Towards unravelling biological mechanisms behind radiation-induced oral mucositis via mass spectrometry-based proteomics.

Objective: Head and neck cancer (HNC) accounts for almost 890,000 new cases per year. Radiotherapy (RT) is used to treat the majority of these patients. A common side-effect of RT is the onset of oral mucositis, which decreases the quality of life and represents the major dose-limiting factor in RT. To understand the origin of oral mucositis, the biological mechanisms post-ionizing radiation (IR) need to be clarified. Such knowledge is valuable to develop new treatment targets for oral mucositis and markers for the early identification of "at-risk" patients. Methods: Primary keratinocytes from healthy volunteers were biopsied, irradiated in vitro (0 and 6 Gy), and subjected to mass spectrometry-based analyses 96 h after irradiation. Web-based tools were used to predict triggered biological pathways. The results were validated in the OKF6 cell culture model. Immunoblotting and mRNA validation was performed and cytokines present in cell culture media post-IR were quantified. Results: Mass spectrometry-based proteomics identified 5879 proteins in primary keratinocytes and 4597 proteins in OKF6 cells. Amongst them, 212 proteins in primary keratinocytes and 169 proteins in OKF6 cells were differentially abundant 96 h after 6 Gy irradiation compared to sham-irradiated controls. In silico pathway enrichment analysis predicted interferon (IFN) response and DNA strand elongation pathways as mostly affected pathways in both cell systems. Immunoblot validations showed a decrease in minichromosome maintenance (MCM) complex proteins 2-7 and an increase in IFN-associated proteins STAT1 and ISG15. In line with affected IFN signalling, mRNA levels of IFNß and interleukin 6 (IL-6) increased significantly following irradiation and also levels of secreted IL-1ß, IL-6, IP-10, and ISG15 were elevated. Conclusion: This study has investigated biological mechanisms in keratinocytes post-in vitro ionizing radiation. A common radiation signature in keratinocytes was identified. The role of IFN response in keratinocytes along with increased levels of pro-inflammatory cytokines and proteins could hint towards a possible mechanism for oral mucositis.

Nuclear Fragility in Radiation-Induced Senescence: Blebs and Tubes Visualized by 3D Electron Microscopy.

Irreparable DNA damage following ionizing radiation (IR) triggers prolonged DNA damage response and induces premature senescence. Cellular senescence is a permanent state of cell-cycle arrest characterized by chromatin restructuring, altered nuclear morphology and acquisition of secretory phenotype, which contributes to senescence-related inflammation. However, the mechanistic connections for radiation-induced DNA damage that trigger these senescence-associated hallmarks are poorly understood. In our in vitro model of radiation-induced senescence, mass spectrometry-based proteomics was combined with high-resolution imaging techniques to investigate the interrelations between altered chromatin compaction, nuclear envelope destabilization and nucleo-cytoplasmic chromatin blebbing. Our findings confirm the general pathophysiology of the senescence-response, with disruption of nuclear lamin organization leading to extensive chromatin restructuring and destabilization of the nuclear membrane with release of chromatin fragments into the cytosol, thereby activating cGAS-STING-dependent interferon signaling. By serial block-face scanning electron microscopy (SBF-SEM) whole-cell datasets were acquired to investigate the morphological organization of senescent fibroblasts. High-resolution 3-dimensional (3D) reconstruction of the complex nuclear shape allows us to precisely visualize the segregation of nuclear blebs from the main nucleus and their fusion with lysosomes. By multi-view 3D electron microscopy, we identified nanotubular channels formed in lamin-perturbed nuclei of senescent fibroblasts; the potential role of these nucleo-cytoplasmic nanotubes for expulsion of damaged chromatin has to be examined.

Ionizing Radiation Protein Biomarkers in Normal Tissue and Their Correlation to Radiosensitivity: A Systematic Review.

