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.

Fine-tuning of DNA damage-dependent ubiquitination by OTUB2 supports the DNA repair pathway choice.

DNA double-strand breaks (DSBs) are deleterious lesions that lead to genetic mutations and cell death. Protein ubiquitination mediated by the E3 ubiquitin ligase RNF8 within the regions surrounding DSBs recruits DNA DSB response (DDR) factors and induces chromatin remodeling, which supports cell survival after DNA damage. Nevertheless, the impact of RNF8-mediated ubiquitination on DNA repair remains to be elucidated. Here, we report that depletion of the deubiquitinating enzyme OTUB2 enhances RNF8-mediated ubiquitination in an early phase of the DDR and promotes faster DSB repair but suppresses homologous recombination. The rapid ubiquitination results in accelerated accumulation of 53BP1 and RAP80 at DSBs, which in turn protects DSB ends from resection in OTUB2-depleted cells. Mechanistically, OTUB2 suppresses RNF8-mediated L3MBTL1 ubiquitination and Lys 63-linked ubiquitin chain formation in a deubiquitinating activity-dependent manner. Thus, OTUB2 fine-tunes the speed of DSB-induced ubiquitination so that the appropriate DNA repair pathway is chosen.

Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair.

CRISPR/Cas9, which generates DNA double-strand breaks (DSBs) at target loci, is a powerful tool for editing genomes when codelivered with a donor DNA template. However, DSBs, which are the most deleterious type of DNA damage, often result in unintended nucleotide insertions/deletions (indels) via mutagenic nonhomologous end joining. We developed a strategy for precise gene editing that does not generate DSBs. We show that a combination of single nicks in the target gene and donor plasmid (SNGD) using Cas9D10A nickase promotes efficient nucleotide substitution by gene editing. Nicking the target gene alone did not facilitate efficient gene editing. However, an additional nick in the donor plasmid backbone markedly improved the gene-editing efficiency. SNGD-mediated gene editing led to a markedly lower indel frequency than that by the DSB-mediated approach. We also show that SNGD promotes gene editing at endogenous loci in human cells. Mechanistically, SNGD-mediated gene editing requires long-sequence homology between the target gene and repair template, but does not require CtIP, RAD51, or RAD52. Thus, it is considered that noncanonical homology-directed repair regulates the SNGD-mediated gene editing. In summary, SNGD promotes precise and efficient gene editing and may be a promising strategy for the development of a novel gene therapy approach.

ALKBH8 promotes bladder cancer growth and progression through regulating the expression of survivin.

Human AlkB homolog 8 (ALKBH8) is highly expressed in high-grade, superficially and deeply invasive bladder cancer. Moreover, ALKBH8 knockdown induces apoptosis in bladder cancer cells. However, the underlying anti-apoptotic mechanism of ALKBH8 in bladder cancer cells has thus far remained unclear. Moreover, there is no direct evidence that highly expressed ALKBH8 is involved in tumor progression in vivo. We here show that ALKBH8 knockdown induced apoptosis via downregulating the protein expression of survivin, an anti-apoptotic factor also exhibiting increased levels in bladder cancer. We also clarify that ALKBH8 transgenic mice showed an accelerated rate of bladder tumor mass and invasiveness in an N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced bladder cancer model. These findings suggest that the high expression of ALKBH8 is critical for the growth and progression of bladder cancer.

Evolutionary changes of multiple visual pigment genes in the complete genome of Pacific bluefin tuna.

Tunas are migratory fishes in offshore habitats and top predators with unique features. Despite their ecological importance and high market values, the open-ocean lifestyle of tuna, in which effective sensing systems such as color vision are required for capture of prey, has been poorly understood. To elucidate the genetic and evolutionary basis of optic adaptation of tuna, we determined the genome sequence of the Pacific bluefin tuna (Thunnus orientalis), using next-generation sequencing technology. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified five common fish visual pigment genes: red-sensitive (middle/long-wavelength sensitive; M/LWS), UV-sensitive (short-wavelength sensitive 1; SWS1), blue-sensitive (SWS2), rhodopsin (RH1), and green-sensitive (RH2) opsin genes. Sequence comparison revealed that tuna's RH1 gene has an amino acid substitution that causes a short-wave shift in the absorption spectrum (i.e., blue shift). Pacific bluefin tuna has at least five RH2 paralogs, the most among studied fishes; four of the proteins encoded may be tuned to blue light at the amino acid level. Moreover, phylogenetic analysis suggested that gene conversions have occurred in each of the SWS2 and RH2 loci in a short period. Thus, Pacific bluefin tuna has undergone evolutionary changes in three genes (RH1, RH2, and SWS2), which may have contributed to detecting blue-green contrast and measuring the distance to prey in the blue-pelagic ocean. These findings provide basic information on behavioral traits of predatory fish and, thereby, could help to improve the technology to culture such fish in captivity for resource management.

