Serological proteome analysis of Staphylococcus aureus isolated from sub-clinical mastitis

Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis. Studies of the molecular epidemiology of S. aureus strongly suggest that some genetic subsets of strains are particularly well adapted for causing infections in cattle. This communication reports the setup of experimental protocols to identify the immunogenic proteins expressed by one of the most common field isolated strain of S. aureus responsible for sub-clinical mastitis cases. The serological proteome analysis (SERPA) approach applied consists of three main steps: two-dimensional electrophoresis-based separation of the proteins contained in field isolated S. aureus extracts enriched for surface proteins, detection of immunogenic spots using anti-serum collected from sub-clinical mastitis cases and identification of antigens by mass spectrometric-based methodologies. The study allowed to identify three immunogenic proteins: DNAase translocase FtsK, ribosomal proteins S1 and a Tell-like protein.     

Quantification of milk fat in chocolate fats by triacylglycerol analysis using gas-liquid chromatography

The development and in-house testing of a method for the quantification of milk fat in chocolate fats is described. A database consisting of the triacylglycerol profiles of 310 genuine milk fat samples from 21 European countries and 947 mixtures thereof with chocolate fats was created under a strict quality control scheme using 26 triacylglycerol reference standards for calibration purposes. Out of the individual triacylglycerol fractions obtained, 1-palmitoyl-2-stearoyl-3-butyroyl-glycerol (PSB) was selected as suitable marker compound for the determination of the proportion of milk fat in chocolate fats. By using PSB values from the standardized database, a calibration function using simple linear regression analysis was calculated to be used for future estimations of the milk fat content. A comparison with the widely used butyric acid method, which is currently used to determine the milk fat content in nonmilk fat mixtures, showed that both methods were equivalent in terms of accuracy. The advantage of the presented approach is that for further applications, i.e., determination of foreign fats in chocolate fats, just a single analysis is necessary, whereas for the same purpose, the C4 method requires two different analytical methods.     

Pistagremic acid,a glucosidase inhibitor from Pistacia integerrima

Pistacia integerrima Stewart in traditionally used as folk remedy for various pathological conditions including diabetes. In order to identify the bioactive compound responsible for its folk use in diabetes, a phytochemical and biological study was conducted. Pistagremic acid (PA) was isolated from the dried galls extract of P. integerrima. Strong α-glucosidase inhibitory potential of PA was predicted using its molecular docking simulations against yeast α-glucosidase as a therapeutic target. Significant experimental α-glucosidase inhibitory activity of PA confirmed the computational predictions. PA showed potent enzyme inhibitory activity both against yeast (IC₅₀: 89.12 ± 0.12 μM) and rat intestinal (IC₅₀: 62.47 ± 0.09 μM) α-glucosidases. Interestingly, acarbose was found to be more than 12 times more potent an inhibitor against mammalian (rat intestinal) enzyme (having IC₅₀ value 62.47 ± 0.09 μM), as compared to the microbial (yeast) enzyme (with IC₅₀ value 780.21 μM). Molecular binding mode was explored via molecular docking simulations, which revealed hydrogen bonding interactions between PA and important amino acid residues (Asp60, Arg69 and Asp 70 (3.11 Å)), surrounding the catalytic site of the α-glucosidase. These interactions could be mainly responsible for their role in potent inhibitory activity of PA. PA has a strong potential to be further investigated as a new lead compound for better management of diabetes.     

<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. Sp. <Emphasis Type="Italic">melonis-</Emphasis>melon interaction<Emphasis Type="Italic">:</Emphasis> Effect of grafting combination on pathogen gene expression

Bread wheat (BW) and durum wheat (DW) are both strongly affected by Septoria tritici blotch caused by the hemibiotrophic fungus Zymoseptoria tritici. However, only the BW-Z. tritici pathosystem has been well studied so far. Here, we compared compatible interactions between Z. tritici and both BW and DW species at the cytological, biochemical and molecular levels. Fungal infection process investigations showed close spore germination and leaf penetration features in both interactions, although differences in the patterns of these events were observed. During the necrotrophic phase, disease severity and sporulation levels were associated in both interactions with increases of the two cell-wall degrading enzyme activities endo-β-1,4-xylanase and endo-β-1,3-glucanase as well as protease. An analysis of plant defense responses during the first five days post inoculation revealed inductions of GLUC, Chi4, POX and PAL and a repression of LOX gene expressions in both wheat species, although differences in kinetics and levels of induction or repression were observed. In addition, peroxidase, catalase, glucanase, phenylalanine ammonia-lyase and lipoxygenase activities were induced in both wheat species, while only weak accumulations of hydrogen peroxide and polyphenols were detected at the fungal penetration sites. Our study revealed overall a similarity in Z. tritici infection process and triggered wheat defense pathways on both pathosystems.     

