Dose-response Assays Research Articles

Annual bluegrass cross resistance to prodiamine and pronamide in the southern United States

Abstract Annual bluegrass is one of the most problematic weeds in the turfgrass industry, exhibiting both cross-resistance and multiple-herbicide resistance. Prodiamine, pronamide, and indaziflam are commonly used preemergence herbicides for the control of this species on golf courses in the southern United States. There have been increasing anecdotal reports of annual bluegrass populations escaping control with these herbicides, but resistance has yet to be confirmed. To evaluate the response of annual bluegrass to three herbicides, populations were collected from golf courses, athletic fields, and landscape areas in Texas and Florida, and a dose-response assay was conducted on populations that were suspected to be resistant to and known to be susceptible to prodiamine, pronamide, and indaziflam. The suspected-resistant populations showed survival to prodiamine at 32 times the recommended field rate (both populations from Florida and Texas) of 736 g ai ha−1, and to pronamide at 32 times (the Florida populations) or 16 times (the Texas populations) the recommended field rate of 1,156 g ha−1. In contrast, the known susceptible populations attained 100% mortality at rates as low as 46 and 578 g ha−1, respectively, from applications of prodiamine and pronamide. For indaziflam, the suspected-resistant populations showed reduced sensitivity up to the recommended field rate of 55 g ha−1, but they were controlled when treated with a rate twice that of the field rate. Overall, annual bluegrass populations with resistance to prodiamine and pronamide, and reduced sensitivity to indaziflam (at the recommended field rate) were confirmed from golf courses in Florida and Texas. In the presence of herbicide-resistant annual bluegrass populations, especially to commonly used herbicides such as prodiamine and pronamide, turfgrass managers should adopt integrated management strategies and frequently rotate herbicide sites of action, rather than relying solely on microtubule-assembly inhibitors or cellulose biosynthesis inhibitors, to control this species.

Inhibition of survivin by 2'-O-methyl phosphorothioate-modified steric-blocking antisense oligonucleotides.

Chemically modified antisense oligonucleotide (ASO) has been established as a successful therapeutic strategy for treating various human diseases. To date, ten ASO drugs, which are capable of either inducing mRNA degradation via RNase H recruitment (fomivirsen, mipomersen, inotersen, volanesorsen and tofersen) or splice modulation (eteplirsen, nusinersen, golodirsen, viltolarsen and casimersen), have been approved by the regulatory agencies for market entry. Nonetheless, none of these approved drugs are prescribed as cancer therapy. Towards this, we have developed steric-blocking ASOs targeting BIRC5 - a well-validated oncogene. Initial screening was performed by transfection of HepG2 cells with seven BIRC5 exon-2 targeting, uniformly 2'-OMe-PS modified ASOs at 400 nM respectively, leading to the identification of two best-performing candidates ASO-2 and ASO-7 in reducing the production of BIRC5 mRNA. Subsequent dose-response assay was conducted via transfection of HepG2 cells by different concentrations (400, 200, 100, 50, 25 nM) of ASO-2 and ASO-7 respectively, showing that both ASOs consistently and efficiently inhibited BIRC5 mRNA expression in a dose-dependent manner. Furthermore, western blot analysis confirmed that ASO-7 could significantly repress survivin production on protein level. Based on our preliminary results, we believe that ASO-7 could be a useful BIRC5 inhibitor for both research purpose and therapeutic development.

The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification.

For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 μM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore's ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 μM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 μM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 μM Dynasore. Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.

An Atypical Somatic Calr Variant Acquired in Cis to a Germline Calr variant Induces Constitutive Signaling of the Thrombopoietin Receptor and Results in Essential Thrombocythemia

