BioID, IP and Characterization of Mitochondrial DNA Associated Proteins in Trypanosoma brucei
1Institute of Cell Biology, University of Bern, Switzerland; 2Sir William Dunn School of Pathology, University of Oxford, United Kingdom; 3Department of Chemistry and Biochemistry, University of Bern, Switzerland
Trypanosoma brucei is a flagellated protozoan responsible for the Human African Trypanosomiasis, also called sleeping sickness. The parasite has some particularities, including the presence of only one mitochondrion, containing only one mitochondrial DNA network, called kinetoplast or kDNA. The kDNA is composed of thousands of minicircles and around 25 maxicircles (Hajduk and Ochsenreiter, RNA biology, 2010). A proper replication and segregation of the kDNA is essential for the parasite. The segregation of the kDNA during cell cycle depends on the Tripartite Attachment Complex (TAC) (Ogbadoyi et al., Molecular Biology of the Cell, 2003). This complex of proteins connects the basal body of the flagellum to the kDNA and can be divided into three parts: the exclusion zone filaments, from the basal body to the outer mitochondrial membrane; the differentiated membranes; and the unilateral filaments, from the inner mitochondrial membrane to the kDNA. Very few proteins of the TAC have been characterized. One of them is TAC102 which localizes to the unilateral filaments (Trikin et al., Plos Pathogens, 2016). In order to identify new TAC components, I am performing BioID using TAC102 as a bait and immunoprecipitation using a monoclonal antibody against TAC102. Potential candidates identified by mass spectrometry are tested by looking at the phenotype upon RNAi induction (growth curve, DAPI staining). I will also analyze two candidates coming from previous screenings. They both have shown a localization to the kDNA and seem to be involved in its maintenance. I am characterizing their putative role in the kDNA replication and/or segregation.
Characterize the Function of Mitochondrial Activity in Lung Cancer Chemotherapy Resistance
1Department for BioMedical Research, University of Bern, Switzerland; 2Division of General Thoracic Surgery, Inselspital, Bern University Hospital, Bern, Switzerland
Introduction: Lung cancer is the most common cause of cancer-related mortality worldwide. This is mainly due to the difficulty of early detection and lack of effective treatment methods, thus more effective treatment options are desperately needed. More than 80% of lung tumors are non-small-cell lung cancers (NSCLC). It was postulated that tumor initiation and propagation are mediated by so called tumor-initiating cells (TICs), which can self-renew, spawn differentiated progeny and are associated with chemotherapy resistance. In NSCLC, high mitochondrial activity correlates with sphere formation capacity and increased tumor growth, which is in agreement with the findings in breast and pancreatic cancer.
Aim: the aim of this study is to further characterize the role of the mitochondrial activity in lung cancer chemotherapy resistance.
Methods: The NSCLC cell line A549 cells were stained with MitoTracker, metabolically fractionated into mito-high (5%) and mito-low (5%) subpopulations according to the mitochondrial activity. Sorted subpopulations were further examined by in vitro colony formation assay. Pemetrexed resistance was assessed by colony formation assay. Mitochondrial activity of A549 after pemetrexed treatment was analyzed by muti-color flow cytometry.
Results: Colony formation capacity was not dependent on mitochondrial activity. However, pemetrexed resistance correlated with mitochondrial activity. Interestingly, pemetrexed treatment increased the average mitochondrial activity over time. In detail, treatment with pemetrexed for 72 hours increased mitochondrial activity 5-fold compared to untreated control.
Conclusions: In summary, our results indicate that mitochondrial activity might affect chemotherapy resistance. Further experiments will be required to confirm these results. We speculate that inhibition of the mitochondrial activity might potentiate the efficiency of current lung cancer therapy.
The Application of Horse Shapes to Assess the Conformation Traits of Franches-Montagnes Stallions
1Institute of Genetics, Vetsuisse Faculty Bern, University of Bern, Switzerland; 2Agroscope – Swiss National Stud Farm, Avenches, Switzerland; 3Equine Sciences Faculty, Veterinary University Vienna, Austria; 4Institute of Animal Breeding and Genetics, Veterinary University Vienna, Austria
Unlike many other livestock species with measurable breeding goals (milk yield, weight gain, etc.), equine breeding has traditionally focused on the evaluation of conformation. Nowadays, many breeding organisations have implemented linear description (LD) instead of judging to minimise subjectivity in scoring, however recent studies have shown that the LD traits show essentially the same problems as traditionally scored traits, such as data converging around the mean value with very small standard deviations. To improve the assessment of LD traits of horses we investigated the application of recently described horse shape space analysis (HSS) based upon digitised photographs of 403 Franches-Montagnes (FM). Repeatability and consistency of the HSS were assessed with Procrustes ANOVA and intra-class correlation coefficients. The principal component scores (PCs) of the HSS were compared to linear description data using linear mixed models. The digitisation process is highly repeatable (R = 0.72 – 0.99, ICC = 0.99) but the photograph, age and placement of the horse have significant effects (p < 0.05) on the HSS and limit its consistency. Comparing the HSS derived PCs describing the shape variation with LD traits, there are significant associations (p(chi2) < 0.05) with traits describing the neck (muscling, length, attachment), type, hind limb muscling, conformation of the legs, length of the back and the withers. The HSS shows variation over time, which is not always the case in LD data. The HSS could thus be a useful tool to improve the assessment of conformation traits in horses, and therefore, their genetic analysis.
