Liquid chromatography-mass spectrometry assay for quantification of Gluten Exorphin B5 in cerebrospinal fluid

A sensitive, precise and accurate method for the quantification of the alimentary opioid peptide Gluten Exorphin B5 (GE-B5, Tyr-Gly-Gly-Trp-Leu) in cerebrospinal fluid (CSF) was developed using liquid chromatography-mass spectrometry (LC-MS). Aliquots (10 microL) of sheep CSF were injected into a LC-MS instrument equipped with a reversed-phase C12 column at a flow rate of 250 microL/min. The mobile phase consisted of Eluent A water with 0.01% acetic acid as an ion-pairing reagent, and Eluent B acetonitrile. The LC-MS system was programmed to divert column flow to waste for 3.5 min after injection, after which time flow was directed into the mass spectrometer that operated in positive ion mode. DADLE (Tyr-D-Ala-Gly-Phe-D-Leu) was used as Internal Standard. No significant interfering peaks were detected at the retention times of GE-B5 in CSF blanks. The calibration curves were linear in the range of 0.39-78.00 ng/mL. The lower limit of detection and the lower limit of quantitation values for GE-B5 in CSF were established at 0.30 and 0.78 ng/mL, respectively. The intra-day and inter-day precision values were <12% relative standard deviation. The intra-day and inter-day accuracy were 99.46-100.86% and 98.95-100.02%, respectively. Recovery of GE-B5 in CSF samples was greater than 80%. Stability studies indicate that GE-B5 in CSF undergoes significant degradation (>55% after 600 min), which is reduced by the addition of protease inhibitors. This is the first reported method for the quantification of GE-B5 in CSF.     

Lipid mediators in marine diatoms

Aquatic Ecology - Diatoms are eukaryotic microalgae representing one of the major groups in the marine phytoplankton, accounting for up to 40% of annual productivity at sea. They are widely...     

Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH₂), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.     

Disparity vs. diversity in Stegosauria (Dinosauria,Ornithischia): cranial and post-cranial sub-dataset provide different signals

The Stegosauria represents an iconic group of ornithischian dinosaurs, with a fossil record spanning the Middle Jurassic to the Late Cretaceous. In this contribution I present the first detailed analysis of the relationship between disparity and diversity through the evolutionary history of the group. The analysis has been performed on a recently published cladistic dataset, allowing the separate study of the signals deriving from discrete characters and from continuous morphometric characters. Whereas the disparity as sum of variance is decoupled with respect to diversity, the sum of ranges provides a signal fairly consistent with the trend in the number of taxa. Both sub-data sets show that evolution of stegosaurs can be considered essentially as symmetrical, i.e. the maximum exploration of the possible morphospace takes place about half way through the history of the group, with subsequent significant decline until extinction in the Upper Cretaceous. An interesting result is a decoupling of the tempo and mode of evolution of the cranium and postcranium in stegosaurs. Specifically, the evolutionary radiation with maximum saturation of morphospace is anticipated in the cranial skeleton, with maximum peak in the Oxfordian; in contrast, the postcranium explores the largest number of morphotypes subsequently during the Kimmeridgian.     

Interactional behaviors of the parasitic beetle Paussus favieri with its ant host Pheidole pallidula: the mimetic role of the acoustical signals

The social parasitic beetle Puussus favieri(Coleoptera,Carabidae,Paussini)performs different types of stridulations,which sclectively mimic those emitted by dif-ferent ant castes of its host Pheidole pallidula(Hymenoptera,Formicidae,Myrmicinae).However,the significance of this acoustical mimicry for the success of the parasitic strat-egy and the behaviors elicited in the host ants by stridulations was unknown.We reared Paussus favieri in Pheidole pallidula colonies and filmed their interacting behaviors.We analyzed in slow motion the behavior of ants near a stridulating beetle.We analyzed sep-arately trains of pulse(Pa+Pb,produced by repeated rubbings)and single pulse(Pc,produced by a single rubbing)of stridulations,clearly recognizable from the shaking up and down of the beetle hind legs.and associated them with differcent ant responscs.The full repertoire of sounds produced by P:favieri elicited benevolent responses both in workers and soldiers.We found that different signals elicit different(sometimes multiplc)bchaviors in ants,with different frequency in the two ant castes.However,Pc(alone or in conjunction with other types of pulses)appears to be the type of acoustic signal mostly responsible for all recorded behaviors.These results indicate that the acoustic channel plays a pivotal role in the host-parasite interaction.Finding that a parasite uses the acoustical channel so intensively,and in such a complicated way to trigger ant bchaviors,indicates that acoustic signals may be more important in ant societies than commonly recognized.     