Acquired mutations in the gene encoding the endoplasmic reticulum (ER) chaperone calreticulin (CALR) are responsible for ~ 30% of essential thrombocythemia (ET) and myelofibrosis (MF) cases. Two CALR mutations, a deletion of 52 base pairs (del52) and an insertion of 5 base pairs (ins5) are the most frequent in ET and MF patients. All CALR mutations result in a +1-frameshift leading to 1) the replacement of the negatively-charged wild-type (WT) C-terminus that binds calcium in the ER by a positively-charged mutant tail including 44 residues that are common to all CALR mutant proteins and 2) the loss of the ER retention KDEL; together leading to the deregulation of calcium homeostasis. The CALR mutants are also secreted and bind to MPL in the ER but induce its constitutive dimerization and downstream signaling pathways at the cell surface. A 16-year-old girl presented with a platelet count of 797 x 10 3/mL rising to 1,228 x 10 3/mL within 2 years. After Informed Consent, we detected two atypical CALR variants in her blood cells by NGS analysis: V1 (c.1122-1125del) at 47% variant allele frequency (VAF) and V2 (c.1227-1231del) in cis of the V1 leading to V1-V2 (c.1122-1125del ; c.1227-1231del) at 5% VAF. The presence of V1 in blood and nails from the propositus and her healthy mother confirmed its germline origin. The V1-V2 was only found in the propositus at 5% in isolated monocytes and at very low levels in NK and B cells, confirming its somatic acquisition. The germline V1 induces a frameshift resulting in the replacement of the CALR WT C-terminus by the positively charged 44 residue-long C-terminal mutant tail present in del52 and ins5 and the loss of KDEL together with an insertion of 10 amino acids not present in CALR del52 nor ins5. In contrast, the V1-V2 generates a similar but shortened C-terminus containing the Arg-rich MPL binding site and KDEL (Fig. 1A). To investigate the functional impact of these CALR variants, we cloned V1 and V1-V2 with a N-terminal hemagglutinin (HA) tag as well as CALR WT and del52 as controls in a retroviral plasmid. Viral particles were produced to transduce Ba/F3 cell lines expressing MPL. Structural modeling using AlphaFold was used to investigate the role of KDEL restoration in V1-V2 in producing an oncogenic protein leading to ET. Both CALR WT and V1-V2 variants that contain the KDEL motif were detected to a similar extent in Ba/F3-MPL cell lysates. In contrast, del52 and V1, that lost the ER-retention KDEL motif, were barely detected in cell lysates. Unexpectedly, despite the presence of the KDEL motif, V1-V2 was found at a similar level than del52 at the Ba/F3-MPL cell surface. In contrast, V1 was barely detectable at the cell surface, like CALR WT. In order to investigate the role of the KDEL in V1-V2 cell surface expression, we generated a CALR WT lacking the KDEL and added the KDEL motif to del52. The presence of the KDEL motif stabilized the intracellular expression of del52 without affecting cell surface localization while the removal of the KDEL motif in CALR WT appeared to destabilize the protein and induce a slight leak to the cell surface. Structural modeling using AlphaFold predicted that the KDEL in V1-V2 structure is accessible to KDEL receptors, suggesting that the motif may be masked by the interaction of V1-V2 with MPL while trafficking to the cell surface. Indeed, cell-surface expression of V1-V2 was decreased in Ba/F3 that do not express MPL. We show that V1-V2, but not V1, constitutively activates the phosphorylation of STATs, ERK and AKT in Ba/F3-MPL. TPO further increases activation of STATs but not of AKT and ERK by V1-V2 and del52. Coherently, V1-V2, but not V1, sustained oncogenic MPL stimulation able to sustain cell autonomous Ba/F3-MPL cell proliferation (Fig. 1B). Both del52 and V1-V2 up-regulated BCL-2 mRNA. Dose-response cytotoxicity assays with the BCL-2 inhibitor venetoclax showed that these two mutants are sensitive to lower doses compared to CALR WT- or V1-expressing Ba/F3-MPL cells. This is the first report of a germline loss-of-function CALR variant (V1) expressed as an unstable protein and that might predispose to the acquisition of a somatic gain-of-function CALR variant (V1-V2) responsible for ET. The presence of KDEL in V1-V2 leads to stabilized CALR expression that does not prevent cell surface localization. Moreover, it leads to induction of MPL signaling and cytokine-independent cell growth.

