Mouse Slfn8 and Slfn9 genes complement human cells lacking SLFN11 during the replication stress response.

The Schlafen (SLFN)11 gene has been implicated in various biological processes such as suppression of HIV replication, replication stress response, and sensitization of cancer cells to chemotherapy. Due to the rapid diversification of the SLFN family members, it remains uncertain whether a direct ortholog of human SLFN11 exists in mice. Here we show that mSLFN8/9 and hSLFN11 were rapidly recruited to microlaser-irradiated DNA damage tracks. Furthermore, Slfn8/9 expression could complement SLFN11 loss in human SLFN11-/- cells, and as a result, reduced the growth rate to wild-type levels and partially restored sensitivity to DNA-damaging agents. In addition, both Slfn8/9 and SLFN11 expression accelerated stalled fork degradation and decreased RPA and RAD51 foci numbers after DNA damage. Based on these results, we propose that mouse Slfn8 and Slfn9 genes may share an orthologous function with human SLFN11. This notion may facilitate understanding of SLFN11's biological role through in vivo studies via mouse modeling.

RNF168 E3 ligase participates in ubiquitin signaling and recruitment of SLX4 during DNA crosslink repair.

SLX4/FANCP is a key Fanconi anemia (FA) protein and a DNA repair scaffold for incision around a DNA interstrand crosslink (ICL) by its partner XPF nuclease. The tandem UBZ4 ubiquitin-binding domains of SLX4 are critical for the recruitment of SLX4 to damage sites, likely by binding to K63-linked polyubiquitin chains. However, the identity of the ubiquitin E3 ligase that mediates SLX4 recruitment remains unknown. Using small interfering RNA (siRNA) screening with a GFP-tagged N-terminal half of SLX4 (termed SLX4-N), we identify the RNF168 E3 ligase as a critical factor for mitomycin C (MMC)-induced SLX4 foci formation. RNF168 and GFP-SLX4-N colocalize in MMC-induced ubiquitin foci. Accumulation of SLX4-N at psoralen-laser ICL tracks or of endogenous SLX4 at Digoxigenin-psoralen/UVA ICL is dependent on RNF168. Finally, we find that RNF168 is epistatic with SLX4 in promoting MMC tolerance. We conclude that RNF168 is a critical component of the signal transduction that recruits SLX4 to ICL damage.

SLFN11 promotes stalled fork degradation that underlies the phenotype in Fanconi anemia cells.

Fanconi anemia (FA) is a hereditary disorder caused by mutations in any 1 of 22 FA genes. The disease is characterized by hypersensitivity to interstrand crosslink (ICL) inducers such as mitomycin C (MMC). In addition to promoting ICL repair, FA proteins such as RAD51, BRCA2, or FANCD2 protect stalled replication forks from nucleolytic degradation during replication stress, which may have a profound impact on FA pathophysiology. Recent studies showed that expression of the putative DNA/RNA helicase SLFN11 in cancer cells correlates with cell death on chemotherapeutic treatment. However, the underlying mechanisms of SLFN11-mediated DNA damage sensitivity remain unclear. Because SLFN11 expression is high in hematopoietic stem cells, we hypothesized that SLFN11 depletion might ameliorate the phenotypes of FA cells. Here we report that SLFN11 knockdown in the FA patient-derived FANCD2-deficient PD20 cell line improved cell survival on treatment with ICL inducers. FANCD2-/-SLFN11-/- HAP1 cells also displayed phenotypic rescue, including reduced levels of MMC-induced chromosome breakage compared with FANCD2-/- cells. Importantly, we found that SLFN11 promotes extensive fork degradation in FANCD2-/- cells. The degradation process is mediated by the nucleases MRE11 or DNA2 and depends on the SLFN11 ATPase activity. This observation was accompanied by an increased RAD51 binding at stalled forks, consistent with the role of RAD51 antagonizing nuclease recruitment and subsequent fork degradation. Suppression of SLFN11 protects nascent DNA tracts even in wild-type cells. We conclude that SLFN11 destabilizes stalled replication forks, and this function may contribute to the attrition of hematopoietic stem cells in FA.

