The synthetic varespladib molecule is a multi-functional inhibitor for PLA2 and PLA2-like ophidic toxins

BackgroundThe treatment for snakebites is early administration of antivenom, which can be highly effective in inhibiting the systemic effects of snake venoms, but is less effective in the treatment of extra-circulatory and local effects. To complement standard-of-care treatments such as antibody-based antivenoms, natural and synthetic small molecules have been proposed for the inhibition of key venom components such as phospholipase A₂ (PLA₂) and PLA₂-like toxins. Varespladib (compound LY315920) is a synthetic molecule developed and clinically tested aiming to block inflammatory cascades of several diseases associated with high PLA₂s. Recent studies have demonstrated this molecule is able to potently inhibit snake venom catalytic PLA₂ and PLA₂-like toxins.MethodsIn vivo and in vitro techniques were used to evaluate the inhibitory effect of varespladib against MjTX-I. X-ray crystallography was used to reveal details of the interaction between these molecules. A new methodology that combines crystallography, mass spectroscopy and phylogenetic data was used to review its primary sequence.ResultsVarespladib was able to inhibit the myotoxic and cytotoxic effects of MjTX-I. Structural analysis revealed a particular inhibitory mechanism of MjTX-I when compared to other PLA₂-like myotoxin, presenting an oligomeric-independent function.ConclusionResults suggest the effectiveness of varespladib for the inhibition of MjTX-I, in similarity with other PLA₂ and PLA₂-like toxins.General significanceVarespladib appears to be a promissory molecule in the treatment of local effects led by PLA₂ and PLA₂-like toxins (oligomeric dependent and independent), indicating that this is a multifunctional or broadly specific inhibitor for different toxins within this superfamily.     

A phylogenetic analysis of species in the Bufo crucifer group (Anura: Bufonidae), based on indolealkylamines and proteins from skin secretions

This research tested the utility of two classes of skin secretion compounds to the phylogeny of the Bufo crucifer group. Skin secretions from specimens of nine populations of B. crucifer group were obtained and submitted to qualitative analysis. We observed a clear difference in the composition of the skin secretion molecules obtained from the species of Bufo studied. Fifty-nine molecules, 16 indolealkylamines and 43 proteins, were used as characters, and 39 of these were parsimonious informative. The tree topology of the skin secretion combined data showed areas of congruence and conflict when compared to an mtDNA phylogeny of the B. crucifer group. We used the Templeton test to evaluate the heterogeneity between the skin secretion and mtDNA data. Although not recommended, we performed a combined analysis with the two partitions. The skin secretion characters from the species of Bufo studied have phylogenetic signal. These data are indicative, at least as a preliminary study, of the phylogenetic relationships among the B. crucifer group taxa.     

Presence in invertebrate genomes of sequences characterized by the repetition of the triplet CCPurine

In Drosophila melanogaster (Dm), polypeptidic domains have been found in different morphogenetic genes. Two types of them are characterized by the repetition of nucleotidic triplets: the M repeat (CAX) and the paired repeat (CAXCCX). In this paper we described a third type of repeat isolated from the genome of a Polychaete annelid: Owenia fusiformis. This repeat is characterized by the repetition of the triplet CCPurine. Phylogenetic studies showed the presence of this repeat in all the invertebrate genomes tested (eight copies in Dm genome) while we failed to detect it in vertebrate genomes.     

An assessment of the breeding range,colony sizes and population of the Westland petrel (Procellaria westlandica)

Abstract

The Westland petrel (Procellaria westlandica) is an endemic New Zealand species and one of the very few burrowing seabird species still breeding on mainland New Zealand. It nests only on a series of coastal ridgelines near to Punakaiki on the West Coast of the South Island. Between 2002 and 2005, surveys were undertaken at 28 of the 29 known colonies. The area occupied by the colonies was 73 ha; most colonies had fewer than 50 burrows, but six colonies had 201–500 burrows and four colonies had more than 1000 burrows. We find that the current breeding range of Westland petrel and the location of individual colonies are similar to those reported in both the 1950s and 1970s. Based on total burrow counts at 28 colonies and burrow occupancy rates determined by annual monitoring, the annual breeding population is estimated to be between 2954 and 5137 breeding pairs.     