HAD2 Regulates Central Carbon Metabolism in Malaria Parasite P. falciparum

Abstract Background The malaria parasite Plasmodium falciparum causes nearly half a million deaths each year. Widespread resistance to antimalarials prompts an urgent need for novel drug targets both unique and essential to the parasite. The methylerythritol (MEP) pathway of isoprenoid biosynthesis fits this profile. Previous studies showed that P. falciparum controls substrates available to the MEP pathway via the phosphatase HAD2. A point mutation in the HAD2 gene was found to misdirect metabolites from glycolysis to the MEP pathway. Elucidating the mechanism by which HAD2 controls central carbon metabolism will inform our understanding of essential parasite biology and the potential for resistance to metabolic inhibitors under development. Methods In this work, we have generated a P. falciparum strain in which HAD2 expression is under control of anhydrous tetracycline (aTc), such that growth in the absence of aTc leads to loss of HAD2 expression. We have used this tunable knockdown strain to regulate expression of HAD2 in asexual-stage parasites to test whether HAD2 is required for glycolytic and isoprenoid biosynthesis homeostasis. Asexual parasite growth rate was measured through flow cytometry, and dose-response assays were performed against a competitive small molecule inhibitor of the MEP pathway. Targeted metabolite profiling with liquid chromatography-tandem mass spectrometry (LC-MS/MS) is in process to quantify glycolytic and isoprenoid metabolites in this knockdown strain. Results Knockdown parasites off aTc exhibited reduced growth rates relative to the wild-type strain. These parasites also exhibited a 4-fold increase in resistance to a competitive inhibitor of the MEP pathway compared to the wild-type [0.755 +/- 0.12 µM vs. 3.201 +/- 0.35 µM; p<0.001). Conclusions Reduced expression of HAD2 confers resistance to a competitive inhibitor of the MEP pathway, consistent with the effect of elevated levels of MEP pathway intermediates as a result of disrupted homeostasis. Ongoing work will use this strain to understand the role of HAD2 on parasite metabolism, parasite growth, and drug resistance.

The use of in silico molecular modelling to screen potential estrogen mimics as part of medicines and agrochemicals development and product license applications.

Estrogen mimics are a diverse group of synthetic and naturally occurring compounds that can interact with estrogen receptors (ERs) in animals, including humans. These interactions rely on key structural features of 17b-estradiol (E2) and if these molecular features are in a similar spatial arrangement on other compounds, they are likely to elicit an agonist (i.e., they are E2 mimics) or antagonist response. The structural diversity of some compounds vis-à-vis analogies with E2 makes it difficult to reliably predict E2 mimicry on simple structural grounds alone. We propose a new approach methodology: in silico molecular modelling augmented by an in vitro transactivation reporter gene assay to predict E2 mimicry and thus further reduce regulatory reliance on animal studies. Transactivation reporter gene assay dose response curves and in silico molecular modelling were used to obtain EC50-values and docking parameters (DockScores), respectively of thirty E2 mimics to assess the reliability of in silico receptor interaction parameters to predict E2 mimicry. A linear relationship (R2 = 0.75) was found between DockScores and EC50s, suggesting molecular modelling is a good tool for predicting E2 mimicry in a regulatory setting.

Enhanced metabolic resistance mechanism endows resistance to metamifop in Echinochloa crus-galli (L.) P. Beauv.

Barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.), one of the worst weeds in paddy fields in China, has been frequently reported evolving resistance to acetyl-CoA carboxylase (ACCase) inhibiting herbicides. However, in the previous research, more attention was paid to target-site resistance (TSR) mechanisms, the non-target-site resistance (NTSR) mechanisms have not been well-established. In this study, the potential mechanism of resistance in a metamifop-resistant E. crus-galli collected from Kunshan city, Jiangsu Province, China was investigated. Dose-response assays showed that the phenotypic resistant population (JS-R) has evolved 4.3-fold resistance to metamifop compared with the phenotypic susceptible population (YN-S). The ACCase CT gene sequencing and relative ACCase gene expression levels studies showed that no mutations were detected in the ACCase CT gene in both YN-S and JS-R, and there was no significant difference in the relative ACCase gene expression between YN-S and JS-R. After the pre-processing of glutathione-S-transferase (GSTs) inhibitor NBD-Cl, the resistance level of JS-R to metamifop was reversed 18.73%. Furthermore, the GSTs activity of JS-R plants was significantly enhanced compared to that of YN-S plants. UPLC-MS/MS revealed that JS-R plants had faster metabolic rates to metamifop than YN-S plants. Meanwhile, the JS-R popultion exhibited resistant to cyhalofop-butyl and penoxsulam. In summary, this study presented a novel discovery regarding the global emergence of metabolic resistance to metamifop in E. crus-galli. The low-level resistance observed in the JS-R population was not found to be related to TSR but rather appeared to be primarily associated with the overexpression of genes in the GSTs metabolic enzyme superfamily.

