Derivation of a Point of Departure using NAMs for application in Quantitative Risk Assessment of fragrance materials.

Replication stress and resulting genome instability, a major driver of cancer progression, stem from perturbations of replication fork progression. The first defense against this stress is activation of "dormant" replication origins, which supports replication completion. To determine whether ATR, in itself, contributes to this compensation process, we submitted human cells to a range of low stresses sufficient to activate ATR, not CHK1. Using molecular combing, we developed a dose-response assay that quantifies compensation efficiencies, enabling accurate comparison of cells with different genotypes. Combined with Repli-seq and OK-seq, this assay revealed that ATR activation is key to compensation triggering. We next asked how TopBP1, the main ATR activator, impacts compensation. In stark contradiction to what would be expected from its checkpoint function, we found that TopBP1 represses compensation and acts downstream of ATR. Instead, the function of TopBP1 in replisome assembly, which remains unclear in mammalian cells where the protein is not essential, well-accounts for our results positing that TopBP1 locks dormant origins at the pre-initiation stage, an intermediate in the assembly process, and that ATR activation allows assembly to resume. TopBP1 engagement in the pre-initiation complex would thus serve as a switch linking replisome assembly to the stress response.

Predator-derived chemical cues suppress pest reproduction through non-consumptive pathways and may be exploitable as semiochemicals in integrated pest management. We evaluated the volatile organic compounds (VOCs) emitted by the predatory mite Pyemotes zhonghuajia, for their inhibitory effects on reproduction in the potato tuber moth (Phthorimaea operculella, PTM). Screening of 34 mite-associated VOCs identified dimethyl trisulfide (DMTS) as the strongest oviposition suppressor. In dose-response assays, DMTS reduced egg-laying (>80% at 0.9 mg mL-1), shortened the oviposition period (from 5.09 to 1.86 days at 0.9 mg mL-1) and decreased female longevity (from 13.48 to 4.80 days), but did not affect the pre-oviposition period. DMTS exposure also lowered mating frequency without affecting copulation duration, thereby compounding fecundity reduction. Offspring from exposed parents hatched slightly earlier (4.00 versus 4.32 days), showed prolonged larval development (14.44 versus 13.61 days), and exhibited higher food consumption (1.66 versus 0.83 g). Ovarian dissections revealed fewer mature oocytes, consistent with inhibited oocyte maturation. DMTS disrupts PTM reproduction by suppressing oviposition and mating, and inhibiting ovarian maturation, with additional, modest carry-over effects on offspring development. These findings support DMTS as a promising predator-derived semiochemical for sustainable PTM management. © 2026 Society of Chemical Industry.

Abstract Background Assembly and activation of the NLRP3 inflammasome is a critical mechanism in renal ischaemia reperfusion injury (IRI). NLRP3 activation leads to caspase-1 dependent release of the pro-inflammatory cytokines IL-1b and IL-18 and pyroptotic cell death. This study investigates the effects of the selective NLRP3 inhibitor MCC950 on renal IRI using normothermic machine perfusion (NMP) as a platform for targeted drug delivery. Methods Dose-response and cytokine release assays were performed using in vitro organ culture. Transplant-declined human kidneys (n = 4 control and n = 4 treatment) were allocated to 4h of NMP using a red cell-based perfusate with or without 100 mM of MCC950. Renal blood flow (RBF) and urine output (UO) were monitored continuously. Renal biopsies taken at the start and end of NMP were studied in an organ culture model of IRI. Results During NMP, MCC950 inhibition of NLRP3 increased RBF and UO (P < 0.050). Perfusate levels of IL-1b and IL-18 and other pro-inflammatory cytokines were decreased in MCC950 treated kidneys (P < 0.050). Organ culture of NMP kidney biopsies submitted to IRI demonstrated that NLRP3 inhibition with MCC950 reduced IL-1b to nearly undetectable levels (P = 0.0002) and significantly reduced IL-18 (P = 0.0373) release into the culture media. Conclusions NLRP3 inflammasome inhibition during NMP reduces renal IRI. The direct delivery of therapies to kidneys during NMP eliminates problem of targeting to the organ of interest and reduces the risk of off-target adverse effects in the recipient. This strategy has potential to better prepare kidneys prior to transplantation and should be evaluated in a clinical trial.

