Effect of the phenylpyrrole fungicide fludioxonil on cell proliferation and cardiac differentiation in mouse embryonic stem cells.

Fludioxnil is extensively used as a fungicide in agricultural application, but its possible impact on embryonic development is not yet well understood. In this study, the potential effect of fludioxonil on cardiac differentiation was evaluated in mouse embryonic stem cells (mESCs). The water-soluble tetrazolium (WST) and colony formation assays were conducted to confirm the effect of fludioxonil on proliferation of mESCs. The effect of fludioxonil on the ability of mESCs to form mouse embryoid bodies (mEBs) was determined by the hanging drop assay, whereas the ability of cardiomyocyte differentiation in the early stage was evaluated by determining the beating ratio (ratio of the number of contracting cells to the number of attached EBs) of cardiomyocytes. The viability of mESCs was significantly decreased (less than 50 %) at 10-5 M fludioxonil. Results of the colony formation assay revealed suppressed colony formation at 10-5 M fludioxonil (about 50 % at 5 days). Furthermore, the expressions of cell-cycle related proteins, i.e., cyclin D1, cyclin E, p21 and p27, were altered and trending towards inhibiting cell growth. Exposure to fludioxonil also resulted in reduced size of the mEB and induced increasing expression levels of the pluripotency markers Oct4, Sox2 and Nanog. Development of the beating ratio in the process of differentiation to cardiomyocytes derived from mESCs was completely inhibited after exposure to 10-5 M fludioxonil during the early stage of differentiation (day 5), whereas the beating ratio gradually increased after 5-day treatment. Simultaneously, expressions of the cardiomyocyte-related proteins, Gata4, Hand1 and cTnI, were inhibited after exposure to 10-5 M fludioxonil. Taken together, these results imply that fludioxonil may impact on the developmental process of mESCs, particularly the cardiac lineage.

Schisandrol B promotes liver enlargement via activation of PXR and YAP pathways in mice.

BACKGROUND: Schisandrol B (SolB) is one of the bioactive components from a traditional Chinese medicine Schisandra chinensis or Schisandra sphenanthera. It has been demonstrated that SolB exerts hepatoprotective effects against drug-induced liver injury and promotes liver regeneration. It was found that SolB can induce hepatomegaly but the involved mechanisms remain unknown. PURPOSE: This study aimed to explore the mechanisms involved in SolB-induced hepatomegaly. METHODS: Male C57BL/6 mice were injected intraperitoneally with SolB (100 mg/kg) for 5 days. Serum and liver samples were collected for biochemical and histological analyses. The mechanisms of SolB were investigated by qRT-PCR and western blot analyses, luciferase reporter gene assays and immunofluorescence. RESULTS: SolB significantly increased hepatocyte size and proliferation, and then promoted liver enlargement without liver injury and inflammation. SolB transactivated human PXR, activated PXR in mice and upregulated hepatic expression of its downstream proteins, such as CYP3A11, CYP2B10 and UGT1A1. SolB also significantly enhanced nuclear translocation of PXR and YAP in human cell lines. YAP signal pathway was activated by SolB in mice. CONCLUSION: These findings demonstrated that SolB can significantly induce liver enlargement, which is associated with the activation of PXR and YAP pathways.

More than additive effects on liver triglyceride accumulation by combinations of steatotic and non-steatotic pesticides in HepaRG cells.

