Dose-response Assays Research Articles

Diatom phytochromes integrate the underwater light spectrum to sense depth.

Aquatic life is strongly structured by the distribution of light, which, besides attenuation in intensity, exhibits a continuous change in the spectrum with depth1. The extent to which these light changes are perceived by phytoplankton through photoreceptors is still inadequately known. We addressed this issue by integrating functional studies of diatom phytochrome (DPH) photoreceptors in model species2 with environmental surveys of their distribution and activity. Here, by developing an in vivo dose-response assay to light spectral variations mediated by DPH, we show that DPH can trigger photoreversible responses across the entire light spectrum, resulting in a change in DPH photoequilibrium with depth. By generating dph mutants in the diatom Thalassiosira pseudonana, we also demonstrate that under simulated low-blue-light conditions of ocean depth, DPH regulates photosynthesis acclimation, thus linking optical depth detection with a functional response. The latitudinal distribution of DPH-containing diatoms from permanently stratified regions to seasonally mixed regions suggests an adaptive value of DPH functions in coping with vertical displacements in the water column. By establishing DPH as a detector of optical depth, this study provides a new view of how information embedded in the underwater light field can be exploited by diatoms to modulate their physiology throughout the photic zone.

Effect of protease inhibitors on the intraerythrocytic development of Babesia microti and Babesia duncani, the causative agents of human babesiosis

AbstractHuman babesiosis is a malaria‐like, tick‐borne infectious disease with a global distribution. Babesiosis is caused by intraerythrocytic, apicomplexan parasites of the genus Babesia. In the United States, human babesiosis is caused by Babesia microti and Babesia duncani. Current treatment for babesiosis includes either the combination of atovaquone and azithromycin or the combination of clindamycin and quinine. However, the side effects of these agents and the resistance posed by these parasites call for alternative approaches for treating human babesiosis. Proteases play several roles in the context of parasitic lifestyle and regulate basic biological processes including cell death, cell progression, and cell migration. Using the SYBR Green‐1 assay, we screened a protease inhibitor library that consisted of 160 compounds against B. duncani in vitro and identified 13 preliminary hits. Dose response assays of hit compounds against B. duncani and B. microti under in vitro conditions identified five effective inhibitors against parasite growth. Of these compounds, we chose ixazomib, a proteasome inhibitor as a potential drug for animal studies based on its lower IC50 and a higher therapeutic index in comparison with other compounds. Our results suggest that Babesia proteasome may be an important drug target and that developing this class of drugs may be important to combat human babesiosis.

Emergence of multiple resistance to EPSPS and ALS herbicides in smooth pigweed (Amaranthus hybridus): a growing concern in Brazil

Abstract Recently, farmers in Brazil have observed a decline in efficacy of glyphosate, chlorimuron, and imazethapyr control of smooth pigweed (Amaranthus hybridus L.). The objectives of this study were to quantify the resistance of Amaranthus in Brazil to glyphosate and acetolactate synthase (ALS)-inhibiting herbicides, elucidate the mechanism of resistance, and assess the frequency of sensitivity shifts to glyphosate and chlorimuron in Brazil. Dose–response assays were conducted in a greenhouse with glyphosate, chlorimuron, and imazethapyr. This was followed by sequencing of the EPSPS and ALS genes. Additionally , 740 Amaranthus populations across several Brazilian states were monitored over 4 yr, subjected to a single discriminatory dose of glyphosate and chlorimuron. The populations BR18Asp051 and BR21Asp205 were resistant to glyphosate, chlorimuron, and imazethapyr. The elevated resistance level to glyphosate in these populations is attributed to multiple amino acid substitutions (TAP-IVS) in the EPSPS gene; and cross-resistance to sulfonylureas and imidazolinones is conferred by the Trp-574-Leu substitution in the ALS gene in both populations. Overall, resistance distribution indicated that 88% of the sampled populations were considered sensitive to glyphosate, while 66% were sensitive to chlorimuron. Furthermore, 10% of the samples demonstrated multiple resistance to both active ingredients. A shift in glyphosate sensitivity was observed in four states in Brazil; however, sensitivity shifts to chlorimuron were more widely dispersed in Brazilian agricultural regions.