Background and objectives: Exposure to ionizing radiation (IR) has increased immensely over the past years, owing to diagnostic and therapeutic reasons. However, certain radiosensitive individuals show toxic enhanced reaction to IR, and it is necessary to specifically protect them from unwanted exposure. Although predicting radiosensitivity is the way forward in the field of personalised medicine, there is limited information on the potential biomarkers. The aim of this systematic review is to identify evidence from a range of literature in order to present the status quo of our knowledge of IR-induced changes in protein expression in normal tissues, which can be correlated to radiosensitivity. Methods: Studies were searched in NCBI Pubmed and in ISI Web of Science databases and field experts were consulted for relevant studies. Primary peer-reviewed studies in English language within the time-frame of 2011 to 2020 were considered. Human non-tumour tissues and human-derived non-tumour model systems that have been exposed to IR were considered if they reported changes in protein levels, which could be correlated to radiosensitivity. At least two reviewers screened the titles, keywords, and abstracts of the studies against the eligibility criteria at the first phase and full texts of potential studies at the second phase. Similarly, at least two reviewers manually extracted the data and accessed the risk of bias (National Toxicology Program/Office for Health Assessment and Translation-NTP/OHAT) for the included studies. Finally, the data were synthesised narratively in accordance to synthesis without meta analyses (SWiM) method. Results: In total, 28 studies were included in this review. Most of the records (16) demonstrated increased residual DNA damage in radiosensitive individuals compared to normo-sensitive individuals based on γH2AX and TP53BP1. Overall, 15 studies included proteins other than DNA repair foci, of which five proteins were selected, Vascular endothelial growth factor (VEGF), Caspase 3, p16INK4A (Cyclin-dependent kinase inhibitor 2A, CDKN2A), Interleukin-6, and Interleukin-1ß, that were connected to radiosensitivity in normal tissue and were reported at least in two independent studies. Conclusions and implication of key findings: A majority of studies used repair foci as a tool to predict radiosensitivity. However, its correlation to outcome parameters such as repair deficient cell lines and patients, as well as an association to moderate and severe clinical radiation reactions, still remain contradictory. When IR-induced proteins reported in at least two studies were considered, a protein network was discovered, which provides a direction for further studies to elucidate the mechanisms of radiosensitivity. Although the identification of only a few of the commonly reported proteins might raise a concern, this could be because (i) our eligibility criteria were strict and (ii) radiosensitivity is influenced by multiple factors. Registration: PROSPERO (CRD42020220064).

Ionizing Radiation Protein Biomarkers in Normal Tissue and Their Correlation to Radiosensitivity: Protocol for a Systematic Review.

Background: Radiosensitivity is a significantly enhanced reaction of cells, tissues, organs or organisms to ionizing radiation (IR). During radiotherapy, surrounding normal tissue radiosensitivity often limits the radiation dose that can be applied to the tumour, resulting in suboptimal tumour control or adverse effects on the life quality of survivors. Predicting radiosensitivity is a component of personalized medicine, which will help medical professionals allocate radiation therapy decisions for effective tumour treatment. So far, there are no reviews of the current literature that explore the relationship between proteomic changes after IR exposure and normal tissue radiosensitivity systematically. Objectives: The main objective of this protocol is to specify the search and evaluation strategy for a forthcoming systematic review (SR) dealing with the effects of in vivo and in vitro IR exposure on the proteome of human normal tissue with focus on radiosensitivity. Methods: The SR framework has been developed following the guidelines established in the National Toxicology Program/Office of Health Assessment and Translation (NTP/OHAT) Handbook for Conducting a Literature-Based Health Assessment, which provides a standardised methodology to implement the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to environmental health assessments. The protocol will be registered in PROSPERO, an open source protocol registration system, to guarantee transparency. Eligibility criteria: Only experimental studies, in vivo and in vitro, investigating effects of ionizing radiation on the proteome of human normal tissue correlated with radio sensitivity will be included. Eligible studies will include English peer reviewed articles with publication dates from 2011-2020 which are sources of primary data. Information sources: The search strings will be applied to the scientific literature databases PubMed and Web of Science. The reference lists of included studies will also be manually searched. Data extraction and results: Data will be extracted according to a pre-defined modality and compiled in a narrative report following guidelines presented as a "Synthesis without Meta-analyses" method. Risk of bias: The risk of bias will be assessed based on the NTP/OHAT risk of bias rating tool for human and animal studies (OHAT 2019). Level of evidence rating: A comprehensive assessment of the quality of evidence for both in vivo and in vitro studies will be followed, by assigning a confidence rating to the literature. This is followed by translation into a rating on the level of evidence (high, moderate, low, or inadequate) regarding the research question. Registration: PROSPERO Submission ID 220064.

Comparison of Two Molecular Assays for Detection and Characterization of Aspergillus fumigatus Triazole Resistance and Cyp51A Mutations in Clinical Isolates and Primary Clinical Samples of Immunocompromised Patients.

