Scientific success stories based on Biocenter Finland technology services
See more success stories in the Annual Report
Recently, Structural Bioinformatics Laboratory (BCT) was contacted by Professor Juha Kere (Karolinska Institute, Kings College and Biomedicum) for help in identifying the DNA binding sequence for a newly-identified transcription factor (TF) that is responsible for the very early 4-8 cell stage of human embryonic development. The DNA sequence was identified using bioinformatics means, model structures were made and demonstrated the most likely residues involved and supported the studies on mutations made on Leutx based on genetic variation in the Icelandic human population (via Decode Genomics). Structural studies on the TF have begun and studies have expanded now to other TFs in early development that were also reported by Juha Kere’s consortium on 9 international laboratories. A manuscript on the genomic studies and modeling have been submitted for publication. The project’s success relied on our ability to rapidly muster the resources of SBL and the IT infrastructure of BCT provided via BF support in 2016.
Lipid droplets (LDs) are ubiquitous cellular organelles that function in regulating lipid metabolism by storing lipids at times of excess and releasing them upon demand. In a collaboration project between University of Helsinki research groups of Elina Ikonen (Department of Anatomy, Faculty of Medicine) and Eija Jokitalo (Institute of Biotechnology), we showed that Seipin, mutated in severe congenital lipodystrophy (BSCL2), is an ER–lipid droplet contact protein, regulates the extent of ER–LD contacts and facilitates incorporation of lipid and protein cargo into maturing LDs. Seipin deficiency increases the heterogeneity of ER–LD contacts, resulting in completely missing, rudimentary, or very extensive contacts. Seipin is required during LD formation for the targeting of ER‐derived fatty acid‐activating enzyme ACSL3 to LDs. In seipin deficiency, the fatty acid flux to neutral lipids becomes compromised when LD formation has been initiated.
Salo VT, Belevich I, Li S, Karhinen L, Vihinen H, Vigouroux C, Magre J, Thiele C, Hölttä-Vuori M, Jokitalo E, Ikonen E. 2016. Seipin regulates ER-lipid droplet contacts and cargo delivery. EMBO J. 35:2699-2716.
The light microscopy platform served a growing number of users, nearly 1000, coming from more than 270 research groups, and continued to have a very significant impact on Finnish life sciences. The services of the platform were used in numerous high-impact scientific publications, a small selection of which is listed below. The contributions of the platform include both routine imaging, advanced imaging and protocol development, and bioimage informatics.
The unit has offered a record service level accounting with an income of around 25 000 € in 2016, this is in part due to the efforts of previous years on public relationships in promoting and publicising the unit, and also for the support of the Faculty of Pharmacy on the upgrade of the SPECT/CT camera, as well as partially covering service contract expenses.
Efforts of this unit, along with the Radiochemistry Unit, have crystallised in the foundation of the Helsinki in vivo animal platform (HAIP), which would provide a more versatile environment and a better situation on the national infrastructure development, and give more international projection.
With the support of core facility services provided by the BF-GWM technology platform, Academy Prof. Johanna Ivaska’s research team made breakthrough novel findings on how surrounding tissue influences growth and malignancy of the cancer cells. The study published by Riina Kaukonen et al. 2016 in Nature Communication revealed that the stiffness of both tumor and the surrounding tissue affects largely expression of the genes in cancer cells and increases the malignancy of the cancer. Importantly, Kaukonen et al. were able to prevent the growth of the tumour by returning stiffness of the surrounding tissue into the normal level found in healthy tissues. Mediator of these effects in cancer cells was found to be an epigenetic regulator JMJD1A enzyme. In collaboration with the medical doctors at University Hospitals in Turku and Helsinki, the group also found that this JMJD1A is expressed at higher levels in those cancers which were surrounded by tissue with higher density. The results by Prof. Ivaska’s group open completely new insights and into the mechanisms how surrounding tissues contribute to the development of cancers. Novel therapies targeting also tissues surrounding the tumor may improve the efficiency of cancer treatments.
