Extended methods for spatial cell classification with DBSCAN-CellX.

Local cell densities and positioning within cellular monolayers and stratified epithelia have important implications for cell interactions and the functionality of various biological processes. To analyze the relationship between cell localization and tissue physiology, density-based clustering algorithms, such as DBSCAN, allow for a detailed characterization of the spatial distribution and positioning of individual cells. However, these methods rely on predefined parameters that influence the outcome of the analysis. With varying cell densities in cell cultures or tissues impacting cell sizes and, thus, cellular proximities, these parameters need to be carefully chosen. In addition, standard DBSCAN approaches generally come short in appropriately identifying individual cell positions. We therefore developed three extensions to the standard DBSCAN-algorithm that provide: (i) an automated parameter identification to reliably identify cell clusters, (ii) an improved identification of cluster edges; and (iii) an improved characterization of the relative positioning of cells within clusters. We apply our novel methods, which are provided as a user-friendly OpenSource-software package (DBSCAN-CellX), to cellular monolayers of different cell lines. Thereby, we show the importance of the developed extensions for the appropriate analysis of cell culture experiments to determine the relationship between cell localization and tissue physiology.

A Hepatitis C virus genotype 1b post-transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo.

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines.

Dynamics and Control of In-Flight Wingtip Docking.

Project Link! is a NASA-led effort to study the feasibility of multi-aircraft aerial docking systems. In these systems, a group of vehicles physically link to each other during flight to form a larger ensemble vehicle with increased aerodynamic performance and mission utility. This paper presents a dynamic model and control architecture for a system of ftxed-wing vehicles with this capability. The dynamic model consists of the 6 degree-of-freedom ftxed-wing aircraft equations of motion, a spring-damper-magnet system to represent the linkage force between constituent vehicles, and the NASA-Burnham-Hallock wingtip vortex model to represent the close-proximity aerodynamic interactions between constituents before the linking occurs. The control architecture consists of a guidance algorithm to autonomously drive the constituents towards their linking partners and an inner-loop angular rate controller. A simulation was constructed from the model, and the flight dynamic modes of the linked system were compared to the individual vehicles. The main contributions of this work are twofold. First is the introduction of close-proximity aerodynamic effects to create a realistic simulation framework for this problem. Second is the application of a sophisticated leader-follower guidance algorithm to achieve in-air wingtip docking. Simulation results for both before and after linking are presented.