The liver is constantly exposed to mixtures of hepatotoxic compounds, such as food contaminants and pesticides. Dose addition is regularly assumed for mixtures in risk assessment, which however might not be sufficiently protective in case of synergistic effects. Especially the prediction of combination effects of substances which do not share a common adverse outcome (AO) might be problematic. In this study, the focus was on the endpoint liver triglyceride accumulation in vitro, an indicator of hepatic fatty acid changes. The hepatotoxic compounds difenoconazole, propiconazole and tebuconazole were chosen which cause hepatic fatty acid changes in vivo, whereas fludioxonil was chosen as a hepatotoxic substance not causing fatty acid changes. Triglyceride accumulation was analyzed for combinations of steatotic and non-steatotic pesticides in human HepaRG hepatocarcinoma cells. Investigations revealed a potentiation of triglyceride accumulation by mixtures of the steatotic compounds with the non-steatotic fludioxonil, as compared to the single compounds. Mathematical modeling of combination effects indicated more than additive effects for the tested combinations if the method by Chou was applied, and a decrease in EC50 values of the steatotic compounds when applied in mixtures. Use of an adverse outcome pathway (AOP)-driven testing strategy for liver steatosis showed interactions of the test compounds with the nuclear receptors AHR, CAR and PXR, as well as a downregulation of ACOX2. An ACOX2-dependent mechanism underlying the observed mixture effect could not be verified using a siRNA approach. By contrast, a toxicokinetic interaction was identified including an inhibition of the metabolic enzyme CYP3A4 by fludioxonil and a decreased metabolic conversion of the CYP3A4 substrate difenoconazole when used in mixture experiments. In conclusion, an interaction by a steatotic and a non-steatotic compound at the toxicokinetic level on the endpoint triglyceride accumulation in vitro was described.

Histopathological, cytotoxicological, and genotoxic effects of the semi-synthetic compound dillapiole n-butyl ether in Balb/C mice.

Dillapiole n-butyl ether is a substance derived from dillapiole, which exhibits potential insecticidal effects on Aedes aegypti, the principal vector of the Dengue fever, Zika, and Chikungunya viruses, as well as Aedes albopictus, a vector of Dengue fever. As these mosquitoes are resistant to synthetic insecticides, dillapiole n-butyl ether may represent a valuable, plant-based alternative for their control. Dillapiole n-butyl ether has insecticidal and genotoxic effects on A. aegypti and A. albopictus, as shown by the reduction in clutch size and egg viability, and increased mortality rates, as well as a high frequency of micronuclei and chromosomal aberrations. However, the potential cytotoxic and genotoxic effects of this substance in mammals are still unknown. In Balb/C mice, structural changes were detected in hepatic, renal, and cardiac tissues, which were directly proportional to the concentration of the dose applied, in both genders. The induction of genotoxic, mutagenic, and cytotoxic effects was also observed at the highest concentrations (150 and 328 mg/kg). Further research will be necessary to better characterize the potential genotoxicity of this substance at lower concentrations, for the evaluation of the potential health risks related to its presence in environmental features, such as drinking water.

Sediment toxicity of the fungicide fludioxonil to benthic macroinvertebrates -evaluation of the tiered effect assessment procedure.

28-Day sediment-spiked laboratory toxicity tests with eight benthic macroinvertebrates and the lipophilic fungicide fludioxonil were conducted to verify the proposed tiered sediment effect assessment procedure as recommended by the European Food Safety Authority (EFSA). The test species were the oligochaetes Lumbriculus variegatus and Tubifex tubifex, the insects Chironomus riparius and Caenis horaria, the crustaceans Hyalella azteca and Asellus aquaticus and the bivalves Corbicula fluminalis and Pisidium amnicum. Toxicity estimates were expressed in terms of total concentration of dry sediment as well as in pore water concentration. Field-collected sediment, also used in a previously performed sediment-spiked microcosm experiment, was used in tests with all species. L. variegatus and C. riparius had similar lowest 28d-L(E)C10 values when expressed in terms of total sediment concentration, but in terms of pore water concentration L. variegatus was more sensitive. Three of the six additional benthic test species (A. aquaticus, C. horaria, C. fluminalis) had 28d-EC10 values a factor of 2-6 lower than that of L. variegatus. Comparing different effect assessment tiers for sediment organisms, i.e. Tier-0 (Modified Equilibrium Partitioning approach), Tier-1 (Standard Test Species approach), Tier-2 (Species Sensitivity Distribution (SSD) approach) and Tier-3 (Model Ecosystem approach), it is concluded that the tiers based on sediment-spiked laboratory toxicity tests provide sufficient protection when compared with the Tier-3 Regulatory Acceptable Concentration (RAC). Differences between Tier-1 and Tier-2 RACs, however, appear to be relatively small and not always consistent, irrespective of expressing the RAC in terms of total sediment or pore water concentration. Derivation of RACs by means of the SSD approach may be a challenge, because it is difficult obtaining a sufficient number of valid chronic EC10 values with appropriate 95% confidence bands for sediment-dwelling macroinvertebrates. Therefore, this paper proposes a Tier-2 Weight-of-Evidence approach to be used in case an insufficient number of valid additional toxicity data is made available. Similar studies with pesticides that differ in fate properties and toxic mode-of-action are necessary for further validation of the tiered effect assessment approach for sediment organisms.

