Investigations on Xenopus laevis body composition and feeding behavior in a laboratory setting.

The African clawed frog, Xenopus laevis, has been used as a laboratory animal for decades in many research areas. However, there is a lack of knowledge about the nutritional physiology of this amphibian species and the feeding regimen is not standardized. The aim of the present study was to get more insights into the nutrient metabolism and feeding behavior of the frogs. In Trial 1, adult female X. laevis were fed either a Xenopus diet or a fish feed. After 4 weeks, they were euthanized, weighed, measured for morphometrics and dissected for organ weights and whole-body nutrient analysis. There were no significant differences between the diet groups regarding the allometric data and nutrient contents. The ovary was the major determinant of body weight. Body fat content increased with body weight as indicator of energy reserves. In Trial 2, 40 adult female frogs were monitored with a specifically developed digital tracking system to generate heat-maps of their activity before and up to 25 min after a meal. Three diets (floating, sinking, floating & sinking) were used. The main feed intake activity was fanning the feed into the mouth, peaking until 20 min after the meal. The different swimming characteristics of the diets thereby influenced the activity of the animals. Our dataset helps to adjust the feeding needs to the physical composition and also to meet the natural behavioral patterns of feed intake as a prerequisite of animal wellbeing and animal welfare in a laboratory setting.

Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions.

Guanylate binding proteins (GBPs) are an evolutionarily ancient family of proteins that are widely distributed among eukaryotes. They belong to the dynamin superfamily of GTPases, and their expression can be partially induced by interferons (IFNs). GBPs are involved in the cell-autonomous innate immune response against bacterial, parasitic and viral infections. Evolutionary studies have shown that GBPs exhibit a pattern of gene gain and loss events, indicative for the birth-and-death model of evolution. Most species harbor large GBP gene clusters that encode multiple paralogs. Previous functional and in-depth evolutionary studies have mainly focused on murine and human GBPs. Since rabbits are another important model system for studying human diseases, we focus here on lagomorphs to broaden our understanding of the multifunctional GBP protein family by conducting evolutionary analyses and performing a molecular and functional characterization of rabbit GBPs. We observed that lagomorphs lack GBP3, 6 and 7. Furthermore, Leporidae experienced a loss of GBP2, a unique duplication of GBP5 and a massive expansion of GBP4. Gene expression analysis by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and transcriptome data revealed that leporid GBP expression varied across tissues. Overexpressed rabbit GBPs localized either uniformly and/or discretely to the cytoplasm and/or to the nucleus. Oryctolagus cuniculus (oc)GBP5L1 and rarely ocGBP5L2 were an exception, colocalizing with the trans-Golgi network (TGN). In addition, four ocGBPs were IFN-inducible and only ocGBP5L2 inhibited furin activity. In conclusion, from an evolutionary perspective, lagomorph GBPs experienced multiple gain and loss events, and the molecular and functional characteristics of ocGBP suggest a role in innate immunity.

Digestibility of crude nutrients and minerals in C57Bl/6J and CD1 mice fed a pelleted lab rodent diet.

In laboratory animals, there is a scarcity of digestibility data under non-experimental conditions. Such data is important as basis to generate nutrient requirements, which contributes to the refinement of husbandry conditions. Digestibility trials can also help to identify patterns of absorption and potential factors that influence the digestibility. Thus, a digestibility trial with a pelleted diet used as standard feed in laboratory mice was conducted. To identify potential differences between genetic lines, inbred C57Bl/6 J and outbred CD1 mice (n = 18 each, male, 8 weeks-old, housed in groups of three) were used. For seven days, the feed intake was recorded and the total faeces per cage collected. Energy, crude nutrient and mineral content of diet and faecal samples were analyzed to calculate the apparent digestibility (aD). Apparent dry matter and energy digestibility did not differ between both lines investigated. The C57Bl/6 J mice had significantly higher aD of magnesium and potassium and a trend towards a lower aD of sodium than the mice of the CD1 outbred stock. Lucas-tests were performed to calculate the mean true digestibility of the nutrients and revealed a uniformity of the linear regression over data from both common laboratory mouse lines. The mean true digestibility of crude nutrients was > 90%, except for fibre, that of the minerals ranged between 66 and 97%.

Comparative analysis of pancreatic amylase activity in laboratory rodents.