CSIG-12. YAP1-TEAD SIGNALING CONTRIBUTES TO LUNG CANCER COLONIZATION OF THE BRAIN

Abstract BACKGROUND Lung cancer is the most common primary tumor to metastasize to the brain. Poor understanding of the brain metastasis process limits therapeutic options. Despite emerging data on the role of YAP signaling pathway in the development of brain tumors, a gap in knowledge remains regarding its role in brain colonization. We hypothesize that YAP1 and the associated TEAD transcriptional factors are responsible for lung cancer progression and that intervention with YAP/TEAD-targeted therapy will prevent brain dissemination. METHODS Lung cancer cells at different stages of cancer progression (VMRC-LCD and NCI-H1975, lung cancer cell lines; NCI-H1915 and LN001, brain metastatic cancer cells) were genetically engineered to overexpress or silence/knockout YAP1 using lentiviral transduction of constructs expressing shRNA/sgRNA or YAP1. We tested several tumor cell capabilities using well-established in vitro assays, including invasion and migration assays, and an in vivo brain metastasis (BM) model to determine the biological effects of YAP1/TEAD suppression at different stages of lung cancer progression. Dose-response assays were performed to determine IC50 of the TEAD inhibitor, verteporfin. RESULTS YAP overexpression enhances the proliferative phenotype in metastatic and BM cell lines but not in non-metastatic cells. However, YAP overexpression only increases the migration ability of BM cell lines such as LN001 and NCI-H1915 compared to the metastatic NCI-H1975 and non-metastatic lung cancer cells. Moreover, we showed the efficacy of the YAP1/TEAD inhibitor in patient-derived lung BM cells. Finally, our in vivo preliminary data showed YAP1 involvement in lung cancer cell colonization of the brain but not in sustaining brain metastasis growth. CONCLUSIONS Our findings showed YAP1 involvement in lung cancer cell colonization of the brain, suggesting YAP1 inhibition is a clinically actionable target for preventing lung cancer dissemination and providing a strong rationale to further evaluate the benefit of a YAP1/TEAD-targeted therapy in in vivo models of BM.

Both chebulagic acid and punicalagin inhibit respiratory syncytial virus entry via multi-targeting glycoprotein and fusion protein.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections, with no currently available small-molecule drugs that are both safe and effective. A major obstacle in antiviral drug development is the rapid emergence of drug-resistant viral strains. Targeting multiple viral compounds may help mitigate the development of resistance. Herein, we conducted a drug screening using the Antiviral Traditional Chinese Medicine Active Compound Library, aiming to identify compounds that simultaneously target the RSV fusion (F) protein, glycoprotein (G), and the host heparan sulfate proteoglycans (HSPGs). From this screening, 10 candidate compounds were identified for their ability to interact with all three targets. Among these 10 candidates, chebulagic acid (CHLA) and punicalagin (PUG) demonstrated the most potent inhibition of RSV replication. In vitro dose-response assays confirmed the antiviral efficacy of CHLA (IC50: 0.07864 µM) and PUG (IC50: 0.08065 µM). Further experiments revealed both CHLA and PUG disrupt RSV attachment and membrane fusion by targeting the RSV-F and G proteins, rather than HSPG. Notably, CHLA and PUG were found to bind to the CX3C motif of the RSV-G protein, with docking assays predicting their binding sites at cysteines 176 and 182. Additionally, CHLA enhanced the conformational stability of the RSV-F protein before fusion. In an in vivo study, both CHLA and PUG were shown to alleviate RSV-induced pulmonary pathology by reducing viral titers, mitigating lung injury, and suppressing the inflammatory responses in the lungs. Our findings suggest that CHLA and PUG hold potential as therapeutic agents for RSV infection.IMPORTANCEA significant challenge in developing anti-respiratory syncytial virus (RSV) agents is the rapid emergence of resistant viral strains. Designing drugs that target multiple viral components can effectively reduce the likelihood of developing resistant strains. In this study, we screened compounds from the Antiviral Traditional Chinese Medicine Active Compound Library, aiming to simultaneously targe the RSV fusion (F) protein, glycoprotein (G), and host heparan sulfate proteoglycans (HSPGs). Our findings revealed that chebulagic acid (CHLA) and punicalagin (PUG) significantly inhibited RSV replication both in vitro and in vivo and interacted with all three targets. Both CHLA and PUG were able to disrupt RSV attachment and membrane fusion. Mechanistically, CHLA and PUG were found to bind to the CX3C motif of the RSV-G protein, with CHLA also enhancing the conformational stability of the RSV-F protein before fusion. In conclusion, our study suggests that CHLA and PUG hold promise as therapeutic agents against RSV infection.