Non-small cell lung cancer (NSCLC) is the leading cause of lung cancer deaths, with resistance to targeted therapies posing a major clinical challenge. Drug-tolerant persister (DTP) cells are key contributors to resistance, and targeting them offers new strategies to enhance existing treatments. MicroRNAs (miRNAs), particularly the tumour-suppressive miR-15/107 family, offer promise due to their ability to target multiple oncogenic pathways. This study evaluated a synthetic consensus miRNA mimic, conmiR-15/107, in NSCLC cell line models. Dose-response assays showed robust, dose-dependent growth inhibition in both EGFR-mutant (PC9) and KRAS-mutant (H358 and A549) lung adenocarcinoma cells, but not in the human bronchial epithelial cell line BEAS-2B. When combined with EGFR inhibitors (osimertinib and gefitinib) in PC9 cells, the mimics showed a higher rate of growth inhibition compared with the controls and reduced IC50 values. Similarly, conmiR-15/107 enhanced growth inhibition by the KRAS inhibitors sotorasib and adagrasib in H358 cells. RT-qPCR confirmed downregulation of conmiR-15/107 targets, including MEK1, BCL2 and BRCA1, suggesting a multi-target mechanism of action. Long-term assays showed that the mimics reduced the survival and delayed the proliferation of DTPs in osimertinib-treated PC9 cells as well as sotorasib-treated H358 cells. These findings support conmiR-15/107 as a potential adjunct to targeted therapy, capable of enhancing treatment efficacy and delaying resistance in lung adenocarcinoma.

Phosphatidic acid (PA) is an essential intermediate generated during phospholipase C (PLC) signaling, but its regulation is complex. PA can be generated by ten different diacylglycerol kinase paralogs (DGKs) and two different phospholipase D paralogs (PLDs) in mammals. Because these enzymes are activated under diverse conditions and at various membranes, understanding paralog-specific contributions to PA production is critical for therapeutic development of drugs that modulate the PLC pathway. To address this, we aimed to characterize the paralog specificity of the DGK inhibitors R59022 and BMS-502 against individual DGK paralogs in cellulo. We found that R59022 and BMS-502 both recruited endogenous DGKalpha to the plasma membrane, and inhibited the catalytic fragment of DGKalpha when ectopically localized to the mitochondrial outer membrane. However, at its effective dose, R59022 paradoxically increased PA levels and was cytotoxic, while BMS-502 functioned as a potent and nontoxic inhibitor. Live-cell imaging experiments using BMS-502 with carbachol stimulation of endogenous muscarinic receptors showed that inhibition of both DGKalpha and the PLDs is needed to substantially reduce PA levels during PLC activation. Our findings both identify paralog-specific druggable targets for modulating PLC signaling events, and establish a new platform that translates typical biochemical dose response assays in cellulo.

CYP-mediated metabolic divergence underpins oxoglaucine selectivity: Detoxification in healthy hepatocytes versus mitochondrial apoptosis in hepatocellular carcinoma.

Padina australis is a brown seaweed that shows promise as a natural source of α-glucosidase inhibitors for diabetes management. This study evaluated the α-glucosidase inhibitory activity of P. australis extracts and examined how extraction solvents influence this activity. It also identified potential active metabolites using gas chromatography–mass spectrometry (GC–MS), molecular docking, and toxicity prediction, thereby providing new evidence on the α-glucosidase inhibitory potential of P. australis. Ultrasonic-assisted extraction with n-hexane, ethyl acetate, and ethanol revealed that the ethyl acetate extract exhibited the highest inhibitory activity at 500 µg∙mL−1 (94.55 ± 0.16%), comparable to the ethanol extract (93.75 ± 2.56%) and higher than the n-hexane extract (45.37 ± 4.59%). GC–MS analysis identified 23 compounds, including loliolide, 2(4H)-benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl, phytol, neophytadiene, and several fatty acids. Docking against yeast α-glucosidase (3A4A), human maltase–glucoamylase (3L4T), and lysosomal α-glucosidase (5NN6) indicated that loliolide and benzofuranone derivatives showed affinities approaching acarbose on the yeast enzyme, while neophytadiene and polyunsaturated fatty acids exhibited the most favorable interactions with the human intestinal enzyme. Binding to lysosomal α-glucosidase (5NN6) was consistently weaker, suggesting a degree of selectivity away from lysosomal targets. ProTox-3.0 predicted low acute toxicity for most metabolites (Classes V-VI) and moderate toxicity for two compounds (Class IV), whereas loliolide was classified as higher-risk (Class II). These findings support Padina australis as a potential source of α-glucosidase-modulating metabolites at a screening level. However, comprehensive studies including dose-response assays, enzyme kinetic characterization, fractionation, and toxicity testing are required to validate and extend these preliminary observations. HIGHLIGHTS Padina australis ethyl acetate extract showed in vitro α-glucosidase inhibition of > 90% at 500 µg∙mL−1. Molecular docking indicated strong binding of key metabolites to yeast (3A4A) and human intestinal (3L4T) α-glucosidase, with consistently weaker binding to the lysosomal isoform (5NN6), suggesting selectivity. GC–MS identified 23 compounds; key metabolites formed favorable interactions with catalytic site residues in docking simulations. In silico toxicity prediction indicated that most compounds were in low-toxicity classes (loliolide was a notable exception, flagged as higher toxicity). These findings support australis as a promising marine source of α-glucosidase-modulating metabolites, though further dose–response and safety studies are warranted. GRAPHICAL ABSTRACT

Crabgrasses (Digitaria spp.) rank among the most invasive upland weeds globally, exhibiting remarkable ecological plasticity. To uncover the genomic basis underlying their weediness and environmental adaptability, we generate a telomere-to-telomere (T2T) reference genome of Digitaria sanguinalis, along with its diploid progenitor D. radicosa and tetraploid progenitor D. milanjiana. In addition, we re-sequence 579 accessions collected from fields of 24 grain-producing provinces in China. A phenotypic classification system for Digitaria species is developed by integrating genome-scale data with detailed morphological observations. Phylogenomic and comparative genomic analyses resolve the evolutionary history of Digitaria and reveal lineage-specific expansions in herbicide-associated gene families. Extensive sympatric introgression is observed within Digitaria, correlated with environmental selection. Through dose-response assay of 196 accessions, we identify 19 candidate genes associated with herbicide resistance, including DsSOH1. Haplotype analysis indicates that the resistant allele at DsSOH1 locus originated from D. ciliaris, suggesting a possible evolutionary contribution of introgression to the distribution of resistance-associated alleles. Together, our findings provide genomic insights into the evolutionary success of D. sanguinalis and offer resources for studying polyploid evolution and environmental adaptation as well as precision weed management in agroecosystems.

One of the major challenges in potato farming across the globe is potato cyst nematodes (PCN). Aeroponic root leachate (ARL),collected from aeroponically grown potato plants, was evaluated for its potential to stimulate Globodera rostochiensis hatching in the absence of a host plant. In vitro assays showed that ARL collected from 30-day-old potato plants induced the highest number of juveniles (J2s) hatching (369 J2s; 48%), far exceeding that induced by root exudate (RE) (105 J2s; 12.6%). Among the tested dilutions, ARL diluted to 50% was most effective (940 J2s; 74.5%), while controls showed no hatching. Pot assays revealed that ARL diluted to 50% and 75% reduced viable eggs by 28.9% and 27.8%, respectively, compared with minimal reductions in controls (tap water; 4.7% and nutrient solution; 6.2%). Field assays (2018-2021) confirmed strong declines in cyst counts across all treatments, with the greatest reduction observed in T3 (ARL diluted to 50%). Initial viable egg populations (235-287 per cyst) declined markedly by 2021, with T3 (ARL diluted to 50%) and T2 (ARL diluted to 75%) showing 46.5% and 44.1% reductions, compared with controls (13.1% in tap water and 11.2% in nutrient solution). In dose-response assays, ARL triggered higher hatching (284 J2s) than α-chaconine (228 J2s at 100µg/ml) and α-solanine (186 J2s at 1µg/ml). Further, ARL-assisted potato farming (ARL-APF) showed lower cultivation costs (643.4 USD/ha), energy inputs (34.5 GJ/ha), carbon inputs (1023.2kg CE/ha), and GHG emissions (3745.9 CO2-e kg/ha) over the conventional potato farming (CPF).

Precise control and measurement of the cellular microenvironment, particularly oxygen concentration, are crucial for developing physiologically relevant in vitro models. However, current methods often lack the spatial resolution and throughput needed to investigate complex, oxygen-dependent biological mechanisms in 3D cell cultures. Here, we present an advanced platform based on microcavity arrays featuring integrated, ratiometric oxygen sensors, so-called SensoSpheres. A unique bevel design at the cavity entrance enables the non-invasive, real-time measurement of pericellular oxygen concentration and oxygen gradients. We established protocols for generating spheroids from various cell lines (e.g., HepG2, HeLa) and characterized their metabolic responses under precisely controlled hypoxic, normoxic, and hyperoxic conditions. Using a dose–response assay, we demonstrate the platform’s sensitivity in capturing distinct metabolic shifts in response to acetaminophen and cisplatin. Furthermore, we introduce the Oxygen Consumption Recovery Rate (OCRR) as a novel parameter to quantify cellular resilience after exposure to toxic compounds such as cisplatin and acetaminophen. This high-throughput-compatible platform represents a significant methodological advancement, enabling detailed studies of oxygen-dependent cellular processes, drug toxicity, and metabolic adaptation. Its potential for integration into microfluidic systems paves the way for more sophisticated organ-on-chip models, ultimately improving the predictive power of preclinical research.

Establishment of novel stable human sinonasal NUT carcinoma cell lines.

Unraveling the Genetic and Molecular Changes Associated with Clopyralid Resistance in Common Ragweed ( <i>Ambrosia artemisiifolia</i> )

Abstract Host competence—the ability to acquire, harbour and transmit infections—drives pathogen spread and persistence in multi‐host communities. Evaluating species‐specific competence is critical for predicting transmission, particularly for generalist fungal pathogens like Batrachochytrium dendrobatidis (Bd). Despite its central role in disease dynamics, we lack an epidemiologically grounded competence metric that rigorously accounts for how infection intensity affects a host's competence. This knowledge gap limits our ability to compare mechanisms across species and assess their roles in pathogen persistence. To address these challenges, we developed a novel, load‐dependent competence metric using host–pathogen Integral Projection Models (IPMs) that integrates variation in susceptibility, within‐host pathogen growth and pathogen shedding dynamics. We applied this metric to laboratory‐based challenge experiments with three common North American amphibians ( Notophthalmus viridescens , Rana clamitans and Rana catesbeianus ) that persist endemically with Bd. Using dose–response assays and repeated pathogen shedding measurements across species, we asked: (i) is there a consistent, non‐linear relationship between infection intensity and pathogen shedding across species? and (ii) which load‐based traits best predict host competence? We quantified four of five components of host competence—susceptibility, pathogen growth, pathogen survival and load‐dependent shedding—and used these to parameterize species‐specific IPMs, integrating competence into a single relative metric across species. We found that Bd shedding increased non‐linearly with infection intensity, contradicting the standard assumption that Bd shedding is linearly related to infection intensity. Notophthalmus viridescens and R. catesbeianus were the most competent hosts but through distinct pathways: high susceptibility in N. viridescens and elevated shedding rates in R. catesbeianus . In contrast, density‐dependent reductions in pathogen growth and shedding limited R. clamitans competence. Thus, species‐level competence is not determined by a single trait, but emerges from interactions among multiple load‐based processes. Our results demonstrate that variation in competence emerges from distinct, species‐specific processes across multiple dimensions of competence. By linking individual infection dynamics to population‐level transmission potential, our integrative framework provides a more mechanistic approach to predicting host contributions to community‐level pathogen persistence. Read the free Plain Language Summary for this article on the Journal blog.

Susceptibility of Anopheles stephensi SDA500 strain to common insecticides and efficacy of glazed tile bioassay for resistance characterization