Combined toxic effects of fludioxonil and triadimefon on embryonic development of zebrafish (Danio rerio).

Pesticides scarcely exist as individual compounds in the water ecosystem, but rather as mixtures of multiple chemicals at relatively low concentrations. In this study, we aimed to explore the mixture toxic effects of fludioxonil (FLU) and triadimefon (TRI) on zebrafish (Danio rerio) by employing different toxicological endpoints. Results revealed that the 96-h LC50 values of FLU to D. rerio at multiple developmental stages ranged from 0.055 (0.039-0.086) to 0.61 (0.33-0.83) mg L-1, which were less than those of TRI ranging from 3.08 (1.84-5.96) to 9.75 (5.99-14.78) mg L-1. Mixtures of FLU and TRI exerted synergistic effects on embryonic zebrafish. Activities of total superoxide dismutase (T-SOD) and catalase (CAT) were markedly altered in most of the individual and pesticide mixture treatments compared with the control. The expressions of 16 genes involved in oxidative stress, cellular apoptosis, immune system and endocrine system displayed that embryonic zebrafish were affected by the individual pesticides and their mixtures, and greater variations of four genes (ERɑ, Tnf, IL and bax) were found when exposed to pesticide mixtures compared with their individual compounds. Therefore, more studies on mixture toxicities among different pesticides should be taken as a priority when evaluating their ecological risk.

Comparing the effects of fludioxonil on non-target soil invertebrates using ecotoxicological methods from single-species bioassays to model ecosystems.

The lower tier toxicity tests used for risk assessment of plant protection products are conducted with single species, only regarding direct effects of the tested substances. However, it is not clear, if lower tier tests are able to protect in situ soil communities, as these tests are not able to account for direct and indirect effects of chemicals on multi-species systems in natural soil communities. This knowledge gap between single-species tests and field studies can be bridged using model ecosystems (microcosms), which allow for the assessment of direct and indirect effects of the compounds under evaluation. In the present study, single-species toxicity tests and soil-spiked microcosms were used to comparatively investigate the toxicity of the non-systemic fungicide fludioxonil (FDO) on non-target soil organisms, with nematodes being the test organisms of choice. The potential effects of FDO on nematodes were investigated in two different test systems: (i) standardized toxicity tests using Caenorhabditis elegans exposed to FDO-spiked soil (FDO concentrations 50-1207 mg/kg soil dry weight) and (ii) in situ nematode communities sampled from microcosms containing FDO-spiked soil (FDO concentrations 75-600 mg/kg soil dry weight). FDO dose-dependently inhibited the reproduction of C. elegans, with an effect concentration (EC50) of 209.9 mg FDO/kg soil dry weight and a no observed effect concentration (NOEC) of 63.0 mg FDO/kg soil dry weight. In the microcosms, FDO significantly affected trait-based indices, such as the Maturity Index (MI25) and the Enrichment Index (EI), which responded already at FDO concentrations of 14.3 and 62.4 mg/kg dry soil. Overall, this study provides new insights into the impact of the non-systemic fungicide FDO on non-target soil organisms and demonstrates the suitability of nematode-based tools, that allow for a quick and cost-effective lower and higher tier risk assessment of plant protection products.

Overexpression of the CORVET complex alleviates the fungicidal effects of fludioxonil on the yeast Saccharomyces cerevisiae expressing hybrid histidine kinase 3.

