Ironing out the wrinkles of neutrophil phagocytosis

Though phagocytosis of microbes by professional phagocytes such as neutrophils is crucial for the survival of the host, it is still unclear how the apparent 'stretching' of the plasma membrane is achieved. Microscopically, pseudopod extension, particulate engulfment and phagosome closure all require seemingly large expansions of the cell surface area. Although actual membrane stretching can be ruled out on the basis of physical properties of lipid bilayers, the addition of new membrane from within the cell, either by exocytosis or phagosomal fusion with endoplasmic reticulum membrane, might provide an explanation. However, these events do not seem to have major roles during phagocytosis by neutrophils. Instead, neutrophils might use a more primitive mechanism, that is, the unfolding of surface membrane wrinkles, to provide the additional membrane for phagocytosis. Here, we briefly discuss why membrane unwrinkling provides a feasible hypothesis for membrane expansion during neutrophil phagocytosis, and suggest a potential molecular mechanism for neutrophil control over membrane surface wrinkles, and the potential signalling route.     

Electrogenerated indium tin oxide-coated glass surface with photosensitive interfaces: surface analysis

We present herein a photo-immobilization technique for the localized and specific conjugation of biochip platforms with different proteinaceous bioreceptors, such as antigen or antibodies. This methodology based on a photoactivable electrogenerated polymer film, pyrrole-benzophenone, allows the covalent immobilization of biomolecules through light mediation. The surface-conductive glass platform electropolymerized with poly(pyrrole-benzophenone) thin film may then be used to affinity-coat the chip with molecular recognition probes. This glass chip electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide (ITO). Thereafter, pyrrole-benzophenone monomers are electropolymerized onto the conductive metal oxide surface and then exposed to an antigen Staphylococcal Enterotoxin B (SEB)) solution and illuminated with UV light (wavelength approximately 345 nm) through a mask. As a result of the photochemical reaction, a pattern thin layer of the antigen was covalently bound to the benzophenone-modified surface. Then the sample to be analyzed, along with its specific target antibody (anti-SEB antibodies), is introduced onto the glass surface and left to react with the previously photo-immobilized antigen. When the immuno-reaction is completed, the specifically attached immunoglobulin analytes are detected by using secondary antibodies conjugated with Fluorescein isothiocyanate (FITC). The fluorescence signal emanating from the biochip surface is then quantified by two methods, using a filtered intensified charge-coupled device (CCD) camera and a grating spectrometer.     

Synthesis, structural analysis, and SAR studies of triazine derivatives as potent, selective Tie-2 inhibitors

A novel class of selective Tie-2 inhibitors was derived from a multi-kinase inhibitor 1. By reversing the amide connectivity and incorporating aminotriazine or aminopyridine hinge-binding moieties, excellent Tie-2 potency and KDR selectivity could be achieved with 3-substituted terminal aryl rings. X-ray co-crystal structure analysis aided inhibitor design. This series was evaluated on the basis of potency, selectivity, and rat pharmacokinetic parameters.     

Predicting future community composition from random soil seed bank sampling – evidence from a drained lake bottom

Questions: What is the accuracy and reliability of the commonly used random soil sampling methodology for predicting seedling density, species richness and composition of the emerging seedling community? Location: Lake Kraenepoel, western Belgium. Methods: We compared density, species composition and observed and rarefactioned species richness of the seedling community emerging on a soft water lake bed exposed after drainage with the seedling community germinating in the laboratory from random soil samples in the same plots. Results: Seedling density did not differ between the two methods and there was a significant correlation between seedling density on the exposed lake bed and in the soil samples. This indicates that future seedling density can be reliably predicted based on soil sampling, in particular for the most abundant species. The most frequently occurring and abundant species among the seedlings in the soil samples were also the most frequent and abundant species germinating on the exposed lake bed. In contrast, species richness was much higher on the exposed lake bed than in the soil samples, and this difference was still significant for annual species after correction for differences in sampling intensity by rarefaction. We found no correlation between the number of species retrieved by the two methods. Although seedlings of rare and target species emerged on the lake bed, random soil sampling clearly failed to detect seeds of most of these species. Conclusions: Random soil sampling at a commonly used intensity and using the standard germination conditions can accurately predict future total seedling density and the density of the most abundant species. However, the method is not reliable for predicting the probability of establishment of populations of uncommon species. When executing a seed bank study, sampling intensity and germination conditions need to be adapted to the nature and the level of detail of the research question to be answered.     

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.     

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.