Genetische Variabilität, Wirt-Assoziation und Pathogenität von Kuhpockenviren (CPXV)

Die Spezies Kuhpockenvirus (CPXV), ein Mitglied des Genus Orthopoxvirus, ist endemisch in weiten Teilen Europas und Asien verbreitet. CPXV besitzt ein sehr breites Wirtsspektrum und zählt zu den zoonotischen Erregern. Phylogenetische Analysen deuten darauf hin, dass CPXV polyphyletisch ist. Die bisher definierten Kladen wurden in vorliegender Arbeit bestätigt. Die 20 neu gewonnenen CPXV-Stämme verschiedenster Wirtsspezies gruppieren vorrangig in die CPXV-like 1 und CPXV-like 2 Kladen. Ein CPXV-Stamm, isoliert von einem Neuweltaffen, erscheint jedoch als single branch und lässt sich keiner bisher bekannten Klade zuordnen. Gegenwärtig ist über die Rolle der Wühlmäuse, die als Reservoirwirt der Kuhpockenviren betrachtet werden, wenig bekannt. In vorliegender Arbeit sollte deshalb das aus einer Feldmaus (Microtus arvalis) stammende CPXV-Isolat FM2292 eingehend charakterisiert werden. CPXV FM2292 weist das bisher längste CPXV-Genom auf, das in die CPXV-like 1 Klade clustert. Neben der Sequenzanalyse sollten vergleichende experimentelle Infektionsstudien in Feldmäusen und Wistar-Ratten durchgeführt werden. Der Krankheitsverlauf nach intranasaler CPXV FM2292-Infektion bei Feldmäusen verlief subklinisch; Wistar-Ratten hingegen zeigten ausgeprägte klinische Symptome. Im Gegensatz dazu verursachte die Infektion mit einem aus einer Schmuseratte isolierten CPXV-Stamm bei den Feldmäusen eine starke Klinik. Daraus lässt sich schließen, dass Feldmäuse gegenüber Wühlmaus-assoziierten Stämmen, wie CPXV FM2292, eine Adaptation entwickelt haben. Die nachgewiesene Virusausscheidung weist auf eine Tröpfchen-basierte Übertragung hin. Neben Feldmäusen sollten auch Rötelmäuse im Tierversuch näher betrachtet werden. Unabhängig vom eingesetzten CPXV-Stamm resultierten die experimentellen CPXV-Infektionsstudien in Rötelmäusen (Myodes glaerolus) in subklinischen Verläufen. Eine nasale Virusausscheidung konnte nicht detektiert werden, was im starken Kontrast zu den Ergebnissen aus experimentell infizierten Feldmäusen steht. Dennoch deuteten die Serokonversionsraten der Rötelmäuse darauf hin, dass eine Replikation im Wirt stattgefunden hat. Zudem entwickelten zwei Kontakttiere ebenfalls OPV-spezifische Antikörper, was auf eine Übertragung des Virus schließen lässt. Wühlmäuse als Reservoirwirt dienen der Übertragung des Kuhpockenvirus auf akzidentielle Wirtsspezies wie Hauskatzen oder Nutztiere. Der in dieser Arbeit betrachtete Fallbericht eines mit CPXV-infizierten Fohlens zeigt, dass CPXV-Infektionen bei akzidentiellen Wirten mit stark ausgeprägter Klinik verbunden sein können. Zudem wird die wachsende Gefahr der Übertragung von CPXV auf Nutztiere und Menschen deutlich. Zusammenfassend unterstreicht die in der vorliegenden Arbeit verwendete phylogenetische Betrachtung die genetische Variabilität der Spezies CPXV. Zudem konnten neue Erkenntnisse zu Feldmäusen und Rötelmäusen als Reservoirwirtsspezies gewonnen werden. CPXV-Infektionen von Wühlmäusen verlaufen subklinisch, im Gegensatz zu dem hier ebenfalls beschriebenen, lethal endenden CPXV-Infektionsverlauf eines akzidentiellen Wirts, eines abortierten Fohlens.The species cowpox virus (CPXV), a member of the genus Orthopoxvirus (OPV), is endemic in parts of Europe and Asia. CPXV has got a broad host range and belongs to the zoonotic diseases. Phylogenetic analyses revealed that the species cowpox virus (CPXV) is polyphyletic. Previously defined clades were confirmed in the present study; the majority of the 20 new CPXV strains clustered in CPXV-like 1 and CPXV-like 2 clades. However, one CPXV strain, isolated from a New-world monkey, appears as single branch and has an unique phylogenetically position which is clearly separated from all other known clades. Currently only little is known about the role of voles as reservoir host of CPXV. To overcome this, the CPXV strain FM2292, which was isolated from a common vole (Microtus arvalis), was analyzed in detail. CPXV FM2292 is characterized by the longest CPXV genome within the CPXV-like 1 clade. Comparative experimental infections of common voles and Wistar rats were performed. Generally, CPXV FM2292 infection in common voles caused subclinical disease, whereas Wistar rats developed respiratory clinical signs and dermal lesions. In contrast, common voles exhibited prominent nasal discharge after intranasal infection with a pet rat derived CPXV strain. Taken together vole-derived strains like CPXV FM2292 induced subclinical infection reflecting most probably a virus-host adaption in accordance to reservoir host conditions. However, virus shedding was generally obtained independently from the CPXV strain used for infection. Moreover, experimental infection studies using bank voles (Myodes glaerolus) were performed. All CPXV-infections of bank voles resulted in subclinical diseases. No virus shedding was detected, which is in strong contrast to the results obtained from experimental infections using common voles. Nevertheless, the rates of seroconversions suggest that virus replicated within the host. Interestingly, two contact animals developed specific antibodies, also demonstrating transmission processes within the voles. CPXV can be transmitted from vole reservoirs to accidental species like cats or farmed animals. The case report of the present study analyzed a foal infected with CPXV. This case clearly shows that CPXV infections of accidental host species might result in severe or lethal disease. In addition this case draws attention towards the increasing danger of CPXV transmission on farmed animals and humans. Taken together, the genetic variability of CPXV is confirmed by the phylogenetic analysis done in the present study. In addition new insights of common and bank voles as reservoir species of CPXV could be obtained. Generally, CPXV infections of voles resulted in subclinical diseases, whereas the infection of a foal, an accidental reservoir species, resulted in an abortion of the foal and seroconversion of the mare