Adjusting the Fitting of Fluorescence-Based Dose-Response Kinase Inhibition Assay to Account for Fluorescent Inhibitors.

Fluorescence is routinely used to monitor kinase inhibition in commercial assays. Occasionally fluorescent compounds can interfere with the fluorescent reading. To address this issue, the problematic data are usually truncated to improve the fit, however, this approach raises ethical and reproducibility concerns. Instead, it is suggested to adjust the fitting formula, to account for the autofluorescence of the compounds and improve the fit of the data compared with a naive approach. Finally, it was noticed that truncating the data can result in a small underestimation of the IC50 values and should therefore be used carefully.

P22-43: The GARDskin Dose-Response assay for determination of a point-of-departure (PoD) for Next Generation Risk Assessment (NGRA) of skin sensitizers: A case study using isocyclocitral

P22-43: The GARDskin Dose-Response assay for determination of a point-of-departure (PoD) for Next Generation Risk Assessment (NGRA) of skin sensitizers: A case study using isocyclocitral

Quizalofop resistance in weedy rice (Oryza sativa L.) is mainly conferred by an Ile1781Leu mutation

Weedy rice (Oryza sativa L.) is an economically important weed species in rice (Oryza sativa L.) cropping systems. Two weedy rice samples (acc7 and acc8) suspected to be resistant to quizalofop-ethyl (quizalofop) were collected in Arkansas. In this research, susceptibility to quizalofop and resistance mechanisms have been explored. Dose-response assays displayed a resistance index of 42- and 58-fold for the acc7 and acc8, respectively. Experiments with metabolism inhibitors demonstrated that NBD-Cl (4-chloro-7-nitrobenzofurazan) increased quizalofop efficacy slightly in acc8, whereas malathion did not improve effectiveness in resistant samples. Sequencing of the ACCase gene displayed an Ile1781Leu substitution in the resistant samples, like the mutation present in Provisia™ rice. In addition, an allele-specific PCR was developed to genotype the Ile1781Leu mutation. The gene copy number of ACCase showed similar values among samples. In the resistant plants, a KASP (Kompetitive Allele Specific PCR) assay to detect the ALSS653D (acetolactate synthase) and HIS1 (HPPD Inhibitor Sensitive 1) traits revealed that 37.5% of plants carried the ALSS653D trait, whereas 25% showed the HIS1 allele. In summary, a target-site mutation is the main resistance mechanism to quizalofop in weedy rice. Results also suggest the presence of herbicide metabolism (a non-target site resistance mechanism) mediated by glutathione-S-transferases (GSTs) in one resistant sample.

Heterologous production of the insecticidal pea seed albumin PA1 protein by Pichia pastoris and protein engineering to potentiate aphicidal activity via fusion to snowdrop lectin Galanthus nivalis agglutinin; GNA)

