Comparison of high-and low-let radiation-induced dna double-strand break processing in living cells

High-linear-energy-transfer (LET) radiation is more lethal than similar doses of low-LET radiation types, probably a result of the condensed energy deposition pattern of high-LET radiation. Here, we compare high-LET α-particle to low-LET X-ray irradiation and monitor double-strand break (DSB) processing. Live-cell microscopy was used to monitor DNA double-strand breaks (DSBs), marked by p53-binding protein 1 (53BP1). In addition, the accumulation of the endogenous 53BP1 and replication protein A (RPA) DSB processing proteins was analyzed by immunofluorescence. In contrast to α-particle-induced 53BP1 foci, X-ray-induced foci were resolved quickly and more dynamically as they showed an increase in 53BP1 protein accumulation and size. In addition, the number of individual 53BP1 and RPA foci was higher after X-ray irradiation, while focus intensity was higher after α-particle irradiation. Interestingly, 53BP1 foci induced by α-particles contained multiple RPA foci, suggesting multiple individual resection events, which was not observed after X-ray irradiation. We conclude that high-LET α-particles cause closely interspaced DSBs leading to high local concentrations of repair proteins. Our results point toward a change in DNA damage processing toward DNA end-resection and homologous recombination, possibly due to the depletion of soluble protein in the nucleoplasm. The combination of closely interspaced DSBs and perturbed DNA damage processing could be an explanation for the increased relative biological effectiveness (RBE) of high-LET α-particles compared to X-ray irradiation

Elucidating the Influence of Tumor Presence on the Polymersome Circulation Time in Mice

The use of nanoparticles as tumor-targeting agents is steadily increasing, and the influence of nanoparticle characteristics such as size and stealthiness have been established for a large number of nanocarrier systems. However, not much is known about the impact of tumor presence on nanocarrier circulation times. This paper reports on the influence of tumor presence on the in vivo circulation time and biodistribution of polybutadiene-polyethylene oxide (PBd-PEO) polymersomes. For thi

Uptake and subcellular distribution of radiolabeled polymersomes for radiotherapy

Polymersomes have the potential to be applied in targeted alpha radionuclide therapy, while in addition preventing release of recoiling daughter isotopes. In this study, we investigated the cellular uptake, post uptake processing and intracellular localization of polymersomes. Methods: High-content microscopy was used to validate polymersome uptake kinetics. Confocal (live cell) microscopy was used to elucidate the uptake mechanism and DNA damage induction. Intracellular distribution of polymersomes in 3-D was determined using super-resolution microscopy. Results: We found that altering polymersome size and concentration affects the initial uptake and overall uptake capacity; uptake efficiency and eventual plateau levels varied between cell lines;

The CST Complex Mediates End Protection at Double-Strand Breaks and Promotes PARP Inhibitor Sensitivity in BRCA1-Deficient Cells

Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells. Loss of members of the CTC1-STN1-TEN1 (CST) complex were found to cause PARPi resistance in BRCA1-deficient cells in vitro and in vivo. We show that CTC1 depletion results in the restoration of end resection and that the CST complex may act downstream of 53BP1/RIF1. These data suggest that, in addition to its role in protecting telomeres, the CST complex also contributes to protecting DSBs from end resection. Using CRISPR/SpCas9-based loss-of-function screens, Barazas et al. show that loss of the CTC1-STN1-TEN1 (CST) complex promotes PARP inhibitor resistance in BRCA1-deficient cells. Mechanistically, the CST complex maintains double-strand break end stability in addition to its role in protecting telomeric ends

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Ionizing Radiation Quality and Dose Effects on DNA Double Strand Break Repai

The use of alpha particles irradiation in clinical practice has gained interest in the past years, for example with the advance of radionuclide therapy. The lack of affordable and easily accessible irradiation systems to study the cell biological impact of alpha particles hampers broad investigation. Here we present a novel alpha particle irradiation set-up for uniform irradiation of cell cultures. By combining a small alpha emitting source and a computer-directed movement stage, we established a new alpha particle irradiation method allowing more advanced biological assays, including large-field local alpha particle irradiation and cell survival assays. In addition, this protocol uses cell culture on glass cover-slips which allows more advanced microscopy, such as super-resolution imaging, for in-depth analysis of the DNA damage caused by alpha particles. This novel irradiation set-up provides the possibility to perform reproducible, uniform and directed alpha particle irradiation to investigate the impact of alpha radiation on the cellular level

Elucidating the Influence of Tumor Presence on the Polymersome Circulation Time in Mice

The use of nanoparticles as tumor-targeting agents is steadily increasing, and the influence of nanoparticle characteristics such as size and stealthiness have been established for a large number of nanocarrier systems. However, not much is known about the impact of tumor presence on nanocarrier circulation times. This paper reports on the influence of tumor presence on the in vivo circulation time and biodistribution of polybutadiene-polyethylene oxide (PBd-PEO) polymersomes. For this purpose, polymersomes were loaded with the gamma-emitter 111In and administered intravenously, followed by timed ex vivo biodistribution. A large reduction in circulation time was observed for tumor-bearing mice, with a circulation half-life of merely 5 min (R2 = 0.98) vs 117 min (R2 = 0.95) in healthy mice. To determine whether the rapid polymersome clearance observed in tumor-bearing mice was mediated by macrophages, chlodronate liposomes were administered to both healthy and tumor-bearing mice prior to the intravenous injection of radiolabeled polymersomes to deplete their macrophages. Pretreatment with chlodronate liposomes depleted macrophages in the spleen and liver and restored the circulation time of the polymersomes with no significant difference in circulation time between healthy mice and tumor-bearing mice pretreated with clodronate liposomes (15.2 ± 1.2% ID/g and 13.6 ± 2.7% ID/g, respectively, at 4 h p.i. with p = 0.3). This indicates that activation of macrophages due to tumor presence indeed affected polymersome clearance rate. Thus, next to particle design, the presence of a tumor can also greatly impact circulation times and should be taken into account when designing studies to evaluate the distribution of polymersomes