Identification of novel SALMFamide neuropeptides in the starfish Marthasterias glacialis

The SALMFamides are a family of neuropeptides found in species belonging to the phylum Echinodermata and which act as muscle relaxants. The first two members of this family to be identified were both isolated from the starfishes Asterias rubens and Asterias forbesi and are known as S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide). However, little is known about the occurrence and characteristics of SALMFamide neuropeptides in other starfish species. Here we report the identification of four SALMFamide neuropeptides in the starfish Marthasterias glacialis: GFNSALMFamide (S1), SGPYSMTSGLTFamide (MagS2), AYHSALPFamide (MagS3), and AYQTGLPFamide (MagS4). Analysis of the effects of MagS2 and MagS3 on cardiac stomach preparations from Asterias rubens revealed that both peptides cause dose-dependent relaxation, consistent with previous studies using S1 and S2. The identification of four SALMFamide neuropeptides in Marthasterias glacialis provides new insights into the diversity and phylogenetic distribution of SALMFamide neuropeptides in the class Asteroidea of the phylum Echinodermata. In particular, the identification of MagS3 and MagS4, in addition to S1 and the S2-like peptide MagS2, has revealed a greater diversity of SALMFamide neuropeptides occurring in a starfish species than any previous studies.     

A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production

A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.     

Application of the secondary structure model of rRNA for phylogeny: D2-D3 expansion segments of the LSU gene of plant-parasitic nematodes from the family Hoplolaimidae Filipjev, 1934

Knowledge of rRNA structure is increasingly important to assist phylogenetic analysis through reconstructing optimal alignment, utilizing molecule features as an additional source of data and refining appropriate models of evolution of the molecule. We describe a procedure of optimization for alignment and a new coding method for nucleotide sequence data using secondary structure models of the D2 and D3 expansion fragments of the LSU-rRNA gene reconstructed for fifteen nematode species of the agriculturally important and diverse family Hoplolaimidae, order Tylenchida. Using secondary structure information we converted the original sequence data into twenty-eight symbol codes and submitted the transformed data to maximum parsimony analysis. We also applied the original sequence data set for Bayesian inference. This used the doublet model with sixteen states of nucleotide doublets for the stem region and the standard model of DNA substitution with four nucleotide states for loops and bulges. By this approach, we demonstrate that using structural information for phylogenetic analyses led to trees with lower resolved relationships between clades and likely eliminated some artefactual support for misinterpreted relationships, such as paraphyly of Helicotylenchus or Rotylenchus. This study as well as future phylogenetic analyses is herein supported by the development of an on-line database, NEMrRNA, for rRNA molecules in a structural format for nematodes. We also have developed a new computer program, RNAstat, for calculation of nucleotide statistics designed and proposed for phylogenetic studies.     

Optimization of backward giant circle technique on the asymmetric bars

The release window for a given dismount from the asymmetric bars is the period of time within which release results in a successful dismount. Larger release windows are likely to be associated with more consistent performance because they allow a greater margin for error in timing the release. A computer simulation model was used to investigate optimum technique for maximizing release windows in asymmetric bars dismounts. The model comprised four rigid segments with the elastic properties of the gymnast and bar modeled using damped linear springs. Model parameters were optimized to obtain a close match between simulated and actual performances of three gymnasts in terms of rotation angle (1.5 degrees ), bar displacement (0.014 m), and release velocities (<1%). Three optimizations to maximize the release window were carried out for each gymnast involving no perturbations, 10-ms perturbations, and 20-ms perturbations in the timing of the shoulder and hip joint movements preceding release. It was found that the optimizations robust to 20-ms perturbations produced release windows similar to those of the actual performances whereas the windows for the unperturbed optimizations were up to twice as large. It is concluded that robustness considerations must be included in optimization studies in order to obtain realistic results and that elite performances are likely to be robust to timing perturbations of the order of 20 ms.     

