Rapid and sensitive large-scale screening of low affinity extracellular receptor protein interactions by using reaction induced inhibition of Gaussia luciferase.

Extracellular protein interactions mediated by cell surface receptors are essential for intercellular communication in multicellular organisms. Assays to detect extracellular interactions must account for their often weak binding affinities and also the biochemical challenges in solubilising membrane-embedded receptors in an active form. Methods based on detecting direct binding of soluble recombinant receptor ectodomains have been successful, but genome-scale screening is limited by the usual requirement of producing sufficient amounts of each protein in two different forms, usually a "bait" and "prey". Here, we show that oligomeric receptor ectodomains coupled to concatenated units of the light-generating Gaussia luciferase enzyme robustly detected low affinity interactions and reduced the amount of protein required by several orders of magnitude compared to other reporter enzymes. Importantly, we discovered that this flash-type luciferase exhibited a reaction-induced inhibition that permitted the use of a single protein preparation as both bait and prey thereby halving the number of expression plasmids and recombinant proteins required for screening. This approach was tested against a benchmarked set of quantified extracellular interactions and shown to detect extremely weak interactions (KDs ≥ μM). This method will facilitate large-scale receptor interaction screening and contribute to the goal of mapping networks of cellular communication

Resurrection of the ancestral RH5 invasion ligand provides a molecular explanation for the origin of P. falciparum malaria in humans.

Many important infectious diseases are the result of zoonoses, in which pathogens that normally infect animals acquire mutations that enable the breaching of species barriers to permit the infection of humans. Our understanding of the molecular events that enable host switching are often limited, and yet this is a fundamentally important question. Plasmodium falciparum, the etiological agent of severe human malaria, evolved following a zoonotic transfer of parasites from gorillas. One gene-rh5-which encodes an essential ligand for the invasion of host erythrocytes, is suspected to have played a critical role in this host switch. Genome comparisons revealed an introgressed sequence in the ancestor of P. falciparum containing rh5, which likely allowed the ancestral parasites to infect both gorilla and human erythrocytes. To test this hypothesis, we resurrected the ancestral introgressed reticulocyte-binding protein homologue 5 (RH5) sequence and used quantitative protein interaction assays to demonstrate that this ancestral protein could bind the basigin receptor from both humans and gorillas. We also showed that this promiscuous receptor binding phenotype of RH5 was shared with the parasite clade that transferred its genome segment to the ancestor of P. falciparum, while the other lineages exhibit host-specific receptor binding, confirming the central importance of this introgression event for Plasmodium host switching. Finally, since its transfer to humans, P. falciparum, and also the RH5 ligand, have evolved a strong human specificity. We show that this subsequent restriction to humans can be attributed to a single amino acid mutation in the RH5 sequence. Our findings reveal a molecular pathway for the origin and evolution of human P. falciparum malaria and may inform molecular surveillance to predict future zoonoses

Genome sequences of five African swine fever virus genotype IX isolates from domestic pigs in Uganda

Complete genome sequences of five African swine fever virus isolates were determined directly from clinical material obtained from domestic pigs in Uganda. Four sequences were essentially identical to each other, and all were closely related to the only known genome sequence of p72 genotype IX

Genome sequences of five African swine fever virus genotype IX isolates from domestic pigs in Uganda

Complete genome sequences of five African swine fever virus isolates were determined directly from clinical material obtained from domestic pigs in Uganda. Four sequences were essentially identical to each other, and all were closely related to the only known genome sequence of p72 genotype IX

A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host–pathogen interactions

Background: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host–pathogen protein–protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host–pathogen protein–protein interactions involved are poorly understood. Methods: To gain a better understanding of the protein–protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. Results: A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. Conclusion: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community

Comparisons among ten models of acoustic backscattering used in aquatic ecosystem research

