Functional asymmetry of the urea transporter UT-B in human red blood cells.

We studied urea, thiourea, and methylurea transport and interaction in human red blood cells (RBCs) under conditions of self-exchange (SE), net efflux (NE), and net influx (NI) at pH 7.2. We combined four methods, a four-centrifuge technique, the Millipore-Swinnex filtering technique, the continuous flow tube method, and a continuous pump method to measure the transport of the 14C-labeled compounds. Under SE conditions, both urea and thiourea show perfect Michaelis-Menten kinetics with half-saturation constants, K½,SE (mM), of ≈300 (urea) and ≈20 (thiourea). The solutes show no concentration-dependent saturation under NE conditions. Under NI conditions, transport displays saturation or self-inhibition kinetics with a K½,NI (mM) of ≈210 (urea) and ≈20 (thiourea). Urea, thiourea, and methylurea are competitive inhibitors of the transport of analog solutes. This study supports the hypothesis that the three compounds share the same urea transport system (UT-B). UT-B functions asymmetrically as it saturates from the outside only under SE and NI conditions, whereas it functions as a high-capacity channel-like transporter under NE conditions. When the red blood cell enters the urea-rich kidney tissue, self-inhibition reduces the urea uptake in the cell. When the cell leaves the kidney, the channel-like function of UT-B implies that intracellular urea rapidly equilibrates with external urea. The net result is that the cell during the passage in the kidney capillaries carries urea to the kidney to be excreted while the urea transfer from the kidney via the bloodstream is minimized.NEW & NOTEWORTHY The kinetics of urea transport in red blood cells was determined by means of a combination of four methods that ensures a high time resolution. In the present study, we disclose that the urea transporter UT-B functions highly asymmetric being channel-like with no saturation under conditions of net efflux and saturable under conditions of net influx and self-exchange in the concentration range 1-1,000 mM (pH 7.2 and 25-38 °C).

A synergistic bacterial pool decomposes tebuthiuron in soil.

This study aimed to propose an eco-compatible strategy to mitigate the possible environmental contamination caused by tebuthiuron. Therefore, we screened potential tebuthiuron-degrading microorganisms from conventional (CS) and no-till (NTS) systems producing sugarcane. Then, they were bioprospected for their ability of decomposing the target-molecule at 2.48 mmol g-1 and 4.96 mmol g-1 into CO2 via respirometry. Integrating microbiota from CS and NTS into an advantageously synergistic bacterial pool produced the highest specific-growth rate of CO2 of 89.60 mg day-1, so outstripped the other inoculum. The bacterial CN-NTS framework notably stabilized the sigmoidal Gompertz curve on microbial degradation earliest and enabled the seeds of Lactuca sativa to germinate healthiest throughout ecotoxicological bioassay for cross-validation. Our study is preliminary, but timely to provide knowledge of particular relevance to progress in the field's prominence in remediating terrestrial ecosystems where residual tebuthiuron can persist and contaminate. The analytical insights will act as an opening of solutions to develop high-throughput biotechnological strategies for environmental decontamination.

Influence of temperature on growth, development and thyroid metabolism of American bullfrog tadpoles (Lithobates catesbeianus) exposed to the herbicide tebuthiuron.

The influence of temperature (25 and 32 °C) on the negative effects of the herbicide tebuthiuron (TBU, 0, 10, 50 and 200 ng.L-1, 16 days) on thyroid function and metamorphosis of Lithobates catesbeianus tadpoles was evaluated. Metamorphosis was accelerated by TBU exposure at 25 ºC, but delayed at 32 ºC with considerable losses of body mass. T3 and T4 levels were not altered. The highest TBU concentrarion at 25 ºC increased TRâ€¯ß and DIO3 transcript levels, which is consistent with development acceleration in tadpoles. At 32 ºC TRâ€¯ß transcript levels were lower than the values recorded at 25 ºC, and those tadpoles exposed to the highest TBU concentration presented increased diameter of thyroid follicles compared to controls at same temperature. This study evidences that TBU at environmentally realistic concentrations is able to disrupt thyroidogenesis in bullfrog tadpoles, impairing their development. These effects are influenced by temperature.

Continuous Flow Acylation of (Hetero)aryllithiums with Polyfunctional N,N-Dimethylamides and Tetramethylurea in Toluene.

