A Two-in-One Strategy: Target and Nontarget Site Mechanisms Both Play Important Role in IMI-Resistant Weedy Rice.

The introduction of Clearfield technology allows the use of imidazolinone (IMI) herbicides to control weedy rice. Imidazolinone herbicides stop the acetolactate synthase (ALS) enzyme from synthesizing branched-chain amino acids, resulting in the death of the plant. Since the launch of Clearfield technology in Malaysia in 2010, many farmers have replaced traditional cultivars with Clearfield (CL) rice lines (MR220-CL1 and MR220-CL2). This technology was initially effective; however, in recent years, local farmers have reported the reduced efficacy of IMI herbicides in controlling the spread of weedy rice. Under IMI herbicide treatment, in previous weedy rice studies, the target-site resistance (TSR) mechanism of the ALS gene has been suggested as a key factor conferring herbicide resistance. In our study, a combination of ALS gene sequencing, enzyme colorimetric assay, and a genome-wide association study (GWAS) highlighted that a non-target-site resistance (NTSR) can be an alternative molecular mechanism in IMI-resistant weedy rice. This is supported by a series of evidence, including a weak correlation between single nucleotide polymorphisms (SNPs) within the ALS exonic region and ALS enzyme activity. Our findings suggest that the adaptability of weedy rice in Clearfield rice fields can be more complicated than previously found in other rice strains.

New insight into the negative impact of imidazolium-based ionic liquid [C10mim]Cl on Hela cells: From membrane damage to biochemical alterations.

As an alternative to volatile organic solvents, ionic liquids (ILs) are known as "green solvents", and widely used in industrial applications. However, due to their high solubility and stability, ILs have tendency to persist in the water environment, thus having potential negative impacts on the aquatic ecosystem. For assessing the environmental risks of ILs, a fundamental understanding of the toxic effects and mechanisms of ILs is needed. Here we evaluated the cytotoxicity of 1-methyl-3-decylimidazolium chloride ([C10mim]Cl) and elucidated the main toxic mechanism of [C10mim]Cl in human cervical carcinoma (Hela) cells. Microstructural analysis revealed that [C10mim]Cl exposure caused the cell membrane breakage, swollen and vacuolated mitochondria, and spherical cytoskeletal structure. Cytotoxicity assays found that [C10mim]Cl exposure increased ROS production, decreased mitochondrial membrane potential, induced cell apoptosis and cell cycle arrest. These results indicated that [C10mim]Cl could induce damage to cellular membrane structure, affect the integrity of cell ultrastructure, cause the oxidative damage and ultimately lead to the inhibition of cell proliferation. Moreover, alterations of biochemical information including the increased ratios of unsaturated fatty acid and carbonyl groups to lipid, and lipid to protein, and the decreased ratios of Amide I to Amide II, and α-helix to ß-sheet were observed in [C10mim]Cl treated cells, suggesting that [C10mim]Cl could affect the structure of membrane lipid alkyl chain and cell membrane fluidity, promote the lipid peroxidation and alter the protein secondary structure. The findings from this work demonstrated that membrane structure is the key target, and membrane damage is involved in [C10mim]Cl induced cytotoxicity.

IMI resistance associated to crop-weed hybridization in a natural Brassica rapa population: characterization and fate.

Wild turnip (Brassica rapa) is a common weed and a close relative to oilseed rape (Brassica napus). The Clearfield® production system is a highly adopted tool which provides an alternative solution for weed management, but its efficiency is threatened by gene transfer from crop to weed relatives. Crop-weed hybrids with herbicide resistance were found in the progeny of a B. rapa population gathered from a weedy stand on the borders of an oilseed rape (B. napus) imidazolinone (IMI)-resistant crop. Interspecific hybrids were confirmed by morphological traits in the greenhouse and experimental field, survival after imazethapyr applications, DNA content through flow cytometry, and pollen viability. The transference of herbicide resistance was demonstrated even in a particular situation of pollen competition between both an herbicide-resistant crop and a non-resistant crop. However, IMI resistance was not found in further generations collected at the same location. These results verify gene transmission from oilseed rape to B. rapa in the main crop area in Argentina where resistant and susceptible varieties are found and seed loss and crop volunteers are common. Hybridization, introgression, and herbicide selection would be associated with the loss of effectiveness of IMI technology.

Results of the Phase I Trial of RG7112, a Small-Molecule MDM2 Antagonist in Leukemia.

PURPOSE: RG7112 is a small-molecule MDM2 antagonist. MDM2 is a negative regulator of the tumor suppressor p53 and frequently overexpressed in leukemias. Thus, a phase I study of RG7112 in patients with hematologic malignancies was conducted. EXPERIMENTAL DESIGN: Primary study objectives included determination of the dose and safety profile of RG7112. Secondary objectives included evaluation of pharmacokinetics; pharmacodynamics, such as TP53-mutation status and MDM2 expression; and preliminary clinical activity. Patients were divided into two cohorts: Stratum A [relapsed/refractory acute myeloid leukemia (AML; except acute promyelocytic leukemia), acute lymphoblastic leukemia, and chronic myelogenous leukemia] and Stratum B (relapsed/refractory chronic lymphocytic leukemia/small cell lymphocytic leukemia; CLL/sCLL). Some Stratum A patients were treated at the MTD to assess clinical activity. RESULTS: RG7112 was administered to 116 patients (96 patients in Stratum A and 20 patients in Stratum B). All patients experienced at least 1 adverse event, and 3 dose-limiting toxicities were reported. Pharmacokinetic analysis indicated that twice-daily dosing enhanced daily exposure. Antileukemia activity was observed in the 30 patients with AML assessed at the MTD, including 5 patients who met International Working Group (IWG) criteria for response. Exploratory analysis revealed TP53 mutations in 14% of Stratum A patients and in 40% of Stratum B patients. Two patients with TP53 mutations exhibited clinical activity. p53 target genes were induced only in TP53 wild-type leukemic cells. Baseline expression levels of MDM2 correlated positively with clinical response. CONCLUSIONS: RG7112 demonstrated clinical activity against relapsed/refractory AML and CLL/sCLL. MDM2 inhibition resulted in p53 stabilization and transcriptional activation of p53-target genes. We provide proof-of-concept that MDM2 inhibition restores p53 function and generates clinical responses in hematologic malignancies.

Synthesis, in silico metabolic and toxicity prediction of some novel imidazolinones derivatives as potent anticonvulsant agents.

A series of 1,2,4-trisubstituted 5-imidazolinone derivatives were synthesized by Erlenmeyer condensation of benzoylglycine (hippuric acid) with different aldehydes in the presence of sodium acetate and acetic anhydride. The derivatives of the compounds were prepared by condensation of some known sulpha drugs with 5-oxazolone derivatives. The anticonvulsant activity of the compounds was determined by the protection of pentylenetetrazole-induced convulsions that was ranged from 10 to 60%. The compounds with p-OCH3, p-OH and o-Cl substitutions in the phenyl ring on 4(th) position of the imidazolinone ring exhibited good anticonvulsant activity. In silico metabolic and toxicity studies showed that all the compounds in the series are not likely to exhibit toxicity except the compounds IIIa, IIIb, VIa and VIb, that is predicted to show 29% mutagenicity and 53% irritation in comparison to the other compounds. The predicted lethal effect and hERG toxicity of the compounds showed that IIa, IVa, Va and Vb might be toxic at higher concentrations. The results successfully establish the synthesized imidazolinone derivatives as novel compounds with anticonvulsant properties, low predicted cardiotoxicity and lethal effects thus can be promising leads for further development as novel anticonvulsants.

Reactive oxygen species-independent activation of the IL-1beta inflammasome in cells from patients with chronic granulomatous disease.

Humans with chronic granulomatous diseases (CGDs) due to mutations in p47-phox have defective NADPH activity and thus cannot generate NADPH-dependent reactive oxygen species (ROS). The role of ROS in inflammation is controversial; some in vitro studies suggest that ROS are crucial for secretion of IL-1beta via inflammasome activation, whereas mice defective for ROS and patients with CGD have a proinflammatory phenotype. In this study, we evaluated activation of the IL-1beta inflammasome in cells from CGD patients. In contrast to previous studies using the small molecule diphenylene iodonium (DPI) as a ROS inhibitor, we found no decrease in either caspase-1 activation or secretion of IL-1beta and IL-18 in primary CGD monocytes. Moreover, activation of CGD monocytes by uric acid crystals induced a 4-fold higher level of IL-1beta secretion compared with that seen in monocytes from unaffected subjects, and this increase was not due to increased synthesis of the IL-1beta precursor. In addition, Western blot analysis of CGD cells revealed that caspase-1 activation was not decreased, but rather was increased compared with control cells. Examination of the effects exerted by the inhibition of ROS activity by DPI revealed that the decrease in IL-1beta secretion by DPI was actually due to inhibition of IL-1beta gene expression. Thus, inconsistent with the proinflammatory role of ROS, the present findings support the concept that ROS likely dampen inflammasome activation. The absence of ROS in CGD monocytes may explain the presence of an inflammatory phenotype characterized by granulomas and inflammatory bowel disease occurring in CGD patients.

The toxic effects of ionic liquids on the activities of acetylcholinesterase and cellulase in earthworms.