BackgroundNew bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide. The purpose of this study was to investigate if Pichia pastoris could be used to express a functional PA1b peptide and if it’s insecticidal activity could be enhanced via engineering to produce a fusion protein comprising the pea albumin protein fused to the mannose-specific snowdrop lectin (Galanthus nivalis agglutinin; GNA).ResultsWe report the production of a recombinant full-length pea albumin protein (designated PAF) and a fusion protein (PAF/GNA) comprised of PAF fused to the N-terminus of GNA in the yeast Pichia pastoris. PAF was orally toxic to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective, Day 5 LC50 values of 54 µM and 105 µM derived from dose–response assays. PAF/GNA was significantly more orally toxic as compared to PAF, with LC50 values tenfold (5 µM) and 3.3-fold (32 µM) lower for pea and peach potato aphids, respectively. By contrast, no phenotypic effects were observed for worker bumble bees (Bombus terristrus) fed PAF, GNA or PAF/GNA in acute toxicity assays. Confocal microscopy of pea aphid guts after pulse-chase feeding fluorescently labelled proteins provides evidence that enhanced efficacy of the fusion protein is attributable to localisation and retention of PAF/GNA to the gut epithelium. In contact assays the fusion protein was also found to be significantly more toxic towards A. pisum as compared to PAF, GNA or a combination of the two proteins.ConclusionsOur results suggest that GNA mediated binding to V-type ATPase pumps acts to potentiate the oral and contact aphicidal activity of PAF. This work highlights potential for the future commercial development of plant protein-based bioinsecticides that offer enhanced target specificity as compared to chemical pesticides, and compatibility with integrated pest management strategies.

Blueberries infected with the fungal pathogen Colletotrichum fioriniae release odors that repel Drosophila suzukii.

Spotted-wing drosophila, Drosophila suzukii, is a serious pest of thin-skinned fruits. Alternative methods to control this pest are needed to reduce insecticide use, including new repellents. Previous research demonstrated that D. suzukii adults use odor cues to avoid blueberries infected with the fungal pathogen Colletotrichum fioriniae, which causes the disease anthracnose. To identify novel D. suzukii repellents, we investigated the volatile emission from experimentally-infected fruit, which were inoculated with C. fioriniae isolates in the laboratory, and from field-collected fruit, which were naturally infected and harvested from a field. We then tested the pathogen-induced volatiles on D. suzukii adult behavior. Volatile emission was similar between all five C. fioriniae strains, with good agreement between experimentally-infected and field-collected berries. In total, 14 volatiles were found to be more abundant in infected versus uninfected fruit headspace. In multiple-choice bioassays, nine of the 14 volatiles elicited repellency responses from adult D. suzukii. These nine volatiles were further evaluated in dual choice assays, where all nine reduced fly capture by 43-96% compared to the control. The most repellent compounds tested were the esters ethyl butanoate and ethyl (E)-but-2-enoate, which were more or equally repellent to the known D. suzukii repellents 1-octen-3-ol, geosmin, and 2-pentylfuran. Dose-response assays identified concentration-dependent effects on D. suzukii repellency and oviposition when applied individually and consistent aversion observed across doses of a 1:1 blend. We report two repellents from C. fioriniae-infected blueberries that could be useful semiochemicals for the behavioral manipulation of D. suzukii in the field. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

The Effects of the Combination of Rhein and Platelet-Rich Plasma on Human Articular Chondrocytes.

The presence of side effects and low bioavailability of rhein has limited its use in the treatment of osteoarthritis. We aimed to evaluate the in vitro response of human articular chondrocytes to the presence of the combination of platelet-rich plasma (PRP) and rhein. Solutions of rhein were prepared to assess solubility and select a working concentration. A stimulus with interleukin-1β (IL-β, 10 ng/mL) was induced for 24 h on human chondrocytes. Five treatment groups were established: control, IL-β control, PRP, rhein, and PRP + rhein. Cell viability, cell migration, nitric oxide (NO) production, tumor necrosis factor-α (TNF-α), and gene expression analyses were carried out. A concentration of 50 mg/L was selected after a dose-response curve assay. Both NO and tumor TNF-α production significantly decreased after PRP and PRP + rhein treatments at 24 and 48 h. The wound healing assay revealed a significant stimulation of migration after 72 h with the PRP and PRP + rhein treatments. Expression of IL-1β, IL-6, MMP-13, and ADAMTS-5 was significantly downregulated, particularly after treatment with the combination of PRP + rhein. Much of the determinations denoted a better performance of the combination of PRP and rhein in decreasing the levels of the different targets evaluated; however, this was not great enough to detect a significant difference in comparison with the PRP treatment alone.

MicroRNAs miR-16 and miR-519 control meningioma cell proliferation via overlapping transcriptomic programs shared with the RNA-binding protein HuR.