USP42 enhances homologous recombination repair by promoting R-loop resolution with a DNA-RNA helicase DHX9.

The nucleus of mammalian cells is compartmentalized by nuclear bodies such as nuclear speckles, however, involvement of nuclear bodies, especially nuclear speckles, in DNA repair has not been actively investigated. Here, our focused screen for nuclear speckle factors involved in homologous recombination (HR), which is a faithful DNA double-strand break (DSB) repair mechanism, identified transcription-related nuclear speckle factors as potential HR regulators. Among the top hits, we provide evidence showing that USP42, which is a hitherto unidentified nuclear speckles protein, promotes HR by facilitating BRCA1 recruitment to DSB sites and DNA-end resection. We further showed that USP42 localization to nuclear speckles is required for efficient HR. Furthermore, we established that USP42 interacts with DHX9, which possesses DNA-RNA helicase activity, and is required for efficient resolution of DSB-induced R-loop. In conclusion, our data propose a model in which USP42 facilitates BRCA1 loading to DSB sites, resolution of DSB-induced R-loop and preferential DSB repair by HR, indicating the importance of nuclear speckle-mediated regulation of DSB repair.

Open-capsule intraocular lens to prevent posterior capsule opacification.

PURPOSE: To develop a single-piece open-capsule intraocular lens (IOL) that can be inserted through a small incision and that prevents posterior capsule opacification (PCO) by expanding the capsule and circulating aqueous humor into the capsular bag. SETTING: Department of Ophthalmology, Dokkyo Medical University, Tochigi, Japan. DESIGN: Experimental study. METHOD: Using the same hydrophobic acrylic material as the NY-60 IOL, a prototype open-capsule IOL was constructed. The IOL has a single optic and 2 haptics, with a 2.8 mm high spacer and holes through which aqueous humor circulates into the capsular bag by separating the anterior capsule from the posterior capsule and expanding the capsule. The open-capsule IOL or NY-60 (as a control group) was inserted in rabbit eyes. Posterior capsule opacification development was evaluated by measuring the thickness of the cell layer at the center of the posterior capsule on histopathologic specimens and statistically comparing the thickness between the open-capsule IOL group and control group. RESULTS: The open-capsule IOL could be inserted through a 3.2 mm corneal incision using a D cartridge. The mean thickness of the cell layer at the center of the posterior capsule was 4.78 µm ± 2.61 (SD) in the open-capsule IOL group and 101.14 ± 25.19 µm in the control group and was significantly smaller in the open-capsule IOL group. CONCLUSION: The prototype single-piece IOL could be implanted through a small incision and prevented PCO by expanding the lens capsule and circulating aqueous humor into the capsular bag.

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype.

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5'-DNA end.

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork.

Defects in homologous recombination repair behind the human diseases: FA and HBOC.

Hereditary breast and ovarian cancer (HBOC) syndrome and a rare childhood disorder Fanconi anemia (FA) are caused by homologous recombination (HR) defects, and some of the causative genes overlap. Recent studies in this field have led to the exciting development of PARP inhibitors as novel cancer therapeutics and have clarified important mechanisms underlying genome instability and tumor suppression in HR-defective disorders. In this review, we provide an overview of the basic molecular mechanisms governing HR and DNA crosslink repair, highlighting BRCA2, and the intriguing relationship between HBOC and FA.

Decreased visual acuity resulting from glistening and sub-surface nano-glistening formation in intraocular lenses: A retrospective analysis of 5 cases.