The earthworm Eisenia foetida was exposed to different concentrations of imidazolium ionic liquids with varying chain lengths according to the method of OECD [OECD, 1984. (The Current Organization of Economic and Cooperative Development Acute Earthworm Toxicity Test) Guidelines for the Testing of Chemicals, No. 207. Earthworm Acute Toxicity Tests]. The acute and subchronic toxic effects of [C(8)mim]Br on the activities of acetylcholinesterase (AChE) and cellulase in earthworms were determined under an artificial soil condition. Using filter paper contact tests, the 48 h-LC(50) values of [C(4)mim]Br, [C(6)mim]Br, [C(8)mim]Br, [C(10)mim]Br and [C(12)mim]Br on the earthworm were 73.33, 28.25, 2.69, 0.37 and 0.02 microg cm(-2), respectively. The 7 d-LC(50) of [C(8)mim]Br was 206.8 mg kg(-1) artificial soil (dry weight) and the 14 d-LC(50) was 159.4 mg kg(-1) artificial soil (dry weight), under the condition of artificial soil. After 1 d and 3 d of acute exposure, the activity of AChE was markedly inhibited when compared to the control, while it was increased at 7d. The cellulase activity was elevated significantly in the treatment groups of 20-160 mg kg(-1) after 3 and 7d of acute exposure. The activity of cellulase was also promoted under the subchronic exposure condition in the 10 and 20 mg kg(-1) groups. The experimental results suggest that [C(8)mim]Br may interfere with the nervous function of the earthworms and increase their cellulase activity. These results indicate that [C(8)mim]Br-exposure can affect the metabolized enzyme activity of earthworms at low concentrations and can even cause worm death at high doses, both of which have potential impacts on the soil environment.

Antiplasmodial, beta-haematin inhibition, antitrypanosomal and cytotoxic activity in vitro of novel 4-aminoquinoline 2-imidazolines.

A novel series of 4-aminoquinoline-containing 2-imidazolines were synthesized via a one-pot 3-component condensation reaction of amine, aldehyde and isocyanoacetate. The products were obtained in high yield as well as purity and were evaluated directly against two strains of Plasmodium falciparum and Trypanosoma brucei. Compound was the most active across all parasites with ED(50) = 3.3 nM against a chloroquine (CQ)-sensitive 3D7 strain, ED(50) = 33 nM against a CQ-resistant K1 strain and ED(50) = 70 nM against T. brucei. Several compounds were able to inhibit formation of beta-haematin in vitro, suggesting haemozoin formation in the malaria parasite as a possible target. On the other hand, evaluation against a human KB cell line revealed that the compounds were generally non-cytotoxic to the host cells.

Acetohydroxyacid synthase mutations conferring resistance to imidazolinone or sulfonylurea herbicides in sunflower.

Wild biotypes of cultivated sunflower ( Helianthus annuus L.) are weeds in corn ( Zea mays L.), soybean ( Glycine max L.), and other crops in North America, and are commonly controlled by applying acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Biotypes resistant to two classes of AHAS-inhibiting herbicides-imidazolinones (IMIs) or sulfonylureas (SUs)-have been discovered in wild sunflower populations (ANN-PUR and ANN-KAN) treated with imazethapyr or chlorsulfuron, respectively. The goals of the present study were to isolate AHAS genes from sunflower, identify mutations in AHAS genes conferring herbicide resistance in ANN-PUR and ANN-KAN, and develop tools for marker-assisted selection (MAS) of herbicide resistance genes in sunflower. Three AHAS genes ( AHAS1, AHAS2, and AHAS3) were identified, cloned, and sequenced from herbicide-resistant (mutant) and -susceptible (wild type) genotypes. We identified 48 single-nucleotide polymorphisms (SNPs) in AHAS1, a single six-base pair insertion-deletion in AHAS2, and a single SNP in AHAS3. No DNA polymorphisms were found in AHAS2 among elite inbred lines. AHAS1 from imazethapyr-resistant inbreds harbored a C-to-T mutation in codon 205 ( Arabidopsis thaliana codon nomenclature), conferring resistance to IMI herbicides, whereas AHAS1 from chlorsulfuron-resistant inbreds harbored a C-to-T mutation in codon 197, conferring resistance to SU herbicides. SNP and single-strand conformational polymorphism markers for AHAS1, AHAS2, and AHAS3 were developed and genetically mapped. AHAS1, AHAS2, and AHAS3 mapped to linkage groups 2 ( AHAS3), 6 ( AHAS2), and 9 ( AHAS1). The C/T SNP in codon 205 of AHAS1 cosegregated with a partially dominant gene for resistance to IMI herbicides in two mutant x wild-type populations. The molecular breeding tools described herein create the basis for rapidly identifying new mutations in AHAS and performing MAS for herbicide resistance genes in sunflower.