Implementation of a de novo genome-wide computational approach for updating Brachypodium miRNAs

Plant microRNAs (miRNAs) are single-stranded 20-22 nt small RNAs (sRNA) that are produced from their own genes. We have developed a de novo genome-wide approach for the computational identification of novel plant miRNAs based on the integration of the complete genome sequence with sRNA libraries. It comprises three modules - the clustering module identifies genomic regions that have two closely-located unidirectional sRNA clusters, the mirplan module explores the secondary structure of the genomic regions, and the duplex module predicts miRNA/miRNA* duplexes. We applied our approach to the Brachypodium genome and publicly available sRNA libraries and predicted 102 miRNAs. Our results extend the list of known miRNAs with 58 novel miRNAs and define the genomic loci of all predicted miRNAs. Because this approach considers specific features of plant miRNAs, it can be employed for the analysis of the genome and sRNA libraries generated for plant species to achieve systematic miRNA discovery.     

Evaluating risks of biological control introductions: A probabilistic risk-assessment approach

We address the need to develop improved quantitative procedures for estimating potential non-target impacts of biological control agents in this paper, and propose a probabilistic risk-assessment approach. This approach employs risk-assessment procedures commonly used in many disciplines. The procedure described here uses precision trees to estimate risk based on probabilities that biological control agents will demonstrate predictable behavior under specific conditions, based on their ecological characteristics. We use Trichogramma ostriniae, an egg parasitoid deployed augmentatively against Ostrina nubilalis in the US as case study to conceptually demonstrate the proposed procedure. We propose that this new approach has potential for widespread use in quantifying non-target risk of biological control introductions prior to introductions being made.     

Sexual Approach in the Praying Mantid Mantis Religiosa (L.)

Males of sexually cannibalistic species are thought to express behaviors that reduce the risk of being killed by the females. In several spider species the male modulates his approach to the female as a function of her feeding behavior. We tested this hypothesis in the preying mantid Mantis religiosa (L.). Males were placed behind perching females in a laboratory arena and their position was video-recorded. Females were presented with a prey item (prey-presentation period) and then either they were allowed to capture and eat the prey (“P” treatment), or the prey was removed before the female could capture it (“NP” treatment). For the next 10 min (between-prey-presentation period) the females ate the prey (P), or perched without moving (NP). Total speed of approach of males was over 6-times higher in the P treatment (2.3 cm/min) than in the NP treatment (0.4 cm/min). Speed in the prey-presentation period was higher in P than in NP (4.4 cm/min and 1.3 cm/min, respectively), which shows that seeing a female striking and/or grasping the prey in itself stimulated faster male approach. Approach speed in the between-prey presentation period was also higher in P than in NP (1.9 cm/min and 0.2 cm/min, respectively), which indicates that seeing a female feeding on the prey and/or cleaning her forelegs also stimulates male approach. We conclude that males of M. religiosa can assess the activity state of the females and respond to this information by modulating their speed of approach, probably reducing the risk of being detected and possibly cannibalized.     

Yeast-based protein delivery to mammalian phagocytic cells is increased by coexpression of bacterial listeriolysin

Yeast-mediated protein delivery to mammalian antigen-presenting cells is a powerful approach for inducing cell-mediated immune responses. We show that coexpression of the pore-forming protein listeriolysin O from Listeria monocytogenes leads to improved translocation of a proteinaceous antigen and subsequent activation of specific T lymphocytes. As the resulting yeast carrier is self-attenuated and killed after antigen delivery without exhibiting any toxic effect on antigen-presenting cells, this novel carrier system suggests itself as promising approach for the development of yeast-based live vaccines.     

Use of RNA interference to dissect the roles of trans-acting factors in alternative pre-mRNA splicing

RNA interference (RNAi) is becoming a popular method for analyzing gene function in a variety of biological processes. We have used RNAi in cultured Drosophila cells to identify trans-acting factors that regulate the alternative splicing of endogenously transcribed pre-mRNAs. We have generated a dsRNA library comprising 70% of the Drosophila genes encoding RNA binding proteins and assessed the function of each protein in the regulation of alternative splicing. This approach not only identifies trans-acting factors regulating specific alternative splicing events, but also can provide insight into the alternative splicing regulatory networks of Drosophila. Here, we describe this RNAi approach to identify alternative splicing regulatory proteins in detail.     

