Automated Image Analysis of Transmission Electron Micrographs: Nanoscale Evaluation of Radiation-Induced DNA Damage in the Context of Chromatin.

BACKGROUND: Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs. METHODS: Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR. RESULTS: Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined. CONCLUSIONS: The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns.

Role of Histone Variant H2A.J in Fine-Tuning Chromatin Organization for the Establishment of Ionizing Radiation-Induced Senescence.

PURPOSE: Radiation-induced senescence is characterized by profound changes in chromatin organization with the formation of Senescence-Associated-Heterochromatin-Foci (SAHF) and DNA-Segments-with-Chromatin-Alterations-Reinforcing-Senescence (DNA-SCARS). Importantly, senescent cells also secrete complex combinations of pro-inflammatory factors, referred as Senescence-Associated-Secretory-Phenotype (SASP). Here, we analyzed the epigenetic mechanism of histone variant H2A.J in establishing radiation-induced senescence. EXPERIMENTAL DESIGN: Primary and genetically-modified lung fibroblasts with down- or up-regulated H2A.J expression were exposed to ionizing radiation and were analyzed for the formation of SAHF and DNA-SCARS by immunofluorescence microscopy. Dynamic changes in chromatin organization and accessibility, transcription factor recruitment, and transcriptome signatures were mapped by ATAC-seq and RNA-seq analysis. The secretion of SASP factors and potential bystander effects were analyzed by ELISA and RT-PCR. Lung tissue of mice exposed to different doses were analyzed by the digital image analysis of H2A.J-immunohistochemistry. RESULTS: Differential incorporation of H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of senescence. Integrative analyses of ATAC-seq and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility of regulatory regions decisively modulates transcription factor recruitment and inflammatory gene expression, resulting in an altered SASP secretome. In lung parenchyma, pneumocytes show dose-dependent H2A.J expression in response to radiation-induced DNA damage, therefore contributing to pro-inflammatory tissue reactions. CONCLUSIONS: The fine-tuned incorporation of H2A.J defines the epigenetic landscape for driving the senescence programme in response to radiation-induced DNA damage. Deregulated H2A.J deposition affects chromatin remodeling, transcription factor recruitment, and the pro-inflammatory secretome. Our findings provide new mechanistic insights into DNA-damage triggered epigenetic mechanisms governing the biological processes of radiation-induced injury.

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.

Exploring the microbial diversity in Jordanian hot springs by comparative metagenomic analysis.

A culture-independent approach was utilized in this study to reveal the microbial diversity in Jordanian hot springs represented by Ma'in and Afra hot springs. Water samples from Ma'in and Afra hot springs were collected in June 2015. The in situ temperature of water samples range was 38-59°C and the pH range was 7.4-8.4. The metagenome was extracted and analyzed using the next generation technology (bTEFAP® ). A total of 314,310 sequences were parsed and 288,452 were then clustered. The sequences were predominated by bacteria (>84%) and the relative abundance of archaea in each sample was <1%. Eukaryotic microorganisms were detected but with varying abundances (0.6%-15%). Because most of the detected sequences were found to belong to the domain of bacteria (196,936 sequences out 288,452), the bacterial sequences were utilized for further microbial analyses. With respect to alpha and beta diversity, samples were rarefied to 30,000 sequences and bootstrapped at 10,000 sequences. The Shannon-Wiener Index curve plot reaches a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity. By examining the relative abundance of phyla detected in each sample, it appears that the biota of both Jordanian hot springs sampled are compositionally similar, with over 50% of the microbial community of each sample being comprised of the phylum Proteobacteria. The second most abundant phylum was the phylum Bacteroidetes which represents more than 13% in each sample. The phylum Firmicutes was also detected with a significant abundance. However, lower abundance of Deinococcus, Verrucomicrobia, Planctomycetes, and Chloroflexi was detected. A principal coordinate analysis plot was generated based upon the weighted UniFrac distance matrix. By utilizing Monte Carlo simulations, we were able to determine that there were no significant differences in the microbial diversity between each sample.

Microbiological analysis, antimicrobial activity, and heavy-metals content of Jordanian Ma'in hot-springs water.

Ma'in hot springs are known as sites of balneotherapy. However, little is known about their microbiology and chemistry. In this study, we aim at evaluating the antimicrobial activity of Ma'in hot-springs water (MHSW), studying its microbiology, and determining its physicochemical properties including the heavy metals content. Therefore, water samples were collected from Ma'in hot springs and tested for antimicrobial activity using agar diffusion method. Water was then cultivated on nutrient agar to isolate and identify the dominant bacteria by chemical and molecular methods. The identified strains were tested by cross streak method to evaluate their antimicrobial activity against different clinical and standard strains. Finally, water samples were chemically analyzed and the heavy-metals content was assessed. Results revealed that MHSW was not active against any of the clinical isolates. Nevertheless, MHSW was found to be active against five standard bacterial strains, namely, Staphylococcus epidermidis ATCC 12228 (inhibition zone: 20mm), Staphylococcus aureus ATCC 29213 (inhibition zone: 19mm), Micrococcus luteus ATCC 9341 (inhibition zone: 15.3mm), and Bacillus cereus ATCC 11778 (inhibition zone: 12.3mm). After cultivation of MHSW, five bacterial isolates were obtained and identified based on 16S rRNA gene analysis as new strains of Anoxybacillus flavithermus (identity percentage ranges between 96-99%). Physicochemical analysis revealed that the in situ temperature was 59°C, pH was 7.8, salinity was 1.6ppt, and dissolved oxygen was 3.8mgl-1. In respect to heavy-metals content in MHSW, the following metals were present in the order: Cr (0.571ppm)>Mn(0.169ppm)>Fe (0.124ppm)>Zn (0.095)>Cu(0.070ppm)>Ni(0.058ppm)>Cd (0.023ppm)>Pb (0ppm). Cd, Cr, Ni and Mn were found to be higher than permissible levels set by international organizations and countries. This study highlights new chemical and microbiological data about Ma'in hot springs.