Investigation of Virulence Candidate Genes of Listeria monocytogenes
1Division of Experimental Clinical Research, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Switzerland; 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland; 3Institute of Veterinary Bacteriology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Switzerland
Listeriosis, responsible of important clinical outbreaks in farm animals and caused by Listeria (L.) monocytogenes is associated with hypervirulence factors from strains present in lineage I, isolated from animal outbreaks, infections and rhombencephalitis and not in lineage II, isolated from the environment and food.
In this study we aim to investigate candidate virulence genes that have been identified by comparative genomics of lineage I and II strains. Seven genes were identified that were present in lineage I strains but not in lineage II. These genes were present in rhombencephalitis strains from lineage I. The proteins encoded by two of the genes are LPXTG motif-containing cell wall-anchored proteins. The impact of these genes on cellular infection was assessed by testing respective deletion mutants of a parental strain from rhombencephalitis in the gentamicin protection assay using CNS derived cell lines (Bovine organotypic brain-slice culture and fetal bovine brain cells) versus non-CNS derived cell lines (Bovine macrophage cell line, the human colon adenocarcinoma cell line and the bovine caruncular epithelial cell line) that represent the various target cells/tissues of L. monocytogenes.
Our current results show that deletion of those two genes does not affect invasion of phagocytic cells (Bovine macrophage cell line) and non-phagocytic cells (Human colon adenocarcinoma cell line, Caco-2) and intracellular replication in comparison to the wild type strain. These preliminary data indicate that those two genes are not involved in the hypervirulence of lineage I strains. However, the effect of those genes and the others on the infection of other cell types remains to be assessed.
IL-33/ST2 Signaling Promotes the Proliferation of AML1/ETO Leukemic Stem Cells
1Tumor Immunology, Department for BioMedical Research, University of Bern, Switzerland; 2Department of Medical Oncology, Inselspital, Bern University Hospital and University of Bern, Switzerland
Interleukin (IL)-33 is crucially involved in type 2 immunity, allergy and inflammation. It is released upon cell necrosis, operating as alarmin by binding to its receptor ST2. It is known that IL-33/ST2 signaling promotes the growth of chronic myeloid leukemia progenitors. However, the role of IL-33/ST2 signaling in acute myeloid leukemia (AML) is under investigation.
Analysis of whole BM cells from AML patients using the gene expression database BloodSpot revealed that ST2 is expressed particularly in the AML1/ETO subtype. We therefore generated a murine AML model that was induced by transplantation of AML1/ETO oncogene transduced hematopoietic stem cells (HSCs). A strong ST2 expression was detectable on transfected HSCs, but not on non-transfected HSCs. Furthermore, AML1/ETO transfected HSCs formed more colonies in a colony forming assay when recombinant IL-33 was added to the media compared to transfected HSCs in media without IL-33.
Subsequently, we analyzed CD34+ leukemic stem cells (LSCs) from human patient samples harboring the AML1/ETO fusion protein. Some samples expressed ST2, while others did not. We sorted LSCs from the ST2-postive samples and cultured them in methylcellulose to assess colony forming capacity. IL-33 addition increased colony formation. Since molecular profiling of the AML LSCs revealed a negative correlation between ST2 and key genes of the Notch pathway, we silenced the Notch pathway in the ST2-negative AML1/ETO LSCs using siRNA. This led to the upregulation of the ST2 protein expression. Furthermore, siRNA treated LSCs formed more colonies when IL-33 was added to the media compared to LSCs in media without IL-33. This difference was not detectable for LSCs that were treated with control RNA.
To summarize, we show for the first time that IL-33/ST2 signaling supports the proliferation of AML1/ETO LSCs. AML induction in ST2- and IL-33-knockout mice will reveal more specifically the importance of IL-33/ST2 signaling for the survival.