Meningiomas are the most common type of primary central nervous system tumors. In about 80% cases, these tumors are benign and grow very slowly, but the remainder 20% can unlock higher proliferation rates and become malignant. In this study we examined two miRs, miR-16 and miR-519, and evaluated their role in tumorigenesis and cell growth in human meningioma. A cohort of 60 intracranial grade 1 and grade 2 human meningioma plus 20 healthy meningeal tissues was used to quantify miR-16 and miR-519 expressions. Cell growth and dose-response assays were performed in two human meningioma cell lines, Ben-Men-1 (benign) and IOMM-Lee (aggressive). Transcriptomes of IOMM-lee cells were measured after both miR-mimics transfection, followed by integrative bioinformatics to expand on available data. In tumoral tissues, we detected decreased levels of miR-16 and miR-519 when compared with arachnoid cells of healthy patients (miR-16: P=8.7e-04; miR-519: P=3.5e-07). When individually overexpressing these miRs in Ben-Men-1 and IOMM-Lee, we observed that each showed reduced growth (P<0.001). In IOMM-Lee cell transcriptomes, downregulated genes, among which ELAVL1/HuR (miR-16: P=6.1e-06; miR-519:P=9.38e-03), were linked to biological processes such as mitotic cell cycle regulation, pre-replicative complex, and brain development (FDR<1e-05). Additionally, we uncovered a specific transcriptomic signature of miR-16/miR-519-dysregulated genes which was highly enriched in HuR targets (>6-fold; 79.6% of target genes). These results were confirmed on several public transcriptomic and microRNA datasets of human meningiomas, hinting that the putative tumor suppressor effect of these miRs is mediated, at least in part, via HuR direct or indirect inhibition.

Herbivore intraguild interactions decrease ectoparasitoid Bracon nigricans parasitism of Phthorimaea absoluta and responses to tomato volatiles

Intraguild interactions are known to trigger competition among phytophagous insects that depend on the same food source, which may have implications on natural enemy performance. Here, we tested the hypothesis that intraguild interactions between the greenhouse whitefly, Trialeurodes vaporariorum, and the South American tomato pinworm, Phthorimaea absoluta on the host plant tomato would affect performance of the pinworm ectoparasitoid Bracon nigricans. In oviposition bioassays, P. absoluta females significantly laid more eggs and were attracted to tomato plants infested with T. vaporariorum than uninfested plants. Additionally, T. vaporariorum infestation decreased B. nigricans parasitism on P. absoluta larvae and attraction to tomato plants. In coupled gas chromatography-electroantennographic detection (GC-EAD) recordings, antennae of P. absoluta and B. nigricans females detected 18 and 15 components, respectively from the herbivore-induced volatiles, which were identified by GC-mass spectrometry (GC-MS) as green leaf volatiles (GLVs) and terpenes. In dose-response behavioral assays, P. absoluta and B. nigricans responses varied with the composition and concentration of blends and individual compounds tested. Whereas P. absoluta was attracted to GLVs and terpenes, B. nigricans avoided most of these chemicals, especially the GLV (E)-2-hexenal and terpenes δ-3-carene, and (E)-caryophyllene. The results suggest that herbivore intraguild interactions can influence the behavioral ecology and performance of natural enemies which may have evolutionary significance. Also, these specific GLVs and terpenes can be exploited in the management of P. absoluta.

Screening the medicine for malaria venture's Pandemic Response Box to identify novel inhibitors of Candida albicans and Candida auris biofilm formation.

Candida spp. are opportunistic yeasts capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. We screened the Pandemic Response Box containing 400 diverse drug-like molecules active against bacteria, viruses or fungi, for inhibitors of Candida albicans and Candida auris biofilm formation. Initial hits were identified based on the demonstration of >70% inhibitory activity. Dose-response assays were used to confirm the antifungal activity of initial hits and establish their potency. The spectrum of antifungal activity of the leading compounds was determined against a panel of medically important fungi, and the in vivo activity of the leading repositionable agent was evaluated in murine models of C. albicans and C. auris systemic candidiasis. The primary screening identified 20 hit compounds, and their antifungal activity and potency against C. albicans and C. auris were validated using dose-response measurements. From these experiments, the rapalog everolimus, emerged as the leading repositionable candidate. Everolimus displayed potent antifungal activity against different Candida spp., but more moderate levels of activity against filamentous fungi. Treatment with everolimus increased survival of mice infected with C. albicans, but not those with C. auris. The screening of the Pandemic Response Box resulted in the identification of several drugs with novel antifungal activity, with everolimus emerging as the main repositionable candidate. Further in vitro and in vivo studies are needed to confirm its potential therapeutic use.

