Asparagine 326 in the extremely C-terminal region of XRCC4 is essential for the cell survival after irradiation.

XRCC4 is one of the crucial proteins in the repair of DNA double-strand break (DSB) through non-homologous end-joining (NHEJ). As XRCC4 consists of 336 amino acids, N-terminal 200 amino acids include domains for dimerization and for association with DNA ligase IV and XLF and shown to be essential for XRCC4 function in DSB repair and V(D)J recombination. On the other hand, the role of the remaining C-terminal region of XRCC4 is not well understood. In the present study, we noticed that a stretch of ∼20 amino acids located at the extreme C-terminus of XRCC4 is highly conserved among vertebrate species. To explore its possible importance, series of mutants in this region were constructed and assessed for the functionality in terms of ability to rescue radiosensitivity of M10 cells lacking XRCC4. Among 13 mutants, M10 transfectant with N326L mutant (M10-XRCC4(N326L)) showed elevated radiosensitivity. N326L protein showed defective nuclear localization. N326L sequence matched the consensus sequence of nuclear export signal. Leptomycin B treatment accumulated XRCC4(N326L) in the nucleus but only partially rescued radiosensitivity of M10-XRCC4(N326L). These results collectively indicated that the functional defects of XRCC4(N326L) might be partially, but not solely, due to its exclusion from nucleus by synthetic nuclear export signal. Further mutation of XRCC4 Asn326 to other amino acids, i.e., alanine, aspartic acid or glutamine did not affect the nuclear localization but still exhibited radiosensitivity. The present results indicated the importance of the extremely C-terminal region of XRCC4 and, especially, Asn326 therein.

Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV.

XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4(K271R) mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4(K210R) mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3'-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4(K271R) was defective in the nuclear localization of itself and LIG4, whereas XRCC4(K210R) was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4(K271R), but not M10-XRCC4(K210R), showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4(WT). The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4(K271R) than in M10-XRCC4(WT), whereas it was only marginally increased in M10-XRCC4(K210R) as compared to M10-XRCC4(WT). The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4.

C-Terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin.

DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

Dosimetric Performance of Poly(vinyl alcohol)/Silver Nanoparticles Hybrid Nanomaterials for Colorimetric Sensing of Gamma Radiation.

A colorimetric liquid sensor based on a poly(vinyl alcohol)/silver nanoparticle (PVA/AgNPs) hybrid nanomaterial was developed for gamma radiation in the range of 0−100 Gy. In this study, gamma rays (Cobalt-60 source) triggered the aggregation of AgNPs in a PVA/silver nitrate (AgNO3) hybrid solution. The color of this solution visibly changed from colorless to dark yellow. Absorption spectra of the PVA/AgNPs solution were analyzed by UV-Vis spectrophotometry in the range of 350−800 nm. Important parameters, such as pH and AgNO3 concentration were optimized. The accuracy, sensitivity, stability, and uncertainty of the sensor were investigated and compared to the reference standard dosimeter. Based on the spectrophotometric results, an excellent positive linear correlation (r = 0.998) between the absorption intensity and received dose was found. For the accuracy, the intra-class correlation coefficient (ICC) between the PVA/AgNPs sensor and the standard Fricke dosimeter was 0.998 (95%CI). The sensitivity of this sensor was 2.06 times higher than the standard dosimeter. The limit of detection of the liquid dosimeter was 13.4 Gy. Moreover, the overall uncertainty of this sensor was estimated at 4.962%, in the acceptable range for routine standard dosimeters (<6%). Based on its dosimetric performance, this new PVA/AgNPs sensor has potential for application as an alternative gamma sensor for routine dose monitoring in the range of 13.4−100 Gy.

A deep learning model (FociRad) for automated detection of γ-H2AX foci and radiation dose estimation.