Lipid Priming of Adipose Mesenchymal Stromal Cells with Docosahexaenoic Acid: Impact on Cell Differentiation, Senescence and the Secretome Neuroregulatory Profile.

Priming strategies that improve the functionality of MSCs may be required to address issues limiting successful clinical translation of MSC therapies. For conditions requiring high trophic support such as brain and spinal cord injuries, priming MSCs to produce higher levels of trophic factors may be instrumental to facilitate translation of current MSC therapies. We developed and tested a novel molecular priming paradigm using docosahexaenoic acid (DHA) to prime adipose tissue-derived mesenchymal stromal cells (ASCs) to enhance the secretome neuroregulatory potential. Comprehensive dose-response and time-course assays were carried to determine an optimal priming protocol. Secretome total protein measurements were taken in association with cell viability, density and morphometric assessments. Cell identity and differentiation capacity were studied by flow cytometry and lineage-specific markers. Cell growth was assessed by trypan-blue exclusion and senescence was probed over time using SA-β-gal, morphometry and gene expression. Secretomes were tested for their ability to support differentiation and neurite outgrowth of human neural progenitor cells (hNPCs). Neuroregulatory proteins in the secretome were identified using multiplex membrane arrays. Priming with 40µM DHA for 72h significantly enhanced the biosynthetic capacity of ASCs, producing a secretome with higher protein levels and increased metabolic viability. DHA priming enhanced ASCs adipogenic differentiation and adapted their responses to replicative senescence induction. Furthermore, priming increased concentrations of neurotrophic factors in the secretome promoting neurite outgrowth and modulating the differentiation of hNPCs. These results provide proof-of-concept evidence that DHA priming is a viable strategy to improve the neuroregulatory profile of ASCs.

Growth form variations of Amaranthus hybridus L. biotypes with EPSPS mutations

AbstractSeveral Amaranthus hybridus L. biotypes present single and triple amino acid substitutions in EPSPS (5‐enolypyruvylshikimate‐3‐phosphate synthase). In resistant and tolerant species to glyphosate, differential susceptibility and alterations in the EPSPS sequence could cause modifications in their morphological–structural characteristics, an issue that is unknown for biotypes of A. hybridus. The aim of the work was to determine the susceptibility to glyphosate, the presence of mutations in EPSPS and the growth form in four biotypes of this weed from north‐central Argentina. To carry out this, a dose–response assay was conducted, to determine the presence of mutations in the EPSPS, DNA was extracted, amplified and sequenced, and finally, to evaluate the morphological–structural variations, the growth form was analysed. Our findings revealed that Ah03, Ah07 and Ah36 biotypes had resistance factors of 22 to 118. The EPSPS sequence remains unaltered in the Ah39 biotype, leading it to be considered a susceptible biotype. Biotypes Ah03 and Ah36 had heterozygous changes in the DNA sequence resulting in amino acid substitutions at positions 102, 103 and 106 being considered resistant biotypes, whereas in Ah07 some individuals showed unaltered DNA sequences and others exhibited heterozygous changes. The growth form was quantitatively different between biotypes, standing the most susceptible biotype that produced more seeds and required less thermal time for the appearance of the terminal inflorescence. The present investigation provides new evidence that resistant biotypes with the triple DNA mutation exhibit variations in the growth form compared to susceptible biotypes of this weed.

Molecular Networking, Docking, and Biological Evaluation of Licarin A from Myristica fragrans as a Potential Cancer Chemopreventive Agent.

