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Plants express an array of receptor-like kinases (RLKs) to control development and communicate with their environment. Many RLKs are uncharacterized and some of them are expected to regulate plant responses to plant growth-promoting rhizobacteria (PGPR). Despite documented effects induced by Caulobacter RHG1, the underlying signaling pathways and the involved RLKs remain uncharted. Through a targeted RLK mutant screening, we aimed to decipher the receptors that steer the Caulobacter RHG1-induced growth promotion in Arabidopsis thaliana. We identified four RLKs that are pivotal in the RHG1-Arabidopsis interaction, including the coreceptors SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1) and BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1/SERK3), which act redundantly in the RHG1-Arabidopsis interaction, possibly by interplaying with the unknown RLK AT3G28040 and the immunity-related ELONGATION FACTOR-TU RECEPTOR (EFR). These results shed new light on the molecular dynamics orchestrating plant responses to PGPR, and concurrently contribute a crucial piece to the intricate puzzle of RLK interactions. Key policy highlights Four RLKs; BAK1, SERK1, EFR, and AT3G28040 (RRHG) are involved in the RHG1-Arabidopsis interaction. BAK1 and SERK1, two well-described co-receptors, act redundantly and play a pivotal role in the RHG1-driven growth promotion, possibly by interplaying with the unknown RLK AT3G28040 and the immunity-related RLK EFR. Most known development – and immunity-related RLKs barely influence RHG1-driven plant growth promotion in Arabidopsis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Utilizing fungicides to protect crops from diseases is an effective method, and novel eco‐friendly plant‐derived fungicides with high efficiency and low toxicity are urgent requirements for sustainable crop protection. RESULT: Two series of rosin‐based fungicides (totally 35) were designed and synthesized. In vitro fungicidal activity revealed that Compound 6a (Co. 6a) effectively inhibited the growth of Valsa mali [median effective concentration (EC50) = 0.627 μg mL−1], and in vivo fungicidal activity suggested a significant protective efficacy of Co. 6a in protecting both apple branches (35.12% to 75.20%) and apples (75.86% to 90.82%). Quantum chemical calculations (via density functional theory) results indicated that the primary active site of Co. 6a lies in its amide structure. Mycelial morphology and physiology were investigated to elucidate the mode‐of‐action of Co. 6a, and suggested that Co. 6a produced significant cell membrane damage, accelerated electrolyte leakage, decreased succinate dehydrogenase (SDH) protein activity, and impaired physiological and biochemical functions, culminating in mycelial mortality. Molecular docking analysis revealed a robust binding energy (ΔE = −7.29 kcal mol−1) between Co. 6a and SDH. Subsequently, biosafety evaluations confirmed the environmentally‐friendly nature of Co. 6a via the zebrafish model, yet toxicological results indicated that Co. 6a at median lethal concentration [LC50(96)] damaged the gills, liver and intestines of zebrafish. CONCLUSION: The above research offers a theoretical foundation for exploiting eco‐friendly rosin‐based fungicidal candidates in sustainable crop protection. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

According to the World Health Organisation and European Commission definitions, substances shall be considered as having endocrine disrupting properties if they show adverse effects, have endocrine activity and the adverse effects are a consequence of the endocrine activity (using a weight-of-evidence approach based on biological plausibility), unless the adverse effects are not relevant to humans or non-target organisms at the (sub)population level. To date, there is no decision logic on how to establish endocrine disruption via the thyroid modality in non-mammalian vertebrates. This paper describes an evidence-based decision logic compliant with the integrated approach to testing and assessment (IATA) concept, to identify thyroid-mediated effect patterns in aquatic vertebrates using amphibians as relevant models for thyroid disruption assessment. The decision logic includes existing test guidelines and methods and proposes detailed considerations on how to select relevant assays and interpret the findings. If the mammalian dataset used as the starting point indicates no thyroid concern, the Xenopus Eleutheroembryonic Thyroid Assay allows checking out thyroid-mediated activity in non-mammalian vertebrates, whereas the Amphibian Metamorphosis Assay or its extended, fixed termination stage variant inform on both thyroid-mediated activity and potentially population-relevant adversity. In evaluating findings, the response patterns of all assay endpoints are considered, including the direction of changes. Thyroid-mediated effect patterns identified at the individual level in the amphibian tests are followed by mode-of-action and population relevance assessments. Finally, all data are considered in an overarching weight-of-evidence evaluation. The logic has been designed generically and can be adapted, e.g. to accommodate fish tests once available for thyroid disruption assessments. It also ensures that all scientifically relevant information is considered, and that animal testing is minimised. The proposed decision logic can be included in regulatory assessments to facilitate the conclusion on whether substances meet the endocrine disruptor definition for the thyroid modality in non-mammalian vertebrates.

ToxTracker is a mammalian cell reporter assay that predicts the genotoxic properties of compounds with high accuracy. By evaluating induction of various reporter genes that play a key role in relevant cellular pathways, it provides insight into chemical mode‐of‐action (MoA), thereby supporting discrimination of direct‐acting genotoxicants and cytotoxic chemicals. A comprehensive interlaboratory validation trial was conducted, in which the principles outlined in OECD Guidance Document 34 were followed, with the primary objectives of establishing transferability and reproducibility of the assay and confirming the ability of ToxTracker to correctly classify genotoxic and non‐genotoxic compounds. Reproducibility of the assay to predict genotoxic MoA was confirmed across participating laboratories and data were evaluated in terms of concordance with in vivo genotoxicity outcomes. Seven laboratories tested a total of 64 genotoxic and non‐genotoxic chemicals that together cover a broad chemical space. The within‐laboratory reproducibility (WLR) was up to 98% (73%–98% across participants) and the overall between‐laboratory reproducibility (BLR) was 83%. This trial confirmed the accuracy of ToxTracker to predict in vivo genotoxicants with a sensitivity of 84.4% and a specificity of 91.2%. We concluded that ToxTracker is a robust in vitro assay for the accurate prediction of in vivo genotoxicity. Considering ToxTracker's robust standalone accuracy and that it can provide important information on the MoA of chemicals, it is seen as a valuable addition to the regulatory in vitro genotoxicity battery that may even have the potential to replace certain currently used in vitro battery assays. [ABSTRACT FROM AUTHOR]