The continuous flow reaction of various aryl or heteroaryl bromides in toluene in the presence of THF (1.0 equiv) with sec-BuLi (1.1 equiv) provided at 25 °C within 40 sec the corresponding aryllithiums which were acylated with various functionalized N,N-dimethylamides including easily enolizable amides at -20 °C within 27 sec, producing highly functionalized ketones in 48-90 % yield (36 examples). This method was well suited for the preparation of α-chiral ketones such as naproxene and ibuprofen derived ketones with 99 % ee. A one-pot stepwise bis-addition of two different lithium organometallics to 1,1,3,3-tetramethyurea (TMU) provided unsymmetrical ketones in 69-79 % yield (9 examples).

The interactions between chiral analytes and chitosan-based chiral stationary phases during enantioseparation.

The goal of the present study was to disclose the interactions between chitosan-type chiral selectors (CSs) and chiral analytes during enantioseparation. Hence, six chitosan 3,6-bis(phenylcarbamate)-2-(cyclohexylmethylurea)s were synthesized and characterized. These chitosan derivatives were employed as CSs with which the corresponding coated-type chiral stationary phases (CSPs) were prepared. According to the nature and position of the substituents on the phenyl group, the CSs and CSPs were divided into three sets. The counterparts of the three sets were 3,5-diMe versus 3,5-diCl, 4-Me versus 4-Cl and 3-Me versus 3-Cl. The enantioseparation capability of the CSPs was evaluated with high-performance liquid chromatography. The CSPs demonstrated a good enantioseparation capability to the tested chiral analytes. In enantioselectivity, the CSs with 3,5-diCl and with 4-Me roughly were better than the counterparts with 3,5-diMe and with 4-Cl respectively. The CS with 3-Me enantiomerically recognized more analytes than the one with 3-Cl, but showed lower separation factors in more enantioseparations. The acidity of the amide hydrogen in the phenylcarbamates was investigated with density functional theory calculations and 1H NMR measurements. The trend of the acidity variation with different substituents on the phenyl group was confirmed by the retention factors of acetone on the CSPs. Compared the retention factors of analytes on every set of the counterparts, the formation of hydrogen bond (HB) in enantioseparation could be outlined as follows: when the CSs interacted with chiral analytes without reactive hydrogen but with lone paired electrons, the carbamate N‒Hs in the CSs were HB donors and the analytes were HB acceptors; if the CSs interacted with analytes with a reactive hydrogen, the role of the CSs in HB formation was related to the acidity of the reactive hydrogen; the patterns of HB formation between the CSs and analytes were also impacted by compositions of mobile phases, in addition to the nature, number and position of substituents on the phenyl group. Based on the discussion, chiral recognition mechanism could be understood in more detail. Besides, the strategy to improve enantioseparation capability of a CSP by introducing a substituent onto phenyl group was clarified and further comprehended.

Determination of azoxystrobin, topramezone, acetamiprid, fluometuron and folpet in their commercially available pesticide formulations by liquid chromatography.

A rapid, simple, precise and accurate high performance liquid chromatographic (HPLC) analytical method was developed and validated for the determination of the active substances (a.s.) azoxystrobin, topramezone, acetamiprid, fluometuron and folpet in their respective commercially available formulations. The method was used for the analysis of samples under the frame of the national quality control program of plant protection products in the Greek market. Chromatographic separation of the active substances from additives and co-formulants is achieved using isocratic elution with acetonitrile and 0.1% phosphoric acid solution (60:40 v/v) at a flow rate of 0.4 mL min-1 on a C18 monolithic column (Chromolith Performance-RP18e 100 × 4.6 mm) and UV detection at 230 nm. Validation parameters of the method fulfilled acceptability criteria. Recovery of all individual compounds was in the range 97.8-100.9%. Precision expressed as relative standard deviation was lower than the theoretical values of the modified Horwitz equation. Correlation coefficients of linearity of response were better than 0.999. The benefits of the proposed method are significant reduction in analysis time and total cost since all analytes were determined with the same extraction procedures and chromatographic system. Analysis of real samples for all active ingredients confirmed fitness for purpose of the suggested method.

Influence of temperature on biomarker responses and histology of the liver of American bullfrog tadpoles (Lithobates catesbeianus, Shaw, 1802) exposed to the herbicide Tebuthiuron.

