Pattern Of DNA Damage Research Articles

Targeted Accumulation of Macrophages Induced by Microbeam Irradiation in a Tissue-Dependent Manner.

Radiation therapy (RT) is a vital component of multimodal cancer treatment, and its immunomodulatory effects are a major focus of current therapeutic strategies. Macrophages are some of the first cells recruited to sites of radiation-induced injury where they can aid in tissue repair, propagate radiation-induced fibrogenesis and influence tumour dynamics. Microbeam radiation therapy (MRT) is a unique, spatially fractionated radiation modality that has demonstrated exceptional tumour control and reduction in normal tissue toxicity, including fibrosis. We conducted a morphological analysis of MRT-irradiated normal liver, lung and skin tissues as well as lung and melanoma tumours. MRT induced distinct patterns of DNA damage, reflecting the geometry of the microbeam array. Macrophages infiltrated these regions of peak dose deposition at variable timepoints post-irradiation depending on the tissue type. In normal liver and lung tissue, macrophages clearly demarcated the beam path by 48 h and 7 days post-irradiation, respectively. This was not reflected, however, in normal skin tissue, despite clear DNA damage marking the beam path. Persistent DNA damage was observed in MRT-irradiated lung carcinoma, with an accompanying geometry-specific influx of mixed M1/M2-like macrophage populations. These data indicate the unique potential of MRT as a tool to induce a remarkable accumulation of macrophages in an organ/tissue-specific manner. Further characterization of these macrophage populations is warranted to identify their organ-specific roles in normal tissue sparing and anti-tumour responses.

Genotoxic Damage During Brain Development Presages Prototypical Neurodegenerative Disease.

Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex (ALS/PDC) is a disappearing prototypical neurodegenerative disorder (tau-dominated polyproteinopathy) linked with prior exposure to phytogenotoxins in cycad seed used for medicine and/or food. The principal cycad genotoxin, methylazoxymethanol (MAM), forms reactive carbon-centered ions that alkylate nucleic acids in fetal rodent brain and, depending on the timing of systemic administration, induces persistent developmental abnormalities of the cortex, hippocampus, cerebellum, and retina. Whereas administration of MAM prenatally or postnatally can produce animal models of epilepsy, schizophrenia or ataxia, administration to adult animals produces little effect on brain structure or function. The neurotoxic effects of MAM administered to rats during cortical brain development (specifically, gestation day 17) are used to model the histological, neurophysiological and behavioral deficits of human schizophrenia, a condition that may precede or follow clinical onset of motor neuron disease in subjects with sporadic ALS and ALS/PDC. While studies of migrants to and from communities impacted by ALS/PDC indicate the degenerative brain disorder may be acquired in juvenile and adult life, a proportion of indigenous cases shows neurodevelopmental aberrations in the cerebellum and retina consistent with MAM exposure in utero. MAM induces specific patterns of DNA damage and repair that associate with increased tau expression in primary rat neuronal cultures and with brain transcriptional changes that parallel those associated with human ALS and Alzheimer’s disease. We examine MAM in relation to neurodevelopment, epigenetic modification, DNA damage/replicative stress, genomic instability, somatic mutation, cell-cycle reentry and cellular senescence. Since the majority of neurodegenerative disease lacks a solely inherited genetic basis, research is needed to explore the hypothesis that early-life exposure to genotoxic agents may trigger or promote molecular events that culminate in neurodegeneration.

Coupling Radiation Transport and Track-Structure Simulations: Strategy Based on Analytical Formulas Representing DNA Damage Yields