[Clinical evaluation of the risk factors for liver abscess after TACE or RFA].

Radiofrequency ablation(RFA)and transcatheter arterial chemoembolization (TACE) are widely enforced as a standard combined therapy for liver cancer. Liver abscess occurs occasionally as a complication. This clinical study was conducted to determine risk factors for liver abscess. We investigated the clinical background of 10 cases complicated by liver abscess in 957 cases of patients who underwent TACE or RFA for liver cancer at Minoh City Hospital between April 2002 and March 2012. Risk factors for liver abscess were analyzed statistically in comparison to a control group without liver abscess. Diabetes and a history of biliary tract organic disease were statistically significant independent risk factors determined by multivariate analysis. We consider patients with a history of biliary tract organic disease, or who have a potential biliary tract infection, and diabetes, to be susceptible to infection. A case presenting with diabetes and a history of biliary tract disease is in a high-risk group, so treatment with TACE or RFA for such cases should be considered carefully.

Prevalence of red sea bream iridovirus among organs of Japanese amberjack (Seriola quinqueradiata) exposed to cultured red sea bream iridovirus.

Red sea bream iridovirus (RSIV) is a representative of the genus Megalocytivirus which causes severe disease to aquaculture fish, mainly in Japan and South-east Asia. However, information to assess the viral kinetics of RSIV in fish is limited since reports on experimental infection by the immersion route, which is the natural infection route, are scarce. In this study, a method to evaluate the titre of RSIV was first developed. Experimental infections were continuously performed using RSIV cell culture as the inoculum to juvenile Japanese amberjack (Seriola quinqueradiata) (initial body weight 12.2 g) by immersion at three different concentrations. In addition, to investigate the prevalence of the virus among the organs of experimentally infected fish, viral DNA was measured at selected times by the real-time PCR method following viral inoculation by immersion. The developed titration method showed a 10(2) increase in sensitivity compared with the conventional method. We demonstrated that grunt fin cells can be used for continuous passage of RSIV. In the experimental infection, fish which were intraperitoneally injected with the RSIV cell culture or immersed with RSIV cell culture at 10(-2) and 10(-3) dilutions showed cumulative mortalities of 100 %. The results of measurements of the viral DNA of several organs from infected fish strongly suggest that the spleen is the target organ of RSIV in Japanese amberjack. Since the viral genome was detected from all the tested organs of two of five surviving fish which appeared to completely recover from the disease, it is suggested that these fish may become carriers.

Effect of acoustic noise reduction technology on image quality: a multivendor study.

The purpose of this study was to clarify the appropriate use of a combination of pulse sequences and acoustic noise reduction technology in general-purpose brain magnetic resonance imaging. Five pulse sequences commonly used in brain magnetic resonance imaging examinations-turbo spin-echo T2-weighted imaging, T1-weighted fluid-attenuated inversion recovery, T2-weighted fluid-attenuated inversion recovery, diffusion-weighted imaging, and magnetic resonance angiography-were performed on healthy participants at three vendors where acoustic noise reduction technology was available. The results showed that acoustic noise reduction technology reduced sound pressure levels and altered image quality in all pulse sequences across all vendors' magnetic resonance imaging scanners. Although T2-weighted imaging and T1-weighted fluid-attenuated inversion recovery resulted in little image quality degradation, T2-weighted fluid-attenuated inversion recovery, diffusion-weighted imaging, and magnetic resonance angiography had significant image degradation. Therefore, acoustic noise reduction technology should be used with caution.

Evaluation of magnetic resonance imaging acoustic noise reduction technology by magnetic gradient waveform control.

