DNA double-strand breaks can be counted as discrete foci by imaging techniques. In personalized medicine and pharmacology, the analysis of counting data is relevant for numerous applications, e.g., for cancer and aging research and the evaluation of drug efficacy. By default, it is assumed to follow the Poisson distribution. This assumption, however, may lead to biased results and faulty conclusions in datasets with excess zero values (zero-inflation), a variance larger than the mean (overdispersion), or both. In such cases, the assumption of a Poisson distribution would skew the estimation of mean and variance, and other models like the negative binomial (NB), zero-inflated Poisson or zero-inflated NB distributions should be employed. The model chosen has an influence on the parameter estimation (mean value and confidence interval). Yet the choice of the suitable distribution model is not trivial.To support, simplify and objectify this process, we have developed the countfitteR software as an R package. We used a Bayesian approach for distribution model selection and the shiny web application framework for interactive data analysis.We show the application of our software based on examples of DNA double-strand break count data from phenotypic imaging by multiplex fluorescence microscopy. In analyzing numerous datasets of molecular pharmacological markers (phosphorylated histone H2AX and p53 binding protein), countfitteR demonstrated an equal or superior statistical performance compared to the usually employed two-step procedure, with an overall power of up to 98%. In addition, it still gave information in cases with no result at all from the two-step procedure. In our data sample we found that the NB distribution was the most frequent, with the Poisson distribution taking second place.countfitteR can perform an automated distribution model selection and thus support the data analysis and lead to objective statistically verifiable estimated values. Originally designed for the analysis of foci in biomedical image data, countfitteR can be used in a variety of areas where non-Poisson distributed counting data is prevalent.
Introduction Infections by oncoviruses are an important risk factor for the development of neoplasms. Particularly, anogenital cancers are strongly associated with persistent infections by high-risk human papillomaviruses (HPV). In the case of cervical cancer the physical state of the virus seems to be an important prognostic marker. Especially the integration of viral DNA into the host cell genome bears the risk of following degenerations. Therefore, the early and sensitive detection is of high interest. Besides Pap-test and PCR, fluorescence in situ hybridization (FISH) is used for a quantitative and qualitative detection of HPV infections and risk assessment. Material and methods For analysis high-risk HPV FISH probes should be developed for standardised, type-specific and quantitative detection of HPV nucleic acid in host cells. Due to the short viral DNA sequence, present with just few copies, conventional FISH probes have little sensitivity. Therefore, we tested specific FISH probes targeting different regions of the HPV genome in combination with signal amplification. PCR-produced FISH probes were biotin labelled by direct incorporation of biotinylated nucleotides or nick translation. Our biotinylated HPV probes were tested on HPV-positive and –negative cells and detected using fluorescence labelled streptavidin or subsequent tyramide signal amplification. In addition to FISH, probes were used for chromogenic in situ hybridization. All analysis were performed with our fully automatized multicolor fluorescence imaging platform VideoScan. Results and discussions The presence of HPV DNA in cell lines was confirmed by (digital) PCR. The protocol for synthesis of biotinylated probes using HPV sequence specific primers and genomic DNA as template was successful. Biotin incorporation was checked with agarose gel electrophoresis and modified dot blot hybridization. In in situ hybridization experiments, our probes showed high signals in HPV high-copy number CaSki cells and a tissue model, compared to HPV negative samples. Conclusion Highly sensitive and specific FISH probes offer a great potential for the detection of HPV DNA in patient samples and thereby for the assessment of tumour risks. Using our probes, we got positive results in hybridization experiments using chromogenic and fluorescent detection methods with or without further amplification steps. Next, comparative FISH experiments will be performed to check probe sensitivity in cells containing few HPV copies and their practicability in biopsy derived tissue samples.
Glycoprotein 2 (GP2) is an autoantigen in Crohn's (CD) and coeliac disease (CeD). We assessed GP2-isoform (GP21-4)-expression in intestinal biopsies of paediatric patients with CD, CeD, ulcerative colitis (UC), and healthy children (HC). Transcription of GP21-4 was elevated in proximal small intestine in CeD and CD patients (only GP22/4) compared to jejunum (CeD/CD) and large bowel (CD). CeD patients demonstrated higher duodenal GP22/4-mRNA levels compared to HC/UC patients whereas CD patients showed higher GP24-mRNA levels compared to UC patients. Duodenal synthesis of only small GP2 isoforms (GP23/4) was demonstrated in epithelial cells in patients/HC and in Brunner glands (also large isoforms) with a more frequent apical location in CD/CeD patients. All four GP2 isoforms interacted with gliadin and phosphopeptidomannan. Gliadin digestion improved binding to GP2 isoforms. GP21-4 binding to CeD/CD-related antigens, elevated duodenal GP21-4-mRNA transcription, and GP2-protein secretion in Brunner glands of CeD/CD patients suggest an autoimmune CeD/CD link.
