N-glycome profiling of Bothrops jararaca newborn and adult venoms

Glycosylation is an important post-translational modification of snake venom proteins and contributes to venom proteome complexity. Many snake venom components are known to be glycosylated, however, very little is known about the carbohydrate structures present in venom glycoproteins. Previous studies showed that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and shift in animal size are associated with changes in the venom proteome of the snake Bothrops jararaca. In this study we explored the composition of the N-glycome released from newborn and adult B. jararaca venom proteins. We used an ion trap mass spectrometer (IT-MS) to disassemble glycan structures based on the use of several pathways of MS (MSn) and demonstrate the presence of some structural isomers in both newborn and adult venom B. jararaca N-glycans. The main N-glycans identified in both venoms are of the hybrid/complex type however some mannose-rich type structures were also detected. The N-glycan composition of newborn and adult venoms did not vary indicating that differences in the utilization of the N-glycosylation motif could be the explanation for the differences in the glycosylation levels indicated by the differential electrophoretic profiles previously reported for B. jararaca newborn and adult venoms.     

The mouths of earth: The dialectical allegories of the Kwakiutl Indians

Summary The profane condition, baxus, is generated as the antithesis of the primordial myth-state by the great transformations. Differentiation of the mutually predatory kinds of creatures, and the increase of their number, unfolds within a temporal frame created by introducing into the world, the concrete opposites of primordial of darkness, wind and water omnipresent in the myth-state. A predominantly coordinate hierarchy among the mythic protokinds turns into obligatory antagonisms among the profane kinds (of baxus). Special individuals, successors of animal and human Ancestors, are set apart to carry the diminished aura of mythic quasi-oneness through time by means of supernatural communions. Within the human social order, this is the religious origin of caste: the presumed limitation on the communions of men and spirits diminishes the communions of men; special privilege, in economy as in ceremony, is thus conferred upon the spiritually blessed.The antagonism and power asymmetry in baxus impels it always toward the dissolution of its diversity, i.e., toward tsetseqa, manifest as winter. The resurrection of baxus, and summer, out of tsetseqa, on the other hand, is miraculous and ultimately ineffable. Kwakiutls presume that something is contributed to this end by the ritual taming of the Baxbakualanuxsiwae. But in the end, it seems, they cede the mystery of regeneration to him alone: Nobody can imitate your dance... great magician...Vernon Kobrinsky is Associate Professor in the Department of Anthropology, The University of Calgary, Canada.     

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.     

Characterisation of the Small RNAs in the Biomedically Important Green-Bottle Blowfly Lucilia sericata

Background

The green bottle fly maggot, Lucilia sericata, is a species with importance in medicine, agriculture and forensics. Improved understanding of this species’ biology is of great potential benefit to many research communities. MicroRNAs (miRNA) are a short non-protein coding regulatory RNA, which directly regulate a host of protein coding genes at the translational level. They have been shown to have developmental and tissue specific distributions where they impact directly on gene regulation. In order to improve understanding of the biology of L. sericata maggots we have performed small RNA-sequencing of their secretions and tissue at different developmental stages.

Results

We have successfully isolated RNA from the secretions of L. sericata maggots. Illumina small RNA-sequencing of these secretions and the three tissues (crop, salivary gland, gut) revealed that the most common small RNA fragments were derived from ribosomal RNA and transfer RNAs of both insect and bacterial origins. These RNA fragments were highly specific, with the most common tRNAs, such as GlyGCC, predominantly represented by reads derived from the 5’ end of the mature maggot tRNA. Each library also had a unique profile of miRNAs with a high abundance of miR-10-5p in the maggot secretions and gut and miR-8 in the food storage organ the crop and salivary glands. The pattern of small RNAs in the bioactive maggot secretions suggests they originate from a combination of saliva, foregut and hindgut tissues. Droplet digital RT-PCR validation of the RNA-sequencing data shows that not only are there differences in the tissue profiles for miRNAs and small RNA fragments but that these are also modulated through developmental stages of the insect.

Conclusions

We have identified the small-RNAome of the medicinal maggots L. sericata and shown that there are distinct subsets of miRNAs expressed in specific tissues that also alter during the development of the insect. Furthermore there are very specific RNA fragments derived from other non-coding RNAs present in tissues and in the secretions. This new knowledge has applicability in diverse research fields including wound healing, agriculture and forensics.     