The NC-UEF is the only laboratory in Nordic countries able to perform combined electroretinogram (ERG) and visual evoked potential (VEP) recordings in mice. These techniques were employed to assess the question whether degenerative brain diseases manifest in the retina of the eye, which is developmentally part of the brain. Indeed, in two genetically modified mouse lines, one modeling Huntington’s disease, and one cerebral neurolipofuscinosis (CLN5), functional ERG changes preceded motor symptoms at an early stage of the disease. Retinal changes were progressive, allowing follow-up studies over several months. On the other hand, the possibility suggested in the literature that progressive impairment in spatial navigation in Alzheimer model mice might be caused by the progressively impaired vision was ruled out: the ERG and VEP responses were intact in middle-aged APP/PS1 mice with documented spatial memory impairment. (Leinonen H, Lipponen A, Gurevicius K, Tanila H. Normal amplitude of ERG and VEP responses in APP/PS1 mice. J Alzheimer’s Dis 2016; 51(1):21-26.)
These findings encourage the use of ERG and VEP in preclinical treatment trials in mice modelling neurodegenerative diseases. They also suggest that ERG, combined with automated fundoscopy, as a minimally invasive and low-cost method could be used as a screening tool for common brain diseases in the risk group of aged individuals.
Non-mammalian model organisms
The most important breakthrough is the current standard use of CRISPR-Cas9 method in zebrafish to create targeted mutants that allows for example phenotypic analysis of novel genes associated with certain diseases. Both in the Helsinki and Tampere unit dozens of mutations are created successfully.
Biocenter Finland SAB evaluated all the platforms and PPN was ranked very high in the evaluation. Furthermore, the user survey indicated the impact of PPN to be high and we received the highest score among the BF platforms. Therefore, it is clear that PPN represents widely recognized source of scientific services. The PPN consortium is well-established and several important contributions were made during 2016. We decided to highlight two success stories, reflecting the technologies important also for the future development.
Example study: A subset of stabilin-1-expressing macrophages prevents fibrosis in chronic liver injury
Rantakari P, Patten DA, Valtonen J, Karikoski M, Gerke H, Dawes H, Laurila J, Ohlmeier S, Elima K, Hübscher SG, Weston CJ, Jalkanen S, Adams DH, Salmi M, Shetty S. Stabilin-1 expression defines a subset of macrophages that mediate tissue homeostasis and prevent fibrosis in chronic liver injury. Proc Natl Acad Sci U S A. 2016, 113(33):9298-303.
BCK Group published an article reporting novel discoveries related to metabolism of type 2 diabetes, including discovery of microbiota-produced compound indolepropionic acid and its protective role against type 2 diabetes, thereby providing direct link between gut microbiota and the disease. The publication gained wide national and international media visibility, e.g.: https://www.sciencedaily.com/releases/2017/04/170411090159.htm
Identified metabolites and their association with the development of T2D in the DPS (N = 200). Closed bars: FDR-P < 0.05 Opened bars: P < 0.05. Phe: phenylalanine GCA: Glycocholic acid TCDC: Taurochenodeoxycholic acid GCDC: Glycochenodeoxycholic acid GDC: Glycodeoxycholic DC: Deoxycholic acid CA: Cholic acid.
Original article: de Mello V, Paananen J, Lindström J, Lankinen MA, Shi L, Kuusisto J, Pihlajamäki J, Auriola S, Lehtonen M, Rolandsson O, Bergdahl IA, Nordin E, Ilanne-Parikka P, Keinänen-Kiukaanniemi S, Landberg R, Eriksson JG, Tuomilehto J, Hanhineva K, Uusitupa M (2017) Indolepropionic acid and novel lipid metabolites are associated with a lower risk of type 2 diabetes in the Finnish Diabetes Prevention Study. Nature Scientific Reports, doi: 10.1038/srep46337.
BCH continued further optimization of reprogramming technology with particular focus on biobanking. We showed that fully reprogrammed iPS cells have similar differentiation propensities regardless of the cell-type of origin. The practical implication of this finding is that bona-fide iPS cell lines derived from different tissues can be combined in the biobank repositories (Kyttälä et al. Stem Cell Reports 2016; 6(2):200-12).