Alpha-amylase is the main enzyme for starch digestion in the mammalian gastrointestinal tract. There are species differences in the enzymatic activity of pancreatic amylase that are related to the digestive strategy and natural diet of a species. This aspect is well investigated in pet and farm animals, while in common laboratory animal rodents, information is scarce. In the context of the 3R concept, detailed knowledge of the digestive physiology should be the basis of adequate nutrition, experimental planning and data interpretation. The present study aimed to obtain reference data on amylase activity in pancreatic tissue and duodenal digesta in laboratory mice, rats and hamsters. In addition, digesta was stained with Lugol's iodine to visualize starch in the process of degradation throughout the gastrointestinal tract. Amylase activity in pancreatic tissue and duodenal digesta was significantly lower in hamsters than rats and mice. The Lugol staining showed intense starch degradation in the hamsters' forestomachs, presumably by microbial fermentation. A possible explanation is that the prae-duodenal microbial starch fermentation enhances digestibility and reduces the need for pancreatic amylase in hamsters. Rats and mice may rely more on pancreatic amylase for prae-caecal starch digestion, while the microbial fermentation is mainly located in the caecum. The results clearly show species differences in the digestive capacity for starch in mice, rats and hamsters that need to be considered in the feeding of these species in the laboratory setting as well as in the use of rodents as translational animal models.

CCL17 Promotes Colitis-Associated Tumorigenesis Dependent on the Microbiota.

Colorectal cancer is one of the most common cancers and a major cause of mortality. Proinflammatory and antitumor immune responses play critical roles in colitis-associated colon cancer. CCL17, a chemokine of the C-C family and ligand for CCR4, is expressed by intestinal dendritic cells in the steady state and is upregulated during colitis in mouse models and inflammatory bowel disease patients. In this study, we investigated the expression pattern and functional relevance of CCL17 for colitis-associated colon tumor development using CCL17-enhanced GFP-knockin mice. CCL17 was highly expressed by dendritic cells but also upregulated in macrophages and intermediary monocytes in colon tumors induced by exposure to azoxymethane and dextran sodium sulfate. Despite a similar degree of inflammation in the colon, CCL17-deficient mice developed fewer tumors than did CCL17-competent mice. This protective effect was abrogated by cohousing, indicating a dependency on the microbiota. Changes in microbiota diversity and composition were detected in separately housed CCL17-deficient mice, and these mice were more susceptible to azoxymethane-induced early apoptosis in the colon affecting tumor initiation. Immune cell infiltration in colitis-induced colon tumors was not affected by the lack of CCL17. Taken together, our results indicate that CCL17 promotes colitis-associated tumorigenesis by influencing the composition of the intestinal microbiome and reducing apoptosis during tumor initiation.

Morphometrics of Xenopus laevis Kept as Laboratory Animals.

Morphometric data that provide information on body conditions can be used to monitor the health and well-being of animals. In laboratory animals, they can help to evaluate the stress due to experiments or treatments, following the 3R principles. The aim of the present study was to obtain morphometric data of male and female African clawed frogs, Xenopus laevis, as the bases for body condition evaluations. Adult frogs (n = 198) were weighed and standardized photographs were taken. The photographs were used to determine several measurements (length, cranial width, caudal width, thigh width). In addition, a triangle was drawn to outline each frog's simplified body form, and the triangle surface was calculated. In conclusion, the triangle surface drawn on the dorsal plane of each frog correlated with the body weight of the females. There were significant differences between the body weights and sizes of male and female frogs, with males being smaller (p < 0.001). Based on the morphometric data, females could be assigned to five groups in which an assessment of the animal's well-being is feasible.

Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma.

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer entities including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity.Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes.We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor entities typically considered to be immunologically 'cold'.Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

Current Feeding Practice of Xenopus laevis in a Laboratory Setting.