Impact of ALS Herbicide-Resistant Perennial Ryegrass (Lolium perenne) Population on Growth Rate and Competitive Ability against Wheat

Three perennial ryegrass (Lolium perenne) populations (R1, R2, and R3) with suspected resistance (R) to acetolactate synthase (ALS) or acetyl-CoA carboxylase (ACCase) herbicides were collected from wheat (Triticum aestivum) fields in northwestern Greece to study the underlying mechanisms of resistance and their impact on growth rate and competitive ability against wheat. Preemergence and postemergence plant dose–response assays showed that the R1 population was cross-resistant to the ALS inhibitors chlorsulfuron, mesosulfuron + iodosulfuron, and pyroxsulam, but susceptible (S) to imazamox. However, all populations were susceptible to the ACCase inhibitors clodinafop-propargyl, clethodim, diclofop-methyl, and pinoxaden. The analysis of the ALS gene sequence revealed a substitution of Pro197 by His or Leu in the ALS enzyme in L. perenne, which is reported for the first time in this weed and indicates a potential mechanism of target site-mediated resistance. The R1 population grown in the absence or presence of wheat competition displayed similar aboveground biomass and tiller number trends, and therefore similar estimated growth rates. In addition, the aboveground biomass of wheat was similarly reduced by both the R1 and S populations, supporting the evidence of their similar competitive ability against wheat. In general, these findings indicate that there is no clear evidence for the fitness advantage of R1 over the S population.

Response surface model to illustrate the benefits of tryptophan, melatonin, and N,N-dimethylglycine in quail chicks exposed to aflatoxin B1

A dose-response assay in a central composite design platform was conducted to investigate the responses (performance, immunity, and meat quality) of quail chicks to dietary tryptophan (Trp), melatonin (MEL), and N,N-dimethylglycine (DMG) exposed to aflatoxin B1 (AFB1). A total of 1,275 quail chicks were randomly allotted to 85-floor pens consisting of 17 treatments with 5 replicates and 15 birds per each pen. Dietary MEL and DMG had a different effect on growth rate and interacted with dietary Trp and AFB1 during the first 4 wk of age, while their effect disappeared at the last week of the experiment. Dietary Trp and AFB1 were only significant on the gain of quail chick after d 28 of the assay. During the second and third weeks of age, the reduction in feed intake caused by AFB1 attenuated by dietary MEL and DMG and dietary Trp profoundly affects feed intake in the last 2 wk of the experiment. Dietary MEL and DMG were effective on feed conversion ratio (FCR) during the second and third weeks of age. AFB1 decreased breast meat yield (BMY) and thigh meat yield (TMY), but the inclusion of either MEL or DMG removed the adverse effects of AFB1. Dietary Trp increased BMY, but it did not affect TMY. Increasing dietary Trp linearly increased the Lactobacillus bacteria (LAB) population, and AFB1 negatively impacts the LAB population. The inclusion of dietary DMG removed that negative effect on LAB. Although AFB1 decreased the antibody production against SRBC-antigen, increasing dietary Trp in intoxicated quails increased the plasma antibody in SRBC-challenged birds. At low levels of dietary Trp (0.15-0.19%), the addition of DMG increased malondialdehyde (MDA) production while increasing Trp reversed this adverse situation. In conclusion, these supplements may interact with AFB1 in younger chicks, and dietary Trp and AFB1 have a significant impact on the growth performance of quail chicks during the fifth and sixth week of age.