Existing radiation codes for biomedical applications face the challenge of dealing with largely different spatial scales, from nanometer scales governing individual energy deposits to macroscopic scales of dose distributions in organs and tissues in radiotherapy. Event-by-event track-structure codes are needed to model energy deposition patterns at cellular and subcellular levels. In conjunction with DNA and chromatin models, they predict radiation-induced DNA damage and subsequent biological effects. Describing larger-scale effects is the realm of radiation transport codes and dose calculation algorithms. A coupling approach with a great potential consists in implementing into radiation transport codes the results of track-structure simulations captured by analytical formulas. This strategy allows extending existing transport codes to biologically relevant endpoints, without the need of developing dedicated modules and running new computationally expensive simulations. Depending on the codes used and questions addressed, alternative strategies can be adopted, reproducing DNA damage in dependence on different parameters extracted from the transport code, e.g., microdosimetric quantities, average linear energy transfer (LET), or particle energy. Recently, a comprehensive database on DNA damage induced by ions from hydrogen to neon, at energies from 0.5 GeV/u down to their stopping, has been created with PARTRAC biophysical simulations. The results have been captured as a function of average LET in the cell nucleus. However, the formulas are not applicable to slow particles beyond the Bragg peak, since these can have the same LET as faster particles but in narrower tracks, thus inducing different DNA damage patterns. Particle energy distinguishes these two cases. It is also more readily available than LET from some transport codes. Therefore, a set of new analytical functions are provided, describing how DNA damage depends on particle energy. The results complement the analysis of the PARTRAC database, widening its potential of application and use for implementation in transport codes.

Genotoxicity of Paragonimus heterotremus Infection in a Rat Model of Simultaneous Pulmonary and Hepatic Paragonimiasis.

Parasites cause numerous health issues in humans, eventually leading to significant social and economic damage; however, the mechanisms of parasite-mediated pathogenesis are not well understood. Nevertheless, it is clearly evidenced that cancerogenic fluke-induced chronic inflammations and cancer are closely associated with oxidative stress. (1) Methods: The Paragonimus heterotremus infection’s genotoxic potential was assessed in a rat model of simultaneous pulmonary and hepatic paragonimiasis by the alkaline version of single-cell gel electrophoresis (comet assay). Statistical analysis of comet parameters was based on the non-parametric Mann–Whitney U test. (2) Results: A clear and statistically significant increase in DNA damage was detected in the helminth-exposed group versus the control rats and the tissue areas adjacent to the parasite capsule versus remote ones; however, differences in DNA damage patterns between different tissues were not statistically significant. Infection resulted in up to 40% cells with DNA damage and an increased genetic damage index. (3) Conclusions: The data obtained contribute to understanding the pathogenesis mechanisms of paragonimiasis, suggesting oxidative stress as the most likely reason for DNA breaks; these findings allow us to consider P. heterotremus as a potentially cancerogenic species, and they are important for the monitoring and treatment of paragonimiasis.

Molecular cloning, sequence, and expression patterns of DNA damage induced transcript 3 (DDIT3) gene in female yaks (Bos grunniens)

Endoplasmic reticulum stress (ERS) plays an important role in regulating the reproductive process of female mammals, mainly involved in follicular atresia and corpus luteum regression. DNA damage induced transcript 3 (DDIT3) is a marker gene of ERS. The objectives of the present study were to clone and analyze the sequence and tissue expression characteristics of DDIT3 gene in female yaks. By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length 507-bp DDIT3-cDNA, encoding for 168-aa protein. Yak DDIT3 exhibited highest and least identity with that of bison and horse, respectively. Real-time PCR analyses revealed that the expression level of DDIT3 gene in ovary was higher than that in heart, liver, kidney, spleen, lung, uterus and oviduct (p < 0.05). DDIT3 expression level in ovary and uterus during pregnancy was higher than that in follicular phase, luteal phase and fetus stage. DDIT3 was highly expressed in metaphase II oocytes and granulosa cells than that in germinal vesicle and metaphase I oocytes (p < 0.05), respectively. This is the first molecular characterization and expression patterns of DDIT3 gene in female yaks. These results indicated that the DDIT3 gene possibly plays an important role in regulating ovary function and pregnancy maintenance in yaks.

First historical genome of a crop bacterial pathogen from herbarium specimen: Insights into citrus canker emergence.

Over the past decade, ancient genomics has been used in the study of various pathogens. In this context, herbarium specimens provide a precious source of dated and preserved DNA material, enabling a better understanding of plant disease emergences and pathogen evolutionary history. We report here the first historical genome of a crop bacterial pathogen, Xanthomonas citri pv. citri (Xci), obtained from an infected herbarium specimen dating back to 1937. Comparing the 1937 genome within a large set of modern genomes, we reconstructed their phylogenetic relationships and estimated evolutionary parameters using Bayesian tip-calibration inferences. The arrival of Xci in the South West Indian Ocean islands was dated to the 19th century, probably linked to human migrations following slavery abolishment. We also assessed the metagenomic community of the herbarium specimen, showed its authenticity using DNA damage patterns, and investigated its genomic features including functional SNPs and gene content, with a focus on virulence factors.

PyDamage: automated ancient damage identification and estimation for contigs in ancient DNA de novo assembly.

DNA de novo assembly can be used to reconstruct longer stretches of DNA (contigs), including genes and even genomes, from short DNA sequencing reads. Applying this technique to metagenomic data derived from archaeological remains, such as paleofeces and dental calculus, we can investigate past microbiome functional diversity that may be absent or underrepresented in the modern microbiome gene catalogue. However, compared to modern samples, ancient samples are often burdened with environmental contamination, resulting in metagenomic datasets that represent mixtures of ancient and modern DNA. The ability to rapidly and reliably establish the authenticity and integrity of ancient samples is essential for ancient DNA studies, and the ability to distinguish between ancient and modern sequences is particularly important for ancient microbiome studies. Characteristic patterns of ancient DNA damage, namely DNA fragmentation and cytosine deamination (observed as C-to-T transitions) are typically used to authenticate ancient samples and sequences, but existing tools for inspecting and filtering aDNA damage either compute it at the read level, which leads to high data loss and lower quality when used in combination with de novo assembly, or require manual inspection, which is impractical for ancient assemblies that typically contain tens to hundreds of thousands of contigs. To address these challenges, we designed PyDamage, a robust, automated approach for aDNA damage estimation and authentication of de novo assembled aDNA. PyDamage uses a likelihood ratio based approach to discriminate between truly ancient contigs and contigs originating from modern contamination. We test PyDamage on both on simulated aDNA data and archaeological paleofeces, and we demonstrate its ability to reliably and automatically identify contigs bearing DNA damage characteristic of aDNA. Coupled with aDNA de novo assembly, Pydamage opens up new doors to explore functional diversity in ancient metagenomic datasets.

Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy.

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

Genomics of radiation-induced damage

Radiation Risks The potential adverse effects of exposures to radioactivity from nuclear accidents can include acute consequences such as radiation sickness, as well as long-term sequelae such as increased risk of cancer. There have been a few studies examining transgenerational risks of radiation exposure but the results have been inconclusive. Morton et al. analyzed papillary thyroid tumors, normal thyroid tissue, and blood from hundreds of survivors of the Chernobyl nuclear accident and compared them against those of unexposed patients. The findings offer insight into the process of radiation-induced carcinogenesis and characteristic patterns of DNA damage associated with environmental radiation exposure. In a separate study, Yeager et al. analyzed the genomes of 130 children and parents from families in which one or both parents had experienced gonadal radiation exposure related to the Chernobyl accident and the children were conceived between 1987 and 2002. Reassuringly, the authors did not find an increase in new germline mutations in this population. Science , this issue p. [eabg2538][1], p. [725][2] [1]: /lookup/doi/10.1126/science.abg2538 [2]: /lookup/doi/10.1126/science.abg2365

Thymidine decreases the DNA damage and apoptosis caused by tumor‐treating fields in cancer cell lines

Tumor-treating fields (TTFields) is an emerging non-invasive cancer-treatment modality using alternating electric fields with low intensities and an intermediate range of frequency. TTFields affects an extensive range of charged and polarizable cellular factors known to be involved in cell division. However, it causes side-effects, such as DNA damage and apoptosis, in healthy cells. To investigate whether thymidine can have an effect on the DNA damage and apoptosis, we arrested the cell cycle of human glioblastoma cells (U373) at G1/S phase by using thymidine and then exposed these cells to TTFields. Cancer cell lines and normal cell (HaCaT) were arrested by thymidine double block method. Cells were seeded into the gap of between the insulated wires. The exposed in alternative electric fields at 120kHz, 1.2V/cm. They were counted the cell numbers and analyzed for cancer malignant such as colony formation, Annexin V/PI staining, γH2AX and RT-PCR. The colony-forming ability and DNA damage of the control cells without thymidine treatment were significantly decreased, and the expression levels of BRCA1, PCNA, CDC25C, and MAD2 were distinctly increased. Interestingly, however, cells treated with thymidine did not change the colony formation, apoptosis, DNA damage, or gene expression pattern. These results demonstrated that thymidine can inhibit the TTFields-caused DNA damage and apoptosis, suggesting that combining TTFields and conventional treatments, such as chemotherapy, may enhance prognosis and decrease side effects compared with those of TTFields or conventional treatments alone.