The hybrid histidine kinase 3 (HHK3) is a highly conserved sensor kinase in fungi that regulates the downstream HOG/p38 mitogen-activated protein kinase (MAPK). In addition to its role in osmoadaptation, HHK3 is involved in hyphal morphogenesis, conidiation, virulence, and cellular adaptation to oxidative stress. However, the molecular mechanisms by which it controls these processes remain obscure. Moreover, HHK3 is a molecular target for antifungal agents such as fludioxonil, which thereby interferes with the HOG/p38 pathway, leading to the abnormal accumulation of glycerol and subsequent cell lysis. Here, we used a chemical genomics approach with the yeast Saccharomyces cerevisiae to better understand the fungicidal action of fludioxonil and the role of HHK3 in fungal growth and physiology. Our results indicated that the abnormal accumulation of glycerol is not the primary cause of fludioxonil toxicity. Fludioxonil appears to impair endosomal trafficking in the fungal cells. We found that the components of class C core vacuole/endosome tethering (CORVET) complex are essential for yeast viability in the presence of a subthreshold dose of fludioxonil and that their overexpression alleviates fludioxonil toxicity. We also noted that by impeding secretory vesicle trafficking, fludioxonil inhibits hyphal growth in the opportunistic fungal pathogen Candida albicans Our results suggest that HHK3 regulates fungal hyphal growth by affecting vesicle trafficking. Together, our results reveal an important role of CORVET complex in the fungicidal action of fludioxonil downstream of HHK3.

Differential sensitivity of developmental stages of the South American toad to a fungicide based on fludioxonil and metalaxyl-M.

Agricultural fungicide application in Argentina has increased twice since 2008, with Maxim® XL (2.5% fludioxonil +1% metalaxyl-M) as one of the most used fungicide formulation. The toxicity of this pesticide on Rhinella arenarum was assessed by means of continuous (from embryo and larval development) and 24-h pulse exposure standardized bioassays. Lethality was concentration- and exposure time-dependent. Maxim® XL caused a progressive lethal effect along the bioassays with higher toxicity on embryos than larvae, obtaining 50% lethal concentrations at 96, 336, and 504 h of 10.85, 2.89, and 1.71 mg/L for embryos, and 43.94, 11.79, and 5.76 mg/L for larvae respectively. Lethal 504-h no observed effect concentration values for embryos and larvae were 1 and 2.5 mg/L respectively. A stage-dependent toxicity of Maxim® XL was also demonstrated within the embryo development, with early stages more sensitive than the later ones, and blastula as the most sensitive developmental stage. The risk quotients obtained for chronic risk assessment determined a potential threat for the survival and continuity of R. arenarum populations under these conditions. The results indicate that the levels of the fungicide reaching amphibian habitats could be risky for the early development of this amphibian species. This study also emphasizes the necessity to evaluate the chronic effects of fungicides in pesticide risk assessment.

Establishment and validation of microsampling techniques in wild rodents for ecotoxicological research.

Compounds and products in the biocide and plant protection sector can only be registered after formal risk assessment to ensure safety for users and the environment. In bird and mammal risk assessment, this is routinely done using generic focal species as models, which are of particular exposure risk. Such a species is the common vole (Microtus arvalis) due to its high food intake relative to the low body weight. For wild species, biological samples, data and hence realistic exposure estimations are particularly difficult to obtain. In recent years, advances have been made in the techniques related to serial microsampling of laboratory mice and rats that allow for a reduction in sampling volumes. Similar progress in wild species sampling is missing. This study presents a proof of concept to dose wild rodents with relevant compounds and to draw serial, low volume blood samples suitable for state-of-the art toxicokinetic analyses. For the first time, the jugular vein of common voles was used to administer compounds (two frequently used fungicidal components). This procedure and the following microsampling of blood (2 × 10 µl six times within 24 hours) from the lateral tail vein did not affect body weight and mortality of voles. Samples were sufficient to detect dissipation patterns of the compounds from blood in toxicokinetic analysis. These results suggest that microsampling can be well translated from laboratory mice to wild rodent species and help to obtain realistic exposure estimates in wild rodents for ecotoxicological studies as well as to promote the 3R concept in studies with wild rodent species.