FRET hybridization probes for the rapid detection of disease resistance alleles in plants: detection of corky root resistance in lettuce

We describe the development of a non-electrophoresis PCR-based assay for allele discrimination at a disease resistance locus. The assay is based on the emission of light by fluorescence resonance energy transfer (FRET) upon annealing of two hybridization probes. The analysis of melting curve profiles of the probes and templates allowed the detection of single nucleotide polymorphisms. The assay was applied to the detection of alleles at the cor locus in lettuce (Lactuca sativa) that confers recessive resistance to corky root disease. Probes and primers for the assay were designed after the characterization of a single nucleotide polymorphism between alleles of PCR products amplified using a linked marker. That polymorphism was validated in a collection of lettuce varieties representing different genetic backgrounds. The FRET hybridization probes approach provided fast and accurate genotyping of breeding material directly in a one-tube reaction. The absence of electrophoresis makes this approach suitable for applications that require automation and high-throughput genotyping analyses such as marker-assisted selection programs.     

Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily that has been used for bone grafting. We were interested in exploring the functions of BMP-2 in other disease areas and focused on expressing and purifying active BMP-2 proteins. We have developed a new approach which involves using FoldIt refolding buffer to refold BMP-2 followed by a heparin affinity column to separate correctly folded dimer from monomer. A high yield of 29.4 mg BMP-2 dimer per gram cell wet weight was achieved. The purified BMP-2 dimer was shown to possess the same level of activity as BMP-2 from CHO cells as tested by the induction of alkaline phosphatase activity in C2C12 cells. This approach has potential application in refolding and purifying other homodimeric proteins.     

Is heterogeneity of catchability in capture–recapture studies a mere sampling artifact or a biologically relevant feature of the population?

The heterogeneity of catchability (HC) among the individuals encountered during a capture–recapture study has long been regarded as a liability. However, heterogeneous capture probabilities may reflect interesting but hidden features of the population, such as social status. The difficulty is to distinguish between this intrinsic heterogeneity and the extrinsic heterogeneity induced by the study itself. So far, population ecologists have not been able to distinguish between these two sources of variation in capture heterogeneity because, in the presence of heterogeneity of capture in the data, they have frequently used a too simple approach. This traditional approach, which consists of incorporating two common sources of lack of fit (transience and trap-dependence), does not directly model the HC and thus cannot investigate its biological meaning. In this context, we propose, for open populations, to directly model the HC by employing multievent models. Multievent models make it possible to break HC into two classes of catchability viewed as uncertain states. With the introduction of a coefficient of heterogeneity to model proportional probabilities of capture over time in the two classes, our approach allows the investigation of HC in a parsimonious way. In this paper, we apply both this new approach and the traditional approach to a long-term data set of male deer mice Peromyscus maniculatus. We then compare 13 candidate models separately for each approach. Our results indicate that the new approach is superior to the traditional approach. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Confirmation of the mode of action of an antibacterial inhibitor using regulated antisense RNA

Summary We previously demonstrated that regulated antisense RNA technology enables us to validate and identify the mode of action for some antibiotics. In this study, we have expanded the application of the regulated antisense approach to track the mode of action for a novel inhibitor of polypeptide deformylase (Pdf), which is an attractive target for the development of novel classes of antibacterial agents. We created a pdf antisense isogenic strain in Staphylococcus aureus using a TetR-regulated expression system. We demonstrated that the partial inhibition of pdf expression significantly increased the susceptibility of S. aureus to Pdf-specific inhibitor. This result provides further evidence that the TetR-regulated antisense technology is a robust tool for tracking the mode of action of novel antibacterial agents.     

Rationally designed neuropeptide antagonists: A novel approach for generation of environmentally friendly insecticides

We report our approach for the generation of a novel type of putative insecticides based on backbone cyclic peptidomimetic antagonists of insect neuropeptides using pheromone biosynthesis activating neuropeptide (PBAN) as a model. This approach, called the backbone cyclic neuropeptide based antagonist (BBC-NBA), includes the following steps: (i) elucidation of the active sequence of the chosen insect neuropeptide; (ii) disclosure of a lead antagonist based on the sequence found in step (i); (iii) design and synthesis of backbone cyclic peptide libraries (cycloscan) based on the sequence of the lead antagonist; and (iv) design and synthesis of a peptidomimetic prototype insecticide. The BBC-NBA approach was applied to PBAN and led to the discovery of a potent linear lead antagonist and a potent backbone cyclic antagonist devoid of agnoistic activity which inhibited sex pheromone biosynthesis inHeliothis peltigera female moths.     