Isolation and characterisation of irinans, androstane-type withanolides from Physalis peruviana L.

Withanolides are steroidal lactones widespread in Nightshade plants with often potent antiproliferative activities. Additionally, the structural diversity of this compound class holds much potential for the discovery of novel biological activity. Here, we report two newly characterised withanolides, named irinans, from Physalis peruviana with highly unusual truncated backbones that resemble mammalian androstane sex hormones. Based on biomimetic chemical reactions, we propose a model that links these compounds to withanolide biosynthesis. Irinans have potent antiproliferative activities, that are however lower than those of 4ß-hydroxywithanolide E. Our work establishes androwithanolides as a new subclass of withanolides

Development of a Methodology for the Determination of Conceptual Automated Disassembly Systems

At a certain point in its life cycle, a product will reach a condition where it partly or completely loses its functionality. When this happens, the disassembly has the ambition to regenerate a product-value or to enable an environmental friendly product recycling. With regard to the high workload and costs for manual labor one approach to increase the productivity of disassembly tasks is the use of automated disassembly systems (ADS). Depending on different life cycle scenarios, requirements on automated disassembly systems vary. Concerning this problem, a general methodology is developed, which enables the determination of a conceptual ADS by assigning automated modules that are processing the product disassembly. In the first place the objective of a disassembly is determined, followed by a closer investigation of the product. Thereby target components are defined, which has to disassembled. By looking at the connections between these target components suitable separation procedures are derived. Finally, modules of the automated disassembly system are determined

Exceptional Clinical Resistance and Variable Reservoir Competence

Cowpox virus (CPXV) is a zoonotic virus and endemic in wild rodent populations in Eurasia. Serological surveys in Europe have reported high prevalence in different vole and mouse species. Here, we report on experimental CPXV infections of bank voles (Myodes glareolus) from different evolutionary lineages with a spectrum of CPXV strains. All bank voles, independently of lineage, sex and age, were resistant to clinical signs following CPXV inoculation, and no virus shedding was detected in nasal or buccal swabs. In- contact control animals became only rarely infected. However, depending on the CPXV strain used, inoculated animals seroconverted and viral DNA could be detected preferentially in the upper respiratory tract. The highest antibody titers and virus DNA loads in the lungs were detected after inoculation with two strains from Britain and Finland. We conclude from our experiments that the role of bank voles as an efficient and exclusive CPXV reservoir seems questionable, and that CPXV may be maintained in most regions by other hosts, including other vole species. Further investigations are needed to identify factors that allow and modulate CPXV maintenance in bank voles and other potential reservoirs, which may also influence spill-over infections to accidental hosts

