the Sulflitobacter, Marinobacter, and Bacillus genera, two of which yielded higher bioconcrete yields in seawater-mimicking media compared to non-ureolytic bacteria utilized in prior literature. Moreover, scanning electron microscopy (SEM)/energy dispersive spectroscopy (EDS) and Fourier transform infrared (FTIR) spectroscopy revealed distinct chemical and structural features of the bioconcrete produced by bacteria in seawater-mimicking medium and between ureolytic and non-ureolytic cultures. Overall, our work establishes a pipeline for the isolation and characterization of novel bioconcrete-forming bacteria from marine samples, with potential for application to marine self-healing materials.

Alternaria toxins are naturally occurring food contaminants with limited and often inconsistent genotoxicity and mutagenicity data. Within the European Partnership for the Assessment of Risks from Chemicals (PARC), an OECD-aligned in vitro testing strategy was applied to fill existing data gaps and to characterize the genotoxic potential of major Alternaria toxins using high-purity test materials. Mutagenicity was assessed using bacterial reverse mutation test (OECD TG 471) and SOS/umu assay, while chromosomal damage was assessed using the in vitro micronucleus (MN) assay (OECD TG 487) in TK6 and HepG2 cells, complemented by fluorescence in situ hybridization (FISH) and {gamma}H2AX assay in HepaRG cells. Alternariol (AOH), alternariol monomethyl ether (AME), and altertoxin-I (ATX-I) showed clear mutagenicity in bacteria, whereas altenuene (ALT), tenuazonic acid (TeA), and tentoxin (TEN) were negative under the tested conditions. In mammalian cells, AOH, AME, and ATX-I induced MN formation in TK6 cells at concentrations [≥]5.5 {micro}M, [≥]2.5 {micro}M, and [≥]0.21 {micro}M, respectively, with FISH analysis supporting a clastogenic mode of action. In HepG2 cells, all tested toxins induced chromosomal damage, with effect threshold ranging from [≥]6.25 {micro}M (AOH) to [≥]50 {micro}M (TeA). {gamma}H2AX induction confirmed DNA damage for AOH and ATX-I, and at higher concentrations for TeA (1000 {micro}M). Overall, the data indicate clear in vitro genotoxic potential for AOH, AME, and ATX-I and provide evidence of chromosomal damage for ALT, TEN, and TeA, thereby reducing critical data gaps for hazard assessment.

The tomato leafminer, Tuta absoluta, poses a severe global agricultural threat due to its rapid leaf-mining behavior and swift development of resistance to conventional chemical pesticides. While microbial chitinases are potent biopesticides, their field efficacy is limited by environmental degradation and the short exposure window before larvae penetrate leaf tissues. This study evaluates a stimuli-responsive, controlled-release nanobiopesticide system utilizing a novel chitinase from newly characterized Serratia marcescens GBS19. A 61.1 kDa chitinase (GBS19_ChiA) was heterologously expressed in Escherichia coli and purified to a specific activity of 215.01 U/mg. The enzyme was immobilized onto starch-coated silica nanoparticles designed for target-triggered release via host alpha-amylase. Genomic profiling and R-based kinetic modeling were integrated to evaluate the efficacy of purified and immobilized forms against T. absoluta. Immobilization enhanced thermal and pH stability, with the nanocarrier maintaining 85% activity over 10 weeks. In larval bioassays, immobilization increased mortality from 21.9% to 59.4% (5000 U/mL) by day 3, reaching 62.5% by day 6. Genomic analysis identified an expansive secretome and a Type VI Secretion System (T6SS), characterizing GBS19 as a multi-pronged pathogen. Kinetic modeling established that while immobilized enzymes are effective, the 2.5-hour exposure time on T. absoluta requires the synergistic action of chitinases (ChiA/B/C) to reach the lethal desiccation threshold before larvae establish protective mines. Starch-coated silica nanoparticles significantly improve chitinase stability and delivery. However, overcoming the rapid penetration of T. absoluta necessitates a whole-cell or multi-enzyme synergistic approach to outpace larval behavioural defences.

Although micro- and nanoplastics have been detected in human atherosclerotic plaques, their mechanistic contribution to disease pathogenesis remains poorly defined. Most experimental studies have used microplastics (particles > 1 micrometer) in non-atherosclerotic animal models or the ApoE KO; mouse, relying on short-term exposure or single-pathway analyses, whereas the chronic cardiovascular effects of nanoplastics (< 100 nm) remain exceedingly scarce, despite their higher biological reactivity and greater tissue penetrance. To address this gap, this study employs a multi-omics approach to investigate the chronic (12-week) oral exposure to 80 nm polystyrene nanoplastics in LDLr KO; mice. We uniquely integrate aortic plaque quantification, hepatic transcriptomics with global alternative splicing profiling, gut microbiome 16S sequencing, and liver untargeted metabolomics to construct a unified host-microbiome-metabolite network. Nanoplastics exposure significantly exacerbates aortic lipid deposition, suppresses hepatic detoxification and anti-atherogenic lipid pathways primarily through transcriptional and post-transcriptional level changes driven by alternative splicing events (e.g., intron retention and isoform switching), and induces gut dysbiosis marked by a reduction in SCFA-producing commensals and enrichment of pro-atherogenic pathobionts-perturbations that correlate with specific hepatic functional modules. Metabolomic changes, including decreased levels of the glutathione precursor gamma-glutamylcysteine and the choline-derived metabolite neurine, implicate oxidative stress and TMAO-related pathways. Cross-species validation using human atherosclerotic transcriptomic and metagenomic datasets supports the clinical translatability. By integrating multi-level biological responses, this work establishes nanoplastics as an environmental cardiovascular risk factor and uncovers novel regulatory mechanisms involving splicing-associated transcriptional reprogramming and gut-liver crosstalk, offering potential early-warning biomarkers and therapeutic targets for nanoplastics-associated cardiovascular disease.