DNA double-strand breaks (DSBs) are the most lethal form of damage to cells from irradiation. γ-H2AX (phosphorylated form of H2AX histone variant) has become one of the most reliable and sensitive biomarkers of DNA DSBs. However, the γ-H2AX foci assay still has limitations in the time consumed for manual scoring and possible variability between scorers. This study proposed a novel automated foci scoring method using a deep convolutional neural network based on a You-Only-Look-Once (YOLO) algorithm to quantify γ-H2AX foci in peripheral blood samples. FociRad, a two-stage deep learning approach, consisted of mononuclear cell (MNC) and γ-H2AX foci detections. Whole blood samples were irradiated with X-rays from a 6 MV linear accelerator at 1, 2, 4 or 6 Gy. Images were captured using confocal microscopy. Then, dose-response calibration curves were established and implemented with unseen dataset. The results of the FociRad model were comparable with manual scoring. MNC detection yielded 96.6% accuracy, 96.7% sensitivity and 96.5% specificity. γ-H2AX foci detection showed very good F1 scores (> 0.9). Implementation of calibration curve in the range of 0-4 Gy gave mean absolute difference of estimated doses less than 1 Gy compared to actual doses. In addition, the evaluation times of FociRad were very short (< 0.5 min per 100 images), while the time for manual scoring increased with the number of foci. In conclusion, FociRad was the first automated foci scoring method to use a YOLO algorithm with high detection performance and fast evaluation time, which opens the door for large-scale applications in radiation triage.

Quantification of histone H2AX phosphorylation in white blood cells induced by ex vivo gamma irradiation of whole blood by both flow cytometry and foci counting as a dose estimation in rapid triage.

A quick, reliable, and reproducible biological assay to distinguish individuals with possible life-threatening risk following radiological or nuclear incidents remains a quest in biodosimetry. In this paper, we examined the use of a γ-H2AX assay as an early dose estimation for rapid triage based on both flow cytometry and image analyses. In the experiment, whole blood from 11 donors was irradiated ex vivo inside a water phantom by gamma rays from Co-60 at 0.51 Gy/min. After the lysis of red blood cells, the white blood cells were collected for immunofluorescence labeling of γ-H2AX, CD45, and nuclear stained for signal collection and visualization. Analysis by flow cytometry showed that the relative γ-H2AX intensities of lymphocytes and granulocytes increased linearly with absorbed doses from 0 to 6 Gy with a large variation among individuals observed above 2 Gy. The relative γ-H2AX intensities of lymphocytes assessed by two different laboratories were highly correlated (ICC = 0.979). Using confocal microscopic images, γ-H2AX foci were observed to be discretely distributed inside the nuclei and to increase proportionally with doses from 0 to 2 Gy, whereas large plagues of merged foci appeared at 4 and 6 Gy, resulting in the saturation of foci counts above 4 Gy. The number of total foci per cell as well as the number of foci per plane were significantly different at 0 vs 1 and 2 vs 4 Gy doses (p < 0.01). Blind tests at 0.5 Gy and 1 Gy doses showed that dose estimation by flow cytometry had a mean absolute difference of less than 0.5 Gy from the actual value. In conclusion, while flow cytometry can provide a dose estimation with an uncertainty of 0.5 Gy at doses ≤ 1 Gy, foci counting can identify merged foci that are prominent at doses ≥ 4 Gy.

The roles of HMGB1-produced DNA gaps in DNA protection and aging biomarker reversal.