Exposure and effects of sediment-spiked fludioxonil on macroinvertebrates and zooplankton in outdoor aquatic microcosms.

Information from effects of pesticides in sediments at an ecosystem level, to validate current and proposed risk assessment procedures, is scarce. A sediment-spiked outdoor freshwater microcosm experiment was conducted with fludioxonil (lipophilic, non-systemic fungicide) to study exposure dynamics and treatment-related responses of benthic and pelagic macroinvertebrates and zooplankton. Besides blank control and solvent control systems the experiment had six different treatment levels (1.7-614mga.s./kg dry sediment) based around the reported 28-d No Observed Effect Concentration (NOEC) for Chironomus riparius (40mga.s./kg dry sediment). Twelve systems were available per treatment of which four were sacrificed on each of days 28, 56 and 84 after microcosm construction. Fludioxonil persisted in the sediment and mean measured concentrations were 53-82% of the initial concentration after 84days. The dissipation rate increased with the treatment level. Also exposure concentrations in overlying water were long-term, with highest concentrations 28days after initiation of the experiment. Sediment-dwelling Oligochaeta and pelagic Rotifera and Cladocera showed the most pronounced treatment-related declines. The most sensitive sediment-dwelling oligochaete was Dero digitata (population NOEC 14.2mga.s./kg dry sediment). The same NOEC was calculated for the sediment-dwelling macroinvertebrate community. The most sensitive zooplankton species was the cladoceran Diaphanosoma brachyurum (NOEC of 1.6µga.s./L in overlying water corresponding to 5.0mga.s./kg dry sediment). At the two highest treatments several rotifer taxa showed a pronounced decrease, while the zooplankton community-level NOEC was 5.6µga.s./L (corresponding to 14.2mga.s./kg dry sediment). Zooplankton taxa calanoid Copepoda and Daphnia gr. longispina showed a pronounced treatment-related increase (indirect effects). Consequently, an assessment factor of 10 to the chronic laboratory NOECs of Chironomus riparius (sediment) and Daphnia magna (water) results in a regulatory acceptable concentration that is sufficiently protective for both the sediment-dwelling and pelagic organisms in the microcosms.

Mechanisms of chromosomal aberrations induced by sesamin metabolites in Chinese hamster lung cells.

Sesamin is a major lignan in sesame seeds and oil. We previously demonstrated that sesamin induces chromosomal aberrations (CA) in Chinese hamster lung (CHL/IU) cells in the presence of a metabolic activation system (S9 mix), although no genotoxicity was detected in vivo. To clarify the mechanism of CA induction by sesamin, we identified its principal active metabolite. A mono-catechol derivative, [2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabi-cyclo[3.3.0]octane (SC-1)], was previously identified in culture medium when sesamin was incubated with S9 mix. In the present study, we show that SC-1 induces CA in CHL/IU cells but not in human hepatoblastoma (HepG2) cells. SC-1 was unstable in culture medium. Addition of glutathione (GSH) to the incubation mixture decreased the rate of decomposition and also suppressed induction of CA in CHL/IU cells. These results indicate that SC-1 itself may not contribute to the induction of CA. Two GSH adducts of SC-1 were identified when SC-1 was incubated with GSH, suggesting that SC-1 was converted to the semiquinone/quinone form and then conjugated with GSH in the culture medium. Sodium sulfite (a quinone-responsive compound) also suppressed CA induction by SC-1. These findings strongly suggest that SC-1 is oxidized to semiquinone/quinone derivatives extracellularly in culture medium, that these derivatives are responsible for the induction of CA in CHL/IU cells, and therefore that the positive results obtained with sesamin in in vitro CA tests using CHL/IU cells may not be relevant to the assessment of in vivo activity.

Larvicidal activity of lignans and alkaloid identified in Zanthoxylum piperitum bark toward insecticide-susceptible and wild Culex pipiens pallens and Aedes aegypti.