Liquid chromatography-tandem mass spectrometric ion-switching determination of chlorantraniliprole and flubendiamide in fruits and vegetables

The anthranilic and phthalic diamides, chlorantraniliprole (CAP) and flubendiamide (FLU), respectively, represent a new class of very effective insecticides that activate the ryanodine-sensitive intracellular calcium release channel (ryanodine receptor). This paper reports an analytical method for the simultaneous determination of the two insecticides on fruits and vegetables by liquid chromatography-electrospray tandem mass spectrometry operated in the positive and negative ionization switching mode. The two diamides were extracted with acetonitrile and separated on a Zorbax Column Eclipse XDB C8 (4.6 mm x 150 mm i.d., 3 microm) by isocratic elution with a mobile phase consisting of acetonitrile and water with 0.1% formic acid pumped at a flow rate of 0.4 mL/min. The diamides were selectively detected by multiple reaction monitoring for transitions of proton adduct precursor ions simultaneously: positive m/z 484.3-->285 for CAP, m/z 445.5-->169 for internal standard, and negative m/z 681.4-->253 for FLU. For CAP calibration in the positive mode was linear over a working range of 2 to 1000 microg/L with r > 0.992. The limit of detection (LOD) and limit of quantification (LOQ) for CAP were 0.8 and 1.6 microg/kg, respectively. For FLU in the negative mode the corresponding values were 1-1000 microg/L for linear working range, with r > 0.996 and 0.4 and 0.8 microg/L for LOD and LOQ, respectively. Moreover, the presence of interfering compounds in the fruit and vegetable extracts was found to be minimal. Due to the linear behavior of the MS detector response for the two analytes, it was concluded that the multiple reaction transitions of molecular ions in the ion-switching mode can be used for analytical purposes, that is, for identification and quantification of diamides in fruit and vegetable extracts at trace levels.     

Applicability of SCAR markers to food genomics: olive oil traceability

DNA analysis with molecular markers has opened a shortcut toward a genomic comprehension of complex organisms. The availability of micro-DNA extraction methods, coupled with selective amplification of the smallest extracted fragments with molecular markers, could equally bring a breakthrough in food genomics: the identification of original components in food. Amplified fragment length polymorphisms (AFLPs) have been instrumental in plant genomics because they may allow rapid and reliable analysis of multiple and potentially polymorphic sites. Nevertheless, their direct application to the analysis of DNA extracted from food matrixes is complicated by the low quality of DNA extracted: its high degradation and the presence of inhibitors of enzymatic reactions. The conversion of an AFLP fragment to a robust and specific single-locus PCR-based marker, therefore, could extend the use of molecular markers to large-scale analysis of complex agro-food matrixes. In the present study is reported the development of sequence characterized amplified regions (SCARs) starting from AFLP profiles of monovarietal olive oils analyzed on agarose gel; one of these was used to identify differences among 56 olive cultivars. All the developed markers were purposefully amplified in olive oils to apply them to olive oil traceability.     

Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schr?t, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.     

ULTRASONOGRAPHIC FINDINGS IN 13 HORSES WITH LYMPHOMA

Ultrasonography and radiography are commonly used for staging of lymphoma in horses, however there is little published information on imaging characteristics for horses with confirmed disease. The purpose of this retrospective, case series study was to describe ultrasonographic and radiographic findings for a group of horses with a confirmed diagnosis of lymphoma. A total of 13 horses were sampled. Lymphadenopathy (8/13), peritoneal effusion (6/13), splenic (6/13), and hepatic (5/13) lesions were the most frequently identified. The predominant splenic and hepatic ultrasonographic lesions were hypoechoic nodules, organomegaly, and changes in echogenicity. Digestive tract lesions were detected in three horses and these included focal thickening and decreased echogenicity of the small (2/13) and large intestinal (2/13) wall. Thoracic lesions were predominantly pleural effusion (4/13), lymphadenopathy (4/13), and lung parenchymal changes (3/13). Enlarged lymph nodes were detected radiographically (4/13) and/or ultrasonographically (2/13) in the thorax and ultrasonographically in the abdomen (7/13) and in the caudal cervical region (4/13). Findings supported the use of abdominal and thoracic ultrasonography for lymphoma staging in horses. Ultrasound landmarks for localizing cecal and caudal deep cervical lymph nodes were also provided.