The SARS-CoV-2 E protein through its C-terminal PDZ-binding motif (PBM) interacts with several host PDZ-containing proteins, including Zonula occludens-1 (ZO-1) protein via its PDZ2 domain, thereby contributing to viral pathogenesis. Targeting this interaction represents a potential therapeutic strategy. In this study, we determined the X-ray structure of the E PBM peptide in complex with the ZO-1 PDZ2 domain at 1.7 Å resolution. The structure revealed a domain-swapped dimer conformation of ZO-1 PDZ2, with the E PBM peptide conventionally bound within the PDZ domain’s canonical binding groove, exhibiting key interactions characteristic of type II PBM/PDZ interactions. To identify potential inhibitors of the E PBM/ZO-1 PDZ2 interaction, we performed a Homogeneous Time-Resolved Fluorescence (HTRF) screening using a protein-protein interaction-focused library of 1000 compounds. This led to the identification of 36 hits that disrupted this interaction. Subsequent cytotoxicity and dose-response assays narrowed the selection to 14 promising compounds. Docking simulations showed that some compounds bind within or near the PBM-binding pocket, supporting a competitive mechanism of interaction inhibition, while others bind at a central interface between the two PDZ monomers, suggesting an inhibition of dimerization, which in turn prevents PBM binding. Thus, the E PBM/ZO-1 PDZ2 interaction can be inhibited through both direct and indirect mechanisms. Finally, antiviral assays using a NanoLuciferase-expressing recombinant SARS-CoV-2 demonstrated that one compound, C19, significantly reduced viral replication, highlighting its potential as a candidate for further therapeutic development.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-31755-y.

Background/Objectives: Chagas disease, caused by Trypanosoma cruzi, remains a major neglected tropical disease with limited therapeutic options restricted to benznidazole and nifurtimox, both associated with significant toxicity and reduced efficacy during chronic infection. Seeking novel, safe, and sustainable chemotherapeutic candidates, two new saturated cardanol-derived phospholipid analogs—LDT10 and LDT119—were rationally designed based on the molecular scaffold of miltefosine and biosourced from cashew nut shell liquid (CNSL). This study aimed to evaluate the pharmacokinetic properties of these compounds in silico and assess their antiparasitic activity, cytotoxicity, and morphological and ultrastructural effects on all developmental forms of T. cruzi in vitro. Materials and Methods: In silico ADMET predictions (SwissADME, pkCSM) were performed to determine bioavailability, pharmacokinetic behavior, CYP inhibition, mutagenicity, and hepatotoxicity. Antiproliferative activity was evaluated in epimastigotes, trypomastigotes, and intracellular amastigotes using dose–response assays and flow cytometry. Cytotoxicity was assessed in HEPG2 and HFF-1 cells using resazurin-based viability assays. Morphological and ultrastructural alterations were investigated through scanning (SEM) and transmission (TEM) electron microscopy. Reactive oxygen species (ROS) generation was quantified with H2DCFDA after 4 h and 24 h of exposure. Results: In silico analyses indicated favorable drug-like profiles, high intestinal absorption (>89%), absence of mutagenicity or hepatotoxicity, and non-penetration of the blood–brain barrier. LDT10 was not a P-gp substrate, and LDT119 acted as a P-gp inhibitor, suggesting reduced efflux and higher intracellular retention. Both compounds inhibited epimastigote proliferation with low IC50 values (LDT10: 0.81 µM; LDT119: 1.2 µM at 48 h) and reduced trypomastigote viability (LD50 LDT10: 2.1 ± 2 µM; LDT119: 1.8 ± 0.8 µM). Intracellular amastigotes were highly susceptible (IC50 LDT10: 0.48 µM; LDT119: 0.3 µM at 72 h), with >90% inhibition at higher concentrations. No cytotoxicity was observed in mammalian cells up to 20 µM. SEM revealed membrane wrinkling, pore-like depressions, rounded cell bodies, and multiple flagella, indicating cell division defects. TEM showed Golgi disorganization, autophagic vacuoles, mitochondrial vesiculation, and abnormal kinetoplast replication, while host cells remained structurally preserved. Both compounds induced significant ROS production in trypomastigotes after 24 h in a dose-dependent manner. Conclusions: LDT10 and LDT119 exhibited potent and selective in vitro activity against all developmental stages of T. cruzi, with low micromolar to submicromolar IC50/LD50 values, minimal mammalian cytotoxicity, and extensive morphological and ultrastructural damage consistent with disruption of phospholipid biosynthesis pathways. Combined with favorable in silico pharmacokinetic predictions, these CNSL-derived phospholipid analogs represent promising candidates for future Chagas disease chemotherapy and warrant further in vivo evaluation.