Despite the availability of currently approved antiviral drugs, infections with human cytomegalovirus (HCMV) still cause clinically challenging, sometimes life-threatening situations. There is an urgent need for enhanced anti-HCMV drugs that offer improved efficacy, reduced dosages and options for long-term treatment without risk of the development of viral drug resistance. Recently, we reported the pronounced anti-HCMV efficacy of pharmacological inhibitors of cyclin-dependent kinases (CDKs), in particular, the potential of utilizing drug synergies upon combination treatment with inhibitors of host CDKs and the viral CDK-like kinase pUL97 (vCDK/pUL97). Here, we expand this finding by further assessing the in vitro synergistic antiviral interaction between vCDK and CDK inhibitors towards HCMV as well as non-human cytomegaloviruses. An extension of this synergy approach was achieved in vivo by using the recombinant MCMV-UL97/mouse model, confirming the high potential of combination treatment with the clinically approved vCDK inhibitor maribavir (MBV) and the developmental CDK7 inhibitor LDC4297. Moreover, mechanistic aspects of this synergistic drug combination were illustrated on the levels of intracellular viral protein transport and viral genome replication. The analysis of viral drug resistance did not reveal resistance formation in the case of MBV + LDC4297 combination treatment. Spanning various investigational levels, these new results strongly support our concept, employing the great potential of anti-HCMV synergistic drug treatment. [ABSTRACT FROM AUTHOR]

A major goal of translational toxicology is to identify adverse chemical effects and determine whether they are conserved or divergent across experimental systems. Translational toxicology encompasses assessment of chemical toxicity across multiple life stages, determination of toxic mode-of-action, computational prediction modeling, and identification of interventions that protect or restore health following toxic chemical exposures. The zebrafish is increasingly used in translational toxicology because it combines the genetic and physiological advantages of mammalian models with the higher-throughput capabilities and genetic manipulability of invertebrate models. Here, we review recent literature demonstrating the power of the zebrafish as a model for addressing all four activities of translational toxicology. Important data gaps and challenges associated with using zebrafish for translational toxicology are also discussed.

ToxTracker is an in vitro mammalian stem cell‐based reporter assay that detects activation of specific cellular signaling pathways (DNA damage, oxidative stress, and/or protein damage) upon chemical exposure using flow cytometry. Here we used quantitative methods to empirically analyze historical control data, and dose–response data across a wide range of reference chemicals. First, we analyzed historical control data to define a fold‐change threshold for identification of a significant positive response. Next, we used the benchmark dose (BMD) combined‐covariate approach for potency ranking of a set of more than 120 compounds; the BMD values were used for comparative identification of the most potent inducers of each reporter. Lastly, we used principal component analysis (PCA) to investigate functional and statistical relationships between the ToxTracker reporters. The PCA results, based on the BMD results for all substances, indicated that the DNA damage (Rtkn, Bscl2) and p53 (Btg2) reporters are functionally complementary and indicative of genotoxic stress. The oxidative stress (Srxn1 and Blvrb) and protein stress (Ddit3) reporters are independent indicators of cellular stress, and essential for toxicological profiling using the ToxTracker assay. Overall, dose–response modeling of multivariate ToxTracker data can be used for potency ranking and mode‐of‐action determination. In the future, IVIVE (in vitro to in vivo extrapolation) methods can be employed to determine in vivo AED (administered equivalent dose) values that can in turn be used for human health risk assessment. [ABSTRACT FROM AUTHOR]

Drimane and coloratane sesquiterpenoids are present in several plants, microorganisms, and marine life. Because of their cytotoxic activity, these sesquiterpenoids have received increasing attention as a source for new anticancer drugs and pharmacophores. Natural drimanes and coloratanes, as well as their semi-synthetic derivatives, showed promising results against cancer cell lines with in vitro activities in the low micro- and nanomolar range. Despite their high potential as novel anticancer agents, the mode of action and structure–activity relationships of drimanes and coloratanes have not been completely enlightened nor systematically reviewed. Our review aims to give an overview of known structures and derivatizations of this class of sesquiterpenoids, as well as their activity against cancer cells and potential modes-of-action. The cytotoxic activities of about 40 natural and 25 semi-synthetic drimanes and coloratanes are discussed. In addition to that, we give a summary about the clinical significance of drimane and coloratane sesquiterpenoids. [ABSTRACT FROM AUTHOR]

Background: Succinate dehydrogenase (SDH) represents a critical target in the development of novel fungicides. To address the growing issue of resistance and safeguard the economic viability of agricultural production, the pursuit of new succinate dehydrogenase inhibitors (SDHIs) has emerged as a significant focus of contemporary research., Results: In this project, 32 arylhydrazine derivatives containing diphenyl ether structural units were synthesized and evaluated for their fungicidal activities against Rhizoctonia solani, Sclerotinia sclerotiorum, Alternaria alternata, Gibberella zeae, Alternaria solani and Colletotrichum gloeosporioides. In an in vitro fungicidal activity assay, compound D6 showed significant inhibitory activity against R. solani with a half-maximum effective concentration (EC 50 ) of 0.09 mg L -1 . The in vivo fungicidal activity demonstrated that compound D6 inhibited R. solani by 95.39% in rice leaves, which was significantly better than that of boscalid (85.76%). The results of SDH enzyme assay, molecular docking simulation, mitochondrial membrane potential assay, cytoplasmic release studies and morphological observations demonstrated that the target compound D6 not only had significant SDH inhibitory activity, but also affected the membrane integrity of mycelium., Conclusion: Bioactivity screening and validation of the mechanism of action indicated that compound D6 was a potentially unique SDHI, acting on SDH while also affecting cell membrane permeability, which deserved further study. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Antibiotics are used to treat bacterial infections in humans and animals and also act as a growth promoter for poultry. Due to incomplete metabolism, these antibiotics are excreted in the environment in their parental forms and accumulates in the aquatic ecosystem. Besides the evolution of antibiotic resistant bacteria, these drugs can damage non-target organisms. Green algae are highly sensitive to different antibiotics. Damage in the algal population will cause imbalances in the ecosystems. Till now, the mechanisms of antibiotic toxicity towards algae have not been completely elucidated. It was observed that antibiotics mainly affected the photosynthetic machinery and decreased the carbon fixation process, finally resulting in algal growth inhibition. This present review deals with antibiotics classification, various routes of antibiotics exposure to the freshwater environment, sensitivity towards the different classes of antibiotics, possible Mode-of-Action (MOA) on algal systems, and gaps that need to be filled. Significant gaps include the unavailability of proper eco-toxicological data for antibiotics. Moreover, they exist in nature as complex mixtures, and their behavior in the ecosystem may vastly differ from the parent molecules. To improve our understanding of antibiotic responses mechanism in real-life scenarios, mixture toxicity studies may be the first step. [ABSTRACT FROM AUTHOR]

