Dose-response Assays Research Articles

Activation of Rho Family GTPases by Small Molecules.

Ras and Ras-related small GTPases are key regulators of diverse cellular functions that impact cell growth, survival, motility, morphogenesis, and differentiation. They are important targets for studies of disease mechanisms as well as drug discovery. Here, we report the characterization of small molecule agonists of one or more of six Rho, Rab, and Ras family GTPases that were first identified through flow cytometry-based, multiplexed high-throughput screening of 200000 compounds. The activators were categorized into three distinct chemical families that are represented by three lead compounds having the highest activity. Virtual screening predicted additional compounds with potential GTPase activating properties. Secondary dose–response assays performed on compounds identified through these screens confirmed agonist activity of 43 compounds. While the lead and second most active small molecules acted as pan activators of multiple GTPase subfamilies, others showed partial selectivity for Ras and Rab proteins. The compounds did not stimulate nucleotide exchange by guanine nucleotide exchange factors and did not protect against GAP-stimulated GTP hydrolysis. The activating properties were caused by a reversible stabilization of the GTP-bound state and prolonged effector protein interactions. Notably, these compounds were active both in vitro and in cell-based assays, and small molecule-mediated changes in Rho GTPase activities were directly coupled to measurable changes in cytoskeletal rearrangements that dictate cell morphology.

Development of rice (Oryza sativa) lines resistant to aryloxyphenoxypropionate herbicides through induced mutation with gamma rays

AbstractThe aryloxyphenoxypropionate herbicides (APPs) are graminicides with excellent control of many grass weeds species, including weedy rice (Oryza sativa L.). These herbicides block the fatty acid biosynthesis by inhibiting the enzyme acetyl‐CoA carboxylase (ACCase), resulting in the death of susceptible plants. Inducing mutation with gamma rays to rice seeds, two lines resistant to APPs herbicides were developed. Plant dose–response assays confirmed the resistance to the APPS herbicides quizalofop‐p‐ethyl and haloxyfop‐p‐methyl. The carboxyltransferase domain fragments of ACCase from the resistant biotype and susceptible control were sequenced and compared. A point mutation was detected in the amino acid position 2,027 (Rice Genome Annotation Project: Os05g22940.1). Results indicated that resistance to APPs is a consequence of an altered ACCase enzyme that confers resistance. The use of APPs herbicide‐resistant rice lines represents an innovative and promising alternative for weedy rice control in paddy rice systems.

Evaluation of allyl isothiocyanate as a soil fumigant against soil-borne diseases in commercial tomato (Lycopersicon esculentum Mill.) production in China.

Root-knot nematodes (Meloidogyne spp.), soil-borne diseases and weeds seriously reduce the commercial yield of tomatoes grown under protected cultivation in China. Allyl isothiocyanate (AITC), a natural product obtained from damaged Brassica tissues, was evaluated as a potential replacement for the fumigant methyl bromide (MB) for use in the greenhouse production of tomatoes in China. The dose-response assay indicates that AITC has high biological activity against major bacterial and fungal pathogens (EC50 of 0.225-4.199 mg L-1 ). The bioassay results indicate that AITC has good efficacy against root-knot nematodes (LC50 of 18.046 mg kg-1 ), and moderate efficacy against fungal pathogens (LC50 of 27.999-29.497 mg kg-1 ) and weeds (LC50 of 17.300-47.660 mg kg-1 ). The potting test indicates that AITC significantly improved plant vigor. Field trials indicate that AITC showed good efficacy against Meloidogyne spp. and Fusarium spp. (both ∼ 80%) as well as Phytophthora spp. and Pythium spp. (both ∼ 70%), and improved plant vigor and marketable yield. AITC used as a soil fumigant (30-50 g m-2 ) effectively controlled major bacterial and fungal pathogens, root-knot nematode, weeds and increased plant vigor, yield and farmers' income in tomato cultivated under protected agriculture in China. © 2018 Society of Chemical Industry.