Identifying Mechanisms of Radiotherapy Response of BRCA1/p53-Mutated Mouse Mammary Tumors
1Institute of Animal Pathology, Vetsuisse Faculty Bern, University of Bern, Switzerland; 2Division of Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands; 3Division of Molecular Pathology and Cancer Genomics, The Netherlands Cancer Institute, Amsterdam, The Netherlands
Radiotherapy (RT) is one of the most widely used anticancer therapies with about 50% of all cancer patients receiving RT during their treatment course. Although RT significantly contributes to cancer cure, the acquirement of resistance as well as tumor-unspecific toxicity remain major obstacles for the success of this treatment. The cytotoxic effect of RT relies on the generation of DNA damage which triggers a complex network of signalling pathways, known as the DNA damage response (DDR) machinery. Over the past years, a growing body of evidence showed that most tumors have an impaired DDR. Impaired DDR as Achilles heel of cancer cells led to the development of new therapeutic strategies to selectively sensitize tumors to DNA-damaging agents, based on the “synthetic lethality” concept: tumors that have lost critical DDR pathways rely more on the remaining pathways, while normal tissues still have all DDR pathways available. Besides synthetic lethality, salvage pathways in the DDR-deficient cells may also cause resistance to DNA-damaging agents such as RT. The aims of this project are to identify genes which are synthetic lethal with RT and to elucidate the molecular mechanisms responsible for RT escape. To achieve these goals, complementary in vivo-in vitro studies using BRCA1/p53-deficient mouse mammary tumor models and haploid cells treated with RT are ongoing. On the one hand, RNA sequencing of matched radio-sensitive and -resistant mammary tumors is performed. On the other hand, we carried out a loss-of-function haploid genetic screen to identify genes that alter RT response. In total, 19 genes were identified as potential synthetic lethal candidate genes with RT. Together with genes that have known functional role on DDR such as DNA-PK, ATM and Artemis, several unknown candidate genes with unknown roles in the DDR were identified. Of these, genes with a novel putative link to RT response, are now further validated.
RNA Oxidation Harms the Ribosome and Differentially Affects the Catalytic Center
Department of Chemistry and Biochemistry, University of Bern, Switzerland
The ribosome is the central ribozyme responsible for protein biosynthesis. In its active site, the peptidyl transferase center (PTC), catalysis relies heavily on the contribution of rRNA, especially the 2’OH of A2451. rRNA bases are oxidized in Alzheimer's, and recent findings indicate this could be a contributing factor to various neurodegenerative diseases. However, the consequences of direct oxidative stress to the ribosome and its effects on translation are unknown. We set out to investigate oxidized nucleobases in the rRNA in general and in the PTC in particular, using extensive chemical engineering experiments. Via the technique of atomic mutagenesis, single modifications can be introduced at any position of the 23S rRNA during in vitro reconstitution of the ribosome. This method surpasses conventional mutagenesis and effectively enables us to alter single atoms in the context of the ribosome, and to insert even non-natural nucleosides. We replaced the five universally conserved PTC nucleobases A2451, A2601, U2585, U2506 and C2063 with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC, respectively. The chemically engineered ribosomes were then studied in various functional assays to investigate the consequences of nucleobase oxidations on translation. We will present our findings on how incorporation of different oxidized bases into the PTC affected ribosome’s functions in diverse ways. For example, individual oxidized nucleobases resulted in either a radical deceleration of peptide bond formation, alteration of tRNA binding to A-site or even an increase of the translation rate. These results expand our current understanding of the roles of PTC residues in ribosomal function. This study is the first investigation of site-specific oxidation of central PTC residues, and provides diverse, interesting findings for both the ribosome field and research on neurodegenerative disease.
