A Survey of Mosquito Populations for Evidence of Lateral Gene Transfer.

Lateral gene transfer is an important evolutionary mechanism between organisms through a variety of processes. In prokaryotes it can be mediated by conjugation, transformation and transduction, and in eukaryotes, transposable elements and viruses play a key role. Additionally, complex evolutionary relationships driven by lateral gene transfer can exist in eukaryotic organisms infected with prokaryotes. One such evolutionary system is seen in mosquitoes carrying Dirofilaria immitis (the causative agent of heartworm) and WO phage-infected Wolbachia pipientis. The studies herein explored evidence of lateral gene transfer in this system of coevolution. Mosquitoes were trapped using resting boxes or light traps and identified taxonomically. Mosquito genomic DNA was used to perform polymerase chain reaction (PCR) to detect the W. pipientis surface protein (wsp) gene. PCR products from Culiseta melanura (856 bp) and A. quadrimaculatus (806 bp) were cloned, sequenced and subjected to genomic analysis. DNA sequencing data showed a 29 bp region corresponding to the upstream primer region at the beginning of the PCR product with 100% homology to the wsp gene. A downstream region of 31 bp at the end of the DNA sequence had 92% homology to the wsp gene. In addition, the 856 bp and 806 bp PCR product was comprised of unidentified DNA with 73-74% homology to Aedes egypti. A detailed analysis is difficult since the genomic DNA sequence for these mosquitoes has not yet been reported. However this data supports that these PCR products have mosquito origin. The data also evidence lateral gene transfer of wsp into these mosquito genomes. Additionally, Anopheles species may also contain W. pipientis DNA. These observations are noteworthy since, evidence of Wolbachia infection and lateral gene transfer of W. pipientis DNA in Anopheles mosquitoes has not been previously reported

Horizon detection in the visible spectrum

In the last few decades, machine learning and computer vision techniques have enabled precise and repeatable image recognition. Computer vision techniques can also recognize star patterns in star trackers for satellite attitude determination. Horizon detection in the visible spectrum was largely discarded for attitude determination in favor of thermal imagery, due to the greater consistency of the earth's thermal radiation. This thesis examines computer vision and machine learning techniques to develop a horizon detection algorithm for the visible spectrum. By examining different features of visual imagery, machine learning techniques were evaluated on the ability to detect a visible horizon and determine its orientation. An empirical analysis of visual imagery from low-earth orbit was conducted to develop a horizon brightness transition model, which allows for consistent and adjustable determination of the horizons location. The final result is a horizon detection and orientation determination algorithm that successfully indicates if a horizon is present in an image with 96% precision and 92% recall. The brightness model correctly identifies the location of the horizon in 85% of the tested image set.http://archive.org/details/horizondetection1094550527Lieutenant, United States NavyApproved for public release; distribution is unlimited

Tidal changes in estuarine systems induced by local geomorphologic modifications

Although rising global sea levels will affect the estuarine flooded areas over the coming decades, the local and regional-scale processes will also induce important changes in these coastal systems. The main aim of this work is to investigate possible tidal changes in estuarine systems induced by local geomorphologic modifications, analysing the particular case of Ria de Aveiro which is in risk of inundation. Located in the Portuguese west coast, this tidally driven lagoon has a large area of mostly abandoned salt pans, which are in progressive degradation caused by the lack of maintenance and by the strong currents which erode their protective walls. To explore possible tidal changes the hydrodynamic model ELCIRC was applied to Ria de Aveiro to simulate and analyse the impact in the lagoon hydrodynamics of this degradation which results in the enlargement of the lagoon flooded area. A high-resolution grid (grid spacing of the order of 1 m) was developed in order to represent the narrow channels adjacent to the salt pans. The hydrodynamic model was then successfully calibrated and assessed for skill for the Aveiro lagoon through comparison between measurements and model results and quantification of the numerical accuracy. The model was subsequently used to investigate the effect of the flooded lagoon area enlargement on tidal propagation in Ria de Aveiro. Simulations were performed for three geomorphologic configurations, representing the reference or present situation and two flooded scenarios. Results were compared through the analysis of tidal currents, tidal asymmetry and tidal prism. The increase of the lagoon flooded area results in an intensification of the tidal currents, tidal prism and tidal asymmetry. Results also indicate that the tidal prism further increases when the flooding depth increases. Otherwise, changes in tidal currents and in tidal asymmetry pattern are negligible with the increase of the flooded area depth. These results indicate that modifications of the flooded area of estuarine systems will result in tidal changes, with an intensification of the actual tidal patterns induced by the enlargement of inundation areas

Proximity of Transmembrane Segments 5 and 8 of the Glutamate Transporter GLT-1 Inferred from Paired Cysteine Mutagenesis