BACKGROUND: To report on five patients with decreased visual acuity due to glistening and severe sub-surface nano-glistening (SSNG) formation within their intraocular lenses (IOLs). DESIGN: Case reports and analysis of extracted IOLs. PARTICIPANTS AND SAMPLES: We report improved visual acuity when IOLs with severe glistening and SSNG were exchanged for clear IOLs in five patients. METHODS: Case reports. MAIN OUTCOME MEASURES: The main outcome measure was visual acuity. The secondary outcome measure was light transmission. Explanted IOLs were subjected to investigation. Pre- and postoperative slit lamp images of the anterior eye and microscopic images of the extracted IOLs were taken and compared. Light transmission of the IOL was measured using a double beam type spectrophotometer. An integrated value of the percentage light transmittance in the visible light spectrum was calculated. RESULTS: We report on five patients whose visual acuity improved when IOLs were exchanged because of severe glistening and SSNG. All of the affected IOLs were MA60BM (Alcon, Forth Wroth Texas, USA) and the original implantation had occurred over a range of 6-15 years prior to the IOL exchange. Light transmission was decreased in all affected lenses compared to a similar control IOL. CONCLUSIONS: Although only a few reports of cases in which glistening and SSNG have progressed to the level of decreased visual function have been published, the likelihood is that this phenomena will increase as the severity and incidence of these inclusions have been shown to increase with time. Appropriate evaluations of visual function in such patients are needed and consideration should be given to IOL exchange in symptomatic patients.

SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells.

Recent studies have shown that homologous recombination (HR) requires chromatin repression as well as relaxation at DNA double strand breaks (DSBs). HP1 and SUV39H1/2 are repressive factors essential for HR. Here, we identify SETDB1 as an additional compacting factor promoting HR. Depletion of HP1, SUV39, SETDB1 or BRCA1 confer identical phenotypes. The repressive factors, like BRCA1, are dispensable for the initiation of resection but promote the extension step causing diminished RPA or RAD51 foci and HR in irradiated G2 cells. Depletion of the compacting factors does not inhibit BRCA1 recruitment but at 8 h post IR, BRCA1 foci are smaller and aberrantly positioned compared to control cells. BRCA1 promotes 53BP1 repositioning to the periphery of enlarged foci and formation of a devoid core with BRCA1 becoming enlarged and localized internally to 53BP1. Depletion of the compacting factors precludes these changes at irradiation-induced foci. Thus, the repressive factors are required for BRCA1 function in promoting the repositioning of 53BP1 during HR. Additionally, depletion of these repressive factors in undamaged cells causes diminished sister chromatid association at centromeric sequences. We propose a model for how these findings may be functionally linked.

Influence of intraocular lens implantation on anterior capsule contraction and posterior capsule opacification.

PURPOSE: To evaluate whether and how intraocular lens (IOL) implantation influences the development of anterior capsule contraction and posterior capsule opacification (PCO). SETTING: Department of Ophthalmology, Dokkyo Medical University, Mibu, Tochigi, Japan. DESIGN: Experimental study. METHODS: Phacoemulsification was performed in 8-week-old white rabbits. A hydrophobic acrylate IOL (12.5 mm) (YA-60BBR) was implanted in 1 eye and no IOL was implanted in the fellow eye. Slitlamp microscopy and anterior segment analysis were performed to evaluate anterior capsule contraction after the surgery. Four weeks postoperatively, sections of the eyes were made, and the thickness of the proliferated lens epithelial cell (LEC) layer at the posterior capsule was measured to assess the PCO. In addition, LECs from white rabbits were cultured in medium containing 50% aqueous humor or in medium containing 50% saline to determine the influence of the aqueous humor on LECs and to compare the degree of LEC proliferation. RESULTS: Starting 2 weeks after surgery, anterior capsule contraction progressed more significantly in the IOL group than in the group without IOLs. Four weeks postoperatively, LEC thickness at the posterior capsule was significantly less in the group without IOLs than in the IOL group. In the culture study, LEC proliferation was more inhibited in the aqueous humor group than in the saline group. CONCLUSIONS: Progression of anterior capsule contraction and PCO is less likely in aphakic eyes than in IOL-implanted eyes. The mechanism of prevention may involve aqueous humor-induced inhibition of LEC proliferation.

Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection.