Bisphenol S (BPS), a widely used substitute for bisphenol A, is increasingly detected in aquatic environments; however, its neurodevelopmental effects remain insufficiently understood. This study investigated whether developmental exposure to an environmentally relevant concentration of BPS disrupts social behaviour and underlying neurobiological pathways in zebrafish (Danio rerio). At 21 days post-fertilization, BPS-exposed larvae exhibited a significant reduction in social preference, indicating impaired conspecific interactions. Neurochemical analysis revealed a marked increase in serotonin (5-HT) levels, whereas lipid peroxidation (MDA) remained unchanged, suggesting the absence of overt oxidative damage. Gene expression profiling demonstrated a dysregulated antioxidant response, suppression of apoptotic signaling, and pronounced upregulation of serotonergic receptors and transporters. To resolve system-level mechanisms, protein-protein interaction (PPI) network analysis identified BDNF and CREB1 as dominant regulatory hubs, with the serotonergic synapse pathway as the most significantly enriched term. Molecular docking further demonstrated direct binding of BPS to multiple serotonergic targets, including HTR1A and TPH2, supporting receptor-level interference. Expanded network and pathway analyses revealed coordinated enrichment of monoamine GPCR, oxidative stress, and inflammatory pathways. These findings demonstrate that BPS induces serotonergic dysregulation and network-level reprogramming rather than significant oxidative damage, leading to behavioural impairment. This study provides a multi-scale mechanistic framework linking molecular perturbations to neurobehavioural outcomes, identifying serotonergic signaling and BDNF-CREB1 pathways as central targets of BPS neurotoxicity.

Biocontainment systems designed to attenuate the spread of mobile DNA are challenging to evaluate within microbiomes of engineered environments. To better understand how toxin-based biocontainment systems affect horizontal gene transfer (HGT) in a microbiome, we evaluated the host range of pairs of plasmids using orthogonal catalytic RNA (cat-RNA) that amend distinct barcodes to 16S rRNA following HGT. We show that mobilizable (5 kb) and self-mobilizable (60 kb) plasmids, which use the same RP4 transfer machinery but different origins of replication, overlap in their host range when conjugated in parallel into a wastewater community, with 127 of the 143 amplicon sequence variants (ASVs) presenting barcoding signals from both plasmids (89%). We also find that mobilizable plasmids with or without the Escherichia coli CcdB toxin overlap in host range in a wastewater community. Among the two most abundant orders, CcdB attenuated the barcoding signal in Aeromonadales more consistently than Enterobacteriales, which have F plasmids containing the CcdB-CcdA toxin-antitoxin system used for biocontainment. Also, CcdB decreased the abundance of the mobilizable plasmid by >100-fold and yielded mutations in 85% of the reads. Together, these findings reveal how pairs of plasmids expressing orthogonal cat-RNA can be used to monitor the effects of plasmid-encoded traits on mobile DNA persistence following HGT. They also highlight challenges when using biocontainment systems containing genes related to those found in the microbiomes targeted for engineering.

Modern agriculture is characterized by substantial fertilizer nitrogen (N) losses from soils, resulting in low crop N-use efficiency. Biological nitrification inhibitors (BNIs) are studied as a strategy to improve N retention in soils by suppressing nitrification. However, the impacts of applying exogenous BNIs to crops with unknown intrinsic BNI capacity remain poorly understood. In this study, we evaluated the impacts of adding three BNIs (methyl 3-(4-hydroxyphenyl) acrylate [MHPA], 6-methoxy-2(3H)-benzoxazolone [MBOA], and limonene), their mixture, and the synthetic nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on barley (Hordeum vulgare L.) growth, plant and soil N dynamics, and soil microbial communities. Using a rhizobox system with planted and bare-soil compartments, combined with 15N isotope tracing and molecular microbial community analyses, we assessed the spatio-temporal dynamics of N transformations, losses, plant N uptake, and microbial community responses in an alkaline agricultural soil. Independent of inhibitor application, the applied fertilizer N was lost primarily through NO- leaching (3-9% of the applied N). In contrast, N2O emissions represented only 0.001-0.028% of the applied N and varied with inhibitor type. MHPA increased dissolved inorganic N soil pools without affecting plant biomass or 15N uptake or strongly shifting microbial community composition. MBOA reduced NO3- concentrations in soil pore water without influencing plant growth or N uptake but shifted soil microbial community composition. In contrast, limonene reduced plant growth and 15N uptake and most significantly altered microbial community composition, without significantly changing N availability. Applying a BNI mixture, as well as limonene alone, was detrimental to plant growth and 15N uptake. DMPP showed only minor effects on N pools, plant growth, plant N uptake and microbial community composition. Overall, our results reveal both the potential and limitations of exogenous BNI application for improving N retention in crop systems.