Purpose: In eukaryotic cells treated with chemotherapeutic drugs DNA double strand breaks (DSB) are the most severe form of DNA damage which usually leads to apoptotic cell death. Cytotoxic chemotherapy is still a mainstay of cancer therapy, but there are few established methods to individually predict efficacy and tolerability. In most regimens dosage is based on the body surface area, a dimension of little biological precision, and individual dose adaptions are usually performed by clinical guessing or in response to adverse events. This project sets out to examine biological parameters of cell damage. Methods: In particular, we measure DNA double strand breaks by an automated yH2AX assay applying the Aklides NUK® system. In preliminary work, PBMCs were stimulated with bendamustine or with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, the components of the R-CHOP protocol, to induce DSB. Results: There was no inter-individual correlation between drug dose and number of foci, whereas does titrations on the cells of each individual donor demonstrated a clear correlation between dose and yH2AX assay result. Conclusion: The lack of correlation across individuals between yH2AX foci and drug dosage based on standard calculation by body surface area suggests that standardized dosing of chemotherapeutic drugs based on gross physical determinants such as weight or body surface area does not correspond to the individual’s biological response and clinical effect. The future plan of this project is the replacement of standard dosage regimens by biologically based, real-time-adapted individual dosage in lymphoma to reduce toxicity while increasing therapeutic efficacy.
Abstract For improving aptamer-ligand binding we have developed a screening system that defines optimal binding buffer composition. Using multiplex assays, one buffer system is needed which guarantees the specific binding of all aptamers. We investigated nine peer-reviewed DNA aptamers. Non-specific binding of aptamers is an obstacle. To address this, we investigated 16 proteins as specificity controls bound covalently to encoded microbeads in a multiplex assay. Increasing the NaCl concentration decreased the binding for all aptamers. Changing pH values by one unit higher or lower did not influence the aptamer binding significantly. However, pH < 5 led to non-specific binding for all aptamers. The Pf LDH-aptamer selected in the absence of divalent cations exhibited doubling of its binding signal by the addition of Ca 2+ and Mg 2+ . We confirmed Ca 2+ and Mg 2+ dependency of the aptamers for streptavidin and thrombin by observing a 90% and 50% binding decrease, respectively. We also achieved a doubling of binding for the streptavidin aptamer when replacing Ca 2+ and Mg 2+ by Mn 2+ . A buffer suitable for all aptamers can have considerable variations in pH or ionic strength, but divalent cations (Ca 2+ , Mg 2+ , Mn 2+ ) are essential.
Neurodegenerative diseases (NDs) are characterized by abnormalities within neurons of the brain or spinal cord that gradually lose function, eventually leading to cell death. Upon examination of affected tissue, pathological changes reveal a loss of synapses, misfolded proteins, and activation of immune cells—all indicative of disease progression—before severe clinical symptoms become apparent. Early detection of NDs is crucial for potentially administering targeted medications that may delay disease advancement. Given their complex pathophysiological features and diverse clinical symptoms, there is a pressing need for sensitive and effective diagnostic methods for NDs. Biomarkers such as microRNAs (miRNAs) have been identified as potential tools for detecting these diseases. We explore the pivotal role of miRNAs in the context of NDs, focusing on Alzheimer’s disease, Parkinson’s disease, Multiple sclerosis, Huntington’s disease, and Amyotrophic Lateral Sclerosis. The review delves into the intricate relationship between aging and NDs, highlighting structural and functional alterations in the aging brain and their implications for disease development. It elucidates how miRNAs and RNA-binding proteins are implicated in the pathogenesis of NDs and underscores the importance of investigating their expression and function in aging. Significantly, miRNAs exert substantial influence on post-translational modifications (PTMs), impacting not just the nervous system but a wide array of tissues and cell types as well. Specific miRNAs have been found to target proteins involved in ubiquitination or de-ubiquitination processes, which play a significant role in regulating protein function and stability. We discuss the link between miRNA, PTM, and NDs. Additionally, the review discusses the significance of miRNAs as biomarkers for early disease detection, offering insights into diagnostic strategies.
