Eliciting density ratio classes

AbstractThe probability distributions of uncertain quantities needed for predictive modelling and decision support are frequently elicited from subject matter experts. However, experts are often uncertain about quantifying their beliefs using precise probability distributions. Therefore, it seems natural to describe their uncertain beliefs using sets of probability distributions. There are various possible structures, or classes, for defining set membership of continuous random variables. The Density Ratio Class has desirable properties, but there is no established procedure for eliciting this class. Thus, we propose a method for constructing Density Ratio Classes that builds on conventional quantile or probability elicitation, but allows the expert to state intervals for these quantities. Parametric shape functions, ideally also suggested by the expert, are then used to bound the nonparametric set of shapes of densities that belong to the class and are compatible with the stated intervals. This leads to a natural metric for the size of the class based on the ratio of the total areas under upper and lower bounding shape functions. This ratio will be determined by the characteristics of the shape functions, the scatter of the elicited values, and the explicit expert imprecision, as characterized by the width of the stated intervals. We provide some examples, both didactic and real, and conclude with recommendations for the further development and application of the Density Ratio Class

Challenges in Exosome Isolation and Analysis in Health and Disease

A growing body of evidence emphasizes the important role exosomes in different physiological and pathological conditions. Exosomes, virus-size extracellular vesicles (EVs), carry a complex molecular cargo, which is actively processed in the endocytic compartment of parental cells. Exosomes carry and deliver this cargo to recipient cells, serving as an intercellular communication system. The methods for recovery of exosomes from supernatants of cell lines or body fluids are not uniformly established. Yet, studies of the quality and quantity of exosome cargos underlie the concept of “liquid biopsy.” Exosomes are emerging as a potentially useful diagnostic tool and a predictor of disease progression, response to therapy and overall survival. Although many novel approaches to exosome isolation and analysis of their cargos have been introduced, the role of exosomes as diagnostic or prognostic biomarkers of disease remains unconfirmed. This review considers existing challenges to exosome validation as disease biomarkers. Focusing on advantages and limitations of methods for exosome isolation and characterization, approaches are proposed to facilitate further progress in the development of exosomes as biomarkers in human disease

CONCEPTUAL DESIGN OF THE USMC FUTURE VERTICAL LIFT (FVL) LIVING LAB

The United States Marine Corps (USMC) is developing the Future Vertical Lift (FVL) system that will rely heavily on Marine-machine teaming, a complex process that requires further development. The development of a living lab (LL)—a multi-function network of simulators that will serve as the platform for testing, experimenting, and training new technologies and ideas for how the FVL will operate—will help mitigate Marine-machine collaboration and trust issues. This capstone studies the options and requirements for developing a LL through interviews, research that focuses on existing technologies and operational concepts, and Model-Based Systems Engineering tools using a systems engineering approach. The report includes a detailed needs and requirements analysis, stakeholder analysis, and functional design. The team presents a conceptual design, that includes the system architecture, comprising of system, function and physical views, system lifecycle, and the evaluation criteria for a LL. The final product is a set of use cases and concepts of operation. The USMC needs a new approach that supports rapid and relevant upgrades that optimizes the system lifecycle and keeps the Marine in mind. This team recommends the USMC consider these findings and continue researching and developing a LL.ONR Arlington, VA 22203Civilian, Department of the NavyCivilian, Department of the NavyCivilian, Department of the NavyCivilian, Department of the NavyCivilian, Department of the NavyCivilian, Department of the NavyApproved for public release. Distribution is unlimited

Activity and interactions of methane seep microorganisms assessed by parallel transcription and FISH-NanoSIMS analyses

To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteobacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A (mcrA), adenosine-5′-phosphosulfate reductase α-subunit (aprA), dinitrogenase reductase (nifH)), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no ^(15)NH^+_4 assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active ‘free-living’ population, and are not dependent on methane or ANME activity. We investigated the possibility of N_2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria. However, nifH expression was methane-dependent. ^(15)N_2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, ^(15)N_2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced ^(15)N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. With this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations

Multiple functional neurosteroid binding sites on GABAA receptors

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action

Phosphate-dependent aggregation of [KL]n_{n} peptides affects their membranolytic activity

In this study, we investigate how the length of amphiphilic β-sheet forming peptides affects their interaction with membranes. Four polycationic model peptides with lengths from 6 to 18 amino acids were constructed from simple Lys-Leu repeats, giving [KL]$_{n=3,5,7,9}$. We found that (1) they exhibit a pronounced antimicrobial activity with an intriguing length dependent maximum for [KL]$_{5}$ with 10 amino acids; (2) their hemolytic effect, on the other hand, increases steadily with peptide length. CD analysis (3) and TEM (4) show that all peptides-except for the short [KL]$_{3}$-aggregate into amyloid-like fibrils in the presence of phosphate ions, which in turn has a critical effect on the results in (1) and (2). In fact, (5) vesicle leakage reveals an intrinsic membrane-perturbing activity (at constant peptide mass) of [KL]$_{5}$ > [KL]$_{9}$ > [KL]7 in phosphate buffer, which changes to [KL]$_{5}$ ≈ [KL]$_{7}$ ≈ [KL]$_{9}$ in PIPES. A specific interaction with phosphate ions thus explains the subtle balance between two counteracting effects: phosphate-induced unproductive pre-aggregation in solution versus monomeric membrane binding and vigorous lipid perturbation due to self-assembly of the bound peptides within the bilayer. This knowledge can now be used to control and optimize the peptides in further applications