Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue

Background  

Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues.     

Increased levels of galactose-deficient anti-Gal immunoglobulin G in the sera of hepatitis C virus-infected individuals with fibrosis and cirrhosis

Hepatitis B and C viruses are major causative agents of liver fibrosis, cirrhosis, and liver cancer. Using comparative glycoproteomics, we identified a glycoprotein that is altered both in amount and in glycosylation as a function of liver fibrosis and cirrhosis. Specifically, this altered glycoprotein is an immunoglobulin G (IgG) molecule reactive to the heterophilic alpha-Gal epitope [Galα-1-3Galβ1-(3)4GlcNAc-R]. While similar changes in glycosylation have been observed in several autoimmune diseases, the specific immunoglobulins and their antigen recognition profiles were not determined. Thus, we provide the first report identifying the specific antigenic recognition profile of an immunoglobulin molecule containing altered glycosylation as a function of liver disease. This change in glycosylation allowed increased reactivity with several fucose binding lectins and permitted the development of a plate-based assay to measure this change. Increased lectin reactivity was observed in 100% of the more than 200 individuals with stage III or greater fibrosis and appeared to be correlated with the degree of fibrosis. The reason for the alteration in the glycosylation of anti-Gal IgG is currently unclear but may be related to the natural history of the disease and may be useful in the noninvasive detection of fibrosis and cirrhosis.     

Affiliation in Four Echimyid Rodent Species based on Intrasexual Dyadic Encounters: Evolutionary Implications

In this work we explored the evolution of sociality in cursorial echimyids by comparing affiliation among three species of Trinomys and one species of Thrichomys. We captured specimens of Trinomys yonenagae, Trinomys albispinus minor, and Thrichomys apereoides in areas of the Brazilian semiarid Caatinga, and Trinomys iheringi denigratus in one area of Atlantic Forest. For each species, we recorded 12 intra‐sexual dyadic encounters in a neutral arena (six between males and six between females) in order to test the hypothesis that species and sex influence level of affiliation. This response variable was assessed based on an affiliation index, calculated as the proportion of the total number of affiliative behaviors to the total number of social behaviors exhibited by the dyad during each encounter. Hypothesis test was performed by means of a parametric two‐way anova . The test was able to detect significant differences only among species, not among sexes. Trinomys yonenagae was the most affiliative species, while T. apereoides and T. albispinus minor were the most agonistic ones. Trinomys iheringi denigratus showed an intermediate pattern. We suggest, based on out‐group comparison, that affiliation in Trinomys increased in the lineage containing T. iheringi denigratus and T. yonenagae and that higher affiliation in the last species can be adaptive to the life in the desert‐like habitat where it lives.     

Transpiration estimation of banana (Musa sp.) plants with the thermal dissipation method

The banana (Musa sp.) plant is one of the largest monocotyledoneous terrestrial herbaceous plants in the world. The measurement of transpiration (Tr) for a whole banana plant is always difficult to perform due to its size. However, the sap flow (SF) of the plant has been successfully measured by using the thermal dissipation probe (TDP) or ‘Granier’ method in the corm of the banana plant (Lu et al. J Exp Bot 53:1771–1779, 2002). The present study aimed to validate their method using a sizable number of banana plants in a greenhouse in Israel. The SF data was compared to the gravimetric measurement of transpiration. The lag time of SF behind Tr was also analyzed. Results showed that the daily SF agreed with the daily Tr when the effective radius for sap flow in the corm was taken as 0.63 R, where R is the radius of the corm. The SF lagged 45 min behind the Tr from 0630 to 1040 hours. Whereas the Tr was not statistically (P>0.05) different from the SF between 1330 and 1700 hours. The reduction in water capacity of banana plant due to SF lag was about 10.5% of the daily Tr, and it recovered gradually in the afternoon. Using more plants can reduce the measurement error of the TDP method. The measured daily SF can be considered as an accurate estimation of the daily Tr for banana plant.     

Shell nacre ultrastructure and depressurisation dissolution in the deep-sea hydrothermal vent mussel <Emphasis Type="Italic">Bathymodiolus azoricus</Emphasis>

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed.     

Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K0.5Na+) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K0.5Na+ at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.     

nAChR‐induced octopamine release mediates the effect of nicotine on a startle response in Drosophila melanogaster

Biogenic amines (BAs) play a central role in the generation of complex behaviors in vertebrates and invertebrates, including the fly Drosophila melanogaster. The comparative advantages of Drosophila as a genetic model to study the contribution of BAs to behaviors stumble upon the difficulty to access the fly brain to ask relevant physiological questions. For instance, it is not known whether the activation of nicotinic acetylcholine receptors (nAChRs) induces the release of BAs in fly brain, a phenomenon associated to several behaviors in vertebrates. Here, we describe a new preparation to study the efflux of BAs in the adult fly brain by in vitro chronoamperometry. Using this preparation we show that nAChR agonists including nicotine induce a fast, transient, dose‐dependent efflux of endogenous BAs, an effect mediated by α‐bungarotoxin‐sensitive nAChRs. By using different genetic tools we demonstrate that the BA whose efflux is induced by nAChR activation is octopamine (Oct). Furthermore, we show that the impairment of a mechanically induced startle response after nicotine exposure is not observed in flies deficient in Oct transmission. Thus, our data show that the efflux of BAs in Drosophila brain is increased by nAChR activation as in vertebrates, and that then AChR‐induced Oct release could have implications in a nicotine‐induced behavioral response.     

The Evolutionary History of the Transposable Element Penelope in the Drosophila virilis Group of Species

We have used phylogenetic techniques to study the evolutionary history of the Penelope transposable element in the Drosophila virilis species group. Two divergent types of Penelope have been detected, one previously described, clade I, and a new one which we have termed clade III. The phylogeny of some copies of the Penelope clade I element was partially consistent with the species phylogeny of the D. montana subphylad, suggesting cospeciation and allowing the estimation of the evolutionary rate of Penelope. Divergence times of elements found in different species are younger than the age of the species, suggesting horizontal transfer events. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Dmitri Petrov]