REM Sleep Reactivation of Lateral Hypothalamus Neurons Associated with Goal-Oriented Behaviors Conditions Food Intake During Wakefulness
1Center for Experimental Neurology (ZEN), Department of Neurology, Inselspital, Bern University Hospital, and Department for BioMedical Research, University of Bern, Switzerland; 2Department of Neurology, Inselspital, Bern University Hospital, Switzerland; 3Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland
The hypothalamus is a key hub for the integration and regulation of the sleep-wake cycle and feeding as it receives information from central and peripheral origins and modulates brain activity through widespread projections. Here, we investigate how single GABAergic and glutamatergic cells in the lateral hypothalamus (LHvgat, LHvglut2, respectively) modulate food intake, sleep and arousal. We first measure the neuronal activity of LHvgat or LHvglut2 neurons during sleep and food intake paradigms by targeting the expression of the calcium sensor GCaMP6s to the LH of vgat-IRES-Cre or BAC-vglut2::Cre mice, respectively, and subsequently imaging Ca2+ transients from single neurons with a miniaturized fluorescence microscope in freely-behaving mice. We found that a large proportion of LHvgat andLHvglut2 neurons show maximal activity during REM sleep, while other subsets of cells were active during wakefulness. When the animals were subjected to a free-access feeding paradigm we found that most LHvgat neurons showed maximal activity during food approach or -intake, whereas LHvglut2 neurons were predominantly active in behaviors unrelated to feeding. When comparing the functional identity of the neurons for sleep and feeding, we found that LHvgat cells associated with food approach or food intake identity were significantly more likely to be active during REM, but not NREM, sleep episodes. No such specialization was observed for LHvglut2 neurons. Interestingly, food-approach and food-intake LHvgat neurons showed a sequential activation profile according to the animal behavior, however, they were reactivated during REM sleep in a random fashion, independently of their functional identities.
These findings indicate that LHvgat neurons are multi-functional encoding aspects of both, sleep- and metabolic function. Furthermore they propose that LHvgat neuron activation in REM sleep may modulate circuits for homeostatic integration via non-experience dependent mechanisms.
Characterization of Equine Papillomavirus Type 2 in vivo and in vitro
1Institute of Virology, Vetsuisse Faculty, University of Zurich, Switzerland; 2Dermatology Department, Vetsuisse Faculty, University of Zurich, Switzerland
Strong epidemiological evidence supports the hypothesis that the infection with equine papillomavirus type 2 (EcPV2) is a crucial factor for the development of genital squamous cell carcinomas (SCC) in horses. As treatment options are sparse and rather aggressive, tools for a better prevention, diagnosis and treatment are needed. Therefore, profound characterization of the virus and its tumour transforming properties are essential.
The aims of this project are on one hand the systematic in vivo categorization based on clinical sample material, while on the other hand the establishment of an in vitro cell culture model to study the influence of the virus and selected viral genes.
To categorize the in vivo sample material, FFPE biopsy samples from affected horses were classified pathohistologically into four different stages: hyperplasia, papilloma, in situ carcinoma and squamous cell carcinoma. These four stages will be further characterized for their expression of markers for cancer/proliferation and virus markers.
To establish an in vitro model for studying the influence and transformation properties of EcPV2 and selected viral genes, equine keratinocyte cell cultures were established from healthy and affected genital mucosa. The cells from healthy mucosa will be transduced with selected viral genes (E1^E4, E6 and E7). Horse cells, which are positive for EcPV2-E7-DNA were successfully established already. Growing these cells as skin resembling raft cultures and compare them to the in vivo situation will be the final goal.
The classification via viral and marker gene expression of in vivo material could be useful to develop new diagnostic or prognostic tools. While the in vitro model might help to study the disease on a molecular basis in more detail.
Phenotypic Variation and Social Dominance Rank Within and Between Cages in SWISS Mice
1Division of Animal Welfare, Veterinary Public Health Institute, Vetsuisse Faculty, University of Bern, Switzerland; 2Department of Biomedical Sciences/Physiology, University of Veterinary Medicine Vienna, Austria
Scientists generally assume that mice housed within the same cage are phenotypically more similar compared to mice from different cages, because of a higher degree of shared environment. This assumption, however, neglects the social context of the environment; for example, social dominance relationships. Previous studies in male mice have indicated that social dominance relationships contribute to individual differences in phenotype, depending on individual rank position, which may mask treatment effects. The current study measured dominance relationships across 3 weeks in adulthood, in 26 groups of male and 27 groups of female SWISS mice housed in same-sex groups of 3, and assessed behavioural and physiological measures of phenotype in the following week. Most groups formed transitive dominance relationships, where each mouse had a unique rank. The remaining groups formed intransitive or despotic dominance relationships. Preliminary analyses tested whether social dominance rank, in groups with transitive dominance relationships, or cage membership could explain phenotypic variation in the open field, novel object test, elevated plus-maze, glucocorticoid metabolites, or body-mass. This analysis indicated that social dominance rank accounted for more phenotypic variation than cage membership for behaviour of males, but not females, in the novel object test and elevated plus-maze; no other differences were observed. These findings, that there are different types of dominance relationships and differences in phenotype related to social dominance rank, do not support the assumption that phenotypic variation is larger between, rather than within, cages of mice. However, more research is necessary to determine whether these findings generalize to other strains of mice and other outcome measures, and whether neglecting social dominance ranks may bias experimental results in biomedical research.