Angiofil®-mediated visualization of the vascular system by microcomputed tomography: a feasibility study

Visualization of the vascular systems of organs or of small animals is important for an assessment of basic physiological conditions, especially in studies that involve genetically manipulated mice. For a detailed morphological analysis of the vascular tree, it is necessary to demonstrate the system in its entirety. In this study, we present a new lipophilic contrast agent, Angiofil®, for performing postmortem microangiography by using microcomputed tomography. The new contrast agent was tested in 10 wild-type mice. Imaging of the vascular system revealed vessels down to the caliber of capillaries, and the digital three-dimensional data obtained from the scans allowed for virtual cutting, amplification, and scaling without destroying the sample. By use of computer software, parameters such as vessel length and caliber could be quantified and remapped by color coding onto the surface of the vascular system. The liquid Angiofil® is easy to handle and highly radio-opaque. Because of its lipophilic abilities, it is retained intravascularly, hence it facilitates virtual vessel segmentation, and yields an enduring signal which is advantageous during repetitive investigations, or if samples need to be transported from the site of preparation to the place of actual analysis, respectively. These characteristics make Angiofil® a promising novel contrast agent; when combined with microcomputed tomography, it has the potential to turn into a powerful method for rapid vascular phenotyping

Quality Assessment of Photogrammetric Methods—A Workflow for Reproducible UAS Orthomosaics

Unmanned aerial systems (UAS) are cost-effective, flexible and offer a wide range of applications. If equipped with optical sensors, orthophotos with very high spatial resolution can be retrieved using photogrammetric processing. The use of these images in multi-temporal analysis and the combination with spatial data imposes high demands on their spatial accuracy. This georeferencing accuracy of UAS orthomosaics is generally expressed as the checkpoint error. However, the checkpoint error alone gives no information about the reproducibility of the photogrammetrical compilation of orthomosaics. This study optimizes the geolocation of UAS orthomosaics time series and evaluates their reproducibility. A correlation analysis of repeatedly computed orthomosaics with identical parameters revealed a reproducibility of 99% in a grassland and 75% in a forest area. Between time steps, the corresponding positional errors of digitized objects lie between 0.07 m in the grassland and 0.3 m in the forest canopy. The novel methods were integrated into a processing workflow to enhance the traceability and increase the quality of UAS remote sensing.This research was funded by the Hessian State Ministry for Higher Education, Research and the Arts, Germany, as part of the LOEWE priority project Nature 4.0—Sensing Biodiversity. The grassland study was funded by the Spanish Science Foundation FECYT-MINECO through the BIOGEI (GL2013- 49142-C2-1-R) and IMAGINE (CGL2017-85490-R) projects, and by the University of Lleida; and supported by a FI Fellowship to C.M.R. (2019 FI_B 01167) by the Catalan Government

Arthropod communities in fungal fruitbodies are weakly structured by climate and biogeography across European beech forests

Aim The tinder fungus Fomes fomentarius is a pivotal wood decomposer in European beech Fagus sylvatica forests. The fungus, however, has regionally declined due to centuries of logging. To unravel biogeographical drivers of arthropod communities associated with this fungus, we investigated how space, climate and habitat amount structure alpha and beta diversity of arthropod communities in fruitbodies of F. fomentarius. Location Temperate zone of Europe. Taxon Arthropods. Methods We reared arthropods from fruitbodies sampled from 61 sites throughout the range of European beech and identified 13 orders taxonomically or by metabarcoding. We estimated the total number of species occurring in fruitbodies of F. fomentarius in European beech forests using the Chao2 estimator and determined the relative importance of space, climate and habitat amount by hierarchical partitioning for alpha diversity and generalized dissimilarity models for beta diversity. A subset of fungi samples was sequenced for identification of the fungus’ genetic structure. Results The total number of arthropod species occurring in fruitbodies of F. fomentarius across European beech forests was estimated to be 600. Alpha diversity increased with increasing fruitbody biomass; it decreased with increasing longitude, temperature and latitude. Beta diversity was mainly composed by turnover. Patterns of beta diversity were only weakly linked to space and the overall explanatory power was low. We could distinguish two genotypes of F. fomentarius, which showed no spatial structuring. Main conclusion Fomes fomentarius hosts a large number of arthropods in European beech forests. The low biogeographical and climatic structure of the communities suggests that fruitbodies represent a habitat that offers similar conditions across large gradients of climate and space, but are characterized by high local variability in community composition and colonized by species with high dispersal ability. For European beech forests, retention of trees with F. fomentarius and promoting its recolonization where it had declined seems a promising conservation strategy

The Leucine Zipper Domains of the Transcription Factors GCN4 and c-Jun Have Ribonuclease Activity

Basic-region leucine zipper (bZIP) proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ) domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3′,5′-phosphodiester bonds with formation of 2′,3′-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins). If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides

Identification of monoclonal antibody variants involved in aggregate formation – Part 2: Hydrophobicity variants

Monoclonal antibodies (mAbs) are valuable tools both in therapy and in diagnostic. Their tendency to aggregate is a serious concern. Since a mAb drug substance (DS) is composed of different variants, it is important for manufacturers to know the behavior and stability not only of the mAb as a whole, but also of the variants contained in the product. We present a method to separate hydrophobicity variants of a mAb and subsequently analyzed these variants for stability and aggregation propensity. We identified a potentially aggregation prone hydrophilic variant which is interrelated with another previously identified aggregation prone acidic charge variant. Additionally, we assessed the risk posed by the aggregation prone variant to the DS by spiking hydrophobicity variants into DS and did not observe an enhanced aggregation propensity. Thus we present an approach to separate, characterize and analyze the criticality of aggregation prone variants in protein DS which is a step forward to further assure drug safety

Identification of Monoclonal Antibody Variants Involved in Aggregate Formation - Part 1: Charge Variants

Biopharmaceutical products contain conformational and chemical variants, that are typically well characterized regarding identity and activity. However, little is known about their self-interaction propensity and tendency to unfold, which are critical characteristics for drug stability and safety. This study aimed to separate and compare charge variants of a monoclonal antibody (mAb) and to identify aggregation prone species. We show a semi-preparative cation exchange method, that we developed to separate the individual acidic and basic variants from the naive mAb. Additionally, we demonstrate, that the yield and purity of the fractionated charge species, extracted by that method, were suffient for subsequent analysis of aggregate content, conformation stability and selfinteraction. Our analysis revealed a differently behaving acidic variant and confirmed its increased aggregation propensity by molecular modeling. During a stability study, the potentially aggregation prone charge variant posed a limited risk to the DS. We are the first to look at the stability of single charge variants of biopharmaceuticals, and thus present manufacturers and regulatory authorities with a method to enhance drug safety

X-Ray Computed Microtomography: Distinguishing Natural Pearls from Beaded and Non-Beaded Cultured Pearls

The distinction of natural from cultured pearls traditionally has been based on X-radiography. X-ray computed microtomography (μ-CT) has recently been applied to gain more insight into pearl structures. Using this technique, this article presents features observed in a selection of natural pearls and beaded and non-beaded cultured pearls. Based on these observations, μ-CT is shown to be a powerful tool for pearl identification