Enantioselective microbial transformation of the phenylpyrazole insecticide fipronil in anoxic sediments

Fipronil, a chiral insecticide, was biotransformed initially to fipronil sulfide in anoxic sediment slurries following a short lag period. Sulfidogenic or methanogenic sediments transformed fipronil with half-lives of approximately 35 and 40 days, respectively. In all microbially active sediment slurries tested, the transformation of fipronil to fipronil sulfide was enantioselective. In the sulfidogenic sediment slurry, the enantiomeric fraction (EF) of fipronil decreased from an initial racemic EF value of 0.46 to a value of 0.22 during the incubation period of active fipronil transformation, indicating preferential transformation of the S-(+)-enantiomer. A previously unidentified product, 5-amino1-[2,6-dichloro-4-(trifluoromethyl)-phenyl]-4-(trifluoromethylthio)-1-H-pyrazole-3-carboxyamide, or fipronil sulfide-amide, was detected in the sulfidogenic slurries and coincided with the loss of fipronil sulfide. Biota from methanogenic freshwater sediment slurries also transformed fipronil enantioselectively but with a preference for the R-(-)-enantiomer. In all microbially inhibited (autoclaved) sediment slurries tested, no changes in the enantiomeric fractions of fipronil were observed and only low levels (< 5% of the added fipronil) of the fipronil sulfide metabolite were detected. In defined (model) chemical experiments, solutions of pyrite (FeS2) and iron sulfide (FeS) non-enantioselectively transformed fipronil primarily to either 2,6-dichloro-4-(trifluoromethyl)-aniline or to fipronil sulfide and fipronil amide, respectively. This report provides the first experimental evidence of enantioselective microbial transformation of fipronil in a natural environment (soil, water, and sediment) as well as identification of a novel fipronil biotransformation product.     

Triplet-sensitized photodegradation of sulfa drugs containing six-membered heterocyclic groups: identification of an SO2 extrusion photoproduct

The aquatic photochemical behavior of a class of sulfa drugs containing six-membered heterocyclic substituents (sulfamethazine, sulfamerazine, sulfadiazine, sulfachloropyridazine, and sulfadimethoxine) was investigated. Photodegradation of the sulfa drugs in a natural water sample was significantly enhanced relative to the degradation in deionized water, with the exception of sulfadimethoxine. This indicated an indirect photochemical process that was identified through the use of quenchers to be attributable to interaction with triplet excited-state dissolved organic matter (3DOM). The direct photolysis rate constant and quantum yield for both the neutral and anionic species of each sulfa drug were calculated using matrix deconvolution methods. The quantum yield values range from 0.01 x 10(-3) for the neutral form of sulfadimethoxine to 5 x 10(-3) for the anionic form of sulfamethazine and are significantly lower than those observed in a previous study for sulfa drugs containing five-membered heterocyclic substituents, although the rate constants are of similar magnitude. The primary product formed in both direct and indirect photodegradation for all five compounds was identified as a sulfur dioxide extrusion product. The predicted environmental half-lives solely attributable to direct photolysis range from 8.6 h in midsummer at 30 degrees latitude in pH 7 surface water for sulfachloropyridazine to 420 h in midwinter at 45 degrees in pH 7 surface water for sulfadimethoxine. These half-lives, except for sulfadimethoxine, will be decreased by interaction with 3DOM.     

Isolation and structural identification of a novel minoxidil analogue in an illegal dietary supplement: triaminodil

A new minoxidil analogue was detected in an illegal dietary supplement advertised as a hair-growth treatment. The analogue was identified using ultra-performance liquid chromatography (UPLC), high-resolution mass spectrometry (LC-HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. The compound was structurally elucidated as a minoxidil analogue in which the piperidinyl group of minoxidil was replaced with a pyrrolidinyl group corresponding to a molecular formula of C₈H₁₃N₅O. The new analogue has been named triaminodil. As this is the first report of the compound, there are no chemical, toxicology or pharmacological data available.     

Expression of a plant serine O-acetyltransferase in Saccharomyces cerevisiae confers osmotic tolerance and creates an alternative pathway for cysteine biosynthesis

Screening of a sugar beet (Beta vulgaris cv. Dita) cDNA library for clones able to confer osmotic tolerance to the osmosensitive gpd1 mutant of Saccharomyces cerevisiae identified a novel serine O-acetyltransferase (BvSAT; EC 2.3.1.30). This enzyme is involved in cysteine biosynthesis in plants and bacteria, producing O-acetylserine, which is converted into cysteine in a reaction catalysed by O-acetylserine sulphydrylase (EC 4.2.99.8). This pathway is not conserved in yeast, where cysteine is synthesized in a four-step pathway starting with homoserine and having O-acetylhomoserine, homocysteine and cystathionine as intermediates. Expression of BvSAT in yeast takes advantage of the activity of yeast O-acetylhomoserine sulphydrylase (MET15/MET17/MET25; EC 4.2.99.10) with O-acetylserine as substrate and induces an alternative cysteine biosynthesis pathway. Our results imply that the resulting increase in cysteine production confers enhanced resistance against osmotic stress in the osmosensitive yeast strain. These data demonstrate that cysteine biosynthesis is a limiting factor in osmotic stress tolerance in yeast.     

Photo-fenton degradation of diclofenac: identification of main intermediates and degradation pathway

In recent years, the presence of pharmaceuticals in the aquatic environment has been of growing interest. These new contaminants are important because many of them are not degraded under the typical biological treatments applied in the wastewater treatment plants and represent a continuous input into the environment. Thus, compounds such as diclofenac are present in surface waters in all Europe and a crucial need for more enhanced technologies that can reduce its presence in the environment has become evident. In this sense, advanced oxidation processes (AOPs) represent a good choice for the treatment of hazardous nonbiodegradable pollutants. This work deals with the solar photodegradation of diclofenac, an antiinflammatory drug, in aqueous solutions by photo-Fenton reaction. A pilot-scale facility using a compound parabolic collector (CPC) reactor was used for this study. Results obtained show rapid and complete oxidation of diclofenac after 60 min, and total mineralization (disappearance of dissolved organic carbon, DOC) after 100 min of exposure to sunlight. Although diclofenac precipitates during the process at low pH, its degradation takes place in the homogeneous phase governed by a precipitation-redissolution-degradation process. Establishment of the reaction pathway was made possible by a thorough analysis of the reaction mixture identifying the main intermediate products generated. Gas chromatography-mass spectrometry (GC/ MS) and liquid chromatography coupled with time-of-flight mass spectrometry (LC/TOF-MS) were used to identify 18 intermediates, in two tentative degradation routes. The main one was based on the initial hydroxylation of the phenylacetic acid moiety in the C-4 position and subsequent formation of a quinone imine derivative that was the starting point for further multistep degradation involving hydroxylation, decarboxylation, and oxidation reactions. An alternative route was based on the transient preservation of the biphenyl amino moiety that underwent a similar oxidative process of C-N bond cleavage. The proposed degradation route differs from those previously reported involving alternative degradation processes (ozonization, UV/H2O2, or photolysis), indicating that diclofenac degradation follows different pathways, depending on the treatment applied.     

Complement factor B and the alternative pathway of complement activation in bovine milk

The contribution of the alternative pathway of complement activation to the capacity of normal milk to deposit C3 fragments on bacteria was tested by attempting to block C3 deposition with antibodies to the alternative pathway component factor B (fB). Factor B was purified and antibodies of the IgY class, which does not activate mammalian complement, were obtained from the egg yolk of immunized laying hens. These antibodies specifically inhibited the deposition of C3. This inhibition and the absence of deposition of C4 demonstrated that C3 deposition in normal milk resulted from the activation of the alternative pathway. Antibodies raised in rabbit were used to develop an ELISA for measuring fB concentrations in milk. The mean concentration of fB was 2.06 microg/ml (+/- 0.18, SEM), 0.57% of the mean value found in serum (360 microg/ml). This proportion was comparable to that of serum albumin (0.63% of serum value) but less than the proportion of C3 in milk (2.71%). Nevertheless, fB was apparently not a limiting factor for the functioning of the alternative pathway, since addition of purified fB to normal milk did not improve C3 deposition. In serum, mild heat-treatment (56 degrees C for 3 min or 50 degrees C for 45 min) blocked the alternative pathway and destroyed fB, as shown by loss of antigenicity in ELISA. In milk, mild heat-treatment did not abrogate C3 deposition, and fB was protected, retaining its functionality and antigenicity. Heating at 56 degrees C for at least 45 min was necessary to completely inhibit C3 deposition in normal milk.     

Naumovozyma castellii: an alternative model for budding yeast molecular biology

Naumovozyma castellii (Saccharomyces castellii) is a member of the budding yeast family Saccharomycetaceae. It has been extensively used as a model organism for telomere biology research and has gained increasing interest as a budding yeast model for functional analyses owing to its amenability to genetic modifications. Owing to the suitable phylogenetic distance to S. cerevisiae, the whole genome sequence of N. castellii has provided unique data for comparative genomic studies, and it played a key role in the establishment of the timing of the whole genome duplication and the evolutionary events that took place in the subsequent genomic evolution of the Saccharomyces lineage. Here we summarize the historical background of its establishment as a laboratory yeast species, and the development of genetic and molecular tools and strains. We review the research performed on N. castellii, focusing on areas where it has significantly contributed to the discovery of new features of molecular biology and to the advancement of our understanding of molecular evolution. Copyright © 2016 John Wiley & Sons, Ltd.     