In G2 phase cells, DNA double-strand break repair switches from DNA non-homologous end-joining to homologous recombination. This switch demands the promotion of resection. We examine the changes in 53BP1 and RAP80 ionizing radiation induced foci (IRIF) in G2 phase, as these are factors that restrict resection. We observed a 2-fold increase in the volume of 53BP1 foci by 8 h, which is not seen in G1 cells. Additionally, an IRIF core devoid of 53BP1 arises where RPA foci form, with BRCA1 IRIF forming between 53BP1 and replication protein A (RPA). Ubiquitin chains assessed using α-FK2 antibodies are similarly repositioned. Repositioning of all these components requires BRCA1's BRCT but not the ring finger domain. 53BP1, RAP80 and ubiquitin chains are enlarged following POH1 depletion by small interfering RNA, but a devoid core does not form and RPA foci formation is impaired. Co-depletion of POH1 and RAP80, BRCC36 or ABRAXAS allows establishment of the 53BP1 and ubiquitin chain-devoid core. Thus, the barriers posed by 53BP1 and RAP80 are relieved by BRCA1 and POH1, respectively. Analysis of combined depletions shows that these represent distinct but interfacing barriers to promote loss of ubiquitin chains in the IRIF core, which is required for subsequent resection. We propose a model whereby BRCA1 impacts on 53BP1 to allow access of POH1 to RAP80. POH1-dependent removal of RAP80 within the IRIF core enables degradation of ubiquitin chains, which promotes loss of 53BP1. Thus, POH1 represents a novel component regulating the switch from non-homologous end-joining to homologous recombination.

Japanese mothers' breastfeeding knowledge and attitudes assessed by the Iowa Infant Feeding Attitudes Scale.

This study describes Japanese mothers' knowledge and attitudes towards breastfeeding using the Iowa Infant Feeding Attitudes Scale (IIFAS). A cross-sectional survey of 1,612 mothers was conducted in Japan in 2007. The participants were recruited at the free health checks conducted for infants at 18 months of age. The survey was self-administered using the Japanese version of the IIFAS. Descriptive statistics were used to summarise sample characteristics and IIFAS score followed by multiple logistic regression to identify association between total IIFAS score and breastfeeding duration. While the IIFAS showed that the majority recognized some benefits of breastfeeding, their overall knowledge and attitudes towards breastfeeding were neutral and more positive towards the use of infant formula. It is important to provide accurate prenatal education that focuses on methods and long-term benefits of infant feeding to mothers, family and health professionals.

[Development of a new method to estimate patient surface dose distributions in IVR].

Recently, the angiography system used for interventional radiology (IVR) provides a device for measuring dose-area product (DAP), which is compulsory in European countries. The usefulness of DAP is that one can observe patient dose in real time during IVR and can obtain an integral dose by overall IVR procedure without a dosimeter directly placed on a patient. It is important to know the most irradiated region (hot spot) of the patient's skin and its maximum value in the dose management of IVR, but this information cannot be obtained only in DAP. In this paper, we describe a new method to estimate patient surface dose distributions in IVR. We devised a sheet dubbed the "Number map", which does not obstruct the IVR procedure, to confirm the hot spot, and we developed software (named PIETA "Patient Information on Exposure Total Dose Analysis in IVR") to analyze the data from the Number map. Using this system, dose distributions of patient's skin were easily obtained, and we could easily perform patient dose management in IVR.

Caffeine yields aneuploidy through asymmetrical cell division caused by misalignment of chromosomes.

Aneuploidy has been implicated as an important step leading to various neoplasias. Although genetic factors that block aneuploidy have been the subject of intense interest, the impact of pharmacological and environmental substances on the development of aneuploidy has not been studied. Here, we show that caffeine induces aneuploidy through asymmetrical cell division. Mitotic exits of HeLa, U2OS, and primary fibroblast cells were significantly delayed by 10 mmol/L caffeine. Most caffeine-treated mitotic cells showed misalignment of chromosomes at the metaphase plates, and were arrested at prometaphase. Mitoticarrest deficient 2 (MAD2) depletion rescued the caffeine-induced delay of mitotic exit, indicating that caffeine-induced prolongation of mitosis was caused by activation of a MAD2-dependent spindle checkpoint. Enumeration of centromeres by fluorescence in situ hybridization revealed that cell division in the presence of caffeine was not symmetrical and resulted in aneuploid cell production. Most of these cells survived and underwent DNA synthesis. Our findings reveal a novel pharmacological effect of a high concentration of caffeine on genomic stability in dividing cells.