Lack of transgenerational effects of ionizing radiation exposure from the Chernobyl accident.

Effects of radiation exposure from the Chernobyl nuclear accident remain a topic of interest. We investigated germline de novo mutations (DNMs) in children born to parents employed as cleanup workers or exposed to occupational and environmental ionizing radiation after the accident. Whole-genome sequencing of 130 children (born 1987-2002) and their parents did not reveal an increase in the rates, distributions, or types of DNMs relative to the results of previous studies. We find no elevation in total DNMs, regardless of cumulative preconception gonadal paternal [mean = 365 milligrays (mGy), range = 0 to 4080 mGy] or maternal (mean = 19 mGy, range = 0 to 550 mGy) exposure to ionizing radiation. Thus, we conclude that, over this exposure range, evidence is lacking for a substantial effect on germline DNMs in humans, suggesting minimal impact from transgenerational genetic effects.

Study of DNA Damage in Diabetic Mothers and Their Newborn

Background: Previous research declares that gestational diabetes mellitus (GDM) affects both mother and infant during pregnancy and in the long term. Objective: To assess the pattern of DNA damage in mothers with gestational diabetes during pregnancy and their possible impact on their offspring. Patients and Methods: A case-control study was designed and conducted on 120 pregnant women and their infants; divided into two groups: 1-Patients group (60 patients had gestational diabetes mellitus during the third trimester of pregnancy and their infants) 2-Control group (60 apparently healthy pregnant women and their infants). Results: DNA damage of mother and their infants was significantly increased among the cases group than controls (p=0.0001). There was a statistically significant increase among infants who had DNA damage group than who hadn't regarding HBA1C, random blood sugar (RBS) 3 hr and RBS 6 hr (p 0.05). Conclusions: Hyperglycemia affects maternal and fetal DNA integrity and DNA damage response differently, gestational and mild gestational hyperglycemia, were all related to increased oxidative DNA damage and DNA repair may be thus considered an important mechanism to prevent the deleterious effects of hyperglycemia in the genetic material.

Study of DNA Damage in Diabetic Mothers and Their Newborn

Background: Previous research declares that gestational diabetes mellitus (GDM) affects both mother and infant during pregnancy and in the long term. Objective: To assess the pattern of DNA damage in mothers with gestational diabetes during pregnancy and their possible impact on their offspring. Patients and Methods: A case-control study was designed and conducted on 120 pregnant women and their infants; divided into two groups: 1-Patients group (60 patients had gestational diabetes mellitus during the third trimester of pregnancy and their infants) 2-Control group (60 apparently healthy pregnant women and their infants). Results: DNA damage of mother and their infants was significantly increased among the cases group than controls (p=0.0001). There was a statistically significant increase among infants who had DNA damage group than who hadn't regarding HBA1C, random blood sugar (RBS) 3 hr and RBS 6 hr (p 0.05). Conclusions: Hyperglycemia affects maternal and fetal DNA integrity and DNA damage response differently, gestational and mild gestational hyperglycemia, were all related to increased oxidative DNA damage and DNA repair may be thus considered an important mechanism to prevent the deleterious effects of hyperglycemia in the genetic material.

Comet Assay analysis of DNA strand breaks after exposure to the DNA-incorporated Auger Electron Emitter Iodine-125

