BackgroundSciuromorpha (squirrels and close relatives) are diverse in terms of body size and locomotor behavior. Individual species are specialized to perform climbing, gliding or digging behavior, the latter being the result of multiple independent evolutionary acquisitions. Each lifestyle involves characteristic loading patterns acting on the bones of sciuromorphs. Trabecular bone, as part of the bone inner structure, adapts to such loading patterns. This network of thin bony struts is subject to bone modeling, and therefore reflects habitual loading throughout lifetime. The present study investigates the effect of body size and lifestyle on trabecular structure in Sciuromorpha.MethodsBased upon high-resolution computed tomography scans, the femoral head 3D inner microstructure of 69 sciuromorph species was analyzed. Species were assigned to one of the following lifestyle categories: arboreal, aerial, fossorial and semifossorial. A cubic volume of interest was selected in the center of each femoral head and analyzed by extraction of various parameters that characterize trabecular architecture (degree of anisotropy, bone volume fraction, connectivity density, trabecular thickness, trabecular separation, bone surface density and main trabecular orientation). Our analysis included evaluation of the allometric signals and lifestyle-related adaptation in the trabecular parameters.ResultsWe show that bone surface density, bone volume fraction, and connectivity density are subject to positive allometry, and degree of anisotropy, trabecular thickness, and trabecular separation to negative allometry. The parameters connectivity density, bone surface density, trabecular thickness, and trabecular separation show functional signals which are related to locomotor behavior. Aerial species are distinguished from fossorial ones by a higher trabecular thickness, lower connectivity density and lower bone surface density. Arboreal species are distinguished from semifossorial ones by a higher trabecular separation.ConclusionThis study on sciuromorph trabeculae supplements the few non-primate studies on lifestyle-related functional adaptation of trabecular bone. We show that the architecture of the femoral head trabeculae in Sciuromorpha correlates with body mass and locomotor habits. Our findings provide a new basis for experimental research focused on functional significance of bone inner microstructure.Electronic supplementary materialThe online version of this article (10.1186/s40851-018-0093-z) contains supplementary material, which is available to authorized users.

The RNA-dependent protein kinase (PKR) has broad antiviral activity inducing translational shutdown of viral and cellular genes and is therefore targeted by various viral proteins to facilitate pathogen propagation. The pleiotropic NS1 protein of influenza A virus acts as silencer of PKR activation and ensures high-level viral replication and virulence. However, the exact manner of this inhibition remains controversial. To elucidate the structural requirements within the NS1 protein for PKR inhibition, we generated a set of mutant viruses, identifying highly conserved arginine residues 35 and 46 within the NS1 N terminus as being most critical not only for binding to and blocking activation of PKR but also for efficient virus propagation. Biochemical and Förster resonance energy transfer (FRET)-based interaction studies showed that mutation of R35 or R46 allowed formation of NS1 dimers but eliminated any detectable binding to PKR as well as to double-stranded RNA (dsRNA). Using in vitro and in vivo approaches to phenotypic restoration, we demonstrated the essential role of the NS1 N terminus for blocking PKR. The strong attenuation conferred by NS1 mutation R35A or R46A was substantially alleviated by stable knockdown of PKR in human cells. Intriguingly, both NS1 mutant viruses did not trigger any signs of disease in PKR ϩ/ϩ mice, but replicated to high titers in lungs of PKR Ϫ/Ϫ mice and caused lethal infections. These data not only establish the NS1 N terminus as highly critical for neutralization of PKR's antiviral activity but also identify this blockade as an indispensable contribution of NS1 to the viral life cycle. IMPORTANCE Influenza A virus inhibits activation of the RNA-dependent protein kinase (PKR) by means of its nonstructural NS1 protein, but the underlying mode of inhibition is debated. Using mutational analysis, we identified arginine residues 35 and 46 within the N-terminal NS1 domain as highly critical for binding to and functional silencing of PKR. In addition, our data show that this is a main activity of amino acids 35 and 46, as the strong attenuation of corresponding mutant viruses in human cells was rescued to a large extent by lowering of PKR expression levels. Significantly, this corresponded with restoration of viral virulence for NS1 R35A and R46A mutant viruses in PKR Ϫ/Ϫ mice. Therefore, our data establish a model in which the NS1 N-terminal domain engages in a binding interaction to inhibit activation of PKR and ensure efficient viral propagation and virulence.KEYWORDS PKR, influenza, innate immunity, virulence regulation

Many songbird species rely on seeds as a primary food source and the process of picking up, positioning, cracking, dehusking, and swallowing seeds is one of the most sophisticated tasks of the beak. Still, we lack understanding about how granivorous songbirds move their beak during the different phases of seed processing. In this study, we used multi-view high speed imaging to analyze the three-dimensional movement of the beak in feeding domestic canaries. Our analysis focuses on correlation of upper and lower beak, frequency of mandibulation, and direction of mandible movement in 3D space. We show that the correlation of maxilla and mandible movement differs among the phases of seed processing. Furthermore, we found that the beak moves at extremely high frequencies, up to 25 Hz, which resembles previously reported maximal syllable rates in singing canaries. Finally, we report that canaries use specific 3D mandible movements during the different phases of seed processing. Kinematic parameters do not differ between male and female canaries. Our findings provide an important biomechanical basis for better understanding the beak as a functional tool.

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Digitization of video recordings often requires the laborious procedure of manually clicking points of interest on individual video frames. Here, we present progressive tracking, a procedure that facilitates manual digitization of markerless videos. In contrast to existing software, it allows the user to follow points of interest with a cursor in the progressing video, without the need to click. To compare the performance of progressive tracking with the conventional frame-wise tracking, we quantified speed and accuracy of both methods, testing two different input devices (mouse and stylus pen). We show that progressive tracking can be twice as fast as frame-wise tracking while maintaining accuracy, given that playback speed is controlled. Using a stylus pen can increase frame-wise tracking speed. The complementary application of the progressive and frame-wise mode is exemplified on a realistic video recording. This study reveals that progressive tracking can vastly facilitate video analysis in experimental research.

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