BMT has mainly focused on using iPSCs for disease modelling and the improvement of analytical methods. Modelling of cardiac electrophysiological defects has progressed without problems, but cardiomyopathies have proven to be more challenging. The phenotype and functional characteristics of iPSC-derived cardiomyocytes were found to be similar to those observed in patients with hypertrophic cardiomyopathy. This paper also described a wide spectrum of functional assays available in the Core (Ojala M, et al, Stem Cells International 2016, ID 1684792).
BCK has developed methods for differentiation of human iPSC derived cells into a variety of CNS and muscle cells. The differentiation protocols developed are exceptionally broad, extending from specific neuronal subtypes (spinal cord motoneurons and midbrain dopaminergic neurons, astrocytes, oligodendrocytes, microglia) to endothelial and two types of muscle cells. We have shown that specific functional pathologies and clinical phenotypes of CNS diseases, such as Parkinson’s disease, Alzheimer’s disease, psychopathy, amyotrophic lateral sclerosis and schizophrenia, as well as familial cardiomyopathies can be recapitulated in iPSC-derived cell models. While most of these results have so far been published only at the abstract level, an example for Parkinson’s disease has been published (https://www.nature.com/articles/npjparkd20169). Another important example of successful exploitation of the results is the identification and validation of IL-33 for therapeutic application in neurodegenerative disease. After patenting the discovery (WO 2014128254 A1), the finding was verified with human iPSC-derived models and licenced by the University of Eastern Finland to an US-based company with a deal worth 10 million euros.
Human parechovirus type 3 is a picornavirus that can cause severe infections in humans, resulting in sepsis and central nervous system disease in newborns. So far the most promising anti-picornaviral drug candidates do not have any effect on the parechovirus, therefore new effective means have to be found. Researchers in the University of Helsinki have determined a high-resolution structure of the human parechovirus type 3. The three-dimensional model was created by collecting thousands of images of virus with an electron microscope under -190 °C. The images were then computationally aligned and combined. “The virus genome is a single-stranded RNA, which is encapsidated in a protein shell. About a quarter of the genome is in close contact with the capsid proteins, leading to highly ordered RNA. This has not been seen in other picornaviruses,” describes Postdoctoral Researcher, Dr Shabih Shakeel in the Institute of Biotechnology. The atomic model of the virus shows a distinct way of how viral proteins interact with each other to stabilize the capsid. The best studied anti-picornaviral drug pleconaril and its derivatives work well against enteroviruses, large group of picornaviruses. The parechovirus type 3 structure demonstrates that pleconaril binding place is blocked in parechoviruses and therefore does not work against this virus group.
Marie Curie Postdoctoral Research Fellow, Dr Ausra Domanska worked on the structure of the same virus in complex with antibody fragments recognising parechovirus type 3. “In the absence of antiviral drugs, developing broadly neutralising monoclonal antibodies as therapeutic antibodies against this virus is one of the most promising treatment options for clinicians in the near future,” she says.
Shakeel S, Westerhuis BM, Domanska A, Konig RI, Matadeen R, Koster AJ, Bakke, AQ, Beaumont T, Wolthers KC, Butcher SJ. 2016. Multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis. Nature Communications 7:11387. doi: 10.1038/ncomms11387.
The research expertise of FIX-UP is a critical component of modern life science research. Our researchers continuously engage in exchanges in discussions with the life science research community. A highlight in this respect has been the EMBO symposium on biocatalysis organized in Oulu, June 12-16, 2016, focusing on fundamental and applied aspects of biocatalysis, with an emphasis on the impact that enzyme research has at the interface of biology and chemistry. The meeting was attended by166 participants who enjoyed the 32 talks by experts in the field. A selected set of papers has been published in a special issue of PEDS (March 2017).
Structural and functional studies of membrane pyrophosphatases
Membrane pyrophosphatases (M-PPases) occur in organisms ranging from plants and protozoan parasites to prokaryotes. They are associated with low-energy stress: overcoming saline or drought conditions in plants or rapid changes in pH or osmotic pressure in protozoan parasites. M-PPases couple pyrophosphate hydrolysis to the pumping of sodium ions and/or protons across the inner membrane of prokaryotes or the vacuole/acidocalcisome membranes of plants/parasites. There are no analogues in mammals, so M-PPases are an important potential drug target. Continuing from our initial M-PPase structure (Kellosalo et al, Science, 2012) using X-ray crystallography, we are completing a model of the catalytic cycle using additional structural and biochemical data. We have solved two new structures of the M-PPase of the thermophilic bacteria: Thermotoga maritima (Tm-PPase) in different catalytic states. Combining this data with previously solved structures of this protein and of a related protein from the plant: Vigna radiata (Vr-PPase) we have generated a plausible model of the complete catalytic cycle
that covers all the major catalytic states (Figure).
Overview of the catalytic cycle model proposed for membrane pyrophosphatases based on structural information obtained via X-ray crystallography. Only key helices and residues are shown for the sake of clarity (Li, K-M., Wilkinson, C., Kellosalo, J., Tsai, J-Y., Kajander, T., Jeuken, L.J.C., Sun, Y-J. and Goldman, A. (2016) Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism. Nat. Commun. 7:13596).
NMR spectroscopy and mass spectrometry
Weak or transient oligomers of proteins are examples of protein-protein interactions, which are difficult to study because the quaternary structure is not stable and protein exist in equilibrium of different forms depending on protein concentrations. Native mass spectrometry is a new effective tool to study mixtures, which consist of different protein species. This methodology has been efficiently used to reveal that inherent feature of allergens is their ability to form transient oligomers (Niemi et al. Sci Rep. 5 (2015) 13841). This is key information in the development hypoallergenic variants of allergens, which can be used in specific immunotherapy of allergic diseases.
The FIMM partner in the consortium has set up a webmicroscopy portal (predect.webmicroscope.net) through which partners in the PREDECT project (predect.eu) funded by Innovative Medicines Initiatives (IMI) of the European Union can access a common repository of digitized tissue samples. More than 28 laboratories around Europe, including 9 pharma companies participate in this 20 million Euro project that is one of the largest public-private partnerships within the biomedical research domain.
The aim of the project is to find better models for cancer drug discovery by integration and comparison of information retrieved from a large series of cancer model (2D and 3D cell culture, mouse xenografts, ex vivo tissue culture and human cancer samples).
All samples are made available on-line for visual inspection, comparisons and quantitative digital image analysis with cloud-based computing support through the portal maintained by FIMM (Figure). Linking the image data from the PREDECT TMAs directly to the sample metadata enables users to assess and antibody staining and tissue morphology.
Interface for PREDECT WebMicroscope database (http://predect.webmicroscope.net) with more than 30,000 scanned tissue samples
The image analysis tools developed at FIMM and IBT can support biomarker research and provide reproducible and high-throughput readout of protein expression and automated morphological characterization of tissue samples. A large number of studies have been published (selected publications reported below) where the platform services have contributed to the results and shown scientific impact.
Interface for PREDECT WebMicroscope database (http://predect.webmicroscope.net) with more than 30,000 scanned tissue samples.
Drug discovery and chemical biology
In a joint project between researchers at the University of Eastern Finland and Eberhard Karls University Tübingen in Germany, a new molecular level mechanism to prevent growth of hepatocellular cancer (HCC, liver cancer) has been identified. In the study, it was revealed that HCC with p53 deficiency is depending on interaction between the kinase Aurora A and MYC. Inhibition of Aurora A with specific inhibitors can prevent this interaction and further kills the HCC cells. Molecular modeling (provided by DDCB) could explain and predict how Aurora A inhibitors should interact with their target and this information is now used to design new anti-cancer compounds. Given that MYC plays a vital role in most types of cancer, these findings are likely to have a broad impact on cancer in general. (Dauch et al, Nat. Med. 2016, 22:744-753).
Viral gene transfer and cell therapy
A new nonviral integrating vector system based on transposons (Sleeping Beauty Transposon system) has been established in A.I. Virtanen Institute virus core facility. This vector system would avoid risks associated with viral gene carriers since it is based on plasmid constructs and it would also be easier to produce due to the simple bioreactor methods available for plasmid production in E. coli suspension cultures. The method was used for liver gene transfer in hypercholesteremic mice using LDL receptor and VLDL receptor transposon constructs. Results revealed a significant decrease in plasma cholesterol levels for extended periods of time after only a single application of the Sleeping Beauty transposon in the liver (Turunen TA et al. Mol Ther. 24:620-35, 2016.).