The Environmental Degradation and Distribution of Saflufenacil, a Fluorinated Protoporphyrinogen IX Oxidase-Inhibiting Herbicide, on a Canadian Winter Wheat Field.

Saflufenacil when applied to a field is susceptible to transport, degradation, and transformation. We used a laboratory-based approach to model the fate of saflufenacil in the environment, the results of which are compared directly with those observed in a field study where saflufenacil was applied to a crop of winter wheat at a standard rate of 63 g of active ingredient/hectare. The water solubility of 2.1 g/L for saflufenacil allows for vertical transport through soil at a rate of 4.3 cm/mL of rainwater, and a soil adsorption coefficient KOC of 28.8 suggests that some of the herbicide will absorb to the soil. Of the saflufenacil in the soil, 78 ± 2.1% (n = 18) partitioned into plants, including nontargeted crop species, where it was found primarily in leaves (78 ± 2.1%, n = 18) and roots (22 ± 1.7%, n = 18). The saflufenacil that does not partition into plants or undergo vertical transport followed a degradation pathway into 3 metabolites: a uracil-ring N-demethylated metabolite (Saf-µCH3 ), a doubly N-demethylated metabolite (Saf-2CH3 ), and a ring-cleavage metabolite (Saf-RC), identified using nontargeted mass spectrometry. In the field, saflufenacil was observed to degrade over 212 d to the persistent metabolite Saf-RC. This metabolite was found at a concentration that was 1/10th of that applied to the field, suggesting that the majority of saflufenacil had undergone transport through the soil, or uptake into the winter wheat crop. Field samples were further examined using F-19 nuclear magnetic resonance and nontargeted mass spectrometry to rule out the potential of other degradation products. Environ Toxicol Chem 2020;39:1918-1928. © 2020 SETAC.

Uptake and Translocation of Triflumezopyrim in Rice Plants.

A new type of mesoionic insecticide triflumezopyrim is mainly used to control rice planthoppers, leafhoppers, etc. In order to study the uptake and translocation characteristics of this new insecticide in rice (Oryza sativa), a method for the detection of triflumezopyrim in rice, soil, and water was established using liquid-liquid extraction and QuEChERS sample pretreatment combined with liquid chromatography-triple quadrupole tandem mass spectrometry. The distribution of triflumezopyrim in rice was investigated after hydroponic treatment and foliar treatment at the concentrations of 2.5 and 5 mg·L-1 within the ranges of 24, 48, and 72 h. The results showed that triflumezopyrim could be absorbed by roots and form a systematic distribution in rice by hydroponic treatment; meanwhile, it could also be absorbed by leaves and transported to the bottom leaves under foliar treatment, but no triflumezopyrim was detected in the roots. Thus, triflumezopyrim exhibited high acropetal translocation within the rice plant. This study provides an important scientific basis for the development of an application strategy of triflumezopyrim to control planthoppers and leafhoppers as well as for the residue detection method and safety evaluation.

Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation.

Riboswitches are a class of nonprotein-coding RNAs that directly sense cellular metabolites to regulate gene expression. They are model systems for analyzing RNA-ligand interactions and are established targets for antibacterial agents. Many studies have analyzed the ligand-binding properties of riboswitches, but this work has outpaced our understanding of the underlying chemical pathways that govern riboswitch-controlled gene expression. To address this knowledge gap, we prepared 15 mutants of the preQ1-II riboswitch-a structurally and biochemically well-characterized HLout pseudoknot that recognizes the metabolite prequeuosine1 (preQ1). The mutants span the preQ1-binding pocket through the adjoining Shine-Dalgarno sequence (SDS) and include A-minor motifs, pseudoknot-insertion helix P4, U·A-U base triples, and canonical G-C pairs in the anti-SDS. As predicted-and confirmed by in vitro isothermal titration calorimetry measurements-specific mutations ablated preQ1 binding, but most aberrant binding effects were corrected by compensatory mutations. In contrast, functional analysis in live bacteria using a riboswitch-controlled GFPuv-reporter assay revealed that each mutant had a deleterious effect on gene regulation, even when compensatory changes were included. Our results indicate that effector binding can be uncoupled from gene regulation. We attribute loss of function to defects in a chemical interaction network that links effector binding to distal regions of the fold that support the gene-off RNA conformation. Our findings differentiate effector binding from biological function, which has ramifications for riboswitch characterization. Our results are considered in the context of synthetic ligands and drugs that bind tightly to riboswitches without eliciting a biological response.

