Nitric oxide production by Biomphalaria glabrata haemocytes: effects of Schistosoma mansoni ESPs and regulation through the extracellular signal-regulated kinase pathway

BACKGROUND: Schistosoma mansoni uses Biomphalaria glabrata as an intermediate host during its complex life cycle. In the snail, the parasite initially transforms from a miracidium into a mother sporocyst and during this process excretory-secretory products (ESPs) are released. Nitric oxide (NO) and its reactive intermediates play an important role in host defence responses against pathogens. This study therefore aimed to determine the effects of S. mansoni ESPs on NO production in defence cells (haemocytes) from schistosome-susceptible and schistosome-resistant B. glabrata strains. As S. mansoni ESPs have previously been shown to inhibit extracellular signal-regulated kinase (ERK) phosphorylation (activation) in haemocytes from susceptible, but not resistant, B. glabrata the regulation of NO output by ERK in these cells was also investigated. RESULTS: Haemocytes from resistant snails challenged with S. mansoni ESPs (20 mug/ml) over 5 h displayed an increase in NO production that was 3.3 times greater than that observed for unchallenged haemocytes; lower concentrations of ESPs (0.1-10 mug/ml) did not significantly increase NO output. In contrast, haemocytes from susceptible snails showed no significant change in NO output following challenge with ESPs at any concentration used (0.1-20 mug/ml). Western blotting revealed that U0126 (1 muM or 10 muM) blocked the phosphorylation (activation) status of ERK in haemocytes from both snail strains. Inhibition of ERK signalling by U0126 attenuated considerably intracellular NO production in haemocytes from both susceptible and resistant B. glabrata strains, identifying ERK as a key regulator of NO output in these cells. CONCLUSION: S. mansoni ESPs differentially influence intracellular NO levels in susceptible and resistant B. glabrata haemocytes, possibly through modulation of the ERK signalling pathway. Such effects might facilitate survival of S. mansoni in its intermediate host

The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster

Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH44) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance. Gene expression and labelled DH44 ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH44 in neurons of the pars intercerebralis and DH44 receptor (DH44-R2) in Malpighian tubule principal cells, indicate that suppression of DH44 signalling improves desiccation tolerance of the intact fly. Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH44 neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH44-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH44 and LK signalling pathways. Finally, although a role for DK in desiccation tolerance was not defined, we demonstrate a novel role for Malpighian tubule cell-specific LKR in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress

A Graphic Method For Depicting Horizontal Direction Data On Vertical Outcrop Photographs

Outcrop photographs which show two-dimensional representations of three-dimensionally dipping surfaces (e.g., bedding planes, cross-bed foresets) are commonly utilized in the description of sedimentary strata. In many instances, accurate depiction of the dip direction of such features is paramount for understanding their interpretation, and for visualizing the true form of three-dimensional bodies (e.g., conceptualizing the form of an architectural element in a cliff-face, preserved as a vertical slice that has been cut oblique to paleocurrent direction). However, as an outcrop photograph often presents information on a vertical plane and directional data refers to a horizontal plane, the accurate co-depiction of both sets of information may be challenging. There is presently no universal method for illustrating such measurements on outcrop photographs: techniques in common usage are often imprecise, and the lack of uniformity hinders comparison between different images. Here we present a method for accurately depicting horizontal direction data on vertical outcrop photographs which permits instant visualization of dip relative to the illustrated outcrop geometry. The method is simple to apply, does not compromise primary data, and is unobtrusive to other visual information within images; thus having utility across a broad spectrum of geological investigations

The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules

Multicellular organisms rely on cell adhesion molecules to coordinate cell–cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. Here we show that Fas2 is essential to brush border maintenance in renal tubules of Drosophila. Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, we demonstrate a radically different role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border

The Stargazin-Related Protein {gamma}7 Interacts with the mRNA-Binding Protein Heterogeneous Nuclear Ribonucleoprotein A2 and Regulates the Stability of Specific mRNAs, Including CaV2.2