Memory enhancement by ferulic acid ester across species

Cognitive impairments can be devastating for quality of life, and thus, preventing or counteracting them is of great value. To this end, the present study exploits the potential of the plant Rhodiola rosea and identifies the constituent ferulic acid eicosyl ester [icosyl-(2E)-3-(4-hydroxy-3-methoxyphenyl)-prop-2-enoate (FAE-20)] as a memory enhancer. We show that food supplementation with dried root material from R. rosea dose-dependently improves odor-taste reward associative memory scores in larval Drosophila and prevents the age-related decline of this appetitive memory in adult flies. Task-relevant sensorimotor faculties remain unaltered. From a parallel approach, a list of candidate compounds has been derived, including R. rosea–derived FAE-20. Here, we show that both R. rosea–derived FAE-20 and synthetic FAE-20 are effective as memory enhancers in larval Drosophila. Synthetic FAE-20 also partially compensates for age-related memory decline in adult flies, as well as genetically induced early-onset loss of memory function in young flies. Furthermore, it increases excitability in mouse hippocampal CA1 neurons, leads to more stable context-shock aversive associative memory in young adult (3-month-old) mice, and increases memory scores in old (>2-year-old) mice. Given these effects, and given the utility of R. rosea—the plant from which we discovered FAE-20—as a memory enhancer, these results may hold potential for clinical applications

Ecology impacts the decrease of Spirochaetes and Prevotella in the fecal gut microbiota of urban humans

Compared to the huge microbial diversity in most mammals, human gut microbiomes have lost diversity while becoming specialized for animal-based diets - especially compared to chimps, their genetically closest ancestors. The lowered microbial diversity within the gut of westernized populations has also been associated with different kinds of chronic inflammatory diseases in humans. To further deepen our knowledge on phylogenetic and ecologic impacts on human health and fitness, we established the herein presented biobank as well as its comprehensive microbiota analysis. In total, 368 stool samples from 38 different animal species, including Homo sapiens, belonging to four diverse mammalian orders were collected at seven different locations and analyzed by 16S rRNA gene amplicon sequencing. Comprehensive data analysis was performed to (i) determine the overall impact of host phylogeny vs. diet, location, and ecology and to (ii) examine the general pattern of fecal bacterial diversity across captive mammals and humans.By using a controlled study design with captive mammals we could verify that host phylogeny is the most dominant driver of mammalian gut microbiota composition. However, the effect of ecology appears to be able to overcome host phylogeny and should therefore be studied in more detail in future studies. Most importantly, our study could observe a remarkable decrease of Spirochaetes and Prevotella in westernized humans and platyrrhines, which is probably not only due to diet, but also to the social behavior and structure in these communities.Our study highlights the importance of phylogenetic relationship and ecology within the evolution of mammalian fecal microbiota composition. Particularly, the observed decrease of Spirochaetes and Prevotella in westernized communities might be associated to lifestyle dependent rapid evolutionary changes, potentially involved in the establishment of dysbiotic microbiomes, which promote the etiology of chronic diseases

Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells

In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm, precardiac or presomitic mesoderm within the first 24 h of differentiation, respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members, antagonist of Nodal signalling LEFTY1 and CER1, are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive, functional role of the BCD, we show its utility as a method to control lineage-specific differentiation. Furthermore, these findings have profound consequences for inter-experimental comparability, reproducibility, bioprocess optimization and scale-up.DFG/REBIRTHDFG/EXC62/1DFG/ZW 64/4-1DFG/MA 2331/16-1BMBF/13N12606BMBF/StemBANCCEU H2020/66872

TFAP2 paralogs facilitate chromatin access for MITF at pigmentation and cell proliferation genes