Tebuthiuron (TBU) is a phenylurea herbicide that is extensively used in sugarcane fields. Owing to the low degradation rate, high water solubility, and leaching potential, TBU is believed to have harmful effects on aquatic organisms, such as anuran tadpoles. Contaminant effects can be influenced by temperature since increases in temperature are often associated with increased metabolic reactions. In this study, we evaluated the influence of temperature on the negative effects of TBU in bullfrog tadpoles (Lithobates catesbeianus) through a multi-biomarker approach. Tadpoles were exposed to 0 (control) 10, 50, and 200 ng L-1 of TBU for 16 days at 25 and 32 °C. TBU increased the transcript levels of genes involved in biotransformation (glutathione S-transferase, GST, and sulfotransferase, SULT) and antioxidant (superoxide dismutase, SOD, and catalase, CAT) enzymes. TBU exposure also increased CAT and glutathione peroxidase (GPx) activities, whereas SOD and carboxylesterase activities were decreased. The highest temperature caused a decrease in the activities of ethoxyresorufin-O-deethylase and SOD but increased the activities of GST, GPx, glucose 6-phosphate dehydrogenase, and acetylcholinesterase. No effects of temperature or TBU exposure were observed in genotoxic markers (frequencies of micronucleous and nuclear abnormalities) or in lipid peroxidation levels. Tadpoles exposed to TBU at all tested concentrations presented a higher index of biomarker responses than that of the control groups. Higher values of severity scores from histological analyses were found in the liver of tadpoles exposed to 50 and 200 ng L-1 of TBU at 32 °C compared with those of the control group at the same temperature. These results indicate that TBU and temperature increases are able to disturb the metabolic homeostasis of L. catesbeianus tadpoles after 16 days of exposure, causing substantial alterations in biomarker responses and liver morphology.

Spatial distribution of sorption and desorption process of 14C-radiolabelled hexazinone and tebuthiuron in tropical soil.

The understanding of the interaction between soil physicochemical attributes and herbicide behavior is fundamental for optimizing the efficient use of PRE-emergence herbicides in a more sustainable approach. However, it is still a poorly studied area within precision agriculture. Thus, the objective of this research was to evaluate the correlation of soil physicochemical attributes with the sorption and desorption processes of hexazinone and tebuthiuron to support application maps considering the field level variability. Soil samples from an agricultural area had their physicochemical attributes analyzed and were submitted to sorption and desorption studies of 14C-tebuthiuron and 14C-hexazinone using the batch equilibrium method. The values of sorption and desorption apparent coefficients (Kd), sorption and desorption percentage and bioavailability were correlated with soil attributes by Pearson's correlation. The Kd values of tebuthiuron and hexazinone sorption ranged from 1.2 to 2.9 mL g-1 and 0.4-0.6 mL g-1, respectively. For desorption of tebuthiuron and hexazinone, Kd values ranged from 3.4 to 4.4 mL g-1 and 2.6-3.0 mL g-1, respectively. A positive correlation among clay content, soil organic matter (OM), and tebuthiuron and hexazinone sorption Kd values were found. Both herbicides had variable retention according to geographic position in the area. The recommendation of application of PRE herbicides, such as tebuthiuron and hexazinone, observing the physicochemical attributes of the soil is an alternative to increase efficiency in weed control and decrease the risk of environmental contamination.

Phytoremediation of quinclorac and tebuthiuron-polluted soil by green manure plants.

Quinclorac and tebuthiuron are residual herbicides that may remain in the soil longer than for the cropping season. The objective of this research was to evaluate the use of green manure plants to remediate soils treated with quinclorac and tebuthiuron. Soils were separately treated with 14C-quinclorac and 14C-tebuthiuron at 266.4 and 132 g ha-1, respectively. After 21 days, four green manure plants, namely Crotalaria spectabilis, Canavalia ensiformis, Stizolobium aterrimum, and Lupinus albus, were separately sown in the treated soils. Overall, all four species absorbed more 14C-tebuthiuron [C. ensiformes (22.49%), S. aterrimum (16.71%), L. albus (15%), and C. spectabilis (4.48%)] than 14C-quinclorac [C. ensiformis (13.44%), L. albus (10.02%), S. aterrimum (6.2%), and C. spectabilis (1.75%)]. Quinclorac translocation in all four plants was greater in young leaves compared to old leaves, cotyledons, or roots, and 14C-tebuthiuron translocation in all four plant species was greater in old leaves and cotyledons compared to young leaves or roots. Regardless of the differences in translocation between the two herbicides, the four green manure plants are capable to remediate soils that have been treated with quinclorac and tebuthiuron. However, C. ensiformis is more efficient for the remediation of tebuthiuron-treated soil compared to the other plants.