African clawed frogs are common animal models used in various research areas. However, husbandry and especially feeding regimens are not nearly as standardized as is established for other laboratory animals. We recorded the diets and feeding protocols commonly used in laboratory practice in a questionnaire (18 responses). The survey revealed a wide variety of housing conditions. Feeding protocols and, in particular, diet composition varied considerably between facilities. While diets tailored to Xenopus were used in the majority, differences in feeding frequency and dietary components were noted. From five responses, the weekly feed intake per frog could be calculated, showing considerable differences in dry matter intake (1.37-5.4 g). The labelled nutrient content of the diets fed in the facilities (n = 10) met the recommendations in most cases, with protein as the major energy source. However, the mineral content varied markedly between diets. Both floating and sinking diets were used, while quickly sinking diets were associated with feed leftovers. Feed processing may likely influence feed intake behavior. Further research is needed to ensure standardization for aquatic species with respect to husbandry systems, feeding regimens, and especially the nutrient composition of feeds. Furthermore, this work will contribute positively to animal welfare and the comparability of research results.

Influence of Strain and Diet on Urinary pH in Laboratory Mice.

Acid base homeostasis and urine pH is influenced by the dietary cation anion balance (DCAB) in many species. Here, a negative DCAB acidifies the urine, while higher DCABs alkalize the urine. The dimension of the DCAB effect can be species-specific, because of differences in urine buffer systems. The aim of the present study was to describe the response of laboratory mice to diets with different DCAB. We used 8-week-old wildtype male mice of the C57Bl/6J inbred strain and CD1 outbred stock. Three groups (n = 15 animals/group) were formed and fed standard diet A for adaptation. For the 7-week feeding trial, mice were either kept on diet A (DCAB -7 mmol/kg dry matter (DM) or switched to diet B (246 mmol/kg DM) or C (-257 mmol/kg DM). Urine pH was measured weekly from a pooled sample per cage. There was a significant difference in the basal urine pH on diet A between C57Bl6/J and CD1 mice. The shift in urine pH was also significantly different between the two groups investigated.

Evaluation of a Configurable, Mobile and Modular Floor-Pen System for Group-Housing of Laboratory Rabbits.

The major responsibility of researchers and laboratory animal facilities is to ensure animal well-being during the time of acclimatization, experiments, and recovery. In this context, animal housing conditions are of utmost importance. Here, we implemented a mobile and modular floor-pen housing system for laboratory rabbits that combines rabbits' natural behavioral requirements and the high hygiene standards needed in biomedical science. Twelve female New Zealand White (NZW) rabbits were single- or group-housed for 12 months in mobile and modular floor-pens. Their general health status was evaluated at the end of the experimental setup. Further, we performed behavioral analysis of six additional NZW females group-housed for eight weeks in pens of two different sizes. We show that our improved housing concept supported species-specific behavioral patterns. Taken together, our housing system provides an optimal setup for rabbits in animal facilities that combines strict requirements for animal experiments with animal welfare.

Helicobacter hepaticus is required for immune targeting of bacterial heat shock protein 60 and fatal colitis in mice.

Gut microbiota and the immune system are in constant exchange shaping both host immunity and microbial communities. Here, improper immune regulation can cause inflammatory bowel disease (IBD) and colitis. Antibody therapies blocking signaling through the CD40-CD40L axis showed promising results as these molecules are deregulated in certain IBD patients. To better understand the mechanism, we used transgenic DC-LMP1/CD40 animals with a constitutive CD40-signal in CD11c+ cells, causing a lack of intestinal CD103+ dendritic cells (DCs) and failure to induce regulatory T (iTreg) cells. These mice rapidly develop spontaneous fatal colitis, accompanied by dysbiosis and increased inflammatory IL-17+IFN-γ+ Th17/Th1 and IFN-γ + Th1 cells. In the present study, we analyzed the impact of the microbiota on disease development and detected elevated IgA- and IgG-levels in sera from DC-LMP1/CD40 animals. Their serum antibodies specifically bound intestinal bacteria, and by proteome analysis, we identified a 60 kDa chaperonin GroEL (Hsp60) from Helicobacter hepaticus (Hh) as the main specific antigen targeted in the absence of iTregs. When re-derived to a different Hh-free specific-pathogen-free (SPF) microbiota, mice showed few signs of disease, normal microbiota, and no fatality. Upon recolonization of mice with Hh, the disease developed rapidly. Thus, the present work identifies GroEL/Hsp60 as a major Hh-antigen and its role in disease onset, progression, and outcome in this colitis model. Our results highlight the importance of CD103+ DC- and iTreg-mediated immune tolerance to specific pathobionts to maintain healthy intestinal balance.