Biological Analysis of Simvastatin-releasing Chitosan Scaffold as a Cell-free System for Pulp-dentin Regeneration

IntroductionThe improvement of biomaterials capable of driving the regeneration of the pulp-dentin complex mediated by resident cells is the goal of regenerative dentistry. In the present investigation, a chitosan scaffold (CHSC) that released bioactive concentrations of simvastatin (SIM) was tested, aimed at the development of a cell-free tissue engineering system. MethodsFirst, we performed a dose-response assay to select the bioactive dose of SIM capable of inducing an odontoblastic phenotype in dental pulp cells (DPCs); after which we evaluated the synergistic effect of this dosage with the CHSC/DPC construct. SIM at 1.0 μmol/L (CHSC-SIM1.0) and 0.5 μmol/L were incorporated into the CHSC, and cell viability, adhesion, and calcium deposition were evaluated. Finally, we assessed the biomaterials in an artificial pulp chamber/3-dimensional culture model to simulate the cell-free approach in vitro. ResultsSIM at 0.1 μmol/L was selected as the bioactive dose. This drug was capable of strongly inducing an odontoblastic phenotype on the DPC/CHSC construct. The incorporation of SIM into CHSC had no deleterious effect on cell viability and adhesion to the scaffold structure. CHSC-SIM1.0 led to significantly higher calcium-rich matrix deposition on scaffold/dentin disc assay compared with the control (CHSC). This biomaterial induced the migration of DPCs from a 3-dimensional culture to its surface as well as stimulated significantly higher expressions of alkaline phosphatase, collagen type 1 alpha 1, dentin matrix acidic phosphoprotein 1, and dentin sialophosphoprotein on 3-dimensional–cultured DPCs than on those in contact with CHSC. ConclusionsCHSC-SIM1.0 scaffold was capable of increasing the chemotaxis and regenerative potential of DPCs.

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices.

Despitenumerous studies that attempt to develop reliable animal modelswhich reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we proposea new procedureadapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer's disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitationswhich could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (α7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (Aβ).

Novel Marine Compounds Modulate Mitochondrial Function in H9c2 Cells: Potential New Pharmaceutical Targets to Control Cardiac Metabolism

IntroductionThe global burden of cardiovascular disease is increasing and is the leading cause of morbidity and mortality worldwide. As the heart is a highly metabolic organ, mitochondrial health is critical to normal cardiac function and is compromised in a number of cardiac diseases. Therefore, new pharmaceutical agents and screening tools to modulate and evaluate cardiac metabolism may offer novel therapeutics for cardiovascular disease.MethodsA number of marine compounds have been curated over the years by faculty at the Scripps Institution of Oceanography. Chemical structures have been largely elucidated through nuclear magnetic resonance spectroscopy and mass spectrometry, and these have been broadly available for biological screening through the UCSD Center for Compound Resources. Seventy‐three promising marine compounds were evaluated by application on H9c2 rat cardiomyoblasts while measuring oxygen consumption rate and extracellular acidification rate with a Seahorse XF96 extracellular flux analyzer. Mitochondrial metabolism was tested at a range of compound concentrations (0.1–1.6 mM). Subsequently, dose response curves of compounds that showed biological activity in the initial screen were performed. Dose response assay concentrations ranged from 0.01 nM to 3 μM with vehicle only as the control.ResultsEight compounds showed significant dose dependent alterations in mitochondrial metabolism in the initial screen. Dose response assays of glycospongosine revealed an increase in ATP linked‐maximal respiration ratio. In contrast, higher glycospongosine concentrations led to a gradual dose dependent decrease in reserve capacity. Injection of leptochelin A first increased basal respiration of the cardiomyoblasts, after the first increase, basal respiration gradually decreased. Increasing concentrations of honaucin A lowered the maximal mitochondrial respiration dose dependently. In addition, kabiramide C decreased maximal respiration normalized to basal respiration.ConclusionWe present here the results of screening multiple marine natural products in a cell based metabolic assay. Marine compounds have a demonstrated potential to modulate cardiac metabolism in a variety of settings; however, further research in disease models is necessary to determine if such modulation via these compounds is beneficial or harmful to cardiovascular disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification/Optimization of Novel Small Molecule Inhibitors of Zika Virus Infection