Application of TMAH thermochemolysis to the detection of nucleobases: Application to the MOMA and SAM space experiment.

Thermochemolysis of seven nucleobases-adenine, thymine, uracil, cytosine, guanine, xanthine, and hypoxanthine-in tetramethylammonium hydroxide (TMAH) was studied individually by pyrolysis gas chromatography mass spectrometry in the frame of the Mars surface exploration. The analyses were performed under conditions relevant to the Sample Analysis at Mars (SAM) instrument of the Mars Curiosity Rover and the Mars Organic Molecule Analyzer (MOMA) instrument of the ExoMars Rover. The thermochemolysis products of each nucleobase were identified and the reaction mechanisms studied. The thermochemolysis temperature was optimized and the limit of detection and quantification of each nucleobase were also investigated. Results indicate that 600°C is the optimal thermochemolysis temperature for all seven nucleobases. The methylated products trimethyl-adenine, 1, 3-dimethyl-thymine, 1, 3-dimethyl-uracil, trimethyl-cytosine, 1, 3, 7-trimethyl-xanthine (caffeine), and dimethyl-hypoxanthine, respectively, are the most stable forms of adenine, thymine, uracil, cytosine, guanine, and xanthine, and hypoxanthine in TMAH solutions. The limits of detection for adenine, thymine, and uracil were 0.075 nmol; the limits of detection for guanine, cytosine, and hypoxanthine were higher, at 0.40, 0.55, and 0.75 nmol, respectively. These experiments allowed to well constrain the analytical capabilities of the thermochemolysis experiments that will be performed on Mars to detect nucleobases.

Detection of adulterated drugs in traditional Chinese medicine and dietary supplements using hydrogen as a carrier gas.

Helium, a minor component of natural gas and radioactive minerals, is most commonly used as a carrier in gas chromatography-mass spectrometry (GC-MS). Its scarcity leads to limited availability and higher costs. In this experiment, hydrogen from a safe source of a hydrogen generator was tested as a substitutive carrier gas for the detection of adulterant in traditional Chinese medicine (TCM) and food supplements by GC-MS analysis. We found that the limits of detection (LODs) of using hydrogen were from 10 to 1000 µg/g. The levels of LODs tested among 170 drugs remain the same whether hydrogen or helium was used as a carrier gas with the exception of 7 drugs-benzbromarone, estradiol benzoate, bezafibrate, mefenamic acid, oxymetholone, piperidenafil and cetilistat. The real sample analysis results using hydrogen were as satisfactory as those using helium. In addition, the retention time was shortened after the chromatographic performance was optimized. In summary, it is worth considering hydrogen as a carrier gas due to its affordable costs, energy efficiency, carbon reduction and chromatographic advantages to detect adulterated drugs in TCM and dietary supplement using GC-MS.

A Robust Liposomal Platform for Direct Colorimetric Detection of Sphingomyelinase Enzyme and Inhibitors.

The enzyme sphingomyelinase (SMase) is an important biomarker for several diseases such as Niemann Pick's, atherosclerosis, multiple sclerosis, and HIV. We present a two-component colorimetric SMase activity assay that is more sensitive and much faster than currently available commercial assays. Herein, SMase-triggered release of cysteine from a sphingomyelin (SM)-based liposome formulation with 60 mol % cholesterol causes gold nanoparticle (AuNP) aggregation, enabling colorimetric detection of SMase activities as low as 0.02 mU/mL, corresponding to 1.4 pM concentration. While the lipid composition offers a stable, nonleaky liposome platform with minimal background signal, high specificity toward SMase avoids cross-reactivity of other similar phospholipases. Notably, use of an SM-based liposome formulation accurately mimics the natural in vivo substrate: the cell membrane. We studied the physical rearrangement process of the lipid membrane during SMase-mediated hydrolysis of SM to ceramide using small- and wide-angle X-ray scattering. A change in lipid phase from a liquid to gel state bilayer with increasing concentration of ceramide accounts for the observed increase in membrane permeability and consequent release of encapsulated cysteine. We further demonstrated the effectiveness of the sensor in colorimetric screening of small-molecule drug candidates, paving the way for the identification of novel SMase inhibitors in minutes. Taken together, the simplicity, speed, sensitivity, and naked-eye readout of this assay offer huge potential in point-of-care diagnostics and high-throughput drug screening.

Toward a high-throughput method for determining vicine and convicine levels in faba bean seeds using flow injection analysis combined with tandem mass spectrometry.

Although faba bean provides environmental and health benefits, vicine and convicine (v-c) limit its use as a source of vegetable protein. Crop improvement efforts to minimize v-c concentration require low-cost, rapid screening methods to distinguish between high and low v-c genotypes to accelerate development of new cultivars and to detect out-crossing events. To assist crop breeders, we developed a unique and rapid screening method that uses a 60 s instrumental analysis step to accurately distinguish between high and low v-c genotypes. The method involves flow injection analysis (FIA) coupled with tandem mass spectrometry (i.e., selective reaction monitoring, SRM). Using seeds with known v-c levels as calibrants, measured v-c levels were comparable with liquid chromatography (LC)-SRM results and the method was used to screen 370 faba bean genotypes. Widespread use of FIA-SRM will accelerate breeding of low v-c faba bean, thereby alleviating concerns about anti-nutritional effects of v-c in this crop.

A practical toxicity bioassay for vicine and convicine levels in faba bean (Vicia faba).

BACKGROUND: Faba bean (Vicia faba) vicine and convicine (V-C) aglycones (divicine and isouramil respectively) provoke an acute hemolytic anemia called favism in individuals with a glucose-6-phosphate dehydrogenase (G6PD) enzyme defect in their red blood cells. Geneticists/plant breeders are working with faba bean to decrease V-C levels to improve public acceptance of this high-protein pulse crop. Here, we present a fast and simple ex vivo in vitro bioassay for V-C toxicity testing of faba bean or faba bean food products. RESULTS: We have shown that 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU)-treated (i.e., sensitized) normal red blood cells, like G6PD-defective blood, displayed (i) continuous glutathione (GSH) depletion with no regeneration as incubation time and the dose of aglycones increased, (ii) progressive accumulation of denatured hemoglobin products into high molecular weight (HMW) proteins with increased aglycone dose, (iii) both band 3 membrane proteins and hemichromes, in HMW protein aggregates. We have also demonstrated that sensitized red blood cells can effectively differentiate various levels of toxicity among faba bean varieties through the two hemolysis biomarkers: GSH depletion and HMW clumping. CONCLUSION: BCNU-sensitized red blood cells provide an ideal model for favism blood, to assess and compare the toxicity of faba bean varieties and their food products. © 2018 Society of Chemical Industry.

Fluorescent-Linked Enzyme Chemoproteomic Strategy (FLECS) for Identifying HSP70 Inhibitors.

