Publication rates in animal research. Extent and characteristics of published and nonpublished animal studies followed up at two German university medical centres

Non-publication and publication bias in animal research is a core topic in current debates on the “reproducibility crisis” and “failure rates in clinical research”. To date, however, we lack reliable evidence on the extent of non-publication in animal research. We collected a random and stratified sample (n = 210) from all archived animal study protocols of two major German UMCs (university medical centres) and tracked their results publication. The overall publication rate was 67%. Excluding doctoral theses as results publications, the publication rate decreased to 58%. We did not find substantial differences in publication rates with regard to i) the year of animal study approval, ii) the two UMCs, iii) the animal type (rodents vs. non-rodents), iv) the scope of research (basic vs. preclinical), or v) the discipline of the applicant. Via the most reliable assessment strategy currently available, our study confirms that the non-publication of results from animal studies conducted at UMCs is relatively common. The non-publication of 33% of all animal studies is problematic for the following reasons: A) the primary legitimation of animal research, which is the intended knowledge gain for the wider scientific community, B) the waste of public resources, C) the unnecessary repetition of animal studies, and D) incomplete and potentially biased preclinical evidence for decision making on launching early human trials. Results dissemination should become a professional standard for animal research. Academic institutions and research funders should develop effective policies in this regard

FAS-Based Cell Depletion Facilitates the Selective Isolation of Mouse Induced Pluripotent Stem Cells

Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF) and mouse pluripotent stem cells (PSC). Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM) and stage-specific embryonic antigen 1 (SSEA1) were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues

Therapeutic concentrations of glucagon-like peptide-1 in cerebrospinal fluid following cell-based delivery into the cerebral ventricles of cats

<p>Abstract</p> <p>Background</p> <p>Neuropeptides may have considerable potential in the treatment of acute and chronic neurological diseases. Encapsulated genetically engineered cells have been suggested as a means for sustained local delivery of such peptides to the brain. In our experiments, we studied human mesenchymal stem cells which were transfected to produce glucagon-like peptide-1 (GLP-1).</p> <p>Methods</p> <p>Cells were packed in a water-permeable mesh bag containing 400 polymeric microcapsules, each containing 3000 cells. The mesh bags were either transplanted into the subdural space, into the brain parenchyma or into the cerebral ventricles of the cat brain. Mesh bags were explanted after two weeks, and cell viability, as well as GLP-1 concentration in the cerebrospinal fluid (CSF), was measured.</p> <p>Results</p> <p>Viability of cells did not significantly differ between the three implantation sites. However, CSF concentration of GLP-1 was significantly elevated only after ventricular transplantation with a maximum concentration of 73 pM (binding constant = 70 pM).</p> <p>Conclusions</p> <p>This study showed that ventricular cell-based delivery of soluble factors has the capability to achieve concentrations in the CSF which may become pharmacologically active. Despite the controversy about the pharmacokinetic limitations of ventricular drug delivery, there might be a niche in this for encapsulated cell biodelivery of soluble, highly biologically-effective neuropeptides of low molecular weight like GLP-1.</p

Novel aspects of age-protection by spermidine supplementation are associated with preserved telomere length

Ageing provokes a plethora of molecular, cellular and physiological deteriorations, including heart failure, neurodegeneration, metabolic maladaptation, telomere attrition and hair loss. Interestingly, on the molecular level, the capacity to induce autophagy, a cellular recycling and cleaning process, declines with age across a large spectrum of model organisms and is thought to be responsible for a subset of age-induced changes. Here, we show that a 6-month administration of the natural autophagy inducer spermidine in the drinking water to aged mice is sufficient to significantly attenuate distinct age-associated phenotypes. These include modulation of brain glucose metabolism, suppression of distinct cardiac inflammation parameters, decreased number of pathological sights in kidney and liver and decrease of age-induced hair loss. Interestingly, spermidine-mediated age protection was associated with decreased telomere attrition, arguing in favour of a novel cellular mechanism behind the anti-ageing effects of spermidine administration

LAMTOR2 (p14) Controls B Cell Differentiation by Orchestrating Endosomal BCR Trafficking

B-cell development and function depend on stage-specific signaling through the B-cell antigen receptor (BCR). Signaling and intracellular trafficking of the BCR are connected, but the molecular mechanisms of this link are incompletely understood. Here, we investigated the role of the endosomal adaptor protein and member of the LAMTOR/Ragulator complex LAMTOR2 (p14) in B-cell development. Efficient conditional deletion of LAMTOR2 at the pre-B1 stage using mb1-Cre mice resulted in complete developmental arrest. Deletion of LAMTOR2 using Cd19-Cre mice permitted analysis of residual B cells at later developmental stages, revealing that LAMTOR2 was critical for the generation and activation of mature B lymphocytes. Loss of LAMTOR2 resulted in aberrant BCR signaling due to delayed receptor internalization and endosomal trafficking. In conclusion, we identify LAMTOR2 as critical regulator of BCR trafficking and signaling that is essential for early B-cell development in mice

The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

A spontaneous mutation leading to the formation of congenital ovarian and testicular tumors was detected in the WKY/Ztm rat strain. The histological evaluation revealed derivatives from all three germ layers, thereby identifying these tumors as teratomas. Teratocarcinogenesis was accompanied by infertility and the underlying mutation was termed ter. Linkage analysis of 58 (WKY-ter×SPRD-Cu3) F2 rats associated the ter mutation with RNO18 (LOD = 3.25). Sequencing of candidate genes detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1), which introduces a premature stop codon assumed to cause a truncation of the Dnd1 protein. Genotyping of the recessive ter mutation revealed a complete penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both genders. Morphologically non-tumorous testes of homozygous ter males were reduced in both size and weight. This testicular malformation was linked to a lack of spermatogenesis using immunohistochemical and histological staining. Our WKY-Dnd1ter/Ztm rat is a novel animal model to investigate gonadal teratocarcinogenesis and the molecular mechanisms involved in germ cell development of both genders

Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells

Effect of mepyramine and JNJ 7777120 on Th2 cytokine and anti-OVA IgE concentrations in sera.

<p>Mice were sensitized to, and provoked with, OVA and treated with DMSO, mepyramine, JNJ 7777120, or mepyramine plus JNJ 7777120 either 30 min before each provocation cycle (<i>A, B</i>) or 30 min before and 2 h after each injection for sensitization (<i>C, D</i>). In sera, collected 24 h after the final provocation, IL-13 concentrations were measured by FlowCytomix (<i>A, C</i>) and anti-OVA IgE titers were determined by ELISA (<i>B, D</i>). Data shown are means ± SD (n_(sham) = 3, n_(DMSO) = 7, n_{(mepyramine, JNJ 7777120, or mepyramine+JNJ 7777120)} = 8; *, p≤0.05; **, p≤0.01; ***, p≤0.005).</p