BACKGROUND: ComforTone is a noise reduction technology used in magnetic resonance imaging (MRI) systems; it suppresses acoustic noise by modifying pulse sequences, which appropriately changes the magnetic field gradient waveforms. Although ComforTone can be used to solve the acoustic noise problems that affect patients who are exposed to acoustic noise from MRI, to the best of our knowledge, the associated technical details have not been published and its effects on acoustic noise reduction remain unclear. PURPOSE: To evaluate the efficacy of acoustic noise reduction and the impact of acoustic noise reduction technology involving magnetic field gradient waveform control on image quality. POPULATION: The study included 18 healthy volunteers (11 males and 7 females; median age, 34 years; age range, 24-51 years). FIELD STRENGTH: 1.5 T Philips Ingenia using a SENSE head-spine coil. ASSESSMENT: The sound pressure level (SPL) and 1/3 octave spectra of MRI acoustic noise with the human head positioned in the iso-center of the MRI system were measured for five different pulse sequences used in clinical MRI. This subjective evaluation of noise included 18 healthy volunteers. The degree of discomfort experienced by the subjects was measured using a visual analog scale. The image quality was assessed objectively and subjectively. For objective assessment, signal-to-noise ratio (SNR) and contrast-to -noise ratio (CNR) of diffusion-weighted images were measured; for subjective assessment, visual evaluation was performed by two radiologists. STATISTICAL TESTS: Data were analyzed using Welch's t-test, and a p value <0.05 defined significance. RESULTS: ComforTone could recognize a decrease in sound pressure, and the sound pressure of the acute high-frequency portion of the auditory characteristics was reduced. As reported by the subjects, discomfort caused by the sound pressure was significantly alleviated with ComforTone (p < 0.01). The sound pressure reduction in the high-frequency region with high audibility characteristics was recognized by ComforTone. The visual evaluation of the image quality of the diffusion-weighted images revealed that although there was no difference between SNR and CNR, the image quality was reduced by distortion artifacts. DATA CONCLUSION: ComforTone reduced the SPL in the frequency range where auditory characteristics were sensitive, suggesting that ComforTone was useful for auditory protection and alleviation of discomfort in patients undergoing MRI. However, because magnetic field gradient waveform control is involved, such noise-reducing techniques should be used by considering their possible influence on the image quality.

Broad ligament hernia successfully repaired by single-incision laparoscopy: A case report.

A 52-year-old woman with a history of two parturitions presented with lower abdominal pain. Multi-detector CT of the abdomen showed discontinuity of the sigmoid colon near the broad ligament on the left side. We assigned a provisional diagnosis of an internal hernia progressing through a defect in the broad ligament. SILS revealed a total broad ligament defect on the left side but no signs of ischemic, necrotic bowel. We successfully repaired the broad ligament defect with suturing. At the 2-month follow-up, the patient remained well with no signs of recurrence. This case appears to be the first report of a broad ligament hernia successfully diagnosed and repaired by SILS.

[Red sea bream iridoviral disease].

The first outbreak of red sea bream iridoviral disease caused by red sea bream iridovirus (RSIV) was recorded in cultured red sea bream Pagrus major in Shikoku Island, Japan in 1990. Since 1991, the disease has caused mass mortalities of cultured marine fishes not only red sea bream but also many other species. The affected fish were lethargic and exhibited severe anemia, petechiae of the gills, and enlargement of the spleen. The causative agent was a large, icosahedral, cytoplasmic DNA virus classified as a member of the family Iridoviridae and was designated as red sea bream iridovirus (RSIV). The genome of RSIV is liner dsDNA and considered to be circularly permitted and terminally redundant like other iridoviruses. The length of physical map of RSIV genome is 112,415bp. An indirect immunofluorescence test with a monoclonal antibody and PCR are commonly used for the rapid diagnosis of RSIV infected fish in the field. For the control of this disease, a formalin-killed vaccine against red sea bream iridoviral disease was developed and now commercially available.

The first symbiont-free genome sequence of marine red alga, Susabi-nori (Pyropia yezoensis).

Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis) using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb), which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35%) are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae.

Megalocytiviruses.

The genus Megalocytivirus, represented by red sea bream iridovirus (RSIV), the first identified and one of the best characterized megalocytiviruses, Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus, and numerous other isolates, is the newest genus within the family Iridoviridae. Viruses within this genus are causative agents of severe disease accompanied by high mortality in multiple species of marine and freshwater fish. To date outbreaks of megalocytivirus-induced disease have occurred primarily in south-east Asia and Japan, but infections have been detected in Australia and North America following the importation of infected ornamental fish. The first outbreak of megalocytiviral disease was recorded in cultured red sea bream (Pagrus major) in Japan in 1990 and was designated red sea bream iridovirus disease (RSIVD). Following infection fish became lethargic and exhibited severe anemia, petechiae of the gills, and enlargement of the spleen. Although RSIV was identified as an iridovirus, sequence analyses of RