Quantitative PCR Method for Sensitive Detection of Ruminant Fecal Pollution in Freshwater and Evaluation of This Method in Alpine Karstic Regions

A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 × 10⁹ marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 10⁵ marker equivalents per liter).     

A cluster of ring stage-specific genes linked to a locus implicated in cytoadherence in Plasmodium falciparum codes for PEXEL-negative and PEXEL-positive proteins exported into the host cell

Blood stages of Plasmodium falciparum export proteins into their erythrocyte host, thereby inducing extensive host cell modifications that become apparent after the first half of the asexual development cycle (ring stage). This is responsible for a major part of parasite virulence. Export of many parasite proteins depends on a sequence motif termed Plasmodium export element (PEXEL) or vacuolar transport signal (VTS). This motif has allowed the prediction of the Plasmodium exportome. Using published genome sequence, we redetermined the boundaries of a previously studied region linked to P. falciparum virulence, reducing the number of candidate genes in this region to 13. Among these, we identified a cluster of four ring stage-specific genes, one of which is known to encode an exported protein. We demonstrate that all four genes code for proteins exported into the host cell, although only two genes contain an obvious PEXEL/VTS motif. We propose that the systematic analysis of ring stage-specific genes will reveal a cohort of exported proteins not present in the currently predicted exportome. Moreover, this provides further evidence that host cell remodeling is a major task of this developmental stage. Biochemical and photobleaching studies using these proteins reveal new properties of the parasite-induced membrane compartments in the host cell. This has important implications for the biogenesis and connectivity of these structures.     

Carbohydrate-based micelle clusters which enhance hydrophobic drug bioavailability by up to 1 order of magnitude

Amphiphilic chitosan-based polymers (Mw < 20 kDa) self-assemble in aqueous media at low micromolar concentrations to give previously unknown micellar clusters of 100-300 nm in size. Micellar clusters comprise smaller 10-30 nm aggregates, and the nanopolarity/drug incorporation efficiency of their hydrophobic domains can be tailored by varying the degree of lipidic derivatization and molecular weight of the carbohydrate. The extent of drug incorporation by these novel micellar clusters is 1 order of magnitude higher than is seen with triblock copolymers, with molar polymer/drug ratios of 1:48 to 1:67. On intravenous injection, the pharmacodynamic activity of a carbohydrate propofol formulation is increased by 1 order of magnitude when compared to a commercial emulsion formulation, and on topical ocular application of a carbohydrate prednisolone formulation, initial drug aqueous humor levels are similar to those found with a 10-fold dose of prednisolone suspension.     

8-Aryl xanthines potent inhibitors of phosphodiesterase 5

In clinical studies, several inhibitors of phosphodiesterase 5 (PDE5) have demonstrated utility in the treatment of erectile dysfunction. We describe herein a series of 8-aryl xanthine derivatives which function as potent PDE5 inhibitors with, in many cases, high levels of selectivity versus other PDE isoforms.     

Abscisic acid and CO2 signalling via calcium sensitivity priming in guard cells, new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses

Background

Stomatal guard cells are the regulators of gas exchange between plants and the atmosphere. Ca²⁺-dependent and Ca²⁺-independent mechanisms function in these responses. Key stomatal regulation mechanisms, including plasma membrane and vacuolar ion channels have been identified and are regulated by the free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt).

Scope

Here we show that CO₂-induced stomatal closing is strongly impaired under conditions that prevent intracellular Ca²⁺ elevations. Moreover, Ca²⁺ oscillation-induced stomatal closing is partially impaired in knock-out mutations in several guard cell-expressed Ca²⁺-dependent protein kinases (CDPKs) here, including the cpk4cpk11 double and cpk10 mutants; however, abscisic acid-regulated stomatal movements remain relatively intact in the cpk4cpk11 and cpk10 mutants. We further discuss diverse studies of Ca²⁺ signalling in guard cells, discuss apparent peculiarities, and pose novel open questions. The recently proposed Ca²⁺ sensitivity priming model could account for many of the findings in the field. Recent research shows that the stomatal closing stimuli abscisic acid and CO₂ enhance the sensitivity of stomatal closing mechanisms to intracellular Ca²⁺, which has been termed ‘calcium sensitivity priming’. The genome of the reference plant Arabidopsis thaliana encodes for over 250 Ca²⁺-sensing proteins, giving rise to the question, how can specificity in Ca²⁺ responses be achieved? Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca²⁺ signal transduction, a topic of central interest in cell signalling research. In this article we further propose an individual stomatal tracking method for improved analyses of stimulus-regulated stomatal movements in Arabidopsis guard cells that reduces noise and increases fidelity in stimulus-regulated stomatal aperture responses ( Box 1). This method is recommended for stomatal response research, in parallel to previously adopted blind analyses, due to the relatively small and diverse sizes of stomatal apertures in the reference plant Arabidopsis thaliana.

Box 1. Improved resolution of stimulus-induced stomatal movements in guard cells by tracking of individual stomatal apertures

Arabidopsis guard cells have become a prime model system for analysing signal transduction, since early research combining genetic and ion channel analyses in this system (Ichida et al., 1997; Pei et al., 1997, 1998; Roelfsema and Prins, 1997). Arabidopsis stomata are small relative to other stomatal model systems and stomatal apertures of various plant types including Arabidopsis are known to show variability in the size of individual stomatal complexes and also variability in the opening apertures of stomata of similar size in a given leaf (Gorton et al., 1988; Mott and Buckley, 2000; Mott and Peak, 2007). Thus stomatal aperture measurements are expected to show a clear degree of statistical variation. Use of blind experiments, in which the genotype and, when possible, the stimulus being applied to guard cells is unknown to the experimenter (Murata et al., 2001) has been employed by several laboratories, has become a standard in the field and has aided in addressing the above limitations of the range of stomatal aperture sizes found under any given condition.Research in our laboratory has shown that a major additional improvement in experiments can be made, by adding imaging of the same individual stomatal apertures over time (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Siegel et al., 2009), while performing blind experiments. In such ‘stomatal tracking’ experiments the lower side of a leaf is attached to a glass coverslip in an extracellular incubation medium (Webb et al., 2001; Young et al., 2006). The mesophyll and upper leaf epidermis are removed surgically for better optical resolution of stomatal apertures in the intact lower leaf epidermis (Young et al., 2006). For stimulus-induced stomatal closing analyses, a field of well-opened stomata is located and images are captured (e.g. using Scion Image software) for later analyses and data storage. The bottom (dry side) of coverslips can be marked with colour marker pens to label grids in the regions where apertures where imaged, for finding these same stomata subsequently if needed. Images of the same stomatal apertures are taken over time and can be stored for later analyses of individual stomatal apertures and for deposition of image files. While this approach has been used as a standard for imposed Ca²⁺ oscillation studies (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Fig. 4), we have found that this method also substantially improves stomatal movement response analyses to any given stimulus (Siegel et al., 2009; see Figs 1 and 4 and, Box Fig. 1). For example, while individual stomata are known to have diverse apertures (e.g. Box Fig. 1C), the relative responses of wide open stomata and smaller stomatal apertures to ABA or to CO₂ were comparable (Fig. 1 and Box Fig. 1; Siegel et al., 2009). Note that this method has previously been proposed and used in Vicia faba (Gorton et al., 1988), for which stomata exhibit relatively weak ABA and CO₂ responses, compared with, for example, Arabidopsis. We propose that this simple image-capturing approach, together with blind analyses, be used as a standard for stomatal response research in arabidopsis. Our research experience with this method shows that this approach will aid in greatly improving resolution and robustness and in defining the functions of individual Ca²⁺-independent and Ca²⁺-dependent components and mechanisms in stomatal response analyses. Open in a separate windowBox Fig. 1.ABA-induced stomatal closing of individually tracked stomatal apertures. (A) Average individually tracked stomatal apertures in the presence of 50 µm Ca²⁺ (open triangles) and in the presence of 200 nm free Ca²⁺ (open squares) in the bath solution from three experiments are shown and were normalized to the stomatal apertures at time = 0. (B, C) ABA-induced stomatal closing in the presence of 50 µm Ca²⁺ in five individually tracked stomatal apertures. In (A; open triangles) normalized stomatal apertures of the same stomata depicted in (B) and (C) are shown. Methods used in these experiments tracking individual stomatal apertures are described in Siegel et al. (2009). ABA-induced stomatal closing experiments are reproduced from Siegel et al. (2009) with permission of the publisher.     

X-ray Crystal Structure and Specificity of the Plasmodium falciparum Malaria Aminopeptidase PfM18AAP

The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.     

Genetic markers for rapid PCR-based identification of gull, Canada goose, duck, and chicken fecal contamination in water

Avian feces contaminate waterways but contribute fewer human pathogens than human sources. Rapid identification and quantification of avian contamination would therefore be useful to prevent overestimation of human health risk. We used subtractive hybridization of PCR-amplified gull fecal 16S RNA genes to identify avian-specific fecal rRNA gene sequences. The subtracters were rRNA genes amplified from human, dog, cat, cow, and pig feces. Recovered sequences were related to Enterobacteriaceae (47%), Helicobacter (26%), Catellicoccus (11%), Fusobacterium (11%), and Campylobacter (5%). Three PCR assays, designated GFB, GFC, and GFD, were based on recovered sequence fragments. Quantitative PCR assays for GFC and GFD were developed using SYBR green. GFC detected down to 0.1 mg gull feces/100 ml (corresponding to 2 gull enterococci most probable number [MPN]/100 ml). GFD detected down to 0.1 mg chicken feces/100 ml (corresponding to 13 Escherichia coli MPN/100 ml). GFB and GFC were 97% and 94% specific to gulls, respectively. GFC cross-reacted with 35% of sheep samples but occurred at about 100,000 times lower concentrations in sheep. GFD was 100% avian specific and occurred in gulls, geese, chickens, and ducks. In the United States, Canada, and New Zealand, the three markers differed in their geographic distributions but were found across the range tested. These assays detected four important bird groups contributing to fecal contamination of waterways: gulls, geese, ducks, and chickens. Marker distributions across North America and in New Zealand suggest that they will have broad applicability in other parts of the world as well.     

Estrogenic plant foods of red colobus monkeys and mountain gorillas in Uganda

Phytoestrogens, or naturally occurring estrogen-mimicking compounds, are found in many human plant foods, such as soybeans (Glycine max) and other legumes. Because the consumption of phytoestrogens may result in both health benefits of protecting against estrogen-dependent cancers and reproductive costs of disrupting the developing endocrine system, considerable biomedical research has been focused on the physiological and behavioral effects of these compounds. Despite this interest, little is known about the occurrence of phytoestrogens in the diets of wild primates, nor their likely evolutionary importance. We investigated the prevalence of estrogenic plant foods in the diets of two folivorous primate species, the red colobus monkey (Procolobus rufomitratus) of Kibale National Park and mountain gorilla (Gorilla beringei) of Bwindi Impenetrable National Park, both in Uganda. To examine plant foods for estrogenic activity, we screened 44 plant items (species and part) comprising 78.4% of the diet of red colobus monkeys and 53 plant items comprising 85.2% of the diet of mountain gorillas using transient transfection assays. At least 10.6% of the red colobus diet and 8.8% of the gorilla diet had estrogenic activity. This was mainly the result of the red colobus eating three estrogenic staple foods and the gorillas eating one estrogenic staple food. All estrogenic plants exhibited estrogen receptor (ER) subtype selectivity, as their phytoestrogens activated ERβ, but not ERα. These results demonstrate that estrogenic plant foods are routinely consumed by two folivorous primate species. Phytoestrogens in the wild plant foods of these two species and many other wild primates may have important implications for understanding primate reproductive ecology.     

Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.