The rate of Zika virus (ZIKV) infection has been increasing since the original 2014 re‐emergence of the virus in Brazil, and the World Health Organization has warned against potential ZIKV epidemics. Furthermore, ZIKV, a flavivirus family member, has been identified as the cause of microcephaly and other developmental neuroimmunopathologies in fetuses of pregnant women and Guillain‐Barre Syndrome in adults, and to date, no therapeutics or vaccines are available against ZIKV infection. In this study, we screened a limited proprietary library of diverse organic small molecules, and identified a number of leads with significant antiviral activity. Briefly, the anti‐ZIKV activity of the compounds was assayed through plaque‐forming assays in A549 cells. Subsequently, dose response assays were performed for the active compounds using the same experimental setup. Furthermore, we performed structure/activity relationship (SAR) analyses and managed to improve the anti‐ZIKV activity with the most active analogs showing activity in low picomolar range. Finally, we constructed a general model of the overall structure of these anti‐ZIKV compounds which is based on the structural similarities of all active hits. These results identify excellent template(s) with potential for further optimization of antiviral activity through rigorous SAR analyses which may be utilized for the development of inexpensive and orally available anti‐ZIKV drugs. These compounds may also be beneficial for treatment of other viruses in the same family including Dengue virus.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P2-07-03: Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line

Abstract Several publications have addressed concerns surrounding drug response screens by pointing out sources of variability and by presenting recommendations for better experimental methods and more robust analytical approaches. In the presented profiling effort, we integrated the latest advances in drug response measurement and focused on data diversity and quality rather than on breadth. We selected 32 breast cancer cell lines with a strong bias towards triple negative lines as well as 4 cell lines established from relevant patient-derived xenografts. We evaluated a panel of clinically relevant kinase inhibitors using a microscopy-based dose response assay to measure drug potency, and to quantify drug efficacy in terms of growth inhibition (GR metrics) and cell death. The use of the GR metrics to quantify drug sensitivity enabled us to identify and study differences between cytostatic and cytotoxic responses. This systematic dose response dataset is complemented by measurements of baseline transcript expression levels by mRNAseq, quantification of absolute abundance of ˜12,000 proteins, and relative phosphoprotein levels by shotgun mass spectrometry across all cell lines. Additionally, the baseline activity of transcription factors and kinases were inferred from the mRNA (using VIPER) and phosphoprotein (using kinase enrichment analysis) data, respectively. The complementarity of these multi-omics data has allowed us to address questions about the landscape of breast cancer cell lines such as: Where do the patient-derived lines lay relative to the conventional cell lines? How consistent are the landscapes defined by each dataset? How does integration across datasets provide mechanistic insight into signaling pathways that are active in each cancer subtypes? The measured and inferred baseline data were used to build predictors of the observed drug responses with the goal of identifying the biological processes responsible for the differences in sensitivity across drugs and cell lines. Overall the dataset that has been collected is a valuable resource for understanding drug response in triple negative breast cancer, and the molecular mechanisms that influence it. Citation Format: Mills CE, Subramanian K, Hafner M, Chung M, Boswell SA, Everley RA, Juric D, Sorger PK. Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-03.

Abstract PD4-02: Advantageous polypharmacology of abemaciclib revealed by omics profiling of CDK4/6 inhibitors

Abstract Small molecule inhibitors of cyclin-dependent kinases (CDK) 4/6 have created new opportunities for the treatment of advanced hormone-receptor positive (HR+) breast cancer and show promise in other malignancies. Three CDK4/6 inhibitors, palbociclib (PD0332991; Ibrance), ribociclib (LEE011; Kisqali), and abemaciclib (LY2835219), which have been approved or are in late stage trials, are reported to be broadly similar although recent data suggest that abemaciclib has distinct single-agent activity in patients and a unique adverse effects profile. Differences in pharmacokinetics and relative potency for CDK4 versus CDK6 are postulated to account for these differences. In this paper, we use molecular and functional profiling by mRNA sequencing, mass spectrometry-based proteomics, and GR-based dose-response assays to obtain complementary views of the mechanisms of action of CDK4/6 inhibitors. We show that abemaciclib, but not ribociclib or palbociclib, is a potent inhibitor of kinases other than CDK4/6, including CDK1/Cyclin B, which appears to cause arrest in the G2 phase of the cell cycle, and CDK2/Cyclin E/A, which is implicated in resistance to palbociclib. Whereas ribociclib and palbociclib induce cytostasis, and cells adapt to these drugs within 2-3 days of exposure, abemaciclib induces cell death and durably blocks cell proliferation. Abemaciclib is active even in retinoblastoma protein (pRb)-deficient cells in which CDK4/6 inhibition by palbociclib or ribociclib is completely ineffective. The degree of polypharmacology of small molecule drugs is increasingly viewed as an important consideration in their design, with implications for efficacy, toxicity, and acquired resistance. In the case of CDK4/6 inhibitors, we propose that abemaciclib polypharmacology elicits unique molecular responses that are likely to be therapeutically advantageous. More generally, we propose that multi-omic approaches are required to fully elucidate the spectrum of targets relevant to drug action in tumor cells. We expect such understanding to assist in stratifying patient populations and ordering sequential therapies when resistance arises. Citation Format: Hafner M, Mills CE, Subramanian K, Chen C, Boswell SA, Everley RA, Juric D, Sorger PK. Advantageous polypharmacology of abemaciclib revealed by omics profiling of CDK4/6 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-02.

Validation of water-borne steroid hormones in a tropical frog (Physalaemus pustulosus)

Minimally invasive methods for estimating hormone concentrations in wild vertebrates offer the opportunity to repeatedly measure behavior and hormone concentrations within individuals while minimizing experimenter interference during sample collection. We examined three steroid hormones (corticosterone, CORT; 17-β estradiol, E2; progesterone, PROG) in túngara frogs (Physalaemus pustulosus) using non-invasive water-borne methods. Using solid-phase extraction of water samples and liquid extraction of plasma and homogenate samples, coupled with enzyme immunoassays, we complimented the conventional validation approaches (parallelism, recovery determination) with dose-response assays that incorporated pharmacological challenges with adrenocorticotropic hormone (ACTH) and human chorionic gonadotropin (HCG). We also compared steroid concentrations in water to those observed in plasma and whole body homogenates. Lastly, we identified the constituent steroids in each sample type with a panel targeting 30 steroid species using high performance liquid chromatography-mass spectrometry (HPLC-MS). We found that a 60-min water-bath captures physiologically relevant changes in concentrations of CORT, E2 and PROG. Peak levels of water-borne CORT were found at approximately 2 h after ACTH injection. Water-borne CORT and E2 concentrations were positively correlated with their plasma and homogenate equivalents, while water-borne PROG was uncorrelated with homogenate PROG concentrations but negatively correlated with homogenate E2 concentrations. Together, our findings indicate that sampling water-borne hormones presents a non-invasive and biologically informative approach that will be useful for behavioral endocrinologists and conservation physiologists.

Development of an Automated Electrophysiology Assay for Kv1.3 using Nanion Syncropatch 384PE

Recently, a couple of giga-seal automated patch clamp (APC) platforms in a standard 384-well plate format have been introduced. These APC instruments can deliver higher throughput measurements with high-quality data for ion channel drug discovery. In this study, we developed an automated electrophysiology assay for the voltage-gated potassium channel, Kv1.3 as a test case on the Nanion SyncroPatch 384PE, one of the latest APC platforms. We achieved a high cell-catch rate (98% wells/plate, defined by membrane resistance >16 MΩ/cell) by screening plate types (number of holes and resistance), and obtained a stable seal (87% wells/plate, defined by initial/final seal resistance >500 MΩ/cell) by optimizing recording solutions. We generated data at an overall success rate (defined by initial peak current >300 pA/cell, initial/final seal resistance >500 MΩ/cell, and current stability <±7% change/min) of 79% on average. Using the same platform, we validated a dose-response assay using clotrimazole, a potent Kv1.3 inhibitor. The assay was robust with Z' factor 0.52 and the success rate 75%. Altogether, our results demonstrated that the SyncroPatch represents a reliable platform for voltage-gated potassium channel assays.