Some aspects of zinc transport across eel (Anguilla anguilla) red blood cell membranes.

Zinc movement across eel and human red blood cell membranes was measured by atomic absorption spectrophotometry. It was observed that:

• 1) In human red blood cells zinc uptake is twice as rapid as in fish red blood cells over a temperature range of 10-40°C. The low rate of zinc uptake in eel red blood cell may be simply the side effect of different surface area to volume ratios for the differences in cell size or, it may be due to the low permeability of bicarbonate through the red blood cell membranes.
• 2) Zinc uptake measured in eel and human red blood cells treated and untreated with external trypsin shows different features. The zinc uptake was reduced by about 40% in treated eel red blood cells with respect to the total uptake of untreated red blood cells. Human red blood cells treated and untreated with trypsin do not show any differences in the amount of zinc transported.
• 3) In fish red blood cells, zinc uptake in NANO₃ medium is markedly reduced, compared with that measured in NaCl medium. The [Zn²⁺_i slightly increases in the presence of bicarbonate. In human red blood cells in NANO₃ medium the zinc uptake is strongly reduced and the presence of bicarbonate marginally increases the zinc influx.
• 4) In eel red blood cells there seem to be two independent pathways for zinc uptake: one DIDS-sensitive and the other DIDS-insensitive. DIDS 10 μM inhibits only 64% of the total zinc transported. Iincreasing the DIDS concentration did not give more inhibition. In human red blood cells only one DIDS-sensitive pathway for zinc transported seems to exist, because, 2,5 μM DIDS inhibits 97% of zinc uptake.

     

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.     

A Trade-Off between Reproduction and Feather Growth in the Barn Swallow (Hirundo rustica)

Physiological trade-offs mediated by limiting energy, resources or time constrain the simultaneous expression of major functions and can lead to the evolution of temporal separation between demanding activities. In birds, plumage renewal is a demanding activity, which accomplishes fundamental functions, such as allowing thermal insulation, aerodynamics and socio-sexual signaling. Feather renewal is a very expensive and disabling process, and molt is often partitioned from breeding and migration. However, trade-offs between feather renewal and breeding have been only sparsely studied. In barn swallows (Hirundo rustica) breeding in Italy and undergoing molt during wintering in sub-Saharan Africa, we studied this trade-off by removing a tail feather from a large sample of individuals and analyzing growth bar width, reflecting feather growth rate, and length of the growing replacement feather in relation to the stage in the breeding cycle at removal and clutch size. Growth bar width of females and length of the growing replacement feather of both sexes were smaller when the original feather had been removed after clutch initiation. Importantly, in females both growth bar width and replacement feather length were negatively predicted by clutch size, and more strongly so for large clutches and when feather removal occurred immediately after clutch completion. Hence, we found strong, coherent evidence for a trade-off between reproduction, and laying effort in particular, and the ability to generate new feathers. These results support the hypothesis that the derived condition of molting during wintering in long-distance migrants is maintained by the costs of overlapping breeding and molt.     

Do Dogs (Canis lupus familiaris) Make Counterproductive Choices Because They Are Sensitive to Human Ostensive Cues?

Dogs appear to be sensitive to human ostensive communicative cues in a variety of situations, however there is still a measure of controversy as to the way in which these cues influence human-dog interactions. There is evidence for instance that dogs can be led into making evaluation errors in a quantity discrimination task, for example losing their preference for a larger food quantity if a human shows a preference for a smaller one, yet there is, so far, no explanation for this phenomenon. Using a modified version of this task, in the current study we investigated whether non-social, social or communicative cues (alone or in combination) cause dogs to go against their preference for the larger food quantity. Results show that dogs' evaluation errors are indeed caused by a social bias, but, somewhat contrary to previous studies, they highlight the potent effect of stimulus enhancement (handling the target) in influencing the dogs' response. A mild influence on the dog's behaviour was found only when different ostensive cues (and no handling of the target) were used in combination, suggesting their cumulative effect. The discussion addresses possible motives for discrepancies with previous studies suggesting that both the intentionality and the directionality of the action may be important in causing dogs' social biases.