Characterization of estrous cycles and pregnancy in Somali wild asses (Equus africanus somaliensis) through fecal hormone analyses

Although reproduction in the domestic horse has been well described, less is known about reproduction in wild equids. This study describes endocrine patterns associated with estrous cycles and pregnancy for Somali wild asses (Equus africanus somaliensis), an endangered African equid. Fecal samples were collected three times per week for more than 2 years from five female Somali wild asses at the Saint Louis Zoo; progestagen and estrogen metabolites were quantified using commercially available immunoassays. Progestagen analysis indicated that cycle lengths were 27.2 ± 1.2 days and females cycled throughout the year. Progestagen levels during early pregnancy were low and not sustained above baseline until approximately 40 weeks prior to partition. Concentrations increased markedly around 16 weeks prior to delivery and peaked 2–3 weeks before birth. Fecal estrogen levels also increased significantly starting 40–45 weeks before parturition and reached their maximal value approximately 20 weeks prior to birth. Neither foal heat nor lactational suppression of estrus was observed, and females cycled within 45 days after delivery. These data are the first to describe the reproductive physiology of Somali wild asses. As the species faces increasing threats in the wild, this information may support conservation efforts by assisting with ex situ breeding programs.     

ATG16 mediates the autophagic degradation of the 19S proteasomal subunits PSMD1 and PSMD2

Autophagy and the ubiquitin proteasome system are the two major cellular processes for protein and organelle recycling and clearance in eukaryotic cells. Evidence is accumulating that these two pathways are interrelated through adaptor proteins. Here, we found that PSMD1 and PSMD2, both components of the 19S regulatory particle of the proteasome, directly interact with Dictyostelium discoideum autophagy 16 (ATG16), a core autophagosomal protein. ATG16 is composed of an N-terminal domain, which is responsible for homo-dimerization and binding to ATG5 and a C-terminal β-propeller structure. Deletion analysis of ATG16 showed that the N-terminal half of ATG16 interacted directly only with PSMD1, while the C-terminal half interacted with both, PSMD1 and PSMD2. RFP-tagged PSMD1 as well as PSMD2 were enriched in large puncta, reminiscent of autophagosomes, in wild-type cells. These puncta were absent in atg16￣ and atg9￣/16￣ cells and weaker and less frequent in atg9￣ cells, showing that ATG16 was crucial and the autophagic process important for their formation. Co-expression of ATG16-GFP or GFP-ATG8a(LC3) with RFP-PSMD1 or RFP-PSMD2, respectively, in atg16￣ or wild-type cells revealed many instances of co-localization, suggesting that RFP-PSMD1 or RFP-PSMD2 positive puncta constitute autophagosomes. LysoTracker^® labeling and a proteolytic cleavage assay confirmed that PSMD1 and PSMD2 were present in lysosomes in wild-type cells. In vivo, ATG16 is required for their enrichment in ATG8a positive puncta, which mature into autolysosomes. We propose that ATG16 links autophagy and the ubiquitin proteasome system.     

Variation in adult stress resistance does not explain vulnerability to climate change in copper butterflies

Ongoing climate change is a major threat to biodiversity. However, although many species clearly suffer from ongoing climate change, others benefit from it, for example, by showing range expansions. However, which specific features determine a species’ vulnerability to climate change? Phenotypic plasticity, which has been described as the first line of defence against environmental change, may be of utmost importance here. Against this background, we here compare plasticity in stress tolerance in 3 copper butterfly species, which differ arguably in their vulnerability to climate change. Specifically, we investigated heat, cold and desiccation resistance after acclimatization to different temperatures in the adult stage. We demonstrate that acclimation at a higher temperature increased heat but decreased cold tolerance and desiccation resistance. Contrary to our predictions, species did not show pronounced variation in stress resistance, though plastic capacities in temperature stress resistance did vary across species. Overall, our results seemed to reflect population—rather than species‐specific patterns. We conclude that the geographical origin of the populations used should be considered even in comparative studies. However, our results suggest that, in the 3 species studied here, vulnerability to climate change is not in the first place determined by stress resistance in the adult stage. As entomological studies focus all too often on adults only, we argue that more research effort should be dedicated to other developmental stages when trying to understand insect responses to environmental change.     

Validation of the live trap ‘Krefelder Fuchsfalle’ in combination with electronic trap sensors based on AIHTS standards

Live traps are established in pest control, fishing and hunting, but they are discussed to compromise animal welfare due to inadequate construction and control of the trap. To assure animal welfare, the Agreement on International Humane Trapping Standards (AIHTS) demands for legal regulations regarding trapping devices. According to AIHTS, the certification of each trap is based on evaluation in 20 individuals of a specific animal species (target species). To this end, more than 80% of the animals must not show specific alterations of physiology and behaviour or potential injuries.The aim of this study was to evaluate the ‘Krefelder Fuchsfalle’, equipped with an electronic trap system, according to AIHTS standards. The raccoon (Procyon lotor) was chosen as target species, as it is an invasive species in urban regions of Central Europe, a potential vector for zoonotic pathogens, and a skilful animal, which potentially is most difficult to meet the AIHTS requirements. The behaviour of the trapped animal was evaluated, before shooting it and performing radiographical, pathological and histological examination.In total, 20 raccoons were trapped within 10 months in a hunting district in Hesse, Germany. Mean period of stay within the trap were 6.39 h. Despite of 14 animals, which demonstrated low-grade excoriations (superficial skin layer) at the paws and rhinarium, no other injuries or alterations of physiology and behaviour were detected.Therefore, this study demonstrates that the ‘Krefelder Fuchsfalle’ fulfilled the AIHTS standards and accomplished trapping according to animal welfare.     

Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites

Interleukin-6 (IL-6) expression and secretion, induced by inflammatory processes, stimulate the acute phase response cascade. The overexpression of IL-6 contributes to a variety of inflammatory diseases, e.g. rheumatoid arthritis, Castleman’s disease, multiple myeloma, and prostate cancer. Screening for high amounts of IL-6 in the patients’ blood serum can be crucial for an adequate treatment. In this study, five novel murine monoclonal antibodies (mAbs) reactive to human IL-6 were generated. The mAbs were characterized for potential diagnostic purposes and recombinant antibodies were derived thereof. Initial epitope mapping using a combination of blocking experiments and Hyper-IL-6, a fusion protein consisting of IL-6 and the soluble IL-6 receptor revealed distinct but overlapping binding sites. At least one of the mAbs was found to interact with the region of IL-6/ IL-R complex formation. Three mAbs were applied successfully in intracellular staining by flow cytometry, whereas one of the mAbs showed comparable binding as a reference reagent. Furthermore, the mAbs were tested for applications in various immunological assays such as ELISA, Western blot and surface plasmon resonance spectroscopy (SPR), using IL-6 from commercial sources as well as in-house produced protein (IL-6_IME). The limit of detection was determined by sandwich ELISA (0.5 ng/mL, SD ±0.005). Our results also demonstrated that the recombinant IL- 6 produced was functional and correctly folded. These findings support the use of the generated mAb clones as promising candidates for application in various immunological assays for diagnostic and scientific purposes.     

Legislating Patient Representation: A Comparison Between Austrian and German Regulations on Self-Help Organizations as Patient Representatives

Governments are increasingly inviting patient organizations (POs) to participate in healthcare policymaking. By inviting POs that claim to represent patients, representation comes into being. However, little is known about the circumstances under which governments accept POs as patient representatives. Based on the analysis of relevant legislation, this article investigates the criteria that self-help organizations (SHOs), a special type of PO, must fulfil in order to be accepted as patient representatives by governments in Austria and Germany. Thereby, it aims to contribute to the discussion on the role of governments in steering SHOs. There are different degrees of regulation (very little in Austria, more in Germany). Governments in both countries not only formulate explicit criteria for SHOs with respect to patient representation but also guide SHOs representing patients through implicit criteria for associations. We discuss the findings against concepts of responsiveness, authorization, and accountability. Our findings indicate that governmental steering is not negative per se as indicated by previous research but—depending on legislative criteria—can promote transparency and democratic quality in patient representation.     

Cytosolic aminoacyl-tRNA synthetases: Unanticipated relocations for unexpected functions

Prokaryotic and eukaryotic cytosolic aminoacyl-tRNA synthetases (aaRSs) are essentially known for their conventional function of generating the full set of aminoacyl-tRNA species that are needed to incorporate each organism's repertoire of genetically-encoded amino acids during ribosomal translation of messenger RNAs. However, bacterial and eukaryotic cytosolic aaRSs have been shown to exhibit other essential nonconventional functions. Here we review all the subcellular compartments that prokaryotic and eukaryotic cytosolic aaRSs can reach to exert either a conventional or nontranslational role. We describe the physiological and stress conditions, the mechanisms and the signaling pathways that trigger their relocation and the new functions associated with these relocating cytosolic aaRS. Finally, given that these relocating pools of cytosolic aaRSs participate to a wide range of cellular pathways beyond translation, but equally important for cellular homeostasis, we mention some of the pathologies and diseases associated with the dis-regulation or malfunctioning of these nontranslational functions.     

Extracellular matrix content and WNT/β-catenin levels of cartilage determine the chondrocyte response to compressive load

During osteoarthritis (OA)-development extracellular matrix (ECM) molecules are lost from cartilage, thus changing gene-expression, matrix synthesis and biomechanical competence of the tissue. Mechanical loading is important for the maintenance of articular cartilage; however, the influence of an altered ECM content on the response of chondrocytes to loading is not well understood, but may provide important insights into underlying mechanisms as well as supplying new therapies for OA. Objective here was to explore whether a changing ECM-content of engineered cartilage affects major signaling pathways and how this alters the chondrocyte response to compressive loading.Activity of canonical WNT-, BMP-, TGF-β- and p38-signaling was determined during maturation of human engineered cartilage and followed after exposure to a single dynamic compression-episode. WNT/β-catenin- and pSmad1/5/9-levels declined with increasing ECM-content of cartilage. While loading significantly suppressed proteoglycan-synthesis and ACAN-expression at low ECM-content this catabolic response then shifted to an anabolic reaction at high ECM-content. A positive correlation was observed between GAG-content and load-induced alteration of proteoglycan-synthesis. Induction of high β-catenin levels by the WNT-agonist CHIR suppressed load-induced SOX9- and GAG-stimulation in mature constructs. In contrast, the WNT-antagonist IWP-2 was capable of attenuating load-induced GAG-suppression in immature constructs.In conclusion, either ECM accumulation-associated or pharmacologically induced silencing of WNT-levels allowed for a more anabolic reaction of chondrocytes to physiological loading. This is consistent with the role of proteoglycans in sequestering WNT-ligands in the ECM, thus reducing WNT-activity and also provides a novel explanation of why low WNT-activity in cartilage protects from OA-development in mechanically overstressed cartilage.     

The 1.25 A crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters.

Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential co-factor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 A in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single-domain alpha/beta-fold with a central four-helix bundle connecting two seven-stranded parallel beta-sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin-substrates, which positions the substrate side chain C1'-carbonyl group near Tyr171 OH and NADP C4'N. The catalytic mechanism of SR appears to consist of a NADPH-dependent proton transfer from Tyr171 to the substrate C1' and C2' carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N-acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N-acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.