Funding Information: This work was supported by grants from the National Institutes of Health (NIH) to RAC (R01-AR062457), a postdoctoral fellowship from the American Association for Anatomy to CK, and grants from the Research Fund of Iceland to ES (207067 & 217768). https://grants.nih.gov/grants/ funding/r01.htm https://www.anatomy.org https:// en.rannis.is/funding/research/icelandic-researchfund/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: © 2022 Kenny et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.In developing melanocytes and in melanoma cells, multiple paralogs of the Activating-enhancer-binding Protein 2 family of transcription factors (TFAP2) contribute to expression of genes encoding pigmentation regulators, but their interaction with Microphthalmia transcription factor (MITF), a master regulator of these cells, is unclear. Supporting the model that TFAP2 facilitates MITF's ability to activate expression of pigmentation genes, single-cell seq analysis of zebrafish embryos revealed that pigmentation genes are only expressed in the subset of mitfa-expressing cells that also express tfap2 paralogs. To test this model in SK-MEL-28 melanoma cells we deleted the two TFAP2 paralogs with highest expression, TFAP2A and TFAP2C, creating TFAP2 knockout (TFAP2-KO) cells. We then assessed gene expression, chromatin accessibility, binding of TFAP2A and of MITF, and the chromatin marks H3K27Ac and H3K27Me3 which are characteristic of active enhancers and silenced chromatin, respectively. Integrated analyses of these datasets indicate TFAP2 paralogs directly activate enhancers near genes enriched for roles in pigmentation and proliferation, and directly repress enhancers near genes enriched for roles in cell adhesion. Consistently, compared to WT cells, TFAP2-KO cells proliferate less and adhere to one another more. TFAP2 paralogs and MITF co-operatively activate a subset of enhancers, with the former necessary for MITF binding and chromatin accessibility. By contrast, TFAP2 paralogs and MITF do not appear to co-operatively inhibit enhancers. These studies reveal a mechanism by which TFAP2 profoundly influences the set of genes activated by MITF, and thereby the phenotype of pigment cells and melanoma cells.Peer reviewe

Higher TIER bumble bees and solitary bees recommendations for a semi-field experimental design

The publication of the proposed EFSA risk assessment guidance document of plant protection products for pollinators highlighted that there are no study designs for non-Apis pollinators available. Since no official guidelines exist for semi-field testing at present, protocols were proposed by the ICPPR non-Apis working group and two years of ring-testing were conducted in 2016 and 2017 to develop a general test set-up. The ringtest design was based on the draft EFSA guidance document, OEPP/EPPO Guideline No. 170 and results of discussions regarding testing solitary bees and bumble bees during the meetings of the ICPPR non-Apis workgroup. Ring-tests were conducted with two different test organisms, one representative of a social bumble bee species (Bombus terrestris L; Hymenoptera, Apidae) and one representative of a solitary bee species (Osmia bicornis L; Hymenoptera, Megachilidae). The species are common species in Europe, commercially available and widely used for pollination services. Several laboratories participated in the higher-tier ring tests. 15 semi-field tests were conducted with bumble bees and 16 semi-field tests were done with solitary bees in 2016 and 2017. Two treatment groups were always included in the ringtests: an untreated control (water treated) and the treatment with dimethoate as a toxic reference item (optional other i.e. brood-affecting substances fenoxycarb or diflubenzuron). The toxic reference items were chosen based on their mode of action and long term experience in honey bee testing. A summary of the ringtest results will be given and the recommendations for the two semi-field test designs will be presented.The publication of the proposed EFSA risk assessment guidance document of plant protection products for pollinators highlighted that there are no study designs for non-Apis pollinators available. Since no official guidelines exist for semi-field testing at present, protocols were proposed by the ICPPR non-Apis working group and two years of ring-testing were conducted in 2016 and 2017 to develop a general test set-up. The ringtest design was based on the draft EFSA guidance document, OEPP/EPPO Guideline No. 170 and results of discussions regarding testing solitary bees and bumble bees during the meetings of the ICPPR non-Apis workgroup. Ring-tests were conducted with two different test organisms, one representative of a social bumble bee species (Bombus terrestris L; Hymenoptera, Apidae) and one representative of a solitary bee species (Osmia bicornis L; Hymenoptera, Megachilidae). The species are common species in Europe, commercially available and widely used for pollination services. Several laboratories participated in the higher-tier ring tests. 15 semi-field tests were conducted with bumble bees and 16 semi-field tests were done with solitary bees in 2016 and 2017. Two treatment groups were always included in the ringtests: an untreated control (water treated) and the treatment with dimethoate as a toxic reference item (optional other i.e. brood-affecting substances fenoxycarb or diflubenzuron). The toxic reference items were chosen based on their mode of action and long term experience in honey bee testing. A summary of the ringtest results will be given and the recommendations for the two semi-field test designs will be presented