Ant Diversity in Dominant Vegetation Types of Southern Cameroon

Ants have been shown as particularly affected by land disturbance through deforestation and conversion of forest to agriculture. The effect of land use change on ant diversity in the Congo Basin is not well known. We conducted intensive sampling along a gradient of increasing vegetation disturbance to test the effect of habitat disturbance on ant diversity and Functional Groups composition. Sampling was conducted in 30 plots (5 study sites × 3 habitat × 2 plots/habitat), replicated six times in 1 year. In each plot, ants were monitored with pitfall traps, quadrats and baits. We recorded 237 ant morphospecies grouped in 10 subfamilies and 43 genera. Myrmicaria opaciventris was the most abundant species followed by Anoplolepis tenella. Forest had greater ant diversity compared with fallows and mixed‐crop fields. Functional groups were dominated by Opportunists, followed by Omnivorous Arboreal Dominants and Generalized Mymicinae. Their composition was not affected by the disturbance, but occurrence of Specialist Predators decreased with increasing disturbance. Occurrence of Generalized Myrmicinae, Opportunists and Subordinate Camponotini increased with disturbance. These results indicate that forest conversion into mixed‐crop fields reduce ant diversity. It can also increase abundance of species with generalized diet that predominates where stress and disturbance limits other ants.     

Parallel declines in survival of adult Northern Fulmars Fulmarus glacialis at colonies in Scotland and Ireland

Assessing broad‐scale changes in seabird populations across the North Atlantic requires an integration of available datasets to understand the spatial extent of potential drivers and demographic change. Here, we compared survival of Northern Fulmars Fulmarus glacialis from a Scottish and an Irish colony from 1974 to 2009. Despite lower recapture probabilities of monel‐ringed Irish birds compared with colour‐ringed Scottish birds, survival probability decreased at both colonies. The extent to which the decline in survival is related to density‐dependent processes or other external drivers remains uncertain, but our results suggest that these changes in survival are possibly indicative of larger‐scale processes and are not confined to local colony dynamics.     

Optimized microRNA purification from TRIzol-treated plasma

Background

MicroRNAs (miRNAs) represent new and potentially informative diagnostic targets for disease detection and prognosis. However, little work exists documenting the effect of TRIzol, a common viral inactivation and nucleic acid extraction reagent, on miRNA purification. Here, we developed an optimized protocol for miRNA extraction from plasma samples by evaluating five different RNA extraction kits, TRIzol phase separation, purification additives, and initial plasma sample volume. This method was then used for downstream profiling of plasma miRNAs found in archived samples from one nonhuman primate (NHP) experimentally challenged with Ebola virus by the aerosol route.

Results

Comparison of real-time RT-PCR results for spiked-in and endogenous miRNA sequences determined extraction efficiencies from five different RNA purification kits. These experiments showed that 50 μL plasma processed using the QIAGEN miRNeasy Mini Kit with 5 μg of glycogen as a co-precipitant yielded the highest recovery of endogenous miRNAs. Using this optimized protocol, miRNAs from archived plasma samples of one rhesus macaque challenged with aerosolized Ebola virus was profiled using a targeted real-time PCR array. A total of 519 of the 752 unique miRNAs assayed were present in the plasma samples at day 0 and day 7 (time of death) post-exposure. Statistical analyses revealed 25 sequences significantly up- or down-regulated between day 0 and day 7 post infection, validating the utility of the extraction method for plasma miRNA profiling.

Conclusions

This study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized an extraction protocol for miRNAs from TRIzol-inactivated plasma samples that can be used for highly pathogenic viruses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1299-5) contains supplementary material, which is available to authorized users.     