The role(s) of the novel stargazin-like {gamma}-subunit proteins remain controversial. We have shown previously that the neuron-specific {gamma}7 suppresses the expression of certain calcium channels, particularly CaV2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of {gamma}7 on CaV2.2 expression is via an increase in the degradation rate of CaV2.2 mRNA and hence a reduction of CaV2.2 protein level. Furthermore, exogenous expression of {gamma}7 in PC12 cells also decreased the endogenous CaV2.2 mRNA level. Conversely, knockdown of endogenous {gamma}7 with short-hairpin RNAs produced a reciprocal enhancement of CaV2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed {gamma}7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C terminus of {gamma}7 is essential for all its effects, and we show that {gamma}7 binds directly via its C terminus to a heterogeneous nuclear ribonucleoprotein (hnRNP A2), which also binds to a motif in CaV2.2 mRNA, and is associated with native CaV2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances CaV2.2 IBa, and this enhancement is prevented by a concentration of {gamma}7 that alone has no effect on IBa. The effect of {gamma}7 is selective for certain mRNAs because it had no effect on {alpha}2{delta}-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride cotransporter, KCC1, which contains a similar hnRNP A2 binding motif to that in CaV2.2 mRNA. Our results indicate that {gamma}7 plays a role in stabilizing CaV2.2 mRNA

Three-dimensional structure of CaV3.1: comparison with the cardiac L-type voltage-gated calcium channel monomer architecture.

Calcium entry through voltage-gated calcium channels has widespread cellular effects upon a host of physiological processes including neuronal excitability, muscle excitation-contraction coupling, and secretion. Using single particle analysis methods, we have determined the first three-dimensional structure, at 23 A resolution, for a member of the low voltage-activated voltage-gated calcium channel family, CaV3.1, a T-type channel. CaV3.1 has dimensions of approximately 115x85x95 A, composed of two distinct segments. The cytoplasmic densities form a vestibule below the transmembrane domain with the C terminus, unambiguously identified by the presence of a His tag being approximately 65 A long and curling around the base of the structure. The cytoplasmic assembly has a large exposed surface area that may serve as a signaling hub with the C terminus acting as a "fishing rod" to bind regulatory proteins. We have also determined a three-dimensional structure, at a resolution of 25 A, for the monomeric form of the cardiac L-type voltage-gated calcium (high voltage-activated) channel with accessory proteins beta and alpha2delta bound to the ion channel polypeptide CaV1.2. Comparison with the skeletal muscle isoform finds a good match particularly with respect to the conformation, size, and shape of the domain identified as that formed by alpha2. Furthermore, modeling of the CaV3.1 structure (analogous to CaV1.2 at these resolutions) into the heteromeric L-type voltage-gated calcium channel complex volume reveals multiple interaction sites for beta-CaV1.2 binding and for the first time identifies the size and organization of the alpha2delta polypeptides

Testing asteroseismology with Gaia DR2: Hierarchical models of the Red Clump

Asteroseismology provides fundamental stellar parameters independent of distance, but subject to systematics under calibration. Gaia DR2 has provided parallaxes for a billion stars, which are offset by a parallax zero-point. Red Clump (RC) stars have a narrow spread in luminosity, thus functioning as standard candles to calibrate these systematics. This work measures how the magnitude and spread of the RC in the Kepler field are affected by changes to temperature and scaling relations for seismology, and changes to the parallax zero-point for Gaia. We use a sample of 5576 RC stars classified through asteroseismology. We apply hierarchical Bayesian latent variable models, finding the population level properties of the RC with seismology, and use those as priors on Gaia parallaxes to find the parallax zero-point offset. We then find the position of the RC using published values for the zero-point. We find a seismic temperature insensitive spread of the RC of ~0.03 mag in the 2MASS K band and a larger and slightly temperature-dependent spread of ~0.13 mag in the Gaia G band. This intrinsic dispersion in the K band provides a distance precision of ~1% for RC stars. Using Gaia data alone, we find a mean zero-point of -41 $\pm$ 10 $\mu$as. This offset yields RC absolute magnitudes of -1.634 $\pm$ 0.018 in K and 0.546 $\pm$ 0.016 in G. Obtaining these same values through seismology would require a global temperature shift of ~-70 K, which is compatible with known systematics in spectroscopy.Comment: Accepted for publication in MNRA

A method for estimating the turbulent kinetic energy dissipation rate from a vertically pointing Doppler lidar, and independent evaluation from balloon-borne in situ measurements

