The role of snail aestivation in transmission of schistosomiasis in changing climatic conditions

Schistosomiasis vector snails are subjected to extreme seasonal changes, particularly in ephemeral rivers and lentic waterbodies. In the tropics, aestivation is one of the adaptive strategies for survival and is used by snails in times of extremely high temperatures and desiccation. Aestivation therefore plays an important role in maintaining the transmission of schistosomiasis. This review assesses the possible impacts of climate change on the temporal and spatial distribution of schistosomiasis-transmitting snails with special emphasis on aestivation, and discusses the effect of schistosome infection on aestivation ability. The impacts of parasite development on snails, as well as physiological changes, are discussed with reference to schistosomiasis transmission. This review shows that schistosome-infected snails have lower survival rates during aestivation, and that those that survive manage to get rid of the infection. In general, snail aestivation ability is poor and survival chances diminish with time. Longer dry periods result in fewer, as well as uninfected, snails. However, the ability of the surviving snails to repopulate the habitats is high.     

Phylogeographic concordance factors quantify phylogeographic congruence among co‐distributed species in the Sarracenia alata pitcher plant system

Comparative phylogeographic investigations have identified congruent phylogeographic breaks in co‐distributed species in nearly every region of the world. The qualitative assessments of phylogeographic patterns traditionally used to identify such breaks, however, are limited because they rely on identifying monophyletic groups across species and do not account for coalescent stochasticity. Only long‐standing phylogeographic breaks are likely to be obvious; many species could have had a concerted response to more recent landscape events, yet possess subtle signs of phylogeographic congruence because ancestral polymorphism has not completely sorted. Here, we introduce Phylogeographic Concordance Factors (PCFs), a novel method for quantifying phylogeographic congruence across species. We apply this method to the Sarracenia alata pitcher plant system, a carnivorous plant with a diverse array of commensal organisms. We explore whether a group of ecologically associated arthropods have co‐diversified with the host pitcher plant, and identify if there is a positive correlation between ecological interaction and PCFs. Results demonstrate that multiple arthropods share congruent phylogeographic breaks with S. alata, and provide evidence that the level of ecological association can be used to predict the degree of similarity in the phylogeographic pattern. This study outlines an approach for quantifying phylogeographic congruence, a central concept in biogeographic research.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.     

The carnivorous plant described as Sarracenia alata contains two cryptic species

Modern methods for species delimitation provide biologists with the power to detect cryptic diversity in nearly any system. To illustrate the application of such methods, we collected data (21 sequence loci) from a carnivorous plant in southeastern North America and applied several recently developed methods (Gaussian clustering, Structurama, BPP, spedeSTEM). The pale pitcher plant Sarracenia alata inhabits the southeastern USA along the northern coast of the Gulf of Mexico. Sarracenia alata populations are separated by the Mississippi River and Atchafalaya Basin, a known biogeographical barrier in this region, but the cohesiveness of S. alata as currently classified has not been tested rigorously. Multiple analytical approaches (including allelic clustering and species trees methods) suggest that S. alata comprises two cryptic lineages that correspond to the eastern and western portions of the plant's distribution. That such clear genetic evidence for cryptic diversity exists within S. alata and is in conflict with other sources of data (e.g. morphology, environmental differentiation) illustrates a conundrum faced by those who investigate species boundaries: genetic data are often the first type of data to accumulate evidence of differentiation, but most existing taxonomic treatments are based on nongenetic data. Our results suggest that S. alata as currently described contains two cryptic species, and we recommend the elevation of the western populations to species status. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 737–746.     

An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach

This protocol describes a method that we developed to adapt the tandem affinity purification (TAP) approach for use in mammalian cells. The protocol involves fusing a protein of interest with a tandem tag consisting of two FLAG tags (FF) followed by two protein-A immunoglobulin G (IgG) binding domains (ZZ). The protocol improves upon previously published TAP approaches by employing FLAG in place of calmodulin binding peptide (CBP) with resulting higher recovery during purification. In addition, we use a bicistronic expression system that ensures recovery of stably transfected cell lines expressing easily detectable levels of the protein of interest. A method is also presented for generating cytoplasmic and nuclear extracts, which extends use of this protocol to identify protein-protein interactions occurring specifically in the cytoplasm or nucleus. This protocol facilitates the preparation of partially purified recombinant protein and identification of protein-protein interactions in mammalian cell culture models. The protocol can be completed in 34 h.     