Preventing secondary cataract and anterior capsule contraction by modification of intraocular lenses.

Advances in intraocular lens (IOL) design have led to the use of lenses with improved performance including tinting, asphericity, multifocality and accommodation. To maximize the visual performance of these IOLs, postoperative complications such as secondary cataracts and anterior capsule contraction must be prevented. Various types of secondary cataracts may occur, each associated with complex biological reactions. Different design configurations, including square-edge IOLs, have been used to prevent secondary cataracts and anterior capsule contraction, but these have not been successful in sufficiently eliminating these complications. We have found that surface modification of IOL surfaces chemically improves surface quality and is also useful in preventing secondary cataracts and anterior capsule contraction. The UV/ozone treatment that we used as a surface modification method is a simple and highly effective method with no safety issues regarding materials following treatment. The combination of square-edge design and surface modification may be able to completely eliminate these postoperative complications.

Early G2/M checkpoint failure as a molecular mechanism underlying etoposide-induced chromosomal aberrations.

Topoisomerase II (Topo II) inhibitors are cell cycle-specific DNA-damaging agents and often correlate with secondary leukemia with chromosomal translocations involving the mixed-lineage leukemia/myeloid lymphoid leukemia (MLL) gene on chromosome 11 band q23 (11q23). In spite of the clinical importance, the molecular mechanism for this chromosomal translocation has yet to be elucidated. In this study, we employed 2-color FISH and detected intracellular chromosomal translocations induced by etoposide treatment. Cells such as ataxia-telangiectasia mutated-deficient fibroblasts and U2OS cells, in which the early G2/M checkpoint after treatment with low concentrations of etoposide has been lost, executed mitosis with etoposide-induced DNA double-strand breaks, and 2-color FISH signals located on either side of the MLL gene were segregated in the postmitotic G1 phase. Long-term culture of cells that had executed mitosis under etoposide treatment showed frequent structural abnormalities of chromosome 11. These findings provide convincing evidence for Topo II inhibitor-induced 11q23 translocation. Our study also suggests an important role of the early G2/M checkpoint in preventing fixation of chromosomal abnormalities and reveals environmental and genetic risk factors for the development of chromosome 11 translocations, namely, low concentrations of Topo II inhibitors and dysfunctional early G2/M checkpoint control.

Detection of ATM gene mutation in human glioma cell line M059J by a rapid frameshift/stop codon assay in yeast.

A yeast-based frameshift/stop codon assay for examining ATM (ataxia telangiectasia mutated) mutations was established. Each of six fragments of a PCR-amplified coding sequence for ATM is inserted in frame by homologous recombination into a yeast URA3 fusion protein gene, and the transformants are assayed for growth in the absence of uracil. The usefulness of this assay was verified in a panel of cell lines derived from individuals with homozygous and heterozygous ATM mutations. The assay was also shown to distinguish between specimens with wild-type alleles and those with truncating mutations: a frameshift mutation or an inserted stop codon. Using this assay M059J cells, which fail to express the catalytic subunit of DNA-dependent protein kinase (PRKDC, also known as DNA-PKcs) and are hypersensitive to ionizing radiation, were found to express two different aberrant ATM transcripts: one characterized by 4776 del 133, which corresponds to the deletion of exon 33, and the other by 4909 ins 116. Subsequent analysis of the intron sequences revealed that 4909 ins 116 is comprised of a nucleotide sequence corresponding to 84013-84128 in intron 33 with a cryptic splice site. Thus the radiosensitive phenotype of M059J cells appears to be due to a defect in PRKDC and a truncating ATM mutation.