Antitrypanosomal, antiplasmodial, and antibacterial activities of extracts from selected Diospyros and Annonaceae species

Aim: To screen methanol extracts from root bark, leaves, and stem bark of selected plant species from the genus Diospyros and some Annonaceae species for antitrypanosomal, antiplasmodial, and antibacterial activities against selected test organisms. Methods: Antitrypanosomal and antiplasmodial assays of methanol extracts from selected plant species were carried out in single concentration screens and in dose-response for active extracts. The minimum inhibitory concentration (MIC) values of selected plant extracts against selected bacterial strains were determined by microplate dilution method in sterile 96-well microtiter plates. Results: In the dose-response antitrypanosomal assay, the most potent extracts tested exhibited activities against Trypanosoma brucei brucei (Lister 427 strain) with IC50 values ranging from 1.28 to 7.85 μg/ml, with methanol extract of Diospyros verrucosa stem bark being the most active with IC50 value of 1.28 μg/ml. In the dose-response antiplasmodial assay, three extracts exhibited activities against Plasmodium falciparum(strain 3D7) with IC50 values ranging from 4.55 to 24.22 μg/ml, with methanol extract of Diospyros capricornuta root bark being the most potent with IC50 value of 4.55 μg/ml. In the antibacterial assay, the investigated extracts exhibited a wide range of activities against Staphylococcus aureus [American Type Culture Collection (ATCC) strain 25923], Bacillus cereus (ATCC strain 11775), and Escherichia coli (ATCC strain 8740) with MIC values ranging from 0.00125 to 0.00625 mg/ml (more active), 0.125 to 0.500 mg/ml (moderately active), and 1.00 to 8.00 mg/ml (less active) while some extracts were inactive at the highest concentration tested of 16.00 mg/ml. Conclusions: Methanol extracts obtained from root bark, leaves, and stem bark of selected plant species from the genus Diospyros and some Annonaceae species that showed good activities in antitrypanosomal, antiplasmodial, and antibacterial assays corroborate reported literature about the traditional medicinal uses of the members of genus Diospyros and some Annonaceae species.

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation–based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

Transgene escape and persistence in an agroecosystem: the case of glyphosate-resistant Brassica rapa L. in central Argentina.

Brassica rapa L. is an annual Brassicaceae species cultivated for oil and food production, whose wild form is a weed of crops worldwide. In temperate regions of South America and especially in the Argentine Pampas region, this species is widely distributed. During 2014, wild B. rapa populations that escaped control with glyphosate applications by farmers were found in this area. These plants were characterized by morphology and seed acidic profile, and all the characters agreed with B. rapa description. The dose-response assays showed that the biotypes were highly resistant to glyphosate. It was also shown that they had multiple resistance to AHAS-inhibiting herbicides. The transgenic origin of the glyphosate resistance in B. rapa biotypes was verified by an immunological test which confirmed the presence of the CP4 EPSPS protein and by an event-specific GT73molecular marker. The persistence of the transgene in nature was confirmed for at least 4years, in ruderal and agrestal habitats. This finding suggests that glyphosate resistance might come from GM oilseed rape crops illegally cultivated in the country or as a seed contaminant, and it implies gene flow and introgression between feral populations of GM B. napus and wild B. rapa. The persistence and spread of the resistance in agricultural environments was promoted by the high selection pressure imposed by intensive herbicide usage in the prevalent no-till farming systems.