Purpose Ionizing radiation causes various types of DNA damage e.g. single strand breaks (SSB) and double strand breaks (DSB), whereby the SSB/DSB ratio is shifted toward the DSB with increasing LET. For the DNA-incorporated Auger electron emitter Iodine-125 a SSB/DSB ratio of 5.4:1 is calculated based on computer simulations. In the presented work the SSB/DSB ratio of DNA-incorporated Iodine-125 was experimentally determined and compared to external homogenous γ-irradiation. Materials and methods Iodine-125-iododeoxyuridine (I-125-UdR) was incorporated into the DNA of SCL-II cells and cells were subsequently frozen for decay accumulation. Accordingly, external γ-irradiation (Cs-137) experiments were performed in frozen cells. After exposure the neutral or alkaline Comet Assay was performed to quantify DSB or DSB and SSB, respectively. Automated quantification of the comets was performed using the Olive Tail Moment (Metafer CometScan; MetaSystems). Calculation of absorbed dose for Auger electrons on cellular level is extremely biased due to the exclusive DNA localization of I-125-UdR. To avoid dose calculation the γ-H2AX assay was used in order to allow the comparison of the Comet Assay data between both investigated radiation qualities. Results For low-LET γ-radiation, a SSB/DSB ratio of 10:1 was determined. In contrast, a lower SSB/DSB ratio of 6:1 was induced by DNA-incorporated Iodine-125 which compares very well to the calculated values of Pomplun and co-authors Conclusion DNA-incorporated Iodine-125 induces a high-LET type DNA damage pattern in respect to SSB/DSB ratio.

Nuclei DNA Damage Due to Extreme High-Temperature Exposure during Forensic Identification Examination

ccurate personal identification is important in investigations because an error in the identification process may bring fatal consequences during trial. The most common identification process is the Deoxyribonucleic acid [DNA] analysis. Degraded DNA sample due to extremely high-temperature exposure may limit DNA analysis. This study aimed to analyze DNA damage patterns caused by an extremely high temperature using STR (short tandem repeat) CODIS marker. This study was conducted at the Forensic and Medicolegal Department, Laboratorium Balai Besar Kesehatan Surabaya, Ministry of Health of the Republic of Indonesia, Human Genetic Study Group of Universitas Airlangga, and Faculty of Science and Technology of Universitas Brawijaya Malang from July until October 2009. Results of PCR visualization using STR CODIS for costae demonstrated that the THO1 detection (+) in 1,2500C - 40’: 25% and the TPOX detection (+) in 1,0000C - 30’: 50% whereas the results from molar teeth showed that the THOI locus detection (+) in 1,2500C - 30’: 25% and TPOX in 1,0000C - 40’: 50%. Results for PCR visualization using mini-STR CODIS for the costae presented that the mini-THOI in 1,2500C - 20’: 50% (+) while for the molar tooth the mini-THOI in 1,2500C - 30’ : 25% (+) and mini-TPOX in 1,0000C - 40’ : 50% (+). All loci were detected on costae and second molar teeth samples of the control group. Thus, extreme high-temperature exposure significantly decreased the DNA level of second costae and second molar tooth. Sequence patterns of STR loci successfully detected were TPOX, THO1, and CSF1PO.

Analysis of Human Decomposition Effect on DNA Quality with Short Tandem Repeats [STRS] Combined Index DNA System [CODIS

Introduction: Personal identification is a problem in criminal or civil cases. Exact determination of personalidentity is very important in investigation as any error can be fatal in the judicial process. The process ofidentification that is often used is through DNA analysis. The problem that often becomes a serious problemfor both forensic DNA expert and other DNA expert is the condition of degraded DNA.Method and Materials: This study was conducted by analysing DNA damage patterns using CODIS STR(Short Tandem Repeat) markers to effect the decay process. The type of research used by experimentallaboratories, with research design used is time series.Results and Discussion: The result of DNA sample extraction from Psoas and Masseter muscle samplesshows the average minimum range of DNA levels for DNA typing is 0.25 ng with purity of 1.8 – 2(1). Theresult of this study prove the effect of decomposition on DNA concentration on each muscle. This study isalso showed a decrease in levels and purity in the samples of Psoas and Masseter muscle tissue buried in soil,sea/salt water and river/fresh water from day 1, 7, 14, 20 and 40.Conclusion: Psoas and Masseter muscle can be alternative material for forensic identification. The successof sequence mapping pattern from STR loci are TPOX, THO1, and CS1PO according to each GC content.GC content of THO1 and TPOX has the same relative value of 0,48, compared to CSF1PO value of 0,33.