Exercise responsive genes measured in peripheral blood of women with chronic fatigue syndrome and matched control subjects

Background  

Chronic fatigue syndrome (CFS) is defined by debilitating fatigue that is exacerbated by physical or mental exertion. To search for markers of CFS-associated post-exertional fatigue, we measured peripheral blood gene expression profiles of women with CFS and matched controls before and after exercise challenge.     

Wing morphology of the active flyer Calliphora vicina (Diptera: Calliphoridae) during its invasion of a sub‐Antarctic archipelago where insect flightlessness is the rule

The cosmopolitan blowfly Calliphora vicina became established in the sub‐Antarctic Kerguelen Islands in the late 1970s, following a warming period that allowed its full development. Although temperature and wind may limit flight activity, the fly invaded the archipelago, reaching sites remote from the introduction point. Most native competitors have converged to flightlessness as a response to stringent environmental conditions and therefore the flight strategy of C. vicina might be either a handicap or a competitive advantage under ongoing climate change. Using geometric morphometrics, we investigated whether the wing had changed over time in C. vicina within the archipelago (1998 vs. 2009) and compared its morphology with that of a continental population from a temperate area (1983 vs. 2009). Wing shape plasticity to temperature was also experimentally investigated. We found no clues of relaxed selection on flight morphology in the range invaded. However, rapid changes of wing shape occurred over time in females from the Kerguelen Islands compared with both males and females of the continental population, despite a shorter time‐lag between samples in the former. The thermal reaction norms for wing shape found for C. vicina from Kerguelen were also different from those of the continental population, but it remains unknown whether this resulted from or preceded the introduction. These combined findings are consistent with a fingerprint of local adaptation in the invasive population. However, the adaptive significance of the changes, in terms of their aerodynamic consequences and the future evolution of C. vicina in the Kerguelen Islands, requires further investigation. From an evolutionary standpoint, sustaining flight capability under the novel sub‐Antarctic conditions might be critical to the invasive success of C. vicina as most competitors are flightless.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.     

A Staining Combination for Phloem and Contiguous Tissues

A technic is described for producing critically stained preparations of phloem tissue. The preparations promise to be relatively stable. Sections of fixed unembedded or of embedded (paraffin or celloidin) phloem, cambium, and xylem are (1) stained in Foster's tannic acid-ferric chloride combination; (2) treated with 1% NaHCOg in 25% or 50% ethyl alcohol for 30 minutes; (3) stained in a saturated solution of lacmoid (made alkaline by adding a few ml. of 1% NaHCO₃ in 25% alcohol) for 12 to 18 hours; (4) dehydrated and cleared in a series composed of 1% solution of NaHCO_s in 50% ethyl alcohol, 80%, 95%, and absolute alcohol, equal proportions of absolute alcohol, clove oil, and xylene, and finally pure xylene; and (5) mounted in a neutral resin. Callose and lignified secondary walls are blue or blue-green in color, cellulose walls and stainable protoplasmic contents are generally light brown. The technic has been successful with sections from 5 to 40μ in thickness, and the staining has been satisfactory for both color and black and white photomicrography.     

Photophosphorylation and related properties of reaggregated vesicles from spinach Photosystem I particles

The small Photosystem I particles prepared from spinach chloroplasts by the action of Triton X-100 (TSF 1 particles) reaggregate into membrane structures when they are incubated with soybean phospholipids and cholate and then subjected to a slow dialysis. The membranes so formed are vesicular in nature and show the capability of catalyzing phenazine methosulfate-mediated cyclic photophosphorylalation at rates which are usually about 20% of those observed with chloroplasts, but higher rates have been obtained. When coupling factor is removed from the chloroplasts by treatment with EDTA, a requirement for coupling factor can be shown for the subsequent ATP formation. The uncouplers carbonylcyanide 3-chlorophenyl-hydrazone, valinomycin, Triton X-100 and NH⁺₄ are effective with the reformed vesicles, which do not show the typical light-induced pH gradient observed with chloroplasts. Incubation of the TSF 1 particles with phospholipids alone allows for the formation of membrane vesicles, but such vesicles are only slightly active in ATP formation. In most properties investigated, the reformed membrane vesicles resemble the original chloroplast membrane so far as phenazine methosulfate-mediated cyclic photophosphorylation is concerned, which indicates a high degree of selectivity in the reaggregation process. The major difference between chloroplasts and the reformed vesicles is the failure of the latter to show a light-induced pH gradient.