Study of effects of fipronil and fipronil sulphone on meat nutritional quality and validation of confirmatory GC-MS/MS method for their analysis

The aim of this study was to validate the method for detection and quantification of fipronil and fipronil sulphone in meat products and study the effects of these pollutants on meat nutritional quality. The determination coefficients (R²) of the analytes were 0.999, while intra-laboratory CV% for repeatability and reproducibility ranged from 0.83% to 19.19%. Meat nutritional quality was assessed before and after the addition of analytes in meat samples. Both total and essential amino acids in meat decreased (P < 0.001) with increasing levels of added analytes, suggesting their detrimental effect on meat amino acid composition. The amino acid composition of meat did not show an univocal behaviour as consequence of fipronil and fipronil sulphone addition. In particular, aspartate, glutamate, arginine (P < 0.05), threonine, alanine, lysine and proline (P < 0.001) showed a significant decrease with increasing levels of added analytes, while serine (P < 0.001) evidenced an opposite trend.     

Lachancea thermotolerans as an alternative yeast for the production of beer

The ability of Lachancea thermotolerans strains to ferment brewer's wort has been investigated. Initial fermentations with three L. thermotolerans strains compared the use of maltose and maltotriose, as well as production of glycerol and lactic acid and pH evolution over the course of the fermentation. The most promising strain was subsequently tested for additional traits important for beer production, including pitching rate, generational capacity, foam stability, hop tolerance, vicinal diketone production, oxygen requirement and flocculation. These tests suggest that L. thermotolerans may be a good choice for producing sour beers in a single fermentation step without the use of lactic acid bacteria. Copyright © 2016 The Institute of Brewing & Distilling     

Soyhulls as an alternative feed for lactating dairy cows: a review

Dairy producers use soyhulls, a byproduct of soybean processing, to replace either grain or forage in diets of lactating dairy cows. In view of the nutritional and economical value of soyhulls it is anticipated that this practice will continue to increase in popularity among nutritionists and producers of ruminant animals. This paper reviews information regarding the nutritional value of soyhulls and the effects of feeding this alternative feed on ruminal fermentation, nutrient digestion and utilization, and performance of dairy cows. Soyhulls can replace corn grain to supply about 30% of the dry matter (DM) in high-grain diets without negatively affecting either the fermentation or digestion of nutrients in the gastrointestinal tract or the performance of dairy cows. Additionally, data suggest that soyhulls might successfully replace forage to supply < or = 25% of the DM in diets of dairy cows when the supply of effective fiber, which includes a chemical and a physical component, remains adequate after including the hulls. However, caution should be exercised when data from different studies are extrapolated to practical situations because the response to feeding soyhulls appears to be largely affected by the type of carbohydrate being replaced by soyhulls; the amount, type, and physical form of the dietary forage; and the incidence of either negative or positive associative effects before and after the addition of soyhulls to the original diet. Unfortunately, the paucity of data from experiments in which soyhulls constituted more than 25 to 30% of the dietary DM restricts the ability to identify the maximum amount of soyhulls that can be used in diets of dairy cows. Information from studies in which > or = 25 to 30% of dietary DM supplied as either cereal grains or forages are replaced with soyhulls is needed to better understand and predict the production of dairy cows fed diets containing the hulls. This knowledge is essential for maximizing the use of soyhulls in diets for dairy cows.     

A novel method for species identification in milk and milk-based products

This study describes a method for species-specific detection of animal DNA from different species (cattle, sheep, goat, water buffalo) in milk and dairy products. A primer set was designed in conserved region on the basis of the alignment of the sequence codifying the genomic kappa-casein gene in order to amplify all four species with a single primer pair. Polymorphisms were detected via minisequencing with extension primers designed in conserved sequences for haplotype determination that allow unambiguous assignment to each species. The method was successfully applied to the detection of raw and pasteurized milk from the four different species considered as well as to cheese products from the retail trade. Estimation of the limit of detection was carried out using a progression of dilutions of genomic DNA as well as DNA isolated from milk of a known number of somatic cells from different species in order to be able to achieve detection rates as low as 0.1% bovine milk mixed with buffalo milk.     