Using Deep Learning Artificial Intelligence Algorithms to Verify N-Nitroso-N-Methylurea and Urethane Positive Control Proliferative Changes in Tg-RasH2 Mouse Carcinogenicity Studies.

In Tg-rasH2 carcinogenicity mouse models, a positive control group is treated with a carcinogen such as urethane or N-nitroso-N-methylurea to test study validity based on the presence of the expected proliferative lesions in the transgenic mice. We hypothesized that artificial intelligence-based deep learning (DL) could provide decision support for the toxicologic pathologist by screening for the proliferative changes, verifying the expected pattern for the positive control groups. Whole slide images (WSIs) of the lungs, thymus, and stomach from positive control groups were used for supervised training of a convolutional neural network (CNN). A single pathologist annotated WSIs of normal and abnormal tissue regions for training the CNN-based supervised classifier using INHAND criteria. The algorithm was evaluated using a subset of tissue regions that were not used for training and then additional tissues were evaluated blindly by 2 independent pathologists. A binary output (proliferative classes present or not) from the pathologists was compared to that of the CNN classifier. The CNN model grouped proliferative lesion positive and negative animals at high concordance with the pathologists. This process simulated a workflow for review of these studies, whereby a DL algorithm could provide decision support for the pathologists in a nonclinical study.

Toxicity of the herbicides diuron, propazine, tebuthiuron, and haloxyfop to the diatom Chaetoceros muelleri.

Conventional photosystem II (PSII) herbicides applied in agriculture can pose significant environmental risks to aquatic environments. In response to the frequent detection of these herbicides in the Great Barrier Reef (GBR) catchment area, transitions towards 'alternative' herbicides are now widely supported. However, water quality guideline values (WQGVs) for alternative herbicides are lacking and their potential ecological impacts on tropical marine species are generally unknown. To improve our understanding of the risks posed by some of these alternative herbicides on marine species under tropical conditions, we tested the effects of four herbicides on the widely distributed diatom Chaetoceros muelleri. The PSII herbicides diuron, propazine, and tebuthiuron induced substantial reductions in both 24 h effective quantum yields (ΔF/Fm') and 3-day specific growth rates (SGR). The effect concentrations, which reduced ΔF/Fm' by 50% (EC50), ranged from 4.25 µg L-1 diuron to 48.6 µg L-1 propazine, while the EC50s for SGR were on average threefold higher, ranging from 12.4 µg L-1 diuron to 187 µg L-1 tebuthiuron. Our results clearly demonstrated that inhibition of ΔF/Fm' in PSII is directly linked to reduced growth (R2 = 0.95) in this species, further supporting application of ΔF/Fm' inhibition as a valid bioindicator of ecological relevance for PSII herbicides that could contribute to deriving future WQGVs. In contrast, SGR and ΔF/Fm' of C. muelleri were nonresponsive to the non-PSII herbicide haloxyfop at the highest concentration tested (4570 µg L-1), suggesting haloxyfop does not pose a risk to C. muelleri. The toxicity thresholds (e.g. no effect concentrations; NECs) identified in this study will contribute to the derivation of high-reliability marine WQGVs for some alternative herbicides detected in GBR waters and support future assessments of the cumulative risks of complex herbicide mixtures commonly detected in coastal waters.

Discovery of 1-(1H-indazol-4-yl)-3-((1-phenyl-1H-pyrazol-5-yl)methyl) ureas as potent and thermoneutral TRPV1 antagonists.

A series of 1-indazol-3-(1-phenylpyrazol-5-yl)methyl ureas were investigated as hTRPV1 antagonists. The structure-activity relationship study was conducted systematically for both the indazole A-region and the 3-trifluoromethyl/t-butyl pyrazole C-region to optimize the antagonism toward the activation by capsaicin. Among them, the antagonists 26, 50 and 51 displayed highly potent antagonism with Ki(CAP) = 0.4-0.5 nM. Further, in vivo studies in mice indicated that these derivatives both antagonized capsaicin induced hypothermia, consistent with their in vitro activity, and themselves did not induce hyperthermia. In the formalin model, 51 showed anti-nociceptive activity in a dose-dependent manner.