Author Posting. © Acoustical Society of America, 2015. This article is posted here by permission of Acoustical Society of America for personal use, not for redistribution. The definitive version was published in Journal of the Acoustical Society of America 138 (2015); 3742, doi:10.1121/1.4937607.Analytical and numerical scatteringmodels with accompanying digital representations are used increasingly to predict acoustic backscatter by fish and zooplankton in research and ecosystem monitoring applications. Ten such models were applied to targets with simple geometric shapes and parameterized (e.g., size and material properties) to represent biological organisms such as zooplankton and fish, and their predictions of acoustic backscatter were compared to those from exact or approximate analytical models, i.e., benchmarks. These comparisons were made for a sphere, spherical shell, prolate spheroid, and finite cylinder, each with homogeneous composition. For each shape, four target boundary conditions were considered: rigid-fixed, pressure-release, gas-filled, and weakly scattering. Target strength (dB re 1 m2) was calculated as a function of insonifying frequency (f = 12 to 400 kHz) and angle of incidence (θ = 0° to 90°). In general, the numerical models (i.e., boundary- and finite-element) matched the benchmarks over the full range of simulation parameters. While inherent errors associated with the approximate analytical models were illustrated, so were the advantages as they are computationally efficient and in certain cases, outperformed the numerical models under conditions where the numerical models did not convergeThis work was supported by the NOAA Fisheries Advanced Sampling Technologies Working Group, the Office of Naval Research, and the National Oceanic Partnership Program. Josiah S. Renfree

Climate Informatics

The impacts of present and potential future climate change will be one of the most important scientific and societal challenges in the 21st century. Given observed changes in temperature, sea ice, and sea level, improving our understanding of the climate system is an international priority. This system is characterized by complex phenomena that are imperfectly observed and even more imperfectly simulated. But with an ever-growing supply of climate data from satellites and environmental sensors, the magnitude of data and climate model output is beginning to overwhelm the relatively simple tools currently used to analyze them. A computational approach will therefore be indispensable for these analysis challenges. This chapter introduces the fledgling research discipline climate informatics: collaborations between climate scientists and machine learning researchers in order to bridge this gap between data and understanding. We hope that the study of climate informatics will accelerate discovery in answering pressing questions in climate science

Field and laboratory validation of remote rover operations Science Team findings: The CanMars Mars Sample Return analogue mission

The CanMars Mars Sample Return Analogue Deployment (MSRAD) was a closely simulated, end-to-end Mars Sample Return (MSR) mission scenario, with instrumentation, goals, and constraints modeled on the upcoming NASA Mars 2020 rover mission; this paper reports on the post-mission validation of the exercise. The exercise utilized the CSA Mars Exploration Science Rover (MESR) rover, deployed to Utah, USA, at a Mars-analogue field site. The principal features of the field site located near Green River, Utah are Late Jurassic inverted, fluvial paleochannels, analogous to features on Mars in sites being considered for the ESA ExoMars rover mission and present within the chosen landing site for the Mars 2020 rover mission. The in-simulation (“in-sim”) mission operations team worked remotely from The University of Western Ontario, Canada. A suite of MESR-integrated and hand-held spectrometers was selected to mimic those of the Mars 2020 payload, and a Utah-based, on-site team was tasked with field operations to carry out the data collection and sampling as commanded by the in-sim team. As a validation of the in-sim mission science findings, the field team performed an independent geological assessment. This paper documents the field team's on-site geological assessment and subsequent laboratory and analytical results, then offers a comparison of mission (in-sim) and post-mission (laboratory) science results. The laboratory-based findings were largely consistent with the in-sim rover-derived data and geological interpretations, though some notable exceptions highlight the inherent difficulties in remote science. In some cases, available data was insufficient for lithologic identification given the absence of other important contextual information (e.g., textural information). This study suggests that the in-sim instruments were largely adequate for the Science Team to characterize samples; however, rover-based field work is necessarily hampered by mobility and time constraints with an obvious effect on efficiency but also precision, and to some extent, accuracy of the findings. The data show a dearth of preserved total organic carbon (TOC) – used as a proxy for ancient biosignature preservation potential – in the fluvial-lacustrine system of this field site, suggesting serious consideration with respect to the capabilities and opportunities for addressing the Mars exploration goals. We therefore suggest a thorough characterization of terrestrial sites analogous to those of Mars rover landing sites, and in-depth field studies like CanMars as important, pre-mission strategic exercises

Capturing protein communities by structural proteomics in a thermophilic eukaryote:Structural systems biology of lysates

The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known-at both the protein and complex levels-this study constitutes another step forward toward a molecular understanding of subcellular organization