In hematological patients, the incidence of invasive aspergillosis (IA) caused by azole resistant Aspergillus fumigatus (ARAf) is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial. In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR) assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A, and M220 in the Aspergillus fumigatus (A. fumigatus) Cyp51A gene by subsequent DNA sequence analysis, we investigated in parallel the commercially available AsperGenius® real time PCR system in detecting the Cyp51A alterations TR34/L98H and Y121F/T289A directly from 52 clinical samples (15 biopsies, 22 bronchoalveolar lavage (BAL), 15 cerebrospinal fluid (CSF) samples) and ARAf isolates (n = 3) of immunocompromised patients. We analyzed DNA aliquots and compared both methods concerning amplification and detection of Aspergillus DNA and Cyp51A alterations. As positive control for the feasibility of our novel Y121F and T289A PCR assays, we used two A. fumigatus isolates with the TR46/Y121F/T289A mutation combination isolated from hematological patients with known Cyp51A alterations and a lung biopsy sample of a patient with acute myeloid leukemia (AML). The rate of positive ARAf PCR results plus successful sequencing using the ARAf PCR assays was 61% in biopsies, 29% in CSF, 67% in BAL samples and 100% in isolates. In comparison the amount of positive PCRs using the AsperGenius® assays was 47% in biopsies, 42% in CSF, 59% in BAL samples and 100% in isolates. Altogether 17 Cyp51A alterations were detected using our ARAf PCRs plus DNA sequencing and therefrom 10 alterations also by the AsperGenius® system. The comparative evaluation of our data revealed that our conventional PCR assays are more sensitive in detecting ARAf in BAL and biopsy samples, whereby differences were not significant. The advantage of the AsperGenius® system is the time saving aspect. We consider non-culture based molecular detection of Aspergillus triazole resistance to be of high epidemiological and clinical relevance in patients with hematological malignancies.

Natural Cubic Spline Regression Modeling Followed by Dynamic Network Reconstruction for the Identification of Radiation-Sensitivity Gene Association Networks from Time-Course Transcriptome Data.

Gene expression time-course experiments allow to study the dynamics of transcriptomic changes in cells exposed to different stimuli. However, most approaches for the reconstruction of gene association networks (GANs) do not propose prior-selection approaches tailored to time-course transcriptome data. Here, we present a workflow for the identification of GANs from time-course data using prior selection of genes differentially expressed over time identified by natural cubic spline regression modeling (NCSRM). The workflow comprises three major steps: 1) the identification of differentially expressed genes from time-course expression data by employing NCSRM, 2) the use of regularized dynamic partial correlation as implemented in GeneNet to infer GANs from differentially expressed genes and 3) the identification and functional characterization of the key nodes in the reconstructed networks. The approach was applied on a time-resolved transcriptome data set of radiation-perturbed cell culture models of non-tumor cells with normal and increased radiation sensitivity. NCSRM detected significantly more genes than another commonly used method for time-course transcriptome analysis (BETR). While most genes detected with BETR were also detected with NCSRM the false-detection rate of NCSRM was low (3%). The GANs reconstructed from genes detected with NCSRM showed a better overlap with the interactome network Reactome compared to GANs derived from BETR detected genes. After exposure to 1 Gy the normal sensitive cells showed only sparse response compared to cells with increased sensitivity, which exhibited a strong response mainly of genes related to the senescence pathway. After exposure to 10 Gy the response of the normal sensitive cells was mainly associated with senescence and that of cells with increased sensitivity with apoptosis. We discuss these results in a clinical context and underline the impact of senescence-associated pathways in acute radiation response of normal cells. The workflow of this novel approach is implemented in the open-source Bioconductor R-package splineTimeR.

Differential Response and Priming Dose Effect on the Proteome of Human Fibroblast and Stem Cells Induced by Exposure to Low Doses of Ionizing Radiation.

It has been suggested that a mechanistic understanding of the cellular responses to low dose and dose rate may be valuable in reducing some of the uncertainties involved in current risk estimates for cancer- and non-cancer-related radiation effects that are inherited in the linear no-threshold hypothesis. In this study, the effects of low-dose radiation on the proteome in both human fibroblasts and stem cells were investigated. Particular emphasis was placed on examining: 1. the dose-response relationships for the differential expression of proteins in the low-dose range (40-140 mGy) of low-linear energy transfer (LET) radiation; and 2. the effect on differential expression of proteins of a priming dose given prior to a challenge dose (adaptive response effects). These studies were performed on cultured human fibroblasts (VH10) and human adipose-derived stem cells (ADSC). The results from the VH10 cell experiments demonstrated that low-doses of low-LET radiation induced unique patterns of differentially expressed proteins for each dose investigated. In addition, a low priming radiation dose significantly changed the protein expression induced by the subsequent challenge exposure. In the ADSC the number of differentially expressed proteins was markedly less compared to VH10 cells, indicating that ADSC differ in their intrinsic response to low doses of radiation. The proteomic results are further discussed in terms of possible pathways influenced by low-dose irradiation.

Development of novel PCR assays to detect azole resistance-mediating mutations of the Aspergillus fumigatus cyp51A gene in primary clinical samples from neutropenic patients.

The increasing incidence of azole resistance in Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediating cyp51A gene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of the Aspergillus fumigatus cyp51A gene to establish PCR assays with consecutive DNA sequence analysis to detect and identify the A. fumigatus cyp51A tandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially diluted A. fumigatus and human DNA, A. fumigatus cyp51A gene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg of A. fumigatus DNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons for A. fumigatus wild-type DNA confirmed the cyp51A wild-type sequence, and PCR products from one azole-resistant A. fumigatus isolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.