Antitumour properties based on the self-assembly of camptothecin and carbamoylmannose conjugates.

Camptothecin (CPT) and its analogues show potent antitumour activity. However, poor water solubility and severe side effects have restricted their applications in clinical practice. In this paper, a novel self-assembly based on camptothecin and carbamoylmannose conjugates (CPT-Man) was constructed. The self-assembly increased the water solubility of camptothecin to 0.64 mg/ml and antitumour activity. Moreover, CPT-Man could induce obvious cancer cell apoptosis. This work provides a new approach for exploring carbohydrate-modified antitumour properties by self-assembled CPT drugs.

Do trifluralin and tebuthiuron impair isolated rat liver mitochondria?

Emerging contaminants, such as the herbicides trifluralin and tebuthiuron, comprise a class of compounds for which toxicological data are lacking, especially data regarding their harmful effects and biomarkers of exposure. Their potential damage to the environment and non-target organisms makes understanding their toxic mechanisms an urgent matter. Mitochondria, which exert an energy production function, play a vital role in maintaining many cellular activities and therefore are reliable predictors of substance toxicity. This study evaluates whether the herbicides trifluralin and tebuthiuron (at concentrations ranging from 1 to 100 µM) affect isolated rat liver mitochondria. The herbicides were analyzed according to their ability to interact with the mitochondrial membrane and induce swelling, lipoperoxidation, ROS formation, and NAD(P)H oxidation; dissipate the membrane potential; dysregulate calcium homeostasis; and alter ATP and GSH/GSSG levels. Tebuthiuron does not disrupt the mitochondrial biochemistry at any of the tested concentrations. In contrast, trifluralin can disturb the mitochondrial respiration, especially at the highest concentration, but it cannot induce oxidative stress. These results suggest that the aforementioned effects can occur as toxic mechanisms of trifluralin in non-target organisms, as well.

An effective configuration for automated magnetic micro solid-phase extraction of phenylurea herbicides from water samples followed by high-performance liquid chromatography.

In this study, a new automated magnetic micro solid-phase extraction was introduced. A Tygon tube was folded and fixed around the pole of a cylindrical magnet. Nanosized magnetic sorbents modified with diphenyldichlorosilane were uniformly immobilized on one side of the inner wall of the tube. Sample solution and desorption solvent were passed through the tube using a peristaltic pump. Four phenylurea herbicides (tebuthiuron, monolinuron, isoproturon, and monuron) were used as the model compounds to evaluate the method performance. HPLC-UV was used to separate and quantify the analytes. The effective parameters influencing the performance of the extraction process (i.e., extraction and desorption flow rates, eluent and sample volumes, type of eluent and sample ionic strength) were investigated. The limit of detection was 0.04 µg L-1 for all studied compounds. Calibration curves were linear in the range of 0.1-500 µg L-1 with a determination coefficient between 0.9988 and 0.9999. Intra-day, inter-day and batch-to-batch precisions expressed as relative standard deviation were less than 7%. Several environmental water samples were investigated to assess the applicability of the method for real sample analysis.

Guiding Lead Optimization for Solubility Improvement with Physics-Based Modeling.

Although there are a number of computational approaches available for the aqueous solubility prediction, a majority of those models rely on the existence of a training set of thermodynamic solubility measurements or/and fail to accurately account for the lattice packing contribution to the solubility. The main focus of this study is the validation of the application of a physics-based aqueous solubility approach, which does not rely on any prior knowledge and explicitly describes the solid-state contribution, in order to guide the improvement of poor solubility during the lead optimization. A superior performance of a quantum mechanical (QM)-based thermodynamic cycle approach relative to a molecular mechanical (MM)-based one in application to the optimization of two pharmaceutical series was demonstrated. The QM-based model also provided insights into the source of poor solubility of the lead compounds, allowing the selection of the optimal strategies for chemical modification and formulation. It is concluded that the application of that approach to guide solubility improvement at the late discovery and/or early development stages of the drug design proves to be highly attractive.