Currently, clinically available cancer chemopreventive drug options are limited to mostly tamoxifen and its derivatives, such as raloxifene, and approved specifically for breast cancer. Thus, the availability of chemopreventive drug molecules for other types of malignant cancers would be desirable. In previous reports, the arils of Myristica fragrans (mace) have been found to exhibit cancer chemopreventive activity. Therefore, the purpose of the present study was to identify a natural product from this species with potential chemopreventive activity guided by chemoinformatic sample analysis via Global Natural Products Social (GNPS) molecular networking and molecular docking. The neolignan licarin A (1) was identified as a potential chemopreventive constituent, and subsequently submitted to several in vitro bioassays and a zebrafish toxicity evaluation. In this work, 1 afforded superior phosphoNF-κBp65 phosphorylation activity in DU-145 prostate cancer cells compared to isoliquiritigenin (2), which was used as a natural product chemopreventive control. Both 1 and 2 showed a longer-lasting reduction in cellular stress in a cell oxidative stress real-time dose-response assay than the positive control using Hepa1c1c7 mouse hepatoma cells. In addition, 1 displayed similar activities to 2, while also being less toxic to zebrafish (Danio rerio) than both this chalcone and the clinically used chemopreventive drug tamoxifen.

Resistance to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in Lolium multiflorum Lam. populations of Argentina.

Italian ryegrass (Lolium multiflorum Lam.) is one of the most troublesome grass weeds in Argentina. The extensive and repetitive use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has induced resistance in this weed species. The objectives of this study were to quantify the resistance levels to ACCase-inhibiting herbicides in two resistant populations and to identify the target-site mutations associated with their resistance. Two resistant Italian ryegrass populations, Roldán and H2, were studied. Roldán was a suspected haloxyfop-resistant population, located in a wheat field from Santa Fe province with a history of ACCase-inhibiting herbicide use. The H2 population was obtained from the susceptible Hernandarias population (H0) after two cycles of selection with the herbicide quizalofop-ethyl. Whole-plant dose-response assays revealed that the resistant populations exhibited a high resistance to haloxyfop, with resistance factors (RF) exceeding 97-fold. Additionally, both populations showed a moderate resistance to pinoxaden (RF > 7), while maintaining susceptibility to clethodim. Partial chloroplastic ACCase sequences revealed isoleucine-to-asparagine substitution at position 2041 (Ile-2041-Asn) in both resistant populations. This work provides a better understanding of cross-resistance to ACCase-inhibiting herbicides in L. multiflorum populations and represents the first report of the target-site mutation Ile-2041-Asn conferring resistance in populations from Argentina. © 2024 Society of Chemical Industry.

Abstract 35: The Dual Endothelin-1 Antagonist Aprocitentan Attenuates Mitochondrial Oxidative Stress in Human Cardiac Fibroblasts

Endothelin-1 (ET-1) is a peptide hormone primarily produced by endothelial cells that functions as a potent vasoconstrictor through two receptors, ETA and ETB. While most research on ET-1 has focused on endothelial dysfunction and hypertension, increasing evidence shows that ET-1 is also involved in other physiological processes such as cell proliferation, inflammation, and tissue remodeling. Dysregulation of ET-1 has been implicated in the pathogenesis of various diseases, including heart failure, atherosclerosis, fibrosis, systemic sclerosis, and cancer. Aprocitentan (also known as ACT-132577) is a relatively new dual ET-1 receptor antagonist that prevents ET-1 from binding to both endothelin receptors and has been shown to significantly reduce blood pressure in patients with resistant hypertension. Our hypothesis was that aprocitentan could reduce the proliferation rate and mitochondrial ROS production induced by TGF-β in human cardiac fibroblasts (HCFs). HCFs were isolated from hearts from fully de-identified cadaveric donors and cultured in cardiac fibroblast growth medium. HCFs were treated with aprocitentan (4 μM) and/or TGF-β1 (10 ng/ml) for 72 hours (doses and times identified in preliminary dose-response and time-course assays). Aprocitentan reduced the TGF-β1-induced proliferation rate in HCFs by approximately 23% ( Figure 1A ). Our study also aimed to determine if aprocitentan could mitigate mitochondrial dysfunction. Since the mitochondrion is a major source of reactive oxygen species (ROS), which can promote myofibroblast differentiation, mitochondrial properties in HCFs were assessed using MitoTracker Green and MitoSOX staining. TGF-β1 treatment increased mitochondrial ROS production, which was significantly reduced by aprocitentan ( Figure 1BC ); furthermore, aprocitentan reduced mitochondrial fragmentation induced by TGF-β1 treatment ( Figure 1D ). To the best of our knowledge, we are the first to demonstrate that aprocitentan can reduce the increased production of mitochondrial ROS, mitochondrial fragmentation, and cell proliferation rate induced by TGF-β1 in HCFs. This discovery could pave the way for new research and therapeutic strategies for treating fibrosis in cardiovascular disorders.