Activation of the heat shock response, and in particular upregulation of stress-inducible Hsp70, herein referred to as Hsp70i, in newly transformed cells, appears to protect against protein damaging stimuli, induction of premature oncogene-induced terminal senescence (OIS), and apoptosis, thereby enabling tumor initiation and progression to an aggressive phenotype. Expressed at very low or undetectable levels in normal tissue, the cytoprotective effects of Hsp70i appear to be mediated through its activity as a molecular chaperone allowing proper folding of mutated proteins, and by blocking cell signaling pathways that regulate OIS and apoptosis. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. However, identification of selective inhibitors of Hsp70i has proven challenging largely because of the affinity of the protein for ATP. Additionally, its chaperone functions do not lend the protein amenable to traditional enzymatic high-throughput screens. Here, we describe the use of fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify Hsp70i inhibitors. The FLECS assay is a simple binding assay that enables proteins tagged with fluorophors to be rapidly and quantitative screened against small-molecule libraries. We show several case history examples of the methodology that led to the discovery of the Fatty acid synthase inhibitor, FASNALL, the DAPK3 inhibitor HS38, and HS72, an allosteric inhibitor selective for Hsp70i.

α,ß-Unsaturated Aldehyde-Induced Delays in Nucleotide Excision Repair and the Contribution of Reactive Oxygen Species.

Aldehydes are widespread environmental and industrial compounds to which humans are frequently exposed. Despite their significant health risk, the mechanisms underlying aldehyde toxicity are poorly understand. We recently demonstrated that cigarette sidestream smoke (CSS) inhibited nucleotide excision repair (NER), and this was attributed to aldehydes in CSS. In the present study, we examined the influence of saturated and unsaturated aldehydes on NER. The human keratinocyte cell line, HaCaT, was treated with aldehydes and then exposed to UVB. Saturated aldehydes did not show toxicity, whereas α,ß-unsaturated aldehydes caused cell death, which was markedly enhanced by UV exposure. The speed of NER was examined by the detection of pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) using ELISA and local UV irradiation assay. The repair of 6-4PPs was markedly reduced by α,ß-unsaturated aldehydes, but not by saturated aldehydes, and this was attributed to a delay in the recruitment of repair proteins (TFIIH and XPG) to DNA damage sites. Reactive oxygen species (ROS) were produced after a treatment with α,ß-unsaturated aldehydes, and hydrogen peroxide (H2O2) inhibited the repair of 6-4PPs, similar to α,ß-unsaturated aldehydes. H2O2 inhibited the accumulation of XPA and XPG at DNA damage sites, whereas TFIIH showed the same recruitment with or without H2O2. These results suggest that an exposure to α,ß-unsaturated aldehydes, not saturated aldehydes inhibits NER by delaying the recruitment of NER proteins to DNA damage sites, and α,ß-unsaturated aldehyde-induced ROS production may partially play a role in this process.

A scalable platform to identify fungal secondary metabolites and their gene clusters.

The genomes of filamentous fungi contain up to 90 biosynthetic gene clusters (BGCs) encoding diverse secondary metabolites-an enormous reservoir of untapped chemical potential. However, the recalcitrant genetics, cryptic expression, and unculturability of these fungi prevent scientists from systematically exploiting these gene clusters and harvesting their products. As heterologous expression of fungal BGCs is largely limited to the expression of single or partial clusters, we established a scalable process for the expression of large numbers of full-length gene clusters, called FAC-MS. Using fungal artificial chromosomes (FACs) and metabolomic scoring (MS), we screened 56 secondary metabolite BGCs from diverse fungal species for expression in Aspergillus nidulans. We discovered 15 new metabolites and assigned them with confidence to their BGCs. Using the FAC-MS platform, we extensively characterized a new macrolactone, valactamide A, and its hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS). The ability to regularize access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

Effects of faba beans with different concentrations of vicine and convicine on egg production, egg quality and red blood cells in laying hens.

