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Summary: The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination. We uncover how the phosphorylation events are tightly regulated in cells and that mimicking their constant phosphorylation leads to an uncontrolled active state of FANCD2, which is loaded onto chromosomes in an unrestrained fashion. Taken together, we describe a mechanism where ATR triggers FANCD2/FANCI loading onto chromosomes.

Abstract Background Malaria is a major global parasitic disease caused by species of the genus Plasmodium. Zygotes of Plasmodium spp. undergo meiosis and develop into tetraploid ookinetes, which differentiate into oocysts that undergo sporogony. Homologous recombination (HR) occurs during meiosis and introduces genetic variation. However, the mechanisms of HR in Plasmodium are unclear. In humans, the recombinases DNA repair protein Rad51 homolog 1 (Rad51) and DNA meiotic recombinase 1 (Dmc1) are required for HR and are regulated by breast cancer susceptibility protein 2 (BRCA2). Most eukaryotes harbor BRCA2 homologs. Nevertheless, these have not been reported for Plasmodium. Methods A Brca2 candidate was salvaged from a database to identify Brca2 homologs in Plasmodium. To confirm that the candidate protein was Brca2, interaction activity between Plasmodium berghei (Pb) Brca2 (PbBrca2) and Rad51 (PbRad51) was investigated using a mammalian two-hybrid assay. To elucidate the functions of PbBrca2, PbBrca2 was knocked out and parasite proliferation and differentiation were assessed in mice and mosquitoes. Transmission electron microscopy was used to identify sporogony. Results The candidate protein was conserved among Plasmodium species, and it was indicated that it harbors critical BRCA2 domains including BRC repeats, tower, and oligonucleotide/oligosaccharide-binding-fold domains. The P. berghei BRC repeats interacted with PbRad51. Hence, the candidate was considered a Brca2 homolog. PbBrca2 knockout parasites were associated with reduced parasitemia with increased ring stage and decreased trophozoite stage counts, gametocytemia, female gametocyte ratio, oocyst number, and ookinete development in both mice and mosquitoes. Nevertheless, the morphology of the blood stages in mice and the ookinete stage was comparable to those of the wild type parasites. Transmission electron microscopy results showed that sporogony never progressed in Brca2-knockout parasites. Conclusions Brca2 is implicated in nearly all Plasmodium life cycle stages, and especially in sporogony. PbBrca2 contributes to HR during meiosis. Graphical Abstract

Abstract Background Breast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination. Results In this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5′ untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5′ UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1. Conclusions This study demonstrates that BRCA2 expression level is regulated via 5′ UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

Abstract The surgical efficacy for supratentorial intracerebral hemorrhage (ICH) remains unknown. We compared the advantages of the widely practiced endoscopic hematoma removal under local anesthesia with that of craniotomy under general anesthesia for ICH. We also focused on our novel operative concept of intentional hematoma leaving technique to avoid further damage to the brain. We retrospectively analyzed 134 consecutive patients (66 endoscopies and 68 craniotomies) who were surgically treated for supratentorial ICH. The characteristics of the 134 patients were as follows: The median (interquartile range) age was 73 (61–82) years. The median Glasgow Coma Scale scores at admission, on day 7, and the median modified Rankin Scale (mRS) score at 6 months were 10 (7–13), 13 (10–14), and 4 (3–5) respectively. The statistical comparison revealed there were no differences in GCS score on day seven between the endoscopy 13 (12–14) and craniotomy group 12 (9–14). No differences were observed in mRS scores at 6 months between the endoscopy 4 (2–5) and craniotomy group 4 (3–5). However, the patients treated with our technique tended to have favorable outcomes. Multivariate analysis revealed the operative time was significantly decreased in the endoscopy group compared to the craniotomy group (p

Mammary tumors are the most prevalent type of tumors in female dogs. Breast cancer 2, early onset (BRCA2) malignant mutations are associated with tumorigenesis in humans and dogs. BRCA2 plays a pivotal role in homologous recombination repair by recruiting RAD51 recombinase to DNA damage sites to maintain genome stability. To recruit RAD51, BRCA2 must interact with RAD51 via BRC repeats, but the regulation of this interaction has been unclear. In this study, we focused on a highly conserved region (HCR) near BRC repeats. Using co-immunoprecipitation and mammalian two-hybrid assay, we found that HCR suppressed the RAD51-interaction activity of BRC repeats and that substitutions of HCR phosphorylation sites affected it. In canine tumor samples, we found ten mutations, including a novel HCR mutation (I1110M) from canine tumor samples. The effect of four HCR mutations, including I1110M, on the RAD51-interaction activity of BRC repeats was tested. One of the HCR mutations found in canine mammary tumors increased the interaction, but the two mutations found in human breast cancers decreased it. This study suggested that the HCR regulated the RAD51-interacting activity of BRC repeats through HCR phosphorylation and that mutations in HCR may be related to tumorigenesis in both dogs and humans.