Exploring antiviral activity of Betanin and Glycine Betaine against dengue virus type-2 in transfected Hela cells

Dengue virus (DENV) infection is a worldwide public health concern infecting approximately 400 million individuals and about 40,000 mortalities yearly. Despite this, no licensed or readily available antiviral medication is currently available specifically for DENV infection, and therapy is typically symptomatic. Therefore, the objective of the study was to investigate the antiviral activity of Beta vulgaris L. phytoconstituents against DENV-2 targeting NS3 protein. The antiviral activity of phytochemicals was examined through virtual ligand-based screening, antiviral inhibition and dosage response assays, western blotting analysis and MD simulations. We conducted toxicological, and pharmacokinetic analysis to assess plant-based natural compound's efficacy, safety, and non-toxic doses. Molecular docking and MD simulation results revealed that the nonstructural protein-3 (NS3) might prove as a funamental target for Betanin and Glycine Betaine against Dengue virus. Betanin and Glycine betaine were initially studied for their non-toxic doses in HeLa, CHO, and Vero cells via MTT assay. HeLa cells were transiently transfected with cloned vector pcDNA3.1/Zeo(+)/DENV-2 NS3 along with non-toxic doses (80 μM–10 μM) of selected phytochemicals. The dose-response assay illustrated downregulated expression of DENV-2 NS3 gene after administration of Betanin (IC50 = 4.35 μM) and Glycine Betaine (IC50 = 4.49 μM). Dose response analysis of Betanin (80 μM–10 μM) depicted the significant inhibition of NS3 protein expression as well. These results suggested downregulated expression of DENV-2 NS3 at mRNA and protein level portraying the DENV replication inhibition. Based on our study findings, NS3 protease is depicted as distinctive DENV-2 inhibitor target. We will channel our study further into in vitro characterization employing the mechanistic study to understand the role of host factors in anti-flavi therapeutic.

In Vitro Prediction of Skin-Sensitizing Potency Using the GARDskin Dose-Response Assay: A Simple Regression Approach.

Toxicological assessments of skin sensitizers have progressed towards a higher reliance on non-animal methods. Current technological trends aim to extend the utility of non-animal methods to accurately characterize skin-sensitizing potency. The GARDskin Dose-Response assay has previously been described; it was shown that its main readout, cDV0 concentration, is associated with skin-sensitizing potency. The ability to predict potency from cDV0 in the form of NESILs derived from LLNAs or human NOELs was evaluated. The assessment of a dataset of 30 chemicals showed that the cDV0 values still correlated strongly and significantly with both LLNA EC3 and human NOEL values (ρ = 0.645-0.787 [p < 1 × 10-3]). A composite potency value that combined LLNA and human potency data was defined, which aided the performance of the proposed model for the prediction of NESILs. The potency model accurately predicted sensitizing potency, with cross-validation errors of 2.75 and 3.22 fold changes compared with NESILs from LLNAs and humans, respectively. In conclusion, the results suggest that the GARDskin Dose-Response assay may be used to derive an accurate quantitative continuous potency estimate of skin sensitizers.

Successful yeast microencapsulation of fluralaner and its potential as a larvicide for vector control

Fly control for any species is most effectively implemented in the immature stages when insects can be eliminated before emerging as adults capable of transmitting pathogens or becoming nuisance pests. Yet a limited number of insecticide classes are available for treating larval development sites for dipteran pest species. The most recently introduced class of insecticides in the United States (US) is the isoxazolines, including fluralaner. In the US, fluralaner is currently exclusively labeled for use against ectoparasites in companion animals. However, research has shown that it has a wider effective target range beyond ectoparasites and could be developed as an insecticide for vector control. Here we tested a novel, proprietary, yeast microencapsulated (YME) formulation of fluralaner against the larvae of three pest species: Musca domestica L. (Diptera: Muscidae), Aedes albopictus Skuse (Diptera: Culicidae), and Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae). These species all naturally consume microorganisms as larvae, including yeasts. Fluralaner was successfully microencapsulated in Saccharomyces cerevisiae yeast. YME fluralaner was reconstituted in water at concentrations of 0.00001–0.1 mg/mL (Aedes and Culicoides) or 1–50 mg/mL (Musca) for use in dose-response assays. For each species, the LC50 at 24 h was estimated using probit analyses. YME fluralaner was highly effective against all species tested (Ae. albopictus LC50 = 0.000077 mg/mL; C. sonorensis LC50 = 0.00067 mg/mL; M. domestica LC50 = 2.58 mg/mL). Additionally, laboratory assays were conducted to determine product reapplication rates using LC50 rates. Reapplication rates to maintain <50 % emergence were five weeks (Ae. albopictus) and greater than eight weeks (C. sonorensis). The results presented here indicate YME fluralaner is a promising candidate for controlling larval insects that naturally feed on detritus, thereby bypassing cuticular penetration barriers and safely delivering the active ingredient to the target species.

Identification of novel bromodomain inhibitors of Trypanosoma cruzi bromodomain factor 2 (TcBDF2) using a fluorescence polarization-based high-throughput assay.

Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and regulate DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi (TcBDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350-ranked compounds were further analyzed in a dose-response assay. From this analysis, seven compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significant trypanocidal activity, all showed bona fide interaction with TcBDF2 with dissociation constants between 1 and 3 µM validating these assays to search for bromodomain inhibitors.

Determining the In Vitro Ligand-Target Interaction by Cellular Thermal Shift Assay and Isothermal Dose-Response Fingerprint Assay.

The cellular thermal shift assay (CETSA) and isothermal dose-response fingerprint assay (ITDRF CETSA) have been introduced as powerful tools for investigating target engagement by measuring ligand-triggered thermodynamic stabilization of cellular target proteins. Yet, these techniques have rarely been used to evaluate the thermal stability of RNA-binding proteins (RBPs) when exposed to ligands. Here, we present an adjusted approach using CETSA and ITDRFCETSA to determine the interaction between enasidenib and RBM45. Our assay is sensitive and time-efficient and can potentially be adapted for studying the interactions of RBM45 protein with other potential candidates. Key features • This protocol builds upon the method developed by Molina et al. and extends its application to new protein classes, such as RBPs.

Cell-intrinsic platinum response and associated genetic and gene expression signatures in ovarian cancer cell lines and isogenic models.