Traditional plastic and glass culture lacks physiological relevance, undermining predictive power in drug discovery. Organoids and organs-on-chip improve biomimicry but do not scale to high-throughput screening (HTS). Even simple hydrogel coatings in HTS plates suffer from curved menisci that disrupt seeding and imaging. We present HYDRA (HYDrogels by Robotic liquid-handling Automation), an automated method to fabricate thin, planar hydrogel films directly in standard plates. Liquid-handlers dispense sub-contact volumes without wall wetting; immediate re-aspiration pins the contact line, leaving a uniform layer with controlled stiffness and thickness. Using fish gelatin hydrogel, HYDRA produces meniscus-free coatings compatible with routine 96- and 384-well workflows and plate-scale quality control. HYDRA was validated through imaging-based dose-response assays with anticancer compounds, engineered epithelial monolayers, and long-term holographic and fluorescence microscopy. It preserved pharmacological sensitivity while supporting high-content imaging on soft, biomimetic substrates, offering a practical bridge between physiological relevance and HTS scalability for early in-vitro drug testing.

Abstract Herbicide resistance poses a significant challenge due to the increasing number of weeds resistant to multiple sites of action (SOAs). Recently, smooth pigweed populations resistant to glyphosate have been confirmed in the KwaZulu-Natal Province in the Republic of South Africa (RSA). This study evaluated herbicide products with different SOAs to provide alternative options for controlling glyphosate-resistant (GR) smooth pigweed populations. Dose-response assays for preemergence and postemergence herbicides were conducted under glasshouse conditions at the University of Pretoria, RSA. Seeds of GR smooth pigweed populations from Bergville and Winterton, and a glyphosate-susceptible (GS) population from Hendrina, were used. For the evaluation of preemergence herbicides (mesotrione, atrazine, imazethapyr, and acetochlor), seeds were sown in pots and herbicides were applied 12 hours after sowing. Postemergence herbicides (mesotrione, atrazine, tembotrione and atrazine tank mixture, and chlorimuron-ethyl) were tested on potted plants at the 6-leaf stage. Herbicides were applied at 0×, 0.5×, 1×, 2×, and 4×, where × is the recommended field rate for the herbicide products representing each SOA. Preemergence herbicides provided greater than 90% control across all populations. For postemergence herbicides, mesotrione effectively controlled all the GR populations, whereas the GS population from Hendrina exhibited reduced sensitivity (&gt;50% survival). Atrazine was effective at rates higher than the recommended field rate in the GR populations. The tank mixture of tembotrione and atrazine had an additive effect compared to the sole application of mesotrione and atrazine. Chlorimuron-ethyl was only effective on the GS population. These results suggest that incorporating effective preemergence and postemergence herbicides into weed management programs could improve control of GR populations of smooth pigweed.

Clostridioides difficile, a leading cause of hospital-acquired diarrhea, exerts its virulence through two co-regulated toxins, TcdA and TcdB. Despite their pivotal roles, the discovery of inhibitors targeting their biosynthesis is underexplored. Here, we present a high-throughput screening (HTS) platform designed to identify toxin synthesis inhibitors (TSIs) that minimally impact bacterial growth. The primary screen utilized a C. difficile reporter strain expressing secreted Nano-luciferase (secNluc) under the tcdA promoter, whereby inhibition of secNluc production indicates toxin biosynthesis inhibition. Screening the Prestwick Chemical Library at 10 and 100 µM identified several compounds that reduced secNluc activity. Through counter-screening, we eliminated compounds that caused spectral interference. Orthogonal dose-response assays assessing the effectiveness of inhibiting toxin production without affecting growth identified meclizine, an antihistamine, as the primary antivirulence candidate. Meclizine was confirmed as a TSI by showing that it reduced TcdA and TcdB protein levels, the cytopathic potential of cultures, and tcdA and tcdB transcription as determined by ELISA, cell-rounding assays, and RT-qPCR, respectively. Meclizine significantly altered central carbon metabolism in C. difficile, upregulating carbohydrate transport systems and the conversion of lactate to pyruvate, while downregulating glycolytic genes. These changes were associated with intracellular accumulation of glucose and pyruvate, metabolites known to negatively impact toxin production. Taken together, our findings underscore the utility of the above HTS platform to identify anti-C. difficile TSIs, which can serve as molecular and cellular probes, as well as chemical starting points for developing novel therapeutics for C. difficile infection.