Reorganization of the nuclear lamina and cytoskeleton in adipogenesis

A thorough understanding of fat cell biology is necessary to counter the epidemic of obesity. Although molecular pathways governing adipogenesis are well delineated, the structure of the nuclear lamina and nuclear-cytoskeleton junction in this process are not. The identification of the ‘linker of nucleus and cytoskeleton’ (LINC) complex made us consider a role for the nuclear lamina in adipose conversion. We herein focused on the structure of the nuclear lamina and its coupling to the vimentin network, which forms a cage-like structure surrounding individual lipid droplets in mature adipocytes. Analysis of a mouse and human model system for fat cell differentiation showed fragmentation of the nuclear lamina and subsequent loss of lamins A, C, B1 and emerin at the nuclear rim, which coincides with reorganization of the nesprin-3/plectin/vimentin complex into a network lining lipid droplets. Upon 18 days of fat cell differentiation, the fraction of adipocytes expressing lamins A, C and B1 at the nuclear rim increased, though overall lamin A/C protein levels were low. Lamin B2 remained at the nuclear rim throughout fat cell differentiation. Light and electron microscopy of a subcutaneous adipose tissue specimen showed striking indentations of the nucleus by lipid droplets, suggestive for an increased plasticity of the nucleus due to profound reorganization of the cellular infrastructure. This dynamic reorganization of the nuclear lamina in adipogenesis is an important finding that may open up new venues for research in and treatment of obesity and nuclear lamina-associated lipodystrophy.     

Knockdown of SPARC leads to decreased cell-cell adhesion and lens cataracts during post-gastrula development in Xenopus laevis

SPARC is a multifunctional matricellular glycoprotein with complex, transient tissue distribution during embryonic development. In Xenopus laevis embryos, zygotic activation of SPARC is first detected during late gastrulation, undergoing rapid changes in its spatiotemporal distribution throughout organogenesis. Injections of anti-sense Xenopus SPARC morpholinos (XSMOs) into 2- and 4-cell embryos led to a dose-dependent dissociation of embryos during neurula and tailbud stages of development. Animal cap explants derived from XSMO-injected embryos also dissociated, resulting in the formation of amorphous ciliated microspheres. At low doses of XSMOs, lens cataracts were formed, phenocopying that observed in Sparc-null mice. At XSMOs concentrations that did not result in a loss of axial tissue integrity, adhesion between myotomes at intersomitic borders was compromised with a reduction in SPARC concentration. The combined data suggest a critical requirement for SPARC during post-gastrula development in Xenopus embryos and that SPARC, directly or indirectly, promotes cell?Ccell adhesion in vivo.     

<Emphasis Type="BoldItalic">In vivo</Emphasis> conditions influence the coding of stimulus features by bursts of action potentials

The functional role of burst firing (i.e. the firing of packets of action potentials followed by quiescence) in sensory processing is still under debate. Should bursts be considered as unitary events that signal the presence of a particular feature in the sensory environment or is information about stimulus attributes contained within their temporal structure? We compared the coding of stimulus attributes by bursts in vivo and in vitro of electrosensory pyramidal neurons in weakly electric fish by computing correlations between burst and stimulus attributes. Our results show that, while these correlations were strong in magnitude and significant in vitro, they were actually much weaker in magnitude if at all significant in vivo. We used a mathematical model of pyramidal neuron activity in vivo and showed that such a model could reproduce the correlations seen in vitro, thereby suggesting that differences in burst coding were not due to differences in bursting seen in vivo and in vitro. We next tested whether variability in the baseline (i.e. without stimulation) activity of ELL pyramidal neurons could account for these differences. To do so, we injected noise into our model whose intensity was calibrated to mimic baseline activity variability as quantified by the coefficient of variation. We found that this noise caused significant decreases in the magnitude of correlations between burst and stimulus attributes and could account for differences between in vitro and in vivo conditions. We then tested this prediction experimentally by directly injecting noise in vitro through the recording electrode. Our results show that this caused a lowering in magnitude of the correlations between burst and stimulus attributes in vitro and gave rise to values that were quantitatively similar to those seen under in vivo conditions. While it is expected that noise in the form of baseline activity variability will lower correlations between burst and stimulus attributes, our results show that such variability can account for differences seen in vivo. Thus, the high variability seen under in vivo conditions has profound consequences on the coding of information by bursts in ELL pyramidal neurons. In particular, our results support the viewpoint that bursts serve as a detector of particular stimulus features but do not carry detailed information about such features in their structure.