Ovarian cancers are still largely treated with platinum-based chemotherapy as the standard of care, yet few biomarkers of clinical response have had an impact on clinical decision making as of yet. Two particular challenges faced in mechanistically deciphering platinum responsiveness in ovarian cancer have been the suitability of cell line models for ovarian cancer subtypes and the availability of information on comparatively how sensitive ovarian cancer cell lines are to platinum. We performed one of the most comprehensive profiles to date on 36 ovarian cancer cell lines across over seven subtypes and integrated drug response and multiomic data to improve on our understanding of the best cell line models for platinum responsiveness in ovarian cancer. RNA-seq analysis of the 36 cell lines in a single batch experiment largely conforms with the currently accepted subtyping of ovarian cancers, further supporting other studies that have reclassified cell lines and demonstrate that commonly used cell lines are poor models of high-grade serous ovarian carcinoma. We performed drug dose response assays in the 32 of these cell lines for cisplatin and carboplatin, providing a quantitative database of IC 50 s for these drugs. Our results demonstrate that cell lines largely fall either well above or below the equivalent dose of the clinical maximally achievable dose (C max ) of each compound, allowing designation of cell lines as sensitive or resistant. We performed differential expression analysis for high-grade serous ovarian carcinoma cell lines to identify gene expression correlating with platinum-response. Further, we generated two platinum-resistant derivatives each for OVCAR3 and OVCAR4, as well as leveraged clinically-resistant PEO1/PEO4/PEO6 and PEA1/PEA2 isogenic models to perform differential expression analysis for seven total isogenic pairs of platinum resistant cell lines. While gene expression changes overall were heterogeneous and vast, common themes were innate immunity/STAT activation, epithelial to mesenchymal transition and stemness, and platinum influx/efflux regulators. In addition to gene expression analyses, we performed copy number signature analysis and orthogonal measures of homologous recombination deficiency (HRD) scar scores and copy number burden, which is the first report to our knowledge applying field-standard copy number signatures to ovarian cancer cell lines. We also examined markers and functional readouts of stemness that revealed that cell lines are poor models for examination of stemness contributions to platinum resistance, likely pointing to the fact that this is a transient state. Overall this study serves as a resource to determine the best cell lines to utilize for ovarian cancer research on certain subtypes and platinum response studies, as well as sparks new hypotheses for future study in ovarian cancer.

Diverse mechanisms confer bensulfuron-methyl resistance in Schoenoplectiella juncoides (Roxb.) lye

The effectiveness of bensulfuron-methyl in controlling Schoenoplectiella juncoides (Roxb.) Lye has significantly decreased in rice fields in China. Hence, a bensulfuron-methyl-resistant S. juncoides population (W15) was collected from Dandong City, Liaoning Province, China, to investigate the underlying resistance mechanisms. Whole-plant dose-response experiments and ALS activity assay confirmed that W15 has evolved high-level resistance to bensulfuron-methyl compared with the susceptible S. juncoides population (W4). Molecular analysis revealed a Pro-197-Ser mutation in ALS1, while there was no significant difference in the relative ALS gene expression between W15 and W4. LC-MS/MS analysis showed W15 metabolized bensulfuron-methyl more rapidly than W4. Furthermore, bensulfuron-methyl resistance in W15 was significantly alleviated by malathion and 4-chloro-7-nitrobenzoxadiazole (NBD-Cl). Glutathione S-transferase activity was higher in W15 than in W4. Meanwhile, W15 displayed cross-resistance to halosulfuron-methyl and multi-resistance to MCPA-Na. In summary, these findings demonstrated for the first time that both target- and non-target-site resistance are relevant in the resistance of S. juncoides to bensulfuron-methyl.

The synergistic effect and mechanism of different adjuvants on pinoxaden efficacy against Lolium multiflorum Lam

Adjuvants play a crucial role in optimizing and improving the efficacy of herbicides. Therefore, adding appropriate adjuvants to herbicides is an important part of weed control. The aim of this study was to investigate the synergistic effect and underlying mechanism of action of three commercial adjuvants (Hasuteng, Penwubao, and Zhenxiao IV) in conjunction with pinoxaden for controlling Lolium multiflorum Lam. The results of synergic effect, which were determined by dose‒response assay showed that the addition of Hasuteng and Penwubao could significantly enhance the efficacy of pinoxaden and decrease the ED50 (the herbicide dose resulting in 50% weed control) on L. multiforum while being safe to wheat. Hasuteng and Penwubao were further selected to investigate the underlying mechanisms by which adjuvants enhanced the efficacy of pinoxaden. The results indicated that the addition of Hasuteng and Penwubao significantly reduced the surface tension and shortened the drying time of the pinoxaden solution; moreover, it significantly increased both expanded diameter and maximum retention. Additionally, ultra high performance liquid chromatography-tandem mass spectrometry (UPLC‒MS/MS) analysis was used to evaluate the recovery rate, absorption level, and rainfastness. These two adjuvants could improve the absorption and metabolism levels of L. multiforum to pinoxaden. However, Hasuteng and Penwubao did not improve the rainfastness of pinoxaden. In summary, Hasuteng and Penwubao enhance the efficacy of pinoxaden by improving its physical properties and absorption level. The studies present a novel approach for enhancing the utilization rate of pinoxaden and provide theoretical guidance toward effectively reducing herbicide application in wheat fields.

A Machine-Learning-Driven Pathophysiology-Based New Approach Method for the Dose-Dependent Assessment of Hazardous Chemical Mixtures and Experimental Validations.

Environmental chemicals, such as PFAS, exist as mixtures and are frequently encountered at varying concentrations, which can lead to serious health effects, such as cancer. Therefore, understanding the dose-dependent toxicity of chemical mixtures is essential for health risk assessment. However, comprehensive methods to assess toxicity and identify the mechanisms of these harmful mixtures are currently absent. In this study, the dose-dependent toxicity assessments of chemical mixtures are performed in three methodologically distinct phases. In the first phase, we evaluated our machine-learning method (AI-HNN) and pathophysiology method (CPTM) for predicting toxicity. In the second phase, we integrated AI-HNN and CPTM to establish a comprehensive new approach method (NAM) framework called AI-CPTM that is targeted at refining prediction accuracy and providing a comprehensive understanding of toxicity mechanisms. The third phase involved experimental validations of the AI-CPTM predictions. Initially, we developed binary, multiclass classification, and regression models to predict binary, categorical toxicity, and toxic potencies using nearly a thousand experimental mixtures. This empirical dataset was expanded with assumption-based virtual mixtures, compensating for the lack of experimental data and broadening the scope of the dataset. For comparison, we also developed machine-learning models based on RF, Bagging, AdaBoost, SVR, GB, KR, DT, KN, and Consensus methods. The AI-HNN achieved overall accuracies of over 80%, with the AUC exceeding 90%. In the final phase, we demonstrated the superior performance and predictive capability of AI-CPTM, including for PFAS mixtures and their interaction effects, through rigorous literature and statistical validations, along with experimental dose-response zebrafish-embryo toxicity assays. Overall, the AI-CPTM approach significantly improves upon the limitations of standalone AI models, showing extensive enhancements in identifying toxic chemicals and mixtures and their mechanisms. This study is the first to develop a hybrid NAM that integrates AI with a pathophysiology method to comprehensively predict chemical-mixture toxicity, carcinogenicity, and mechanisms.

Abolished clustering of MeCP2T158M can be partially reverted with small molecules

Rett syndrome (OMIM 312750) is a rare neurodevelopmental disorder caused by de novo mutations in the Methyl-CpG Binding Protein 2 (MeCP2) gene located on the X-chromosome, typically affecting girls. Rett syndrome symptoms, characterized by microcephaly and lack of motor coordination, first appear between 6 to 18 months of age. The disease continues to progress until adulthood at which point it reaches a stationary phase. Currently, available therapy for Rett Syndrome is only symptomatic. More than 800 mutations causing Rett syndrome have been described, the most common being T158M (9% prevalence) located in the Methyl-Binding Domain (MBD) of MeCP2. Due to its importance for DNA binding through recognition of methylated CpG, mutations in the MBD have a significant impact on the stability and function of MeCP2. MeCP2 is a nuclear protein and accumulates in liquid–liquid phase condensates visualized as speckles in NIH3T3 cells by microscopy. This speckled pattern is lost with MeCP2 mutations in the MBD such as T158M. We developed a high content phenotypic assay, detecting fluorescent MeCP2 speckles in NIH3T3 cells. The assay allows the identification of small molecules that stabilize MeCP2-T158M and phenotypically rescue speckle formation. To validate the assay, a collection of 3572 drugs was screened, including FDA-approved drugs, compounds in clinical trials and biologically annotated tool compounds. 18 hits showed at least 25% rescue of speckles in the mutant cell line while not affecting wild-type MeCP2 speckles. Primary hits were confirmed in a dose response assay, a thermal shift assay with recombinant MeCP2 and by testing the MeCP2 expression levels. One class of identified hits represents histone deacetylase inhibitors (HDACis) showing 25% speckle rescue of mutant MeCP2 without toxicity. This screening strategy can be expanded to additional compound libraries and will support novel drug discovery.