Faecal genetic analysis to determine the presence and distribution of elusive carnivores: design and feasibility for the Iberian lynx

Noninvasive methods using genetic markers have been suggested as ways to overcome difficulties associated with documenting the presence of elusive species. We present and assess a novel, reliable and effective molecular genetic technique for the unequivocal genetic identification of faeces from the endangered Iberian lynx (Lynx pardinus). From mitochondrial DNA (mtDNA) cytochrome b and D-loop region sequences, we designed four species-specific primers (for products 130-161 bp long) that were considered to be likely to amplify degraded DNA. We compared two DNA extraction methods, various DNA amplification conditions and the robustness and specificity of the primer pairs with 87 lynx samples from 5 potentially different lynx populations and with 328 samples of other carnivore species. The utility of the identification technique was tested with faeces of different ages, with faeces from controlled field experiments, and with faeces collected from locales with possible lynx populations from throughout the state of Andalusia, Spain (8052 km2). Faecal mtDNA extraction was more efficient using PBS wash of the faeces instead of a faeces homogenate. Our assay increased from 92.6 to 99% efficiency with a second amplification and a reduction in template concentration to overcome polymerase chain reaction (PCR) inhibition. Our assay never produced false positives, and correctly identified all lynx faeces. Of 252 faeces samples of unknown species collected throughout Andalusia, 26.6% (from three different areas) were classified as Iberian lynx, 1.4% showed evidence of PCR inhibition and 1.2% were of uncertain origin. This method has proven to be a reliable technique that can be incorporated into large-scale surveys of Iberian lynx populations and exemplifies an approach that can easily be extended to other species.     

IL-11 and IL-11Rα immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor α (IL-11Rα). The present study examined immunoreactive IL-11 and IL-11Rα in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Rα increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Rα was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Rα was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.     

Removal of N-glycans from cell surface proteins induces apoptosis by reducing intracellular glutathione levels in the rhabdomyosarcoma cell line S4MH

Expression of determined Asn-bound glycans (N-glycans) in cell surface glycoproteins regulates different processes in tumour cell biology. Specific patterns of N-glycosylation are displayed by highly metastatic cells and it has been shown that inhibition of N-glycan processing restrains cell proliferation and induces cell death via apoptosis. However, the mechanisms by which different N-glycosylation states may regulate cell viability and growth are not understood. Since malignant cells express high levels of intracellular glutathione (GSH) and a reduction of intracellular GSH induces cell death via apoptosis, we investigated whether GSH was involved in the induction of apoptosis by removal of cell surface N-glycans. We found that removal of N-glycans from cell surface proteins by treating the rhabdomyosarcoma cell line S4MH with tunicamycin or N-glycosidase resulted in a reduction in intracellular GSH content and cell death via apoptosis. Moreover, GSH depletion caused by the specific inhibitor of GSH synthesis BSO induced apoptosis in S4MH cells. This data indicates that adequate N-glycosylation of cell surface glycoproteins is required for maintenance of intracellular GSH levels that are necessary for cell survival and proliferation.     

Mitochondrial DNA variability in the Canary Islands honeybees (Apis mellifera L.)

The mitochondrial DNA (mtDNA) of individuals from 79 colonies of Apis mellifera from five Canary Islands was studied using the Dra I test based on the restriction of PCR products of the tRNAleu–COII intergenic region. Five haplotypes of the African (A) lineage and one of the west European (C) lineage were found. The haplotypes A14 and A15 are described for the first time. These haplotypes have a new P sequence named P₁. The wide distribution and high frequency of haplotype A15 suggest that it is characteristic of the Canarian Archipelago. Sources of haplotype variability of honeybee mtDNA in the Canary Islands (waves of colonization from Africa, queen importations, habitat diversification) are discussed.     

Economic analysis of royalactin production under uncertainty: Evaluating the effect of parameter optimization

Royalactin is a protein with several different potential uses in humans. Research, in insects and in mammalian cells, has shown that it can accelerate cell division and prevent apoptosis. The method of action is through the use of the epidermal growth factor receptor, which is present in humans. Potential use in humans could be to lower cholesterolemic levels in blood, and to elicit similar effects to those seen in bees, e.g., increased lifespan. Mass production of Royalactin has not been accomplished, though a recent article presented a Pichia pastoris fermentation and recovery by aqueous two‐phase systems at laboratory scale as a possible basis for production. Economic modelling is a useful tool with which compare possible outcomes for the production of such a molecule and in particular, to locate areas where additional research is needed and optimization may be required. This study uses the BioSolve software to perform an economic analysis on the scale‐up of the putative process for Royalactin. The key parameters affecting the cost of production were located via a sensitivity analysis and then evaluated by Monte Carlo analysis. Results show that if titer is not optimized the strategy to maintain a low cost of goods is process oriented. After optimization of this parameter the strategy changes to a product‐oriented and the target output becomes the critical parameter determining the cost of goods. This study serves to provide a framework for the evaluation of strategies for future production of Royalactin, by analyzing the factors that influence its cost of manufacture. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:744–749, 2015