Ocimum basilicum essential oil combined with deltamethrin to improve the management of Spodoptera frugiperda

ABSTRACT For an important and expensive crop such as corn, the resistance of Spodoptera frugiperda J.E. Smith to various pesticides has led to research throughout the world for a potential insecticide from a natural source. For the management of pest resistance, natural compounds associated with synthetic insecticides can be a promising tool because they can reduce the application of the synthetics molecules while maintaining their effectiveness and promoting the control of the pests. Linalool is a potential insecticide that is easily obtained because it is found in high concentrations in the essential oil of Ocimum basilicum L. Therefore, the present study aimed to evaluate the toxicity of this essential oil and its combination with deltamethrin to control S. frugiperda. Through dose response assays, the acute toxicities (LD50) of the essential oil and deltamethrin were estimated. Additionally, the combination of these materials was also assessed, attaining a reduction of 80% of the LD50 of deltamethrin while obtaining the same result as when the pyrethroid was administered alone. From these results, it is expected that the combination of natural compounds and synthetic insecticides will be a promising practice, helping to manage resistance while reducing the environmental impact of toxic compounds.

Investigation of the acaricidal activity of the acetone and ethanol extracts of 12 South African plants against the adult ticks of Rhipicephalus turanicus.

The acaricidal activity of acetone and ethanol extracts of 12 plant species was evaluated using the contact method on Rhipicephalus turanicus (Acari: Ixodidae) ticks at an initial concentration of 20% (200 mg/mL). Eight of the 12 plants had mortality greater than 50% and the acetone extracts had better acaricidal activity than the ethanol extracts. The acetone extract of Calpurnia aurea (leaves and flowers) had the highest corrected mortality (CM) of 92.2% followed by Schkuhria pinnata (whole plant) with a CM of 88.9%, Ficus sycomorus (bark and stems) 86.7% and Senna italica subsp. arachoides (roots, leaves and fruits) 83.3%. Selected extracts were tested at five different concentrations using the adult immersion test. From dose–response assays, EC50 values of 61.82 mg/mL, 115.21 mg/mL and 161.02 mg/mL were obtained for the acetone extracts of S. pinnata (whole plant), S. italica subsp. arachoides (roots, leaves and fruits) and C. aurea (leaves and flowers) respectively. The ethanol extract of Monsonia angustifolia (whole plant) had the highest CM of 97.8% followed by S. pinnata (whole plant) with a CM of 86.7%, C. aurea (leaves and flowers) 81.1% and Cleome gynandra (leaves) 77.8%. There is potential for the development of environmentally benign botanicals as natural acaricides against R. turanicus.

Comparative toxicological evaluation of nonylphenol and nonylphenol polyethoxylates using human keratinocytes

Nonylphenol polyethoxylates (NPEOs) are a major group of nonionic surfactants widely used in various detergents, cleaners, plastics, papers, and agro-chemical products. Nonylphenol (NP), which is a final degraded metabolite derived from NPEOs, has been reported as an endocrine disrupter, known to mimic or disturb reproductive hormone functions. Concern about the hazards of NP and NPEOs has generated legal restrictions and action plans worldwide. Considering the fact that NP and NPEOs are majorly used in the production of products such as detergents, shampoos, and cosmetics which frequently come into contact with the skin, we investigated the effects of NP and NPEOs on a human keratinocyte cell line (HaCaT). In this study, the toxicity of NP and NPEOs was screened in HaCaT cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue assay and Western blotting. The potential cytotoxicity of substitutes was assessed by dose-response assays, relative cell viability, and genotoxicity caused by specific alterations in DNA damage response proteins (including ataxia-telangiectasia mutated, p53, Chk1, Chk2, and Histone H2A.X). We demonstrated that NP and NPEOs are toxic to HaCaT cells, as revealed by the decreased cell viability after 24 h treatment. NPs and NPEOs also induced apoptosis and DNA damage as shown by the activation of Poly(ADP-ribose) polymerase, Caspase-3, and Histone H2A.X.

Target-site resistance to acetolactate synthase (ALS)-inhibiting herbicides in Amaranthus palmeri from Argentina.

Herbicide-resistant weeds are a serious problem worldwide. Recently, two populations of Amaranthus palmeri with suspected cross-resistance to acetolactate synthase (ALS)-inhibiting herbicides (R1 and R2) were found by farmers in two locations in Argentina (Vicuña Mackenna and Totoras, respectively). We conducted studies to confirm and elucidate the mechanism of resistance. We performed in vivo dose-response assays, and confirmed that both populations had strong resistance to chlorimuron-ethyl, diclosulam and imazethapyr when compared with a susceptible population (S). In vitro ALS activity inhibition tests only indicated considerable resistance to imazethapyr and chlorimuron-ethyl, indicating that other non-target mechanisms could be involved in diclosulam resistance. Subsequently, molecular analysis of als nucleotide sequences revealed three single base-pair mutations producing substitutions in amino acids previously associated with resistance to ALS inhibitors, A122, W574, and S653. This is the first report of als resistance alleles in A. palmeri in Argentina. The data support the involvement of a target-site mechanism of resistance to ALS-inhibiting herbicides. © 2017 Society of Chemical Industry.

First Case of Multiple Resistance to Glyphosate and PPO-inhibiting Herbicides in Rigid Ryegrass (Lolium rigidum) in Spain

Five rigid ryegrass populations suspected of being resistant to both glyphosate and oxyfluorfen were collected in southern Spain and tested under laboratory-controlled conditions. Four populations (Depuradora, Condado, AlamoRasilla, and Portichuelo) were treated with glyphosate for at least 15 consecutive years, and treatments during the last 5 yr were mixed with oxyfluorfen. The fifth population (4alamos) followed the same glyphosate treatment, although oxyfluorfen was never used to control it. Dose–response assays confirmed glyphosate resistance in all populations, with resistance indexes ranging from 11.7 to 37.5 (GR90). Shikimate accumulation assays consistently supported these data, as the most glyphosate-resistant populations (Depuradora and Condado) displayed the lowest shikimate levels. Surprisingly, four populations (Depuradora, Condado, AlamoRasilla, and Portichuelo) displayed 7.93- to 70.18-fold more resistance (GR90) to oxyfluorfen, despite limited selection pressure, showing a similar resistance pattern as that for glyphosate. The 4alamos population displayed oxyfluorfen GR90values that were similar to those observed in susceptible plants; however, this population was significantly more resistant in terms of plant survival (LD90). Protoporphyrin IX accumulation assays supported the results of dose–response assays, in that the most oxyfluorfen-resistant populations accumulated less protoporphyrin IX. Although more studies are needed, it seems that these five glyphosate-resistant weed populations display a natural tendency to easily develop resistance to oxyfluorfen, with the populations that have higher resistance to glyphosate also having higher resistance to oxyfluorfen.

Pro-106-Ser mutation and EPSPS overexpression acting together simultaneously in glyphosate-resistant goosegrass (Eleusine indica)

Glyphosate has been used for more than 15 years for weed management in citrus groves in the Gulf of Mexico, at up to 3–4 applications per year. Goosegrass (Eleusine indica (L.) Gaertn.) control has sometimes failed. In this research, the mechanisms governing three goosegrass biotypes (Ein-Or from an orange grove, and Ein-Pl1 and Ein-Pl2 from Persian lime groves) with suspected resistance to glyphosate were characterized and compared to a susceptible biotype (Ein-S). Dose-response and shikimate accumulation assays confirmed resistance of the resistant (R) biotypes. There were no differences in glyphosate absorption, but the R biotypes retained up to 62–78% of the herbicide in the treated leaf at 96 h after treatment (HAT), in comparison to the Ein-S biotype (36%). The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity in the Ein-Or and Ein-S biotypes was over 100-fold lower than the Ein-Pl1 and Ein-Pl2 ones. The latter showed a high EPSPS-basal activity, a mutation at Pro-106-Ser position in the EPSPS gene, and EPSPS overexpression. The EPSPS basal and EPSPS overexpression were positively correlated. The R goosegrass biotypes displayed poor glyphosate translocation. Furthermore, this grassweed showed, for the first time, two mechanisms at the target-site level (Pro-106-Ser mutation + EPSPS overexpression) acting together simultaneously against glyphosate.