The faba bean (Vicia faba L.) is a potential source of proteins for poultry, mainly for laying hens whose protein requirements are lower than those of other birds such as growing broilers and turkeys. However, this feedstuff contains anti-nutritional factors, that is, vicine (V) and convicine (C) that are already known to reduce laying hen performance. The aim of the experiment reported here was to evaluate the effects of a wide range of dietary V and C concentrations in laying hens. Two trials were performed with laying hens fed diets including 20% or 25% of faba bean genotypes highly contrasting in V+C content. In Trial 1, faba beans from two tannin-containing cultivars, but with high or low V+C content were dehulled in order to eliminate the tannin effect. In addition to the contrasting levels of V+C in the two cultivars, two intermediate levels of V+C were obtained by mixing the two cultivars (70/30 and 30/70). In Trial 2, two isogenic zero-tannin faba bean genotypes with high or low V+C content were used. In both trials, a classical corn-soybean diet was also offered to control hens. Each experimental diet was given to 48 laying hens for 140 (Trial 1) or 89 (Trial 2) days. Laying performance and egg quality were measured. The redox sensitivity of red blood cells (RBCs) was assessed by measuring hemolysis and reduced glutathione (GSH) concentration in these cells. Egg weight was significantly reduced by the diets containing the highest concentrations of V+C (P<0.0001) in Trial 1 and slightly reduced (P<0.10) in Trial 2, but only weak linear relationships between egg weight and dietary V+C concentration were established. No negative effect of V+C level was observed for egg quality parameters. In contrast, certain parameters (i.e. Haugh units, yolk color) were improved by feeding low V+C diets (P<0.05). Hemolysis of RBCs was higher in hens fed high V+C diets. A decrease in GSH concentration in RBCs of hens fed the highest levels of V+C was observed. Faba bean genotypes with low concentrations of V+C can therefore be used in laying hen diets up to 25% without any detrimental effects on performance levels or egg characteristics, without any risk of hemolysis of RBCs.

Determination and stability of divicine and isouramil produced by enzymatic hydrolysis of vicine and convicine of faba bean.

The aglycones of vicine and convicine, divicine and isouramil, are the causative agents of favism and, therefore, should be analysed along with vicine and convicine in research seeking to eliminate them. This study investigated the stability of the aglycones produced by hydrolysis with ß-glucosidase. Reversed-phase, high-performance liquid chromatography (HPLC) with UV detection was shown to be able to observe both aglycone formation and further reactions in isolated fractions and extract made from faba bean and in faba bean suspension. Divicine and isouramil were unstable and degraded almost completely in extract in 60min and completely in fractions in 120min at a pH of 5 at 37°C. Adding sodium ascorbate delayed degradation of divicine. Divicine was more stable at 20°C than at 37°C. Being able to show formation and degradation of the aglycones, the proposed method allows monitoring of the vicine and convicine detoxification process.

Drug Concentration in Rat Plasma, Bladder, and Prostate After Mirodenafil Administration in a Chronic Pelvic Ischemia Model.

OBJECTIVE: To evaluate the distribution of a daily phosphodiesterase type 5 inhibitor dose (mirodenafil) in rat plasma and bladder and prostate tissue in a model of atherosclerosis-induced chronic pelvic ischemia. METHODS: Thirty-two 18-week-old male Sprague Dawley rats were divided into two groups. Group I (n = 16) comprised a chronic pelvic ischemia model treated with mirodenafil and group II (n = 16) comprised a sham-operated model also treated with mirodenafil. The mirodenafil concentrations in each organ were measured at specific time points after 14 days of daily mirodenafil administration. The drug distribution ratio of group I to group II of each organ was measured, and the bladder tissue-to-plasma and prostate tissue-to-plasma ratios were calculated. RESULTS: The mean drug concentration in the bladder of the rats in group I did not differ significantly from that of group II after mirodenafil administration. In the prostate, the mean drug concentration of group I was significantly higher than that of group II at 1 and 4 hours after drug administration. The drug concentration was higher in the bladder tissue than in the prostate tissue and the bladder tissue-to-plasma ratio was significantly higher than the prostate tissue-to-plasma ratio. CONCLUSION: Our results suggest that mirodenafil levels might be sufficient in the target tissue after daily treatment in an ischemia-induced aging model. Considering the difficulties of tissue distribution study in human subjects, the results of this investigation provided meaningful evidence of the application of daily doses of mirodenafil for treating lower urinary tract symptoms in an aging population.