Application of the novel Droplet digital PCR technology for identification of meat species

The authenticity and traceability of meat products are issues of primary importance to ensure food safety. Unfortunately, food adulteration (e.g. the addition of inexpensive cuts to minced meat products) and mislabelling (e.g. the inclusion of meat from species other than those declared) happens frequently worldwide. The aim of this study was to apply a droplet digital PCR assay for the detection and quantification (copies μL⁻¹) of the beef, pork, horse, sheep, chicken and turkey in meat products. The analysis conducted on commercial meat showed the presence of traces of DNA from other animal species than those declared. We show that the method is highly sensitive, specific and accurate (accuracy = 100%). This method could be adopted by competent food safety authorities to verify compliance with the labelling of meat products and to ensure quality and safety throughout the meat supply chain, from primary production to consumption.     

一株好氧反硝化细菌的分离与鉴定

为研究活性污泥中的反硝化细菌，从实验室驯化培养的活性污泥反应器中，分离获得1株反硝化细菌P7，进行了菌株形态观察及生理生化测定，并在此基础上，经PCR扩增后测定该菌株的16SrRNA基因序列，由16SrRNA基因序列比较可知，菌株P7与睾丸酮假单胞菌（Comamonas testosteroni）和稻草假单胞菌（Pseudomonas straminea）亲缘关系最为接近，16SrRNA基因相似性达到99．65％。可初步判定其为丛毛单胞菌属（Comamonas sp．）。结合生理生化实验结果，综合分析后，将其鉴定为丛毛单胞菌属（Comamonas sp．）的睾丸酮假单胞菌（Comamonastes tosteroni）。     

一种新型产纤维素酶菌种的分离鉴定

从江西生产豆渣菌的作坊采样,检测到一株分解纤维素的真菌(MC1),分离纯化后,再利用以纤维素为唯一碳源的选择性培养基培养,根据形态特征可鉴定为面包串珠霉(MoniliasitophilaMont.)。摇床发酵产酶实验表明,在30℃、160r/min的条件下摇瓶培养6d后,其所产纤维素酶系中的以羧甲基纤维素钠为底物的CMC酶活为4119U/mL,以微晶纤维素为底物的C1酶活为6833U/mL,以水杨素为底物的βG酶活为451U/mL。     

贵长猕猴桃腐烂菌的侵染途径及分离鉴定

为了明确贵长猕猴桃腐烂的病原菌种类及其可能的侵染途径,选取其花、鲜果及腐烂果为研究对象,进行菌种分离、形态学和分子生物学鉴定、致病性试验。 结果显示,从猕猴桃花中分离到6种真菌,分别为漏斗多孔菌（Polyporusarcularius）、平革菌属（Phanerochaetesp.）、曲霉属（Aspergillussp.）、橘青霉（Penicilliumcitrinum）、枝孢菌属（Cladosporiumsp.）、镰刀菌属（Fusariumsp.）;从猕猴桃鲜果果肉中分离到裂褶菌（Schizophyllumcommune）,鲜果果皮中分离到3种真菌,分别是蔡氏轮层炭壳菌（Daldiniachildiae）、链格孢菌（Alternariasp.）、烟曲霉（Aspergillusfumigates）;从腐烂果中分离到6种真菌,分别是小新壳梭孢菌（Neofusicoccumparvum）、拟茎点霉（Phomopsissp.）、尖孢镰刀菌（Fusariumoxysporum）和3株青霉菌（Penicilliumsp.）。 其中小新壳梭孢菌和拟茎点霉是贵长猕猴桃腐烂的主要病原菌,贵长猕猴桃病原菌的侵染途径可能主要是在采收、运输和包装过程中进入猕猴桃内,引起猕猴桃的腐烂。     

Microarray technology offers a novel tool for the diagnosis and identification of therapeutic targets for male infertility

Male infertility is now a major reproductive health problem because of an increasing number of environmental pollutants and chemicals, which eventually result in gene mutations. Genetic alterations caused by environmental factors account for a significant percentage of male infertility. Microarray technology is a powerful tool capable of measuring simultaneously the expression of thousands of genes expressed in a single sample. Eventually, advances in genetic technology will allow for the diagnosis of patients with male infertility due to congenital reasons or environmental factors. Since its introduction in 1994, microarray technology has made significant advances in the identification and characterization of novel or known genes possibly correlated with male infertility in mice, as well as in humans. This provides a rational basis for the application of microarray to establishing molecular signatures for the diagnosis and gene therapy targets of male infertility. In this review, the differential gene expression patterns characterized by microarray in germ and somatic cells at different steps of development or in response to stimuli, as well as a number of novel or known genes identified to be associated with male infertility in mice and humans, are addressed. Moreover, issues pertaining to measurement reproducibility are highlighted for the application of microarray data to male infertility.