The endogenous DNA damage triggering an aging progression in the elderly is prevented in the youth, probably by naturally occurring DNA gaps. Decreased DNA gaps are found during chronological aging in yeast. So we named the gaps "Youth-DNA-GAPs." The gaps are hidden by histone deacetylation to prevent DNA break response and were also reduced in cells lacking either the high-mobility group box (HMGB) or the NAD-dependent histone deacetylase, SIR2. A reduction in DNA gaps results in shearing DNA strands and decreasing cell viability. Here, we show the roles of DNA gaps in genomic stability and aging prevention in mammals. The number of Youth-DNA-GAPs were low in senescent cells, two aging rat models, and the elderly. Box A domain of HMGB1 acts as molecular scissors in producing DNA gaps. Increased gaps consolidated DNA durability, leading to DNA protection and improved aging features in senescent cells and two aging rat models similar to those of young organisms. Like the naturally occurring Youth-DNA-GAPs, Box A-produced DNA gaps avoided DNA double-strand break response by histone deacetylation and SIRT1, a Sir2 homolog. In conclusion, Youth-DNA-GAPs are a biomarker determining the DNA aging stage (young/old). Box A-produced DNA gaps ultimately reverse aging features. Therefore, DNA gap formation is a potential strategy to monitor and treat aging-associated diseases.

B1 siRNA Increases de novo DNA Methylation of B1 Elements and Promotes Wound Healing in Diabetic Rats.

Alu (B1 in rodents) hypomethylation, commonly found in diabetes mellitus patients, increases DNA damage and, consequently, delays the healing process. Alu siRNA increases Alu methylation, reduces DNA damage, and promotes cell proliferation. Aim: To explore whether B1 siRNA treatment restores B1 hypomethylation, resulting in a reduction in DNA damage and acceleration of the healing process in diabetic rat wounds. Methods: We generated splinted-excisional wounds in a streptozotocin (STZ)-induced type I diabetic rat model and treated the wounds with B1 siRNA/Ca-P nanoparticles to generate de novo DNA methylation in B1 intersperse elements. After treatment, we investigated B1 methylation levels, wound closure rate, wound histopathological structure, and DNA damage markers in diabetic wounds compared to nondiabetic wounds. Results: We reported that STZ-induced diabetic rat wounds exhibited B1 hypomethylation, wound repair defects, anatomical feature defects, and greater DNA damage compared to normal rats. We also determined that B1 siRNA treatment by Ca-P nanoparticle delivery restored a decrease in B1 methylation levels, remedied delayed wound healing, and improved the histological appearance of the wounds by reducing DNA damage. Conclusion: B1 hypomethylation is inducible in an STZ-induced type I diabetes rat model. Restoration of B1 hypomethylation using B1 siRNA leads to increased genome stability and improved wound repair in diabetes. Thus, B1 siRNA intervention may be a promising strategy for reprogramming DNA methylation to treat or prevent DNA damage-related diseases.

A model of modified meta-iodobenzylguanidine conjugated gold nanoparticles for neuroblastoma treatment.

Iodine-131 meta-iodobenzylguanidine (131I-mIBG) has been utilized as a standard treatment to minimize adverse side effects by targeting therapies to bind to the norepinephrine transporter (NET) expressed on 90% of neuroblastoma cells. However, only a minority of patients who receive 131I-mIBG radiotherapy have clinical responses, and these are usually not curative. In this study, novel ligand-conjugated gold nanoparticles (GNPs) based on mIBG were synthesized and evaluated biologically with neuroblastoma cells in vitro. To induce specific internalization to the tumor cells and utilize it as a model for radioenhancement, 127I-modified mIBG was successfully synthesized and grafted covalently to the surface of carboxylated PEG-GNPs. 49.28% of the novel mIBG derivative was grafted on carboxylated PEG-GNPs. The particles were stable and not toxic to the normal fibroblast cell line, L929, even at the highest concentration tested (1013 NPs per mL) at 24, 48, and 72 h. Moreover, the cellular uptake of the model was decreased significantly in the presence of a NET inhibitor, suggesting that there was specific internalization into neuroblastoma cells line (SH-SY5Y) via the NET. Therefore, this model provides useful guidance toward the design of gold nanomaterials to enhance the efficiency of 131I-mIBG treatment in neuroblastoma patients. However, the investigation of radio-therapeutic efficiency after radioisotope 131I substitution will be further conducted in a radiation safety laboratory using an animal model.

Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity.

Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs*68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs*68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4's DSB repair function in normal development.