Simultaneous detection of flumethasone, dl-methylephedrine, and 2-hydroxy-4,6-dimethylpyrimidine in porcine muscle and pasteurized cow milk using liquid chromatography coupled with triple-quadrupole mass spectrometry.

A simple analytical method based on liquid chromatography coupled with triple-quadrupole mass spectrometry was developed for detection of the veterinary drugs flumethasone, dl-methylephedrine, and 2-hydroxy-4,6-dimethylpyrimidine in porcine muscle and pasteurized cow milk. The target drugs were extracted from samples using 10mM ammonium formate in acetonitrile followed by clean-up with n-hexane and primary secondary amine sorbent (PSA). The analytes were separated on an XBridge™ hydrophilic interaction liquid chromatography (HILIC) column using 10mM ammonium formate in ultrapure water and acetonitrile. Good linearity was achieved over the tested concentrations in matrix-fortified calibrations with correlation coefficients (R(2))≥0.9686. Recovery at two spiking levels ranged between 73.62-112.70% with intra- and inter-day precisions of ≤20.33%. The limits of quantification ranged from 2-10ng/g in porcine muscle and pasteurized cow milk. A survey of market samples showed that none of them contained any of the target analytes. Liquid-liquid purification using n-hexane in combination with PSA efficiently removed the interferences during porcine and milk sample extraction. The developed method is sensitive and reliable for detection of the three target drugs in a single chromatographic run. Furthermore, it exhibits high selectivity and low quantification limits for animal-derived food products destined for human consumption.

Characterization of TPN729 metabolites in humans using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

TPN729 has been reported as a novel phosphodiesterase type 5 (PDE5) inhibitor to treat erectile dysfunction, and is currently being tested in clinical trials. In addition to the potent inhibition against PDE5, TPN729 is regarded as a better alternative to provide fewer side effects and better patient compliance. Given the potential therapeutic benefits of TPN729, it is of great importance to elucidate its metabolic characteristics in drug development. This study is the first to investigate the metabolic fate of TPN729 in humans. A rapid and reliable analytical method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was established to investigate the metabolic profiles of TPN729 in human plasma, urine, and feces after its oral administration. As a result, a total of 22 metabolites were identified, of which seven were confirmed in comparison with the reference substances. The incubations of the metabolite references in human hepatocytes and aldehyde trapping experiment were further conducted to investigate its metabolic pathways. The results of the present study indicated the extensive metabolism of TPN729 in humans, including oxidative deamination, oxidative ring opening, N-dealkylation, N-oxidation, hydroxylation, dehydrogenation, lactam formation, and glucuronidation. M3 resulting from N-dealkylation was the major circulating substance detected in human plasma. The principal metabolites detected in human feces were products of oxidative deamination and oxidative ring opening. The parent drug was identified as the major component in urine. Taken together, this study provided valuable information on the metabolic fate of TPN729 in humans, and applicable analytical strategies for rapid metabolic elucidation in complex matrix samples through the useful and reliable UPLC/Q-TOF MS technique.

Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication.

UNLABELLED: Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed arenavirus vaccines and current antiarenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name: N-[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong anti-LCMV activity in the absence of cell toxicity. Mechanism-of-action studies revealed that F3406 inhibited LCMV cell entry by specifically interfering with the pH-dependent fusion in the endosome compartment that is mediated by LCMV glycoprotein GP2 and required to release the virus ribonucleoprotein into the cell cytoplasm to initiate transcription and replication of the virus genome. We identified residue M437 within the transmembrane domain of GP2 as critical for virus susceptibility to F3406. IMPORTANCE: Hemorrhagic fever arenaviruses (HFA) are important human pathogens that cause high morbidity and mortality in areas where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Concerns posed by arenavirus infections are aggravated by the lack of U.S. Food and Drug Administration-licensed arenavirus vaccines and current antiarenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. Here we describe a novel recombinant LCMV and its use to develop a cell-based assay suitable for HTS to rapidly identify inhibitors arenavirus multiplication. The concepts and experimental strategies we describe in this work provide the bases for the rapid identification and characterization of novel anti-HFA therapeutics.

Effect of air classification and fermentation by Lactobacillus plantarum VTT E-133328 on faba bean (Vicia faba L.) flour nutritional properties.

The effects of air classification and lactic acid bacteria fermentation on the reduction of anti-nutritional factors (vicine and convicine, trypsin inhibitor activity, condensed tannins and phytic acid) and in vitro protein and starch digestibility of faba bean flour were studied. Free amino acid (FAA) profile analysis was also carried out. Air classification allowed the separation of the flour into protein and starch rich fractions, showing different chemical compositions and microstructures. Lactobacillus plantarum growth and acidification in faba bean flour and its fractions were assessed. The anti-nutritional compounds were separated mostly to the fine protein-rich fraction. Fermentation caused the decrease of vicine and convicine contents by more than 91% and significantly reduced trypsin inhibitor activity and condensed tannins (by more than 40% in the protein-rich fraction). No significant (P>0.05) variation was observed for total phenols and phytic acid content. Fermentation increased the amount of FAA, especially of the essential amino acids and γ-aminobutyric acid, enhanced the in vitro protein digestibility and significantly lowered the hydrolysis index. This work showed that the combination of air classification and fermentation improved nutritional functionality of faba bean flour which could be utilized in various food applications.

Simultaneous determination of saflufenacil and its two metabolites in soil samples by ultra-performance liquid chromatography with tandem mass spectrometry.

Saflufenacil is a new protoporphyrinogen-IX-oxidase inhibitor herbicide. When used, it can enter the soil and has a high risk to reach and contaminate groundwater and aquatic systems. A rapid and sensitive method of ultra-performance LC with MS/MS was developed for the simultaneous determination of saflufenacil and its two metabolites in soil samples. A modified quick, easy, cheap, effective, rugged, and safe method was applied as the pretreatment procedure. The method was validated by five types of soil samples collected from several regions of China, which all showed good linearity (R(2) ≥ 0.9914) and precision (RSD ≤ 26.2%). The average recoveries of the three analytes ranged between 74.1 and 118.9% at spiking levels of 3-300 µg/kg. The method limits of detection (S/N 3:1) and method limits of quantification (S/N 10:1) achieved are in the ranges of 0.25-2.75 and 0.83-9.16 µg/kg, respectively. This indicated that the developed ultra-performance LC with MS/MS method is a promising analytical tool for monitoring the environmental risks posed by saflufenacil.

A simple and sensitive biosensor strain for detecting toxoflavin using ß-galactosidase activity.

In this study, we developed a simple and sensitive biosensor for the determination of toxoflavin (which is toxic to various plants, fungi, animals, and bacteria) in natural samples based on ß-galactosidase activity. The proposed toxoflavin detection method for toxin-producing bacteria or toxin-contaminated foods is simple and cost effective. Burkholderia glumae, a species known to cause rice grain rot and wilt in various field crops, produces toxoflavin under the control of a LysR-type transcriptional regulator ToxR and its ligand toxoflavin. As the expression of toxoflavin biosynthetic genes requires toxoflavin as a co-activator of ToxR, a novel biosensor stain was constructed based on lacZ reporter gene integration into the first gene of the toxoflavin biosynthesis operon, toxABCDE of B. glumae. The biosensor was composed of a sensor strain (COK71), substrates (X-gal or ONPG), and culture medium, without any complex preparation process. We demonstrated that the biosensor strain is highly specific to toxoflavin, and can quantify relative amounts of toxoflavin compared with known concentrations of toxoflavin. The proposed method was reliable and simple; samples containing 50-500 nM of toxoflavin could be analyzed. More importantly, the proposed biosensor strain could identify toxoflavin-producing bacteria in real samples. The excellent performance of this biosensor is useful for diagnostic purposes, such as detecting toxoflavin-contaminated foods and environmental samples.