Due to the high incidence of mammary tumors in dogs, it is important to elucidate the pathogenesis of these tumors in veterinary medicine. Radiation therapy is often used to treat mammary tumors that target DNA lesions. RAD51 is a key molecule that repairs DNA damage via homologous recombination. We examined the relationship between RAD51 expression and radiosensitivity in mammary tumor cell lines. CHMp and CHMm from the same individual were selected based on the differences in RAD51 expression. The radiosensitivity of both cell lines was examined using MTT and scratch assays; CHMm, which has high RAD51 expression, showed higher sensitivity to radiation than CHMp. However, the nuclear focus of RAD51 during DNA repair was formed normally in CHMp, but not in most of CHMm. Since irradiation resulted in the suppression of cell cycle progression in CHMp, the expression of p21, a cell cycle regulatory factor, was detected in CHMp after 15 Gy irradiation but not in CHMm. These results indicate that functional expression is more important than the quantitative expression of RAD51 in canine mammary tumor cells in response to DNA damage.

These data present the 141 intracranial arterial branches' visibilities near the 72 cerebral aneurysms in postoperative 58 patients treated with titanium or cobalt-chromium-nickel-molybdenum (CCNM) alloy clips. The visibilities were evaluated using time-of-flight magnetic resonance angiography (TOF-MRA), pointwise encoding time reduction with radial acquisition (PETRA)-MRA, which uses MRA with ultrashort echo time (UTE-MRA) and subtraction technique between saturated and non-saturated images, and three-dimensional computed tomography angiography (3DCTA). We retrospectively acquired the data from the medical records of Suwa Red Cross Hospital. Each method's appearance was compared, and associations between visibility on PETRA-MRA, arterial diameter, clip numbers, clip shapes, clip materials, and amounts of hematoma were summarized. Our article on PETRA-MRA's usefulness for proximal and branched arteries evaluation after cerebral aneurysm clipping [1] was based on these data. This dataset would be useful for reference value for other neurosurgeons or radiologists for further analysis on PETRA-MRA and another UTE-MRA like SILENT-MRA after cerebral aneurysm clipping.

Objective: Time-of-flight magnetic resonance angiography (MRA) is limited by clip-induced artifacts after cerebral aneurysmal clipping. Recently, ultrashort echo time was shown to reduce metal artifacts. We assessed the pointwise encoding time reduction with radial acquisition (PETRA) sequence in subtraction-based MRA as an ultrashort echo time method during follow-up for clipping surgery. Methods: We retrospectively evaluated 114 branches of 63 aneurysms in 56 patients treated with titanium clips using MRA and 3-dimensional computed tomography angiography. The appearance using each method was compared, and the associations between visibility on PETRA-MRA, clip number and shape, and amount of hematoma were examined. Furthermore, the visibility of the aneurysm remnants and 2 clipping cases with cobalt-chromium-nickel-molybdenum clips were evaluated. Results: No branches were visible using time-of-flight-MRA, but 79 of 114 branches (69.3%) were visible on PETRA-MRA. PETRA-MRA was effective for follow-up imaging in 33 of 63 aneurysms (52.4%). The median vessel diameters were 1.67 mm (interquartile range, 1.24–2.62 mm) and 0.96 mm (interquartile range, 0.59–1.53 mm) in the visible and invisible groups, respectively. Only the vessel diameter correlated significantly (P < 0.001) with the visibility on PETRA-MRA. A receiver operating characteristic curve for the association between the vessel diameter and visibility on PETRA-MRA showed a cutoff value of 1.26 mm for vessel diameter. Cobalt-chromium-nickel-molybdenum clips produced a strong artifact, even on PETRA-MRA. All 4 residual aneurysms were visible on PETRA-MRA. Conclusions: PETRA-MRA can be useful for follow-up aneurysm imaging when the diameter of vessels adjacent to the clip exceeds 1.26 mm. However, its usefulness is limited to titanium clips.

These data present the characteristics of 148 intracerebral hemorrhage (ICH) patients surgically treated. We retrospectively collected data from the medical records of Suwa Red Cross Hospital, including neurological and physiological symptoms, laboratory data, radiological data on admission, complication rate, Glasgow Coma Scale scores on admission or postoperative day 7, and modified Rankin Scale scores at 6 months. Our two articles on the endoscopic hematoma removal and craniotomy for ICH were based on these data [1,2]. This dataset includes detailed laboratory data and radiological features, and it would be useful for reference value for other neurosurgeons or further analysis.