Bitrophic toxicity of Cry1Ac to Cycloneda sanguinea,a predator in Brazilian cotton

Insect predators are exposed to the Cry1Ac toxin in Bt cotton fields through several pathways. In this study, we investigated the effects of activated Cry1Ac added to a diet on Cycloneda sanguinea (L.) (Coleoptera: Coccinellidae), which is one of the main predators of non‐target pests in Brazilian cotton. Direct bitrophic exposure of C. sanguinea to Cry1Ac was done by feeding beetles with Aphis gossypii (Glover) (Hemiptera: Aphidae) sprayed with 500 μg per ml Cry1Ac solution. Larval and pupal survival, development time, aphid consumption, and adult longevity were recorded daily. Couples within the same experimental treatment were paired and numbers of eggs laid and hatched per female were recorded daily. Net replacement rate was calculated for each female. During development, a C. sanguinea larva consumed on average 1.8 μg of activated Cry1Ac. No significant differences due to Cry1Ac were observed for any of the response variables, except aphid consumption. Larvae receiving Cry1Ac consumed more aphids than larvae receiving distilled water alone. Additional statistical analyses were conducted to evaluate independence of responses, and for the independent responses, a simple meta‐analysis was conducted to test the null hypothesis that all responses were zero. Nearly all of the response variables were statistically independent. Two pairs of responses were not independent, but the associated multivariate tests were not significant. The meta‐analysis suggested that all effects were not different from random variation around zero and no cumulative effects could be detected. Our results indicated that bitrophic exposure to activated Cry1Ac is likely to have little or no adverse ecological effect on C. sanguinea.     

Novel model for “calcium paradox” in sympathetic transmission of smooth muscles: Role of cyclic AMP pathway

It is well established that reduction of Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channel (L-type VDCC), or increase of cytosolic cAMP concentration ([cAMP]c), inhibit contractile activity of smooth muscles in response to transmitters released from sympathetic nerves. Surprisingly, in this work we observed that simultaneous administration of L-type VDCC blocker (verapamil) and [cAMP]c enhancers (rolipram, IBMX and forskolin) potentiated purinergic contractions evoked by electrical field stimulation of rat vas deferens, instead of inhibiting them. These results, including its role in sympathetic transmission, can be considered as a “calcium paradox”. On the other hand, this potentiation was prevented by reduction of [cAMP]c by inhibition of adenylyl cyclase (SQ 22536) or depletion of Ca²⁺ storage of sarco-endoplasmic reticulum by blockade of Ca²⁺ reuptake (thapsigargin). In addition, cytosolic Ca²⁺ concentration ([Ca²⁺]_c) evaluated by fluorescence microscopy in rat adrenal medullary slices was significantly reduced by verapamil or rolipram. In contrast, simultaneous incubation of adrenal slices with these compounds significantly increased [Ca²⁺]_c. This effect was prevented by thapsigargin. Thus, a reduction of [Ca²⁺]_c due to blockade of Ca²⁺ influx through L-type VDCC could stimulate adenylyl cyclase activity increasing [cAMP]c thereby stimulating Ca²⁺ release from endoplasmic reticulum, resulting in augmented transmitter release in sympathetic nerves and contraction.     

Advances in genetic modification of pluripotent stem cells

Genetically engineered stem cells aid in dissecting basic cell function and are valuable tools for drug discovery, in vivo cell tracking, and gene therapy. Gene transfer into pluripotent stem cells has been a challenge due to their intrinsic feature of growing in clusters and hence not amenable to common gene delivery methods. Several advances have been made in the rapid assembly of DNA elements, optimization of culture conditions, and DNA delivery methods. This has lead to the development of viral and non-viral methods for transient or stable modification of cells, albeit with varying efficiencies. Most methods require selection and clonal expansion that demand prolonged culture and are not suited for cells with limited proliferative potential.