BACKGROUND: Fluazaindolizine is a new compound for the control of plant‐parasitic nematodes (PPNs) with an unknown and novel mode‐of‐action. This compound is very effective against important PPNs. However, investigations elucidating the impact of sublethal fluazaindolizine doses on early nematode virulence and plant–nematode interaction parameters are lacking. RESULTS: The effect of direct exposure of Heterodera schachtii juveniles to 50 ppm fluazaindolizine was negligible. Infection assays revealed a 57% reduction in adult females at 1.25 ppm and a 46% reduction in offspring at 40 ppm when juveniles were soaked in the compound for 48 h and subsequently inoculated onto Arabidopsis thaliana. Pre‐incubation of A. thaliana roots with fluazaindolizine was not effective against H. schachtii. Conversely, supplementing the plant growth medium with fluazaindolizine led to a significant reduction of adults (−35%), females (−75%) and female size at 1.25 ppm and nearly completely inhibited nematode parasitism at 5 ppm. The impact of fluazaindolizine on A. thaliana was dependent on plant age, compound concentration and duration of contact. Very low sublethal fluazaindolizine concentrations, 5 or 10 ppm, did not interfere with nematode mobility, host finding, penetration, and induction of the feeding site, but specifically inhibited sedentary nematode development inside the root in a concentration‐dependent manner. CONCLUSION: Fluazaindolizine does not have direct toxicity against PPN infective juveniles, but has a clear effect on nematodes during sedentary development. The formation of females and the development of offspring are strongly reduced. It will be interesting to identify the underlying mechanism in the future. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The widespread presence of tolerance to copper in Xanthomonas species has resulted in the need to develop alternative approaches to control plant diseases caused by xanthomonads. In recent years, nanotechnological approaches have resulted in the identification of novel materials to control plant pathogens. With many metal-based nanomaterials having shown promise for disease control, an important question relates to the mode of action of these new materials. In this study, we used several approaches, such as scanning electron microscopy, propidium monoazide quantitative polymerase chain reaction, epifluorescence microscopy, and RNA sequencing to elucidate the mode of action of a Cu/Zn hybrid nanoparticle against copper-tolerant strains of Xanthomonas euvesicatoria. We demonstrate that Cu/Zn did not activate copper resistance genes (i.e., copA and copB ) in the copper-tolerant bacterium but functioned by disrupting the bacterial cell structure and perturbing important biological processes such as cell respiration and chemical homeostasis., Competing Interests: The author(s) declare no conflict of interest.

BACKGROUND: Emamectin benzoate (EB) has recently been successfully applied as a trunk injection for preventative control of the pine wilt disease (PWD) caused by Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. Here, a whole‐organism transcriptomic analysis provides comprehensive insights into the adverse effects of EB on B. xylophilus. RESULTS: A large set of differentially expressed genes (DEGs) were found, demonstrating the antagonistic effects of EB on B. xylophilus embryonic and larval development, reproduction, nervous and motor systems, and pathogenesis. In toxicity assays with EB, the number of eggs laid, hatching rate, thrashing frequency, and developmental rate of B. xylophilus were significantly suppressed at low concentrations (0.1 μg mL−1). Moreover, the transcriptional changes validated by real‐time quantitative PCR showed downregulated transcript levels of the genes encoding pectate lyases, β‐1,4‐endoglucanases, and upregulated the genes encoding glutamate‐gated chloride channel, γ‐aminobutyric acid type β receptor, uridine 5′‐diphospho‐glucuronosyl transferase, ATP‐binding cassette transporter. The potential responses of B. xylophilus to EB included the upregulation of several genes putatively contributing to oocyte protection, stem cell renewal, and xenobiotic degradation, implying the potential for drug resistance to develop. CONCLUSION: Our findings further our understanding of the effects of EB for managing the PWD and may help to improve the pesticide‐use strategies for controlling B. xylophilus. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Arsenic exposure disturbs brain development in humans. Although developmental neurotoxicity (DNT) of arsenic has been studied in vivo and in vitro , its mode-of-action (MoA) is not completely understood. Here, we characterize the adverse neurodevelopmental effects of sodium arsenite on developing human and rat neural progenitor cells (hNPC, rNPC). Moreover, we analyze the involvement of reactive oxygen species (ROS) and the role of the glutathione (GSH)-dependent antioxidative defense for arsenite-induced DNT in a species-specific manner. We determined IC 50 values for sodium arsenite-dependent (0.1–10 μM) inhibition of hNPC and rNPC migration (6.0 μM; >10 μM), neuronal (2.7 μM; 4.4 μM) and oligodendrocyte (1.1 μM; 2.0 μM) differentiation. ROS involvement was studied by quantifying the expression of ROS-regulated genes , measuring glutathione (GSH) levels, inhibiting GSH synthesis and co-exposing cells to the antioxidant N-acetylcysteine. Arsenite reduces NPC migration, neurogenesis and oligodendrogenesis of differentiating hNPC and rNPC at sub-cytotoxic concentrations. Species-specific arsenite cytotoxicity and induction of antioxidative gene expression is inversely related to GSH levels with rNPC possessing >3-fold the amount of GSH than hNPC. Inhibition of GSH synthesis increased the sensitivity towards arsenite in rNPC > hNPC. N-acetylcysteine antagonized arsenite-mediated induction of HMOX1 expression as well as reduction of neuronal and oligodendrocyte differentiation in hNPC suggesting involvement of oxidative stress in arsenite DNT. hNPC are more sensitive towards arsenite-induced neurodevelopmental toxicity than rNPC, probably due to their lower antioxidative defense capacities. This species-specific MoA data might be useful for adverse outcome pathway generation and future integrated risk assessment strategies concerning DNT. • Arsenite affects oligodendrocyte differentiation in developing hNPC and rNPC. • hNPC have a lower Glutathione dependent antioxidative defense compared to rNPC. • hNPC are more sensitive towards arsenite-induced neurodevelopmental toxicity. [ABSTRACT FROM AUTHOR]

Combination chemotherapy can increase treatment efficacy and suppress drug resistance. Knowledge of how to engineer rational, mechanism-based drug combinations, however, remains lacking. Although studies of drug activity have historically focused on the primary drug-target interaction, growing evidence has emphasized the importance of the subsequent consequences of this interaction. Bedaquiline (BDQ) is the first new drug for tuberculosis (TB) approved in more than 40 y, and a species-selective inhibitor of the Mycobacterium tuberculosis (Mtb) ATP synthase. Curiously, BDQ-mediated killing of Mtb lags significantly behind its inhibition of ATP synthase, indicating a mode of action more complex than the isolated reduction of ATP pools. Here, we report that BDQ-mediated inhibition of Mtb's ATP synthase triggers a complex metabolic response indicative of a specific hierarchy of ATP-dependent reactions. We identify glutamine synthetase (GS) as an enzyme whose activity is most responsive to changes in ATP levels. Chemical supplementation with exogenous glutamine failed to affect BDQ's antimycobacterial activity. However, further inhibition of Mtb's GS synergized with and accelerated the onset of BDQ-mediated killing, identifying Mtb's glutamine synthetase as a collateral, rather than directly antimycobacterial, metabolic vulnerability of BDQ. These findings reveal a previously unappreciated physiologic specificity of ATP and a facet of mode-of-action biology we term collateral vulnerability, knowledge of which has the potential to inform the development of rational, mechanism-based drug combinations. [ABSTRACT FROM AUTHOR]

Repeated dose toxicity evaluation aims at assessing the occurrence of adverse effects following chronic or repeated exposure to chemicals. Non-animal approaches have gained importance in the last decades because of ethical considerations as well as due to scientific reasons calling for more human-based strategies. A critical aspect of this challenge is linked to the capacity to cover a comprehensive set of interdependent mechanisms of action, link them to adverse effects and interpret their probability to be triggered in the light of the exposure at the (sub)cellular level. Inherent to its structured nature, an ontology addressing repeated dose toxicity could be a scientific and transparent way to achieve this goal. Additionally, repeated dose toxicity evaluation through the use of a harmonized ontology should be performed in a reproducible and consistent manner, while mimicking as accurately as possible human physiology and adaptivity. In this paper, the outcome of a series of workshops organized by Cosmetics Europe on this topic is reported. As such, this manuscript shows how experts set critical elements and ways of establishing a mode-of-action ontology model as a support to risk assessors aiming to perform animal-free safety evaluation of chemicals based on repeated dose toxicity data. • A repeated dose toxicity mode-of-action ontology model consisting of 4 pillars is proposed. • Pillar 1 and 2 relate to kinetic and chemical aspects, respectively. • Pillar 3 and 4 relate to mechanistic and toxicological aspects, respectively. • The repeated dose toxicity mode-of-action ontology model can serve safety evaluation purposes. [ABSTRACT FROM AUTHOR]

BACKGROUND Bifenthrin is usually considered a Type I pyrethroid, because it lacks an α‐CN group present in Type II pyrethroids, but some previous studies suggest a mixed Type I/II mode‐of‐action. Results are presented for bifenthrin in a rat developmental neurotoxicity (DNT) study along with effects on Na currents in human VGSC subtypes. Molecular modeling comparisons were also made for bifenthrin and other pyrethroids. RESULTS: In a rat DNT study, bifenthrin produced tremors and clonic convulsions in dams and pups and slightly reduced acoustic startle response amplitude, and increased Tmax, at PND20 in females. Similar blood levels of bifenthrin were measured in dams and pups at each dose level i.e. no concentration in pups. In human VGSC experiments, using the Nav1.8 subtype, bifenthrin's effects on inactivation were slight, as for Type II pyrethroids, but without large prolongation of the tail current (deactivation) seen with Type II. Molecular modeling of bifenthrin indicates that the o‐Me group may occupy a similar space to the α‐CN group of cypermethrin and fenpropathrin. CONCLUSION: In a DNT study and on human Nav1.8 tail currents bifenthrin showed Type I and II effects, similar to some published studies. Overall, bifenthrin acts as a mixed Type I/II pyrethroid. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. Bifenthrin was previously considered a Type I pyrethroid. However, data presented here, including a rat DNT study and one on patch‐clamped human VGSCs, indicate mixed Type I and II properties. [ABSTRACT FROM AUTHOR]

The fast growing evidences have shown that the plant-derived compound honokiol is a promising candidate for treating multiple human diseases, such as inflammation and cancer. However, the mode-of-action (MoA) of honokiol remains largely unclear. Here, we studied the antifungal activity of honokiol in fission yeast model, with the goal of understanding the honokiol’s mechanism of action from the molecular level. We found that honokiol can inhibit the yeast growth at a dose-dependent way. Microarray analysis showed that honokiol has wide impacts on the fission yeast transcription levels (in total, 512 genes are up-regulated, and 42 genes are down-regulated). Gene set enrichment analysis indicated that over 45% up-regulated genes belong to the core environmental stress responses category. Moreover, network analysis suggested that there are extensive gene–gene interactions amongst the co-expression gene lists, which can assemble several biofunctionally important modules. It is noteworthy that several key components of central carbon metabolism, such as glucose transporters and metabolic enzymes of glycolysis, are involved in honokiol’s MoA. The complexity of the honokiol’s MoA displayed in previous studies and this work demonstrates that multiple omics approaches and bioinformatics tools should be applied together to achieve the complete scenario of honokiol’s antifungal function.

RNA viruses have gained plenty of attention during recent outbreaks of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Zika virus (ZIKV), and Ebola virus. ZIKV is a vector borne Flavivirus that is spread by mosquitoes and it mainly infects neuronal progenitor cells. One hallmark of congenital ZIKV disease is a reduced brain size in fetuses, leading to severe neurological defects. The World Health Organization (WHO) is urging the development of new antiviral treatments against ZIKV, as there are no efficient countermeasures against ZIKV disease. Previously, we presented a new class of host-targeting antivirals active against a number of pathogenic RNA viruses, such as SARS-CoV-2. Here, we show the transfer of the image-based phenotypic antiviral assay to ZIKV-infected brain cells, followed by mechanism-of-action studies and a proof-of-concept study in a three-dimensional (3D) organoid model. The novel antiviral compounds showed a therapeutic window against ZIKV in several cell models and rescued ZIKV-induced neurotoxicity in brain organoids. The compound’s mechanism-of-action was pinpointed to late steps in the virus life cycle, impairing the formation of new virus particles. Collectively, in this study, we expand the antiviral activity of new small molecule inhibitors to a new virus class of Flaviviruses, but also uncover compounds’ mechanism of action, which are important for the further development of antivirals.

The kinetoplastids are protozoa characterized by the presence of a distinctive organelle, called the kinetoplast, which contains a large amount of DNA (kinetoplast DNA (kDNA)) inside their single mitochondrion. Kinetoplastids of medical and veterinary importance include Trypanosoma spp. (the causative agents of human and animal African Trypanosomiasis and of Chagas disease) and Leishmania spp. (the causative agents of the various forms of leishmaniasis). These neglected diseases affect millions of people across the globe, but drug treatment is hampered by the challenges of toxicity and drug resistance, among others. Propolis (a natural product made by bees) and compounds isolated from it are now being investigated as novel treatments of kinetoplastid infections. The anti-kinetoplastid efficacy of propolis is probably a consequence of its reported activity against kinetoplastid parasites of bees. This article presents a review of the reported anti-kinetoplastid potential of propolis, highlighting its anti-kinetoplastid activity in vitro and in vivo regardless of geographical origin. The mode of action of propolis depends on the organism it is acting on and includes growth inhibition, immunomodulation, macrophage activation, perturbation of the cell membrane architecture, phospholipid disturbances, and mitochondrial targets. This gives ample scope for further investigations toward the rational development of sustainable anti-kinetoplastid drugs.

Pharmaceuticals and chemicals produce hemangiosarcomas (HS) in mice, often by nongenotoxic, proliferative mechanisms. A mode-of-action (MOA) for hemangiosarcoma was proposed based on information presented at an international workshop (Cohen et al., Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18.). Five key elements of the MOA were articulated and included hypoxia, macrophage activation, increased angiogenic growth factors, dysregulated angiogenesis/erythropoiesis, and endothial cell proliferation. The goal of the current study was to add to the weight-of-evidence for the proposed MOA by assessing these key elements with 3 different compounds of varying potency for HS induction: fenretinide (high), troglitazone (intermediate), and elmiron (low). Multiple endpoints, including hypoxia (hyproxyprobe, transcriptomics), endothelial cell (EC) proliferation, and clinical and anatomic pathology, were assessed after 2, 4, and 13-weeks of treatment in B6C3F1 mice. All 3 compounds demonstrated strong evidence for dysregulated erythropoiesis (decrease in RBC and a failure to increase reticulocytes) and macrophage activation (4- to 11-foId increases); this pattern of hematological changes in mice might serve as an early biomarker to evaluate EC proliferation in suspected target organs for potential HS formation. Fenretinide demonstrated all 5 key elements, while troglitazone demonstrated 4 and elmiron demonstrated 3. Transcriptomics provided support for the 5 elements of the MOA, but was not any more sensitive than hypoxyprobe immunohistochemistry for detecting hypoxia. The overall transcriptional evidence for the key elements of the proposed MOA was also consistent with the potency of HS induction. These data, coupled with the previous work with 2-butoxyethanol and pregablin, increase the weight-of- evidence for the proposed MOA for HS formation. [ABSTRACT FROM AUTHOR]

The bacterial pigment prodigiosin has various biological activities; it is, for instance, an effective antimicrobial. Here, we investigate the primary site targeted by prodigiosin, using the cells of microbial pathogens of humans as model systems: Candida albicans, Escherichia coli, Staphylococcus aureus. Inhibitory concentrations of prodigiosin; leakage of intracellular K+ ions, amino acids, proteins and sugars; impacts on activities of proteases, catalases and oxidases; and changes in surface appearance of pathogen cells were determined. Prodigiosin was highly inhibitory (30% growth rate reduction of C. albicans, E. coli, S. aureus at 0.3, 100 and 0.18 μg ml−1, respectively); caused leakage of intracellular substances (most severe in S. aureus); was highly inhibitory to each enzyme; and caused changes to S. aureus indicative of cell-surface damage. Collectively, these findings suggest that prodigiosin, log Poctanol–water 5.16, is not a toxin but is a hydrophobic stressor able to disrupt the plasma membrane via a chaotropicity-mediated mode-of-action. [ABSTRACT FROM AUTHOR]

Bacteriocin-producing (Bac+) lactic acid bacteria (LAB) comprising selected strains of Lactobacillus curvatus, Lactococcus lactis, Pediococcus acidilactici, and Enterococcus faecium and thailandicus were examined for inhibition of Listeria monocytogenes during hotdog challenge studies. The Bac+ strains, or their cell-free supernatants (CFS), were grouped according to mode-of-action (MOA) as determined from prior studies. Making a mixture of as many MOAs as possible is a practical way to obtain a potent natural antimicrobial mixture to address L. monocytogenes contamination of RTE meat products (i.e., hotdogs). The heat resistance of the bacteriocins allowed the use of pasteurization to eliminate residual producer cells for use as post-process surface application or their inclusion into hotdog meat emulsion during cooking. The use of Bac+ LAB comprising 3× MOAs directly as co-inoculants on hotdogs was not effective at inhibiting L. monocytogenes. However, the use of multiple MOA Bac+ CFS mixtures in a variety of trials demonstrated the effectiveness of this approach by showing a >2-log decrease of L. monocytogenes in treatment samples and 6-7 log difference vs. controls. These data suggest that surface application of multiple mode-of-action bacteriocin mixtures can provide for an Alternative 2, and possibly Alternative 1, process category as specified by USDA-FSIS for control of L. monocytogenes on RTE meat products. [ABSTRACT FROM AUTHOR]

Aim To determine uncovered antifungal activity of lichen-derived compound, vulpinic acid, by using chemical-genetic analyses. Methods and Results Haploinsufficiency and homozygous-profiling assays were performed, revealing that strains lacking GLC7, MET4, RFC2, YAE1 and PRP18 were sensitive to three concentrations (12·5, 25 and 50% of inhibitory concentration) of vulpinic acid and independently validated. To verify inhibition of those genes, cell cycle analysis using flow cytometry was performed and relative expressions were measured. Under vulpinic acid-treated condition, cell cycle was arrested in S and G2/M phases and sensitive strains' relative expressions were significantly lower than the wild type yeast. Conclusions Vulpinic acid mainly affects cell cycle, glycogen metabolism, transcription and translation to fungi. Significance and Impact of the Study Although lichen-derived compounds are commercially valuable, few studies have determined their modes of action. This study used a chemogenomic approach to gain insight into the mechanisms of one of well-known lichen-derived compound, vulpinic acid. [ABSTRACT FROM AUTHOR]

The fast growing evidences have shown that the plant-derived compound honokiol is a promising candidate for treating multiple human diseases, such as inflammation and cancer. However, the mode-of-action (MoA) of honokiol remains largely unclear. Here, we studied the antifungal activity of honokiol in fission yeast model, with the goal of understanding the honokiol’s mechanism of action from the molecular level. We found that honokiol can inhibit the yeast growth at a dose-dependent way. Microarray analysis showed that honokiol has wide impacts on the fission yeast transcription levels (in total, 512 genes are up-regulated, and 42 genes are down-regulated). Gene set enrichment analysis indicated that over 45% up-regulated genes belong to the core environmental stress responses category. Moreover, network analysis suggested that there are extensive gene–gene interactions amongst the co-expression gene lists, which can assemble several biofunctionally important modules. It is noteworthy that several key components of central carbon metabolism, such as glucose transporters and metabolic enzymes of glycolysis, are involved in honokiol’s MoA. The complexity of the honokiol’s MoA displayed in previous studies and this work demonstrates that multiple omics approaches and bioinformatics tools should be applied together to achieve the complete scenario of honokiol’s antifungal function. [ABSTRACT FROM PUBLISHER]

In this article, we discuss our recent work in elucidating the mode-of-action of compounds used in traditional medicine including Ayurvedic medicine. Using computational (′in silico′) approach, we predict potential targets for Ayurvedic anti-cancer compounds, obtained from the Indian Plant Anticancer Database given its chemical structure. In our analysis, we observed that: (i) the targets predicted can be connected to cancer pathogenesis i.e. steroid-5-alpha reductase 1 and 2 and estrogen receptor-β, and (ii) predominantly hormone-dependent cancer targets were predicted for the anti-cancer compounds. Through the use of our in silico target prediction, we conclude that understanding how traditional medicine such as Ayurveda work through linking with the ′western′ understanding of chemistry and protein targets can be a fruitful avenue in addition to bridging the gap between the two different schools of thinking. Given that compounds used in Ayurveda have been tested and used for thousands of years (although not in the same approach as Western medicine), they can potentially be developed into potential new drugs. Hence, to further advance the case of Ayurvedic medicine, we put forward some suggestions namely: (a) employing and integrating novel analytical methods given the advancements of ′omics′ and (b) sharing experimental data and clinical results on studies done on Ayurvedic compounds in an easy and accessible way.

Bacteriocin-producing (Bac+) lactic acid bacteria (LAB) comprising selected strains of Lactobacillus curvatus, Lactococcus lactis, Pediococcus acidilactici, and Enterococcus faecium and thailandicus were examined for inhibition of Listeria monocytogenes during hotdog challenge studies. The Bac+ strains, or their cell-free supernatants (CFS), were grouped according to mode-of-action (MOA) as determined from prior studies. Making a mixture of as many MOAs as possible is a practical way to obtain a potent natural antimicrobial mixture to address L. monocytogenes contamination of RTE meat products (i.e., hotdogs). The heat resistance of the bacteriocins allowed the use of pasteurization to eliminate residual producer cells for use as post-process surface application or their inclusion into hotdog meat emulsion during cooking. The use of Bac+ LAB comprising 3× MOAs directly as co-inoculants on hotdogs was not effective at inhibiting L. monocytogenes. However, the use of multiple MOA Bac+ CFS mixtures in a variety of trials demonstrated the effectiveness of this approach by showing a >2-log decrease of L. monocytogenes in treatment samples and 6–7 log difference vs. controls. These data suggest that surface application of multiple mode-of-action bacteriocin mixtures can provide for an Alternative 2, and possibly Alternative 1, process category as specified by USDA-FSIS for control of L. monocytogenes on RTE meat products.

Combining natural product fragments to design new scaffolds with unprecedented bioactivity is a powerful strategy for the discovery of tool compounds and potential therapeutics. However, the choice of fragments to couple and the biological screens to employ remain open questions in the field. By choosing a primary fragment containing the A/B ring system of estradiol and fusing it to nine different secondary fragments, we were able to identify compounds that modulated four different phenotypes: inhibition of autophagy and osteoblast differentiation, as well as potassium channel and tubulin modulation. The latter two were uncovered by using unbiased morphological profiling with a cell-painting assay. The number of hits and variety in bioactivity discovered validates the use of recombining natural product fragments coupled to phenotypic screening for the rapid identification of biologically diverse compounds., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma characterized by malignant skin-homing T cells with an increasing bias towards the T helper cell type 2 (Th2) during disease progression. Patients with advance CTCL have a high disease burden and the majority of CTCL patients suffer from severe itching (pruritus). A key challenge in the treatment of advanced CTCL is to maintain and to stabilize initial therapeutic responses after systemic treatment. Addressing this clinical need, the oral class I, IIb, IV HDAC inhibitor resminostat is currently under clinical evaluation for disease control after systemic therapy (RESMAIN, NCT02953301). Here, the mode-of-action of resminostat was explored. Several CTCL cell lines representing mycosis fungoides (MF) and Sézary syndrome (SS) were used in cell biological and molecular assays. HDAC inhibitors are epigenetic drugs that alter the chromatin landscape and the expression of genes. Indicating an interference with epigenetic processes, resminostat induced hyperacetylation on specific histone residues linked with transcriptional regulation. Whole transcriptome analysis upon resminostat treatment uncovered changes in oncogenic signaling pathways and in the expression of genes associated with the pathogenesis of CTCL. The expression of several skin-homing receptors, which mediate the infiltration of malignant T cells into the skin, was reduced by resminostat. Furthermore, resminostat up-regulated a gene expression signature representative of Th1 cell type and down-regulated genes of the Th2 cell type, thus favoring the beneficial Th1 T cell phenotype. In agreement with this observation, resminostat decreased the mRNA level and protein secretion of the Th2 and itch-mediating cytokine IL-31 suggesting that resminostat might improve pruritus. Moreover, resminostat treatment resulted in anti-proliferative and pro-apoptotic effects in both MF and SzS cells. In addition to counteracting intrinsic pathogenic pathways in malignant CTCL cells, we found that resminostat enhanced natural killer cell-mediated lysis, thus promoting the innate immune response towards malignant cells. In contrast to reports for other HDAC inhibitors, resminostat did not reduce the viability or function of NK cells but directly positively affected their cytolytic function. Moreover, resminostat up-regulated the expression NKG2D-ligands on cancer cells and, thereby, facilitates the recognition of cancer cells by NK cells. Interestingly, the combination of resminostat with opsonizing antibodies (e.g. Mogamulizumab, IPH4102) improved the lysis of CTCL cells in NK cell assays, suggesting a potential of resminostat's combinations with opsonizing antibodies. Taken together, these data corroborate that resminostat has the potential to counteract the malignant T cell population in CTCL. In conclusion, our preclinical data support the hypothesis that resminostat treatment will improve or stabilize CTCL and its symptoms. [ABSTRACT FROM AUTHOR]

Plant protection products (PPPs) and the active substance(s) contained within them are rigorously and comprehensively tested prior to registration to ensure that human health is not impacted by their use. In recent years, there has been a widespread drive to have more relevant testing strategies (e.g., ILSI/HESI-ACSA and new EU Directives), which also take account of animal welfare, including the 3R (replacement, refinement, and reduction) principles. The toxicity potential of one such new active substance, sulfoxaflor, a sulfoximine insecticide (CAS #946578-00-3), was evaluated utilizing innovative testing strategies comprising: (1) an integrated testing scheme to optimize information obtained from as few animals as possible (i.e., 3R principles) through modifications of standard protocols, such as enhanced palatability study design, to include molecular endpoints, additional neurotoxicity and immunotoxicity parameters in a subchronic toxicity study, and combining multiple test guidelines into one study protocol; (2) generation of toxicokinetic data across dose levels, sexes, study durations, species, strains and life stages, without using satellite animals, which was a first for PPP development, and (3) addition of prospective mode of action (MoA) endpoints within repeat dose toxicity studies as well as proactive inclusion of specific MoA studies as an integral part of the development program. These novel approaches to generate key data early in the safety evaluation program facilitated informed decision-making on the need for additional studies and contributed to a more relevant human health risk assessment. This supplement also contains papers which describe in more detail the approach taken to establish the MoA and human relevance framework related to toxicities elicited by sulfoxaflor in the mammalian toxicology studies: [ABSTRACT FROM AUTHOR]

Abstract: The Kv7 channels, a family of voltage-dependent K+ channels (Kv7.1–Kv7.5), have gained much attention in drug discovery especially because four members are genetically linked to diseases. For disorders of the CNS focus was originally on epilepsy and pain, but it is becoming increasingly evident that Kv7 channels can also be valid targets for psychiatric disorders, such as anxiety and mania. The common denominator is probably neuronal hyperexcitability in different brain areas, which can be successfully attenuated by pharmacological increment of Kv7 channel activity. This perspective attempts to review the current status and challenges for CNS drug discovery based on Kv7 channels as targets for neurological and psychiatric indications with special focus on selectivity and mode-of-actions. [Copyright &y& Elsevier]

Background To assess the risk of all nanomaterials (NMs) on a case-by-case basis is challenging in terms of financial, ethical and time resources. Instead a more intelligent approach to knowledge gain and risk assessment is required. Methods A framework of future research priorities was developed from the accorded opinion of experts covering all major stake holder groups (government, industry, academia, funders and NGOs). It recognises and stresses the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling approaches as key components of the current and future risk assessment of NMs. Results The framework for future research has been developed from the opinions of over 80 stakeholders, that describes the research priorities for effective development of an intelligent testing strategy (ITS) to allow risk evaluation of NMs. In this context, an ITS is a process that allows the risks of NMs to be assessed accurately, effectively and efficiently, thereby reducing the need to test NMs on a case-by-case basis. For each of the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling, key-priority research areas are described via a series of stepping stones, or hexagon diagrams structured into a time perspective. Importantly, this framework is flexible, allowing individual stakeholders to identify where their own activities and expertise are positioned within the prioritisation pathway and furthermore to identify how they can effectively contribute and structure their work accordingly. In other words, the prioritisation hexagon diagrams provide a tool that individual stakeholders can adapt to meet their own particular needs and to deliver an ITS for NMs risk assessment. Such an approach would, over time, reduce the need for testing by increasing the reliability and sophistication of in silico approaches. The manuscript includes an appraisal of how this framework relates to the current risk assessment approaches and how future risk assessment could adapt to accommodate these new approaches. A full report is available in electronic format (pdf) at www.its-nano.eu. Conclusion ITS-NANO has delivered a detailed, stakeholder driven and flexible research prioritisation (or strategy) tool, which identifies specific research needs, suggests connections between areas, and frames this in a time-perspective. [ABSTRACT FROM AUTHOR]

Background: Ginkgo biloba L. is one of the oldest trees on earth, and its leaves have been used since ages as herbal medicine to treat cerebrovascular disorders. It is worth noting that in addition to the widely concerned flavonoids and terpenoids, it also contains various thus far neglected biflavonoids. In fact, biflavonoids are flavonoids consisting of apigenin or its derivatives as monomeric scaffold, and are linked via C-C or C-O-C bond.Purpose: Based on the structural similarity of flavonoids, we hypothesized that biflavonoids may play a potential role in the treatment of cerebrovascular diseases. Here, we describe the effectiveness and underlying mechanisms for prevention and treatment of atherosclerosis (AS) by biflavonoids.Study Design and Methods: Four main biflavonoids in Ginkgo biloba leaves were screened by oleic acid-induced lipid production in HepG2 cells. The non-covalent effects of biflavonoids on the potential targets of atherosclerosis were screened by reverse targeting and molecular dynamics simulation. The interactions between biflavonoids and potential targets were evaluated by an exogenous cell model, which verified the consistency of the simulation results.Conclusion: Among all four biflavonoids, ginkgetin significantly inhibited oleic acid-induced lipid production in HepG2 cells and reduced total cholesterol and triglyceride levels. The interaction of ginkgetin with CDK2 through π-alkyl and hydrogen bonds increased the binding of molecules and proteins. Ginkgetin arrested the cells in the G1-S phase, which significantly inhibited abnormal cell growth which closely related to the occurrence and development of atherosclerosis. Biflavonoids could be a promising natural medicine for the treatment of atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: Cancer is still an all-times issue due to a large and even increasing number of deaths. Impaired genes regulating cell proliferation and apoptosis are targets for the development of novel cancer treatments.Hypothesis: Increased transcription of NADPH oxidase activator (NOXA), Bcl2-like11 (BIM), BH3-only proteins and p53 unregulated apoptosis modulator (PUMA) is caused by the imbalance between pro- and anti-apoptotic Bcl-2 proteins due to endoplasmic reticulum (ER) stress. The membranous network of ER is present in all eukaryotic cells. ER stress facilitates the interaction between Bax and PUMA, triggering the release of cytochrome C. As a main intracellular organelle, ER is responsible for translocation as well as post-translation modification and protein folding.Results: Hesperetin is a cytoprotective flavonone, which acts against ER stress and protects from cell damage induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Hesperetin inhibits lipid peroxidation induced by Fe2+ and l-ascorbic acid in rat brain homogenates.Conclusion: This review deals with the anticancer effects of hesperetin regarding the regulation of ER stress as a principal mechanism in the pathogenesis of tumors. [ABSTRACT FROM AUTHOR]

Abstract: This work tests the mode-of-action (MOA) hypothesis that maternal and developmental triclosan (TCS) exposure decreases circulating thyroxine (T4) concentrations via up-regulation of hepatic catabolism and elimination of T4. Time-pregnant Long-Evans rats received TCS po (0–300mg/kg/day) from gestational day (GD) 6 through postnatal day (PND) 21. Serum and liver were collected from dams (GD20, PND22) and offspring (GD20, PND4, PND14, PND21). Serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) concentrations were measured by radioimmunoassay. Ethoxy-O-deethylase (EROD), pentoxyresorufin-O-depentylase (PROD) and uridine diphosphate glucuronyltransferase (UGT) enzyme activities were measured in liver microsomes. Custom Taqman® qPCR arrays were employed to measure hepatic mRNA expression of select cytochrome P450s, UGTs, sulfotransferases, transporters, and thyroid hormone-responsive genes. TCS was quantified by LC/MS/MS in serum and liver. Serum T4 decreased approximately 30% in GD20 dams and fetuses, PND4 pups and PND22 dams (300mg/kg/day). Hepatic PROD activity increased 2–3 fold in PND4 pups and PND22 dams, and UGT activity was 1.5 fold higher in PND22 dams only (300mg/kg/day). Minor up-regulation of Cyp2b and Cyp3a expression in dams was consistent with hypothesized activation of the constitutive androstane and/or pregnane X receptor. T4 reductions of 30% for dams and GD20 and PND4 offspring with concomitant increases in PROD (PND4 neonates and PND22 dams) and UGT activity (PND22 dams) suggest that up-regulated hepatic catabolism may contribute to TCS-induced hypothyroxinemia during development. Serum and liver TCS concentrations demonstrated greater fetal than postnatal internal exposure, consistent with the lack of T4 changes in PND14 and PND21 offspring. These data support the MOA hypothesis that TCS exposure leads to hypothyroxinemia via increased hepatic catabolism; however, the minor effects on thyroid hormone metabolism may reflect the low efficacy of TCS as thyroid hormone disruptor or highlight the possibility that other MOAs may also contribute to the observed maternal and early neonatal hypothyroxinemia. [Copyright &y& Elsevier]