Determination of tylosins A, B, C and D in bee larvae by liquid chromatography coupled to ion trap-tandem mass spectrometry

A LC-MS/MS method has been developed to simultaneously quantify tylosins A, B, C and D in bee larvae, compounds currently used to treat one of the most lethal diseases affecting honey bees around the world, American Foulbrood (AFB). The influence of different aqueous media, temperature and light exposure on the stability of these four compounds was studied. The analytes were extracted from bee larvae with methanol and chromatographic separation was achieved on a Luna C(18) (150 × 4.6 mm i.d.) using a ternary gradient composed of a diluted formic acid, methanol and acetonitrile mobile phase. To facilitate sampling, bee larvae were initially dried at 60°C for 4h and afterwards, they were diluted to avoid problems of pressure. MSD-Ion Trap detection was employed with electrospray ionization (ESI). The calibration curves were linear over a wide range of concentrations and the method was validated as sensitive, precise and accurate within the limits of quantification (LOQ, 1.4-4.0 ng/g). The validated method was successfully employed to study bee larvae in field tests of bee hives treated with two formulations containing tylosin. In both cases it was evident that the minimal inhibitory concentration (MIC) had been reached.     

C terminus of the P2X7 receptor: treasure hunting

P2X receptor (P2XR) is a family of the ATP-gated ion channel family and can permeabilize the plasma membrane to small cations such as potassium, sodium, and calcium, resulting in cellular depolarization. There are seven P2XR that have been described and cloned, with 45% identity in amino acid sequence. Each P2X receptors has two transmembrane domains that are separated by an extracellular loop and an intracellular N and C terminus. Unlike the other P2X receptors, the P2X7R has a larger C terminus with an extra 200 amino acid residues compared with the other receptors. The C terminus of the P2X7R has been implicated in regulating receptor function including signaling pathway activation, cellular localization, protein–protein interactions, and post-translational modification (PTM). In the present review, we discuss the role of the P2X7R C terminus in regards to receptor function, describe the specific domains and motifs found therein and compare the C terminus sequence with others proteins to discover predicted domains or sites of PTM.     

Effects of diadenosine tetraphosphate on FGF9-induced chloride flux changes in achondroplastic chondrocytes

Achondroplasia, the most common type of dwarfism, is characterized by a mutation in the fibroblast growth factor receptor 3 (FGFR3). Achondroplasia is an orphan pathology with no pharmacological treatment so far. However, the possibility of using the dinucleotide diadenosine tetraphosphate (Ap₄A) with therapeutic purposes in achondroplasia has been previously suggested. The pathogenesis involves the constitutive activation of FGFR3, resulting in altered biochemical and physiological processes in chondrocytes. Some of these altered processes can be influenced by changes in cell volume and ionic currents. In this study, the action of mutant FGFR3 on chondrocyte size and chloride flux in achondroplastic chondrocytes was investigated as well as the effect of the Ap₄A on these processes triggered by mutant FGFR3. Stimulation with the fibroblast growth factor 9 (FGF9), the preferred ligand for FGFR3, induced an enlarged achondroplastic chondrocyte size and an increase in the intracellular chloride concentration, suggesting the blockade of chloride efflux. Treatment with the Ap₄A reversed the morphological changes triggered by FGF9 and restored the chloride efflux. These data provide further evidence for the therapeutic potential of this dinucleotide in achondroplasia treatment.     

Phylogeography, genetic structure, and gene flow in the endemic freshwater shrimp Palaemonetes suttkusi from Cuatro Ci��negas, Mexico

The valley of Cuatro Ciénegas in Mexico has the highest degree of local endemicity of any habitat in North America. A large number of endemic aquatic species occur both in the Cuatro Ciénegas basin and in the upper parts of the Río Salado de los Nadadores drainage, located immediately to the east of the valley. No natural surface flow occurs between these basins but artificial canals connect both areas. It is not clear whether the sharing of endemics between the Cuatro Ciénegas basin and the Río Salado is due to migration through canals. We conducted a phylogeographic study of mitochondrial haplotypes of the endemic shrimp Palaemonetes suttkusi to determine the evolutionary distinctiveness of the population found in Río Salado. We discovered that P. suttkusi likely has occurred naturally in both basins well into the distant past. Based on molecular clock analyses of the COI gene, the populations in the Río Salado and much of the Cuatro Ciénegas basin likely began diverging from each other between 1.9 and 11.2 Myr ago. The general levels of divergence are substantial but our results suggest there has also likely been recent gene flow between these basins. This is consistent with migration through human-made canals, but also consistent with the occurrence of natural gene flow during intermittent wet periods in the past million years. We also found significant differentiation of the Pozas Azules area from the rest of the Cuatro Ciénegas basin, a finding that is concordant with several phylogeographic studies on other aquatic endemics in Cuatro Ciénegas. We recommend that the upper parts of the Río Salado, the Pozas Azules area, and the rest of the Cuatro Ciénegas basin should each be considered independent evolutionarily significant units for conservation, and that migration of species through human-made canals should be monitored and controlled.