BACKGROUND: GLT-1 is a glial glutamate transporter which maintains low synaptic concentrations of the excitatory neurotransmitter enabling efficient synaptic transmission. Based on the crystal structure of the bacterial homologue Glt(Ph), it has been proposed that the reentrant loop HP2, which connects transmembrane domains (TM) 7 and 8, moves to open and close access to the binding pocket from the extracellular medium. However the conformation change between TM5 and TM8 during the transport cycle is not clear yet. We used paired cysteine mutagenesis in conjunction with treatments with Copper(II)(1,10-Phenanthroline)(3) (CuPh), to verify the predicted proximity of residues located at these structural elements of GLT-1. METHODOLOGY/PRINCIPAL FINDINGS: To assess the proximity of transmembrane domain (TM) 5 relative to TM8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. A complete inhibition of transport by Copper(II)(1,10-Phenanthroline)(3) is observed in the double mutants I295C/I463C and G297C/I463C, but not in the corresponding single mutants. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of I295C/I463C and G297C/I463C by CuPh. Transport by the double mutants I295C/I463C and G297C/I463C also was inhibited by Cd(2+). CONCLUSIONS/SIGNIFICANCE: Our results suggest that TM5 (Ile-295, Gly-297) is in close proximity to TM8 (Ile-463) in the mammalian transporter, and that the spatial relationship between these domains is altered during the transport cycle

Shaping a screening file for maximal lead discovery efficiency and effectiveness: elimination of molecular redundancy

High Throughput Screening (HTS) is a successful strategy for finding hits and leads that have the opportunity to be converted into drugs. In this paper we highlight novel computational methods used to select compounds to build a new screening file at Pfizer and the analytical methods we used to assess their quality. We also introduce the novel concept of molecular redundancy to help decide on the density of compounds required in any region of chemical space in order to be confident of running successful HTS campaigns

Translating Clinical Findings into Knowledge in Drug Safety Evaluation - Drug Induced Liver Injury Prediction System (DILIps)

Drug-induced liver injury (DILI) is a significant concern in drug development due to the poor concordance between preclinical and clinical findings of liver toxicity. We hypothesized that the DILI types (hepatotoxic side effects) seen in the clinic can be translated into the development of predictive in silico models for use in the drug discovery phase. We identified 13 hepatotoxic side effects with high accuracy for classifying marketed drugs for their DILI potential. We then developed in silico predictive models for each of these 13 side effects, which were further combined to construct a DILI prediction system (DILIps). The DILIps yielded 60–70% prediction accuracy for three independent validation sets. To enhance the confidence for identification of drugs that cause severe DILI in humans, the “Rule of Three” was developed in DILIps by using a consensus strategy based on 13 models. This gave high positive predictive value (91%) when applied to an external dataset containing 206 drugs from three independent literature datasets. Using the DILIps, we screened all the drugs in DrugBank and investigated their DILI potential in terms of protein targets and therapeutic categories through network modeling. We demonstrated that two therapeutic categories, anti-infectives for systemic use and musculoskeletal system drugs, were enriched for DILI, which is consistent with current knowledge. We also identified protein targets and pathways that are related to drugs that cause DILI by using pathway analysis and co-occurrence text mining. While marketed drugs were the focus of this study, the DILIps has a potential as an evaluation tool to screen and prioritize new drug candidates or chemicals, such as environmental chemicals, to avoid those that might cause liver toxicity. We expect that the methodology can be also applied to other drug safety endpoints, such as renal or cardiovascular toxicity

The Metalloprotease Meprinβ Processes E-Cadherin and Weakens Intercellular Adhesion

BACKGROUND: Meprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used MDCK and Caco-2 cells stably transfected with meprin alpha and or meprin beta to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprin beta, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins beta-catenin and plakoglobin were processed by an intracellular protease, whereas alpha-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprin beta and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprin beta-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates. CONCLUSIONS/SIGNIFICANCE: By identifying E-cadherin as a substrate for meprin beta in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprin beta in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression

Spinning Gland Transcriptomics from Two Main Clades of Spiders (Order: Araneae) - Insights on Their Molecular, Anatomical and Behavioral Evolution

Characterized by distinctive evolutionary adaptations, spiders provide a comprehensive system for evolutionary and developmental studies of anatomical organs, including silk and venom production. Here we performed cDNA sequencing using massively parallel sequencers (454 GS-FLX Titanium) to generate ∼80,000 reads from the spinning gland of Actinopus spp. (infraorder: Mygalomorphae) and Gasteracantha cancriformis (infraorder: Araneomorphae, Orbiculariae clade). Actinopus spp. retains primitive characteristics on web usage and presents a single undifferentiated spinning gland while the orbiculariae spiders have seven differentiated spinning glands and complex patterns of web usage. MIRA, Celera Assembler and CAP3 software were used to cluster NGS reads for each spider. CAP3 unigenes passed through a pipeline for automatic annotation, classification by biological function, and comparative transcriptomics. Genes related to spider silks were manually curated and analyzed. Although a single spidroin gene family was found in Actinopus spp., a vast repertoire of specialized spider silk proteins was encountered in orbiculariae. Astacin-like metalloproteases (meprin subfamily) were shown to be some of the most sampled unigenes and duplicated gene families in G. cancriformis since its evolutionary split from mygalomorphs. Our results confirm that the evolution of the molecular repertoire of silk proteins was accompanied by the (i) anatomical differentiation of spinning glands and (ii) behavioral complexification in the web usage. Finally, a phylogenetic tree was constructed to cluster most of the known spidroins in gene clades. This is the first large-scale, multi-organism transcriptome for spider spinning glands and a first step into a broad understanding of spider web systems biology and evolution