BACKGROUND: The yellow fever mosquito, Aedes aegypti, and the common house mosquito, Culex pipiens pallens, transmit dengue fever and West Nile virus diseases, respectively. This study was conducted to determine the toxicity of the three lignans (-)-asarinin, sesamin and (+)-xanthoxylol-γ,γ-dimethylallylether (XDA), and the alkaloid pellitorine from Zanthoxylum piperitum (Rutaceae) bark to third-instar larvae from insecticide-susceptible C. pipiens pallens and Ae. aegypti as well as wild C. pipiens pallens resistant to deltamethrin, cyfluthrin, fenthion, and temephos. METHODS: The toxicities of all isolates were compared with those of mosquito larvicide temephos. LC50 values for each species and their treatments were significantly different from one another when their 95% confidence intervals did not overlap. RESULTS: XDA was isolated from Z. piperitum as a new larvicidal principle. XDA (LC50, 0.27 and 0.24 mg/l) was 4, 53, and 144 times and 4, 100, and 117 times more toxic than pellitorine, sesamin, and asarinin toward larvae from susceptible C. pipiens pallens and Ae. aegypti, respectively. Overall, all the isolates were less toxic than temephos (LC50, 0.006 and 0.009 mg/l). These constituents did not differ in toxicity to larvae from the two Culex strains. The present finding indicates that the lignans and alkaloid and the insecticides do not share a common mode of larvicidal action or elicit cross-resistance. CONCLUSION: Naturally occurring Z. piperitum bark-derived compounds, particularly XDA, merit further study as potential mosquito larval control agents or as lead compounds for the control of insecticide-resistant mosquito populations.

Sesamin encouraging effects on chondrogenic differentiation of human amniotic fluid-derived mesenchymal stem cells.

Worldwide, the most recognized musculoskeletal degenerative disease is osteoarthritis (OA). Sesamin, a major abundant lignan compound present in Sesamun Indicum Linn, has been described for its various pharmacological effects and health benefits. However, the promoting effects of sesamin on chondrogenic differentiation have not yet been observed. Herein, the aim of this study was to investigate the effects of sesamin on cell cytotoxicity and the potent supporting effects on chondrogenic differentiation of human amniotic fluid-derived mesenchymal stem cells (hAF-MSCs). The results indicated that sesamin was not toxic to hAF-MSCs after sesamin treatment. When treating the cells with a combination of sesamin and inducing factors, sesamin was able to up-regulate the expression level of specific genes which play an essential role during the cartilage development process, including SOX9, AGC, COL2A1, COL11A1, and COMP and also simultaneously promote the cartilage extracellular protein synthesis, aggrecan and type II collagen. Additionally, histological analysis revealed a high amount of accumulated sGAG staining inside the porous scaffold in the sesamin co-treating group. In conclusion, the results of this study have indicated that sesamin can be considered a chondrogenic inducing factor and a beneficial dietary supplement for cartilage repair.

Thiamethoxam Toxicity and Effects on Consumption Behavior in Orius insidiosus (Hemiptera: Anthocoridae) on Soybean.

Neonicotinoid residues can be present in soybean vegetative tissue, prey insects, and flower tissues, possibly making them toxic to pollinators and natural enemies. Baseline information on the toxicity of neonicotinoids to beneficial insects other than pollinators through multiple routes of insecticide exposure is limited. The objectives of this study were 1) to evaluate the toxicity of thiamethoxam to the hemipteran predator, Orius insidiosus Say, exposed to residues through treated vegetative tissue and insect prey, and 2) to evaluate the effect of thiamethoxam on the abundance of this predator species in soybean fields. Predators were exposed to thiamethoxam in soybean leaves and Aphis glycines Matsumura using a systemic bioassay. Abundance of the predator was evaluated in thiamethoxam seed-treated fields during two different soybean seasons. Our results indicate that concentrations required to kill >50% of the evaluated insects were higher than the concentrations that the insects are likely to encounter in the field. Consumption of A. glycines by O. insidiosus was affected at 10 ng/ml and 5 ng/ml of thiamethoxam at 24 h of evaluation. There was significant mortality for O. insidiosus at 24 h after exposure to thiamethoxam-treated aphids at these concentrations. In soybean fields, there were no significant differences in O. insidiosus number between the plots treated with thiamethoxam and the control. Thiamethoxam may have significant effects on the predators if O. insidiosus feeds on early soybean vegetative tissue or contaminated prey. These results suggest that the compatibility of thiamethoxam with IPM programs for A. glycines needs further evaluation.

