Kamilla Vandsø Petersen’s research while affiliated with Aarhus University and other places

With the increasing need for effective compounds against cancer or pathogen-borne diseases, the development of new tools to investigate the enzymatic activity of biomarkers is necessary. Among these biomarkers are DNA topoisomerases, which are key enzymes that modify DNA and regulate DNA topology during cellular processes. Over the years, libraries of natural and synthetic small-molecule compounds have been extensively investigated as potential anti-cancer, anti-bacterial, or anti-parasitic drugs targeting topoisomerases. However, the current tools for measuring the potential inhibition of topoisomerase activity are time consuming and not easily adaptable outside specialized laboratories. Here, we present rolling circle amplification-based methods that provide fast and easy readouts for screening of compounds against type 1 topoisomerases. Specific assays for the investigation of the potential inhibition of eukaryotic, viral, or bacterial type 1 topoisomerase activity were developed, using human topoisomerase 1, Leishmania donovani topoisomerase 1, monkeypox virus topoisomerase 1, and Mycobacterium smegmatis topoisomerase 1 as model enzymes. The presented tools proved to be sensitive and directly quantitative, paving the way for new diagnostic and drug screening protocols in research and clinical settings.

Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable for many cloning and sequencing procedures. Methods for specific detection of restriction endonuclease activities can therefore find broad use for many purposes. In the current study, we demonstrate proof-of-concept for a new principle for the detection of restriction endonuclease activities. The method is based on rolling circle amplification of circular DNA products that can only be formed upon restriction digestion of specially designed DNA substrates. By combining the activity of the target restriction endonuclease with the highly specific Cre recombinase to generate DNA circles, we demonstrate specific detection of selected restriction endonuclease activities even in crude cell extracts. This is, to our knowledge, the first example of a sensor system that allows activity measurements of restriction endonucleases in crude samples. The presented sensor system may prove valuable for future characterization of bacteria species or strains based on their expression of restriction endonucleases as well as for quantification of restriction endonuclease activities directly in extracts from recombinant cells.

With the increased effort for identification of anticancer compounds, there is a growing need for tools to investigate the activity of enzyme biomarkers. Human topoisomerase 1 is the only target of the camptothecin derivatives, and the cellular drug response depends on the enzyme activity. Here we use the colon cancer cell line Caco2 to investigate the topoisomerase 1 activity using a simple and improved version of our rolling circle enhanced enzyme activity detection, the REEAD assay. We present two fast readout methods that do not require the use of specialized training or equipment. In this setup, topoisomerase 1 converts specific DNA substrates to closed circles. The circles are amplified by rolling circle amplification in the presence of biotinylated nucleotides allowing for the detection of the products using horse radish peroxidase conjugated anti-biotin antibodies. The visualization occurs by either ECL or by color development through the precipitation of the TMB onto the surface. The presented readouts allow for fast and sensitive screening of topoisomerase 1 activity in extracts from Caco2 cells, potentially enabling the patients’ stratification and the prediction of the chemotherapeutic response for individualized treatment. For these reasons, we believe that the presented method would be easily adaptable to the clinical settings.

Background Prediction of the pattern of organ involvement in patients with diffuse cutaneous systemic sclerosis (dcSSc) is supported by the specificity of autoantibodies. These autoantibodies are generally mutually exclusive and highly specific for dcSSc ¹ . The autoantibody reactivity allows patients to be stratified early in the disease course, leading to a tailored approach and management. Anti-topoisomerase I antibodies (ATA) are predictors of the development of interstitial lung disease (ILD) and digital ulcers but appear to be protective against pulmonary artery hypertension (PAH). Objectives The aim of this project was to elucidate the functioning of ATA and their role in the pathogenicity of the disease. Methods Sera from healthy volunteers (HV) and dcSSc patients with and without ATA were collected from Aarhus University Hospital. Total IgG was purified from the sera using a Protein G column. ATA was further purified from the total IgG fraction by passing it through a column packed with the functional domains of human topoisomerase-1 (TOP1) expressed from E.coli. Sera, total IgG and purified ATA fractions were utilized in preliminary experiments. The ability of ATA to inhibit TOP1 was tested by a in house developed REEAD assay in which the generation of closed DNA circles, by the cleavage-ligation activity of TOP1, is monitored after rolling circle amplification ² . Results Serum from patients with dcSSc, but not from HV, could inhibit TOP1 activity at various dilutions. Similarly, purified total IgG fractions from dcSSc patients could also inhibit TOP1 activity. ATA was successfully separated from IgG from dcSSc patients who were positive for ATA. This could inhibit TOP1 activity even at lower concentrations than total IgG. This was the case for both mitochondrial and nuclear TOP1 activity. Fully in line with presence of ATA, PBMC’s from dcSSc patients contained a lower TOP1 activity compared to HVs PBMCs. Conclusion To our knowledge we are the first group to be able to successfully separate ATA from total IgG from dcSSc patients. our unique in house developed REEAD assay clearly demonstrates the function of ATA and provides the opportunity to better understand References [1]Role of autoantibodies in the diagnosis and prognosis of interstitial lung disease in autoimmune rheumatic disorders. Masataka Kuwana, Albert Gil-Vila and Albert Selva-O’Callaghan, Ther Adv Musculoskel Dis 2021, Vol. 13: 1–17 DOI: 10.1177/ [2]Rolling circle amplification based detection of human topoisomerase I activity on magnetic beads. Zuccaro, Laura; Tesauro, Cinzia; Cerroni, Barbara; Ottaviani, Alessio; Knudsen, Birgitta Ruth; Balasubramanian, Kannan; Desideri, Alessandro. Analytical Biochemistry, Bind 451, 15.04.2014, s. 42-44. Disclosure of Interests None declared

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells.

Background: Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomerase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoisomerase 1 activity is affected. Methods: Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results: We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-molecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Conclusion: REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.