A method of estimating dissipation rates from a vertically pointing Doppler lidar with high temporal and spatial resolution has been evaluated by comparison with independent measurements derived from a balloon-borne sonic anemometer. This method utilizes the variance of the mean Doppler velocity from a number of sequential samples and requires an estimate of the horizontal wind speed. The noise contribution to the variance can be estimated from the observed signal-to-noise ratio and removed where appropriate. The relative size of the noise variance to the observed variance provides a measure of the confidence in the retrieval. Comparison with in situ dissipation rates derived from the balloon-borne sonic anemometer reveal that this particular Doppler lidar is capable of retrieving dissipation rates over a range of at least three orders of magnitude. This method is most suitable for retrieval of dissipation rates within the convective well-mixed boundary layer where the scales of motion that the Doppler lidar probes remain well within the inertial subrange. Caution must be applied when estimating dissipation rates in more quiescent conditions. For the particular Doppler lidar described here, the selection of suitably short integration times will permit this method to be applicable in such situations but at the expense of accuracy in the Doppler velocity estimates. The two case studies presented here suggest that, with profiles every 4 s, reliable estimates of ϵ can be derived to within at least an order of magnitude throughout almost all of the lowest 2 km and, in the convective boundary layer, to within 50%. Increasing the integration time for individual profiles to 30 s can improve the accuracy substantially but potentially confines retrievals to within the convective boundary layer. Therefore, optimization of certain instrument parameters may be required for specific implementations

Desiccation, thermal stress and associated mortality in Drosophila fruit flies induced by neuropeptide analogue treatment

Drosophila suzukii is a serious pest of soft fruit worldwide. With the global over-dependence on broad-spectrum pesticides, a strong imperative exists for more environmentally friendly and targeted methods of control. One promising avenue involves employing synthetic neuropeptide analogues as insecticidal agents to reduce pest fitness. Neuropeptides, central to the regulation of physiological and behavioural processes, play a vital role in cold and desiccation survival. Building upon this, the current study investigated the effects of biostable kinin, the cardioacceleratory peptide CAP2b and pyrokinin (PK) analogues (the latter of which have previously displayed cross-talk with the capa receptor), on desiccation, starvation and cold stress tolerance of the pest, D. suzukii, and the closely related non-pest, D. melanogaster. Results demonstrated analogues of the superfamily (CAP2b and PK derived) significantly impacted survival of the target insect under conditions of desiccation stress. However, these peptides enhanced desiccation stress survival in relation to controls, suggesting that they may act as antagonists of the capa signalling pathway in the Malpighian tubules. Of particular note was the ability of analogues 1895 (2Abf-Suc-FGPRLa) and 1902 (2Abf-Suc-FKPRLa) to impact D. suzukii but not D. melanogaster. A focus on native Drosophila CAP2b/PK and kinin sequences in analogue development may yield pure agonists with diuretic action that may reduce desiccation stress survival in the pest flies. In highlighting the PRXamide neuropeptide superfamily more generally, and the structures of promising analogues more specifically, this research will feed the evolution of next-generation analogues and drive forward the development of neuropeptidomimetic-based agents

The most reactive amide as a transition-state mimic for cis-trans interconversion.

1-Azatricyclo[3.3.1.1(3,7)]decan-2-one (3), the parent compound of a rare class of 90°-twisted amides, has finally been synthesized, using an unprecedented transformation. These compounds are of special interest as transition-state mimics for the enzyme-catalyzed cis-trans rotamer interconversion of amides involved in peptide and protein folding and function. The stabilization of the amide group in its high energy, perpendicular conformation common to both systems is shown for the rigid tricyclic system to depend, as predicted by calculation, on its methyl group substitution pattern, making 3 by some way the most reactive known "amide".Financial assistance from Enamine Ltd. (www.enamine.net) is gratefully acknowledged. We also thank Dr. V. Stepanenko for valuable advice and encouragement which helped to find the nonstandard solutions to the synthetic problems, and Vitaliy Bilenko for drawing our attention to the instructive EI mass spectrum of compound 8.This is the final published version of the article, originally published in the Journal of the American Chemical Society, 2015, 137 (2), pp 926–93, DOI: 10.1021/ja511460