The interactive effect of fungicide residues and yeast assimilable nitrogen on fermentation kinetics and hydrogen sulfide production during cider fermentation.

BACKGROUND: Fungicide residues on fruit may adversely affect yeast during cider fermentation, leading to sluggish or stuck fermentation or the production of hydrogen sulfide (H2 S), which is an undesirable aroma compound. This phenomenon has been studied in grape fermentation but not in apple fermentation. Low nitrogen availability, which is characteristic of apples, may further exacerbate the effects of fungicides on yeast during fermentation. The present study explored the effects of three fungicides: elemental sulfur (S0 ) (known to result in increased H2 S in wine); fenbuconazole (used in orchards but not vineyards); and fludioxonil (used in post-harvest storage of apples). RESULTS: Only S0 led to increased H2 S production. Fenbuconazole (≥0.2 mg L-1 ) resulted in a decreased fermentation rate and increased residual sugar. An interactive effect of yeast assimilable nitrogen (YAN) concentration and fenbuconazole was observed such that increasing the YAN concentration alleviated the negative effects of fenbuconazole on fermentation kinetics. CONCLUSION: Cidermakers should be aware that residual fenbuconazole (as low as 0.2 mg L-1 ) in apple juice may lead to stuck fermentation, especially when the YAN concentration is below 250 mg L-1 . These results indicate that fermentation problems attributed to low YAN may be caused or exacerbated by additional factors such as fungicide residues, which have a greater impact on fermentation performance under low YAN conditions. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Optimization of Caged Electrophiles for Improved Monitoring of Cysteine Reactivity in Living Cells.

Cysteine residues play critical roles in protein function and are susceptible to numerous post-translational modifications (PTMs) that serve to modulate the activity and localization of diverse proteins. Many of these PTMs are highly transient and labile, thus necessitating methods to study these modifications directly within the context of living cells. We previously reported a caged electrophilic probe, CBK1, that can be activated by UV for temporally controlled covalent modification of cysteine residues in living cells. To improve upon the number of cysteine residues identified in cellular cysteine-profiling studies, the reactivity and uncaging efficiency of a panel of caged electrophiles were explored. We identified an optimized caged electrophilic probe, CIK4, that affords significantly improved coverage of cellular cysteine residues. The broader proteome coverage afforded by CIK4 renders it a useful tool for the biological investigation of cysteine-reactivity changes and PTMs directly within living cells and highlights design elements that are critical to optimizing photoactivatable chemical probes for cellular labeling.

Evaluating the teratogenicity of the selective ß3-adrenoceptor agonist, CL 316.243 hydrate by employing FETAX (frog embryo teratogenesis assay).

In this study, the frog embryo teratogenesis assay (FETAX - Xenopus) technique was employed to evaluate the potential teratogenicity of the selective ß-adrenoceptor (AR) agonist, CL 316.243. In this context, CL 316.243 was applied to the South African clawed frog (Xenopus laevis) embryos. The media containing the CL 316.24-exposed embryos were monitored and changed/replaced once every 24 hours. Using FETAX, we determined the minimum concentrations to inhibit growth (MCIG) for CL 316.243. The 96-hour no observable adverse effect concentration (NOAEC), the 96-hour lowest observable adverse effect concentration (LOAEC), the 96-hour EC50 (malformation) and the 96-hour LC50 (lethal concentration) for mortality and malformation could not be determined because the used concentrations did not affect viability or the presence of abnormalities. On the other hand, the MCIG of CL 316.243 was determined as 1 mg/L. Our results demonstrated that CL 316.243 administration was associated with no of teratogenic and toxic effects. However, from first concentration we used (1 to 5 mg/L) length of embryos reduced significantly (p < 0.001) when compared to control of Xenopus embryos. Further studies should be conducted with different concentrations in order to investigate the optimal concentrations for treating preterm labor with these substances.