Zoonotic bacterial populations, gut fermentation characteristics and methane production in feedlot steers during oral nitroethane treatment and after the feeding of an experimental chlorate product

Nitroethane inhibits the growth of certain zoonotic pathogens such as Campylobacter and Salmonella spp., foodborne pathogens estimated to cause millions of human infections each year, and enhances the Salmonella- and Escherichia coli-killing effect of an experimental chlorate product being developed as a feed additive to kill these bacteria immediately pre-harvest. Limited studies have shown that nitroethane inhibits ruminal methane production, which represents a loss of 2-12% of the host's gross energy intake and contributes to global warming and destruction of the ozone layer. The present study was conducted to assess the effects of 14-day oral nitroethane administration, 0 (0X), 80 (1X) or 160 (2X)mg nitroethane/kg body weight per day on ruminal and fecal E. coli and Campylobacter, ruminal and fecal methane-producing and nitroethane-reducing activity, whole animal methane emissions, and ruminal and fecal fermentation balance in Holstein steers (n=6 per treatment) averaging 403+/-26 (SD) kg BW. An experimental chlorate product was fed the day following the last nitroethane administration to determine effects on E. coli and Campylobacter. The experimental chlorate product decreased (P<0.001) fecal, but not ruminal (P>0.05) E. coli concentrations by 1000- and 10-fold by 24 and 48 h, respectively, after chlorate feeding when compared to pre-treatment concentrations (>5.7 log(10) colony forming units/g). No effects (P>0.05) of nitroethane or the experimental chlorate product were observed on fecal Campylobacter concentrations; Campylobacter were not recovered from ruminal contents. Nitroethane treatment decreased (P<0.01) ruminal (8.46, 7.91 and 4.74+/-0.78 micromol/g/h) and fecal (3.90, 1.36 and 1.38+/-0.50 micromol/g/h) methane-producing activity for treatments 0X, 1X and 2X, respectively. Administration of nitroethane increased (P<0.001) nitroethane-reducing activity in ruminal, but not fecal samples. Day of study affected ruminal (P<0.0001) but not fecal (P>0.05) methane-producing and nitroethane-reducing activities (P<0.01); treatment by day interactions were not observed (P>0.05). Ruminal accumulations of acetate decreased (P<0.05) in 2X-treated steers when compared with 0X- and 1X-treated steers, but no effect (P>0.05) of nitroethane was observed on propionate, butyrate or the acetate to propionate ratio. Whole animal methane emissions, expressed as L/day or as a proportion of gross energy intake (%GEI), were unaffected by nitroethane treatment (P>0.05), and were not correlated (P>0.05) with ruminal methane-producing activity. These results demonstrate that oral nitroethane administration reduces ruminal methane-producing activity but suggest that a microbial adaptation, likely due to an in situ enrichment of ruminal nitroethane-reducing bacteria, may cause depletion of nitroethane, at least at the 1X administration dose, to concentrations too low to be effective. Further research is warranted to determine if the optimization of dosage of nitroethane or related nitrocompouds can maintain the enteropathogen control and anti-methanogen effect in fed steers.     

Heterogeneous ribonucleoprotein m is a splicing regulatory protein that can enhance or silence splicing of alternatively spliced exons

Splicing of fibroblast growth factor receptor 2 (FGFR2) alternative exons IIIb and IIIc is regulated by the auxiliary RNA cis-element ISE/ISS-3 that promotes splicing of exon IIIb and silencing of exon IIIc. Using RNA affinity chromatography, we have identified heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a splicing regulatory factor that binds to ISE/ISS-3 in a sequence-specific manner. Overexpression of hnRNP M promoted exon IIIc skipping in a cell line that normally includes it, and association of hnRNP M with ISE/ISS-3 was shown to contribute to this splicing regulatory function. Thus hnRNP M, along with other members of the hnRNP family of RNA-binding proteins, plays a combinatorial role in regulation of FGFR2 alternative splicing. We also determined that hnRNP M can affect the splicing of several other alternatively spliced exons. This activity of hnRNP M included the ability not only to induce exon skipping but also to promote exon inclusion. This is the first report demonstrating a role for this abundant hnRNP family member in alternative splicing in mammals and suggests that this protein may broadly contribute to the fidelity of splice site recognition and alternative splicing regulation.     

Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA

A restriction fragment library containing Autographa californica nuclear polyhedrosis virus (AcNPV) DNA was constructed by using the pBR322 plasmid as a vector. The library, which is representative of more than 95% of the viral genome, consists of 2 of the 7 BamHI fragments, 12 of the 24 HindIII fragments, and 23 of the 24 EcoRI fragments. The cloned fragments were characterized and used to generate physical maps of the genome by hybridizing nick-translated recombinant plasmid to Southern blots of AcNPV DNA digested with SmaI, BamHI, XhoI, PstI, HindIII, and EcoRI restriction endonucleases. This information was used to define our strain of AcNPV (HR3) with respect to other strains for which physical maps have been previously published. The hybridization data also indicate that reiteration of DNA sequences occurs at the HindIII-L and -Q regions of the genome.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.