Heavy ion space radiation triggers ongoing DNA base damage by downregulating DNA repair pathways

Long-duration space missions outside low earth orbit will expose astronauts to a cumulative dose of high-energy particle radiation especially to highly damaging heavy ion radiation, which poses considerable risk to astronauts’ health. The purpose of the current study was to quantitatively identify oxidatively induced DNA base modifications and assess status of the repair pathways involved in removing the modified bases in mouse intestinal cells after exposure to γ-rays and iron radiation. Mice (C57BL/6J; 6 to 8 weeks; female) were exposed to 0.5 Gy of either γ-rays or iron radiation and control mice were sham-irradiated. Intestinal tissues were collected 2 months after radiation. DNA base lesions were measured using gas chromatography-tandem mass spectrometry with isotope‑dilution. Base excision repair (BER) and nucleotide excision repair (NER) pathways were assessed using PCR and immunoblotting. Effects of iron radiation were compared to γ-rays and sham-irradiated controls. Exposure to iron radiation resulted in significantly higher levels of several DNA base lesions relative to control animals and those exposed to γ radiation. Assessment of BER and NER showed downregulation of pathway factors both at the RNA as well as at the protein levels. Our results not only provide important insight into DNA damage pattern in intestinal cells in response to iron radiation, but they also confirm our previous immunohistochemistry data on oxidatively induced DNA damage. We suggest that downregulation of the BER and NER pathways is contributing to ongoing DNA base damages long time after radiation exposure and has implications for chronic diseases including gastrointestinal diseases after heavy ion radiation exposure during space travel.

High-precision image-guided proton irradiation of mouse brain sub-volumes

Background and purposeProton radiotherapy offers the potential to reduce normal tissue toxicity. However, clinical safety margins, range uncertainties, and varying relative biological effectiveness (RBE) may result in a critical dose in tumor-surrounding normal tissue. To assess potential adverse effects in preclinical studies, image-guided proton mouse brain irradiation and analysis of DNA damage repair was established. Material and methodsWe designed and characterized a setup to shape proton beams with 7 mm range in water and 3 mm in diameter and commissioned a Monte Carlo model for in vivo dose simulation. Cone-beam computed tomography and orthogonal X-ray imaging were used to delineate the right hippocampus and position the mice. The brains of three C3H/HeNRj mice were irradiated with 8 Gy and excised 30 min later. Initial DNA double-strand breaks were visualized by staining brain sections for cell nuclei and γH2AX. Imaged sections were analyzed with an automated and validated processing pipeline to provide a quantitative, spatially resolved radiation damage indicator. ResultsThe analyzed DNA damage pattern clearly visualized the radiation effect in the mouse brains and could be mapped to the simulated dose distribution. The proton beam passed the right hippocampus and stopped in the central brain region for all evaluated mice. ConclusionWe established image-guided proton irradiation of mouse brains. The clinically oriented workflow facilitates (back-) translational studies. Geometric accuracy, detailed Monte Carlo dose simulations, and cell-based assessment enable a biologically and spatially resolved analysis of radiation response and RBE.

Uncovering Signatures of DNA Methylation in Ancient Plant Remains From Patterns of Post-mortem DNA Damage

The ultra-short DNA molecules still preserved in archaeological remains can provide invaluable genetic information about past individuals, species and communities within the half to one-million-year time range. The sequence data are, however, generally affected by post-mortem DNA damage and include specific patterns of nucleotide mis-incorporations, which can help data authentication. Recent work in ancient mammals has shown that such patterns can also help assess past levels of DNA methylation in CpG contexts. Despite pioneering work in barley and sorghum, ancient epigenetic marks have received limited attention in plants and it remains unknown whether ancient epigenetic signatures can be retrieved in any of the three main sequence contexts (CG, CHG and CHH). To address this question, we extended a statistical methylation score originally proposed to trace cytosine methylation in mammal sequence data to accommodate the three plant-specific methylation contexts. We applied this score to a range of tissues (wood, cobs and grains) and species (oak, maize and barley), spanning both desiccated and waterlogged archaeological samples. Ancient sequence data obtained for USER-treated DNA extracts yielded methylation scores on par with DNA methylation levels of modern organellar and nuclear genomes. At the quantitative level, scores were (1) positively correlated to post-mortem cytosine deamination, and (2) replicated relative contributions of CG, CHG and CHH contexts to DNA methylation assessed by bisulfite DNA sequencing of modern plant tissues. This demonstrates that genuine DNA methylation signatures can be characterized in ancient plant remains, which opens new avenues for investigating the plant evolutionary response to farming, pollution, epidemics and changing environmental conditions.

Molecular Analysis of Mutations in the Human HPRT Gene.

The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and the products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.