Dimethylolurea as a Novel Slow-Release Nitrogen Source for Nitrogen Leaching Mitigation and Crop Production.

Rapid hydrolysis of urea results in further fertilization frequency and excessive nitrogen (N) input. A modified urea, dimethylolurea (DMU), was synthesized in this study. The structure of the sample was characterized by Fourier transform infrared and nuclear magnetic resonance analysis, manifesting the formation of DMU. N release investigation confirmed that DMU enabling provided a gradual N supply. The N leaching experiment indicated that increasing the applied DMU significantly reduced the NH4+-N, NO3--N, and total N leaching, compared with urea application alone. The application effect on maize and wheat was evaluated. The results revealed that singly applied DMU with 100% or 80% N input, irrespective of the amount, promoted crop yield and agronomic characteristic and N use efficiency (NUE) of maize and wheat, beyond urea with two split applications at the recommended rate. Thus, the potential availability of DMU was proven; this could be widely used in agricultural fields as a slow-release fertilizer.

Enzyme-Functionalized Piezoresistive Hydrogel Biosensors for the Detection of Urea.

Urea is used in a wide variety of industrial applications such as the production of fertilizers. Furthermore, urea as a metabolic product is an important indicator in biomedical diagnostics. For these applications, reliable urea sensors are essential. In this work, we present a novel hydrogel-based biosensor for the detection of urea. The hydrolysis of urea by the enzyme urease leads to an alkaline pH change, which is detected with a pH-sensitive poly(acrylic acid-co-dimethylaminoethyl methacrylate) hydrogel. For this purpose, the enzyme is physically entrapped during polymerization. This enzyme-hydrogel system shows a large sensitivity in the range from 1 mmol/L up to 20 mmol/L urea with a high long-term stability over at least eight weeks. Furthermore, this urea-sensitive hydrogel is highly selective to urea in comparison to similar species like thiourea or N-methylurea. For sensory applications, the swelling pressure of this hydrogel system is transformed via a piezoresistive pressure sensor into a measurable output voltage. In this way, the basic principle of hydrogel-based piezoresistive urea biosensors was demonstrated.

D2 Dopamine Receptor G Protein-Biased Partial Agonists Based on Cariprazine.

Functionally selective G protein-coupled receptor ligands are valuable tools for deciphering the roles of downstream signaling pathways that potentially contribute to therapeutic effects versus side effects. Recently, we discovered both Gi/o-biased and ß-arrestin2-biased D2 receptor agonists based on the Food and Drug Administration (FDA)-approved drug aripiprazole. In this work, based on another FDA-approved drug, cariprazine, we conducted a structure-functional selectivity relationship study and discovered compound 38 (MS1768) as a potent partial agonist that selectively activates the Gi/o pathway over ß-arrestin2. Unlike the dual D2R/D3R partial agonist cariprazine, compound 38 showed selective agonist activity for D2R over D3R. In fact, compound 38 exhibited potent antagonism of dopamine-stimulated ß-arrestin2 recruitment. In our docking studies, compound 38 directly interacts with S1935.42 on TM5 but has no interactions with extracellular loop 2, which appears to be in contrast to the binding poses of D2R ß-arrestin2-biased ligands. In in vivo studies, compound 38 showed high D2R receptor occupancy in mice and effectively inhibited phencyclidine-induced hyperlocomotion.

Urinary Pesticide Levels in Children and Adolescents Residing in Two Agricultural Communities in Mexico.

The use of pesticides in agricultural activities has increased significantly during the last decades. Several studies have reported the health damage that results from exposure to pesticides. In Mexico, hundreds of communities depend economically on agricultural activities. The participation of minors in this type of activity and their exposure to pesticides represents a potential public health problem. A cross-sectional study was conducted, in which urine samples (first-morning urine) were taken from children under 15 years of age in both communities. A total of 281 urine samples obtained in both communities were processed for the determination of pesticides with high-performance liquid chromatography together with tandem mass spectrometry. In 100% of the samples, at least two pesticides of the 17 reported in the total samples were detected. The presence of malathion, metoxuron, and glyphosate was remarkable in more than 70% of the cases. Substantial differences were detected regarding the other compounds. It is necessary to carry out long-term studies to determine the damage to health resulting from this constant exposure and to inform the health authorities about the problem in order to implement preventive measures.