In vitro effects of undifferentiated callus extracts from Plantago major L, Rhodiola rosea L and Silybum marianum L in normal and malignant human skin cells

Background and objectivesThe occurrence of non-melanoma and melanoma skin cancers is currently increasing rapidly with one in every three cancers diagnosed as a skin cancer. A useful strategy to control the progression of skin cancer could be the use of plant flavonoids that suppress pro-inflammatory cytokines involved in tumor initiation and progression. In this study, the anti-inflammatory and antioxidant activity of undifferentiated callus extracts from Plantago major L, Silybum marianum L and Rhodiola rosea L was investigated both in normal and malignant skin cells. MethodsAntioxidant activity of the extracts was analyzed by using the Trolox Equivalent Antioxidant Capacity (TEAC) assay. High-Performance Thin-Layer Chromatography (HPTLC) was performed to demonstrate the phytochemical profile, and the total flavonoid content was analyzed with an aluminum chloride colorimetric method. The anti-inflammatory effect was investigated by cell treatments using the plant extracts. Thereafter, the possible suppression of induced IL-6 response was measured from the cultured skin cancer cell lines A2058 and A431, and normal primary keratinocytes with Enzyme-Linked Immunosorbent Assay (ELISA). ResultsThe HPTLC analysis assessed that the extracts contained a complex phytochemical profile that was rich in phenolic and flavonoid compounds. Dose response assays showed that concentrations between 15 and 125 μg/mL of all three plant extracts could be used to investigate an effect on the IL-6 production. The S. marianum extract had the most pronounced anti-inflammatory effect, which significantly inhibited induced IL-6 production in both normal keratinocytes and skin cells derived from epidermal carcinoma. The extract from S. marianum also had the highest flavonoid content and showed the highest antioxidant activity of the three extracts tested. ConclusionAll in all, we have confirmed that undifferentiated callus extracts of S. marianum possess properties such as antioxidant and anti-inflammatory activities in both normal and malignant keratinocytes, and thus could be a promising agent controlling the pro-inflammatory IL-6 production.

Discrimination between protoporphyrinogen oxidase–inhibiting herbicide-resistant and herbicide-susceptible redroot pigweed (Amaranthus retroflexus) with spectral reflectance

AbstractThe current assays to confirm herbicide resistance can be time- and labor-intensive (dose–response) or require a skill set/technical equipment (genetic sequencing). Stakeholders could benefit from a rapid assay to confirm herbicide-resistant weeds to ensure sustainable crop production. Because protoporphyrinogen oxidase (PPO)-inhibiting herbicides rapidly interfere with chlorophyll production/integrity; we propose a new, rapid assay utilizing spectral reflectance to confirm resistance. Leaf disks were excised from two PPO-inhibiting herbicide-resistant (target-site [TSR] and non–target site [NTSR]) and herbicide-susceptible redroot pigweed (Amaranthus retroflexus L.) populations and placed into a 24-well plate containing different concentrations (0 to 10 mM) of fomesafen for 48 h. A multispectral sensor captured images from the red (668 nm), green (560 nm), blue (475 nm), and red edge (717 nm) wavebands after a 48-h incubation period. The green leaf index (GLI) was utilized to determine spectral reflectance ratios of the treated leaf disks. Clear differences of spectral reflectance were observed in the red edge waveband for all populations treated with the 10 mM concentration in the dose–response assays. Differences of spectral reflectance were observed for the NTSR population compared with the TSR and susceptible populations treated with the 10 mM concentration in the green waveband and the GLI in the dose–response assay. Leaf disks from the aforementioned A. retroflexus populations and two additional susceptible populations were subjected to a similar assay with the discriminating concentration (10 mM). Spectral reflectance was different between the PPO-inhibiting herbicide-resistant and herbicide-susceptible populations in the red, blue, and green wavebands. Spectral reflectance was not distinctive between the populations in the red edge waveband and the GLI. The results provide a basis for rapidly (∼48 h) detecting PPO-inhibiting herbicide-resistant A. retroflexus via spectral reflectance. Discrimination between TSR and NTSR populations was possible only in the dose–response assay, but the assay still has utility in distinguishing herbicide-resistant plants from herbicide-susceptible plants.