Liquid chromatography tandem mass spectrometry analysis of synthetic coccidiostats in eggs

Coccidiostats are synthetic drugs administered to animals, especially to poultry, to cure coccidiosis. In this paper, we present a selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyze residues of five synthetic coccidiostats in eggs: clazuril, diclazuril, robenidine, nicarbazin, toltrazuril and its two metabolites. The extraction efficiency was evaluated by testing several solvents, pH, different volumes and time of extraction. The clean-up procedures were optimized using different solid phase extraction cartridges and different eluants. The chromatographic separation was achieved in reversed phase using a gradient of 0.1% formic acid in water and acetonitrile, whereas the MS detection was performed in negative electrospray ionization (ESI) for all the analytes, except for the robenidine. The developed method has been validated according to Commission Decision 2002/657/CE. The validation parameters, as linearity, precision, recovery, specificity, decision limit (CC alpha), detection capability (CC beta), and robustness have been determined. The proposed method resulted simple, fast, and suitable for screening and confirmation purposes

Determination of Sugar Content in Commercial Fruit Juices by Refractometric, Volumetric and Chromatographic Methods

In this paper several approaches are discussed for the direct analysis of the main sugars in different fruit juices. Refractometry, thin layer chromatography, volumetric analysis and high performance liquid chromatography with refractive index detector were tested and the results compared, discussing the advantages and disadvantages of each of them. Whereas the first method gives generically indications on the whole content of sugar and it doesn't require any prior manipulation of the sample, thin layer chromatography is useful only for qualitative purpose, on the other hand the third method, after removal of interferences, makes possible the determination of the reducing and not reducing sugar, and the last one allows the qualitative and quantitative determination of the saccharides singularly. It's very important to have not only knowledge about the chemical analysis of carbohydrates and their physicochemical properties, but especially how the methods can be used in product development for benefit of the public. In the wide range of options for the determination of the mono and disaccharides in beverages, the approach selected must be robust, accurate, powerful and reproducible

The phase diagram of the three-dimensional Z2 gauge Higgs system at zero and finite temperature

We study the effect of adding a matter field to the Z2 gauge model in three dimensions at zero and finite temperature. Up to a given value of the parameter regulating the coupling, the matter field produces a slight shift of the transition line without changing the universality class of the pure gauge theory, as seen by finite size scaling analysis or by comparison, in the finite temperature case, to exact formulas of conformal field theory. At zero temperature the critical line turns into a first-order transition. The fate of this kind of transition in the finite temperature case is discussed.Comment: 6 pages, 10 figures, based on a poster by AR and a talk by FG at Lattice2002(higgssusy

PU.1 regulates the commitment of adult hematopoietic progenitors and restricts granulopoiesis

Although the transcription factor PU.1 is essential for fetal lymphomyelopoiesis, we unexpectedly found that elimination of the gene in adult mice allowed disturbed hematopoiesis, dominated by granulocyte production. Impaired production of lymphocytes was evident in PU.1-deficient bone marrow (BM), but myelocytes and clonogenic granulocytic progenitors that are responsive to granulocyte colony-stimulating factor or interleukin-3 increased dramatically. No identifiable common lymphoid or myeloid progenitor populations were discernable by flow cytometry; however, clonogenic assays suggested an overall increased frequency of blast colony-forming cells and BM chimeras revealed existence of long-term self-renewing PU.1-deficient cells that required PU.1 for lymphoid, but not granulocyte, generation. PU.1 deletion in granulocyte-macrophage progenitors, but not in common myeloid progenitors, resulted in excess granulocyte production; this suggested specific roles of PU.1 at different stages of myeloid development. These findings emphasize the distinct nature of adult hematopoiesis and reveal that PU.1 regulates the specification of the multipotent lymphoid and myeloid compartments and restrains, rather than promotes, granulopoiesis

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F1, Atp9p-ring, unassembled alpha-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane

Nodular Thyroid Disease in the Era of Precision Medicine

Management of thyroid nodules in the era of precision medicine is continuously changing. Neck ultrasound plays a pivotal role in the diagnosis and several ultrasound stratification systems have been proposed in order to predict malignancy and help clinicians in therapeutic and follow-up decision. Ultrasound elastosonography is another powerful diagnostic technique and can be an added value to stratify the risk of malignancy of thyroid nodules. Moreover, the development of new techniques in the era of "Deep Learning," has led to a creation of machine-learning algorithms based on ultrasound examinations that showed similar accuracy to that obtained by expert radiologists. Despite new technologies in thyroid imaging, diagnostic surgery in 50-70% of patients with indeterminate cytology is still performed. Molecular tests can increase accuracy in diagnosis when performed on "indeterminate" nodules. However, the more updated tools that can be used to this purpose in order to "rule out" (Afirma GSC) or "rule in" (Thyroseq v3) malignancy, have a main limitation: the high costs. In the last years various image-guided procedures have been proposed as alternative and less invasive approaches to surgery for symptomatic thyroid nodules. These minimally invasive techniques (laser and radio-frequency ablation, high intensity focused ultrasound and percutaneous microwave ablation) results in nodule shrinkage and improvement of local symptoms, with a lower risk of complications and minor costs compared to surgery. Finally, ultrasound-guided ablation therapy was introduced with promising results as a feasible treatment for low-risk papillary thyroid microcarcinoma or cervical lymph node metastases

Molecular Basis of the Pathogenic Mechanism Induced by the m.9191T>C Mutation in Mitochondrial ATP6 Gene

International audienceProbing the pathogenicity and functional consequences of mitochondrial DNA (mtDNA) mutations from patient's cells and tissues is difficult due to genetic heteroplasmy (co-existence of wild type and mutated mtDNA in cells), occurrence of numerous mtDNA polymorphisms, and absence of methods for genetically transforming human mitochondria. Owing to its good fermenting capacity that enables survival to loss-of-function mtDNA mutations, its amenability to mitochondrial genome manipulation, and lack of heteroplasmy, Saccharomyces cerevisiae is an excellent model for studying and resolving the molecular bases of human diseases linked to mtDNA in a controlled genetic background. Using this model, we previously showed that a pathogenic mutation in mitochondrial ATP6 gene (m.9191T>C), that converts a highly conserved leucine residue into proline in human ATP synthase subunit a (aL222P), severely compromises the assembly of yeast ATP synthase and reduces by 90% the rate of mitochondrial ATP synthesis. Herein, we report the isolation of intragenic suppressors of this mutation. In light of recently described high resolution structures of ATP synthase, the results indicate that the m.9191T>C mutation disrupts a four α-helix bundle in subunit a and that the leucine residue it targets indirectly optimizes proton conduction through the membrane domain of ATP synthase