Indigenous microbes and their soluble factors differentially modulate intestinal glycosylation steps in vivo

It has been shown that Bacteroides thetaiotaomicron, a representative member of the gut microflora, signals intestinal epithelial cells both in vivo and in vitro and modulate specific glycosylation processes that may mediate intestinal functions. However it is not known whether these modulations depend on the presence of live bacteria or may be elicited by soluble factors produced in vitro by this bacterium. We used lectins and an histochemical approach to survey tissue sections prepared from various cellular compartments of the small and large intestine of NRMI/KI mice grown under gnotobiotic conditions. We compared the results obtained with bacterial culture supernatant and live B. thetaiotaomicron to those obtained from germ-free mice or mice having a conventional microflora. This approach allowed us to conclude that (1) a small but specific number of glycan patterns were restored after treatment with bacterial culture supernatant and (2) the B. thetaiotaomicron associated mice restored a larger number of patterns, however, the complete conventional mice pattern must be a function of the whole microflora in the gut. The possibility to modulate this complex glycosylation pattern by introducing exogenous bacteria and bacterial products should be considered as a promising approach towards understanding the molecular basis of microbial-host interactions.     

DNA methylation increases throughout Arabidopsis development

We used amplified fragment length polymorphisms (AFLP) to analyze the stability of DNA methylation throughout Arabidopsis development. AFLP can detect genome-wide changes in cytosine methylation produced by DNA demethylation agents, such as 5-azacytidine, or specific mutations at the DDM1 locus. In both cases, cytosine demethylation is associated with a general increase in the presence of amplified fragments. Using this approach, we followed DNA methylation at methylation sensitive restriction sites throughout Arabidopsis development. The results show a progressive DNA methylation trend from cotyledons to vegetative organs to reproductive organs.     

Community homogenization and the invasiveness of commensal species in Mediterranean afforested landscapes

The ecological consequences of homogenization remain relatively unexplored. One example of landscape-homogenizing is the establishment of plantations. We studied the effect of human-made forests by contrasting plant and small-mammal community composition between planted tree stands and adjacent natural habitat in two different Mediterranean habitats in Israel: (1) inland habitat where we focused on pine (Pinus halepensis) and carob (Ceratonia siliqua) stands, and (2) coastal sand dune habitat where we focused on planted acacia (Acacia saligna) stands. We first wanted to verify whether planted trees modify plant species composition, and second, if and how the small-mammal community is affected by the different habitat conditions created in plantations with different canopy cover. We were especially interested in the abundance of the commensal house mouse (Mus musculus). All tree stands underwent biotic homogenization indicated by abundance of house mice coupled with lower diversity of indigenous vegetation and small-mammal abundances and diversities. Habitat structural diversity was positively related with small-mammals diversity and was lower in artificial tree stands in both habitats. Our results suggest that using the abundance of commensal generalist species such as the house mouse relative to other more specialist small-mammals is a good approach to determine ecosystem integrity. Pre-commercial thinning treatment is a potential management tool to maintain a proportion of native tree species within the canopy of planted tree stands. However, until sufficient data is available for making generalizations, the exact level of thinning necessary to reverse the homogenization processes in man-made plantations and keeping indigenous small-mammal communities diverse and less prone to invasion must be determined empirically.     

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

Development and testing of an optimized method for DNA-based identification of jaguar (<Emphasis Type="Italic">Panthera onca</Emphasis>) and puma (<Emphasis Type="Italic">Puma concolor</Emphasis>) faecal samples for use in ecological and genetic studies

The elusive nature and endangered status of most carnivore species imply that efficient approaches for their non-invasive sampling are required to allow for genetic and ecological studies. Faecal samples are a major potential source of information, and reliable approaches are needed to foster their application in this field, particularly in areas where few studies have been conducted. A major obstacle to the reliable use of faecal samples is their uncertain species-level identification in the field, an issue that can be addressed with DNA-based assays. In this study we describe a sequence-based approach that efficiently distinguishes jaguar versus puma scats, and that presents several desirable properties: (1) considerably high amplification and sequencing rates; (2) multiple diagnostic sites reliably differentiating the two focal species; (3) high information content that allows for future application in other carnivores; (4) no evidence of amplification of prey DNA; and (5) no evidence of amplification of a nuclear mitochondrial DNA insertion known to occur in the jaguar. We demonstrate the reliability and usefulness of this approach by evaluating 55 field-collected samples from four locations in the highly fragmented Atlantic Forest biome of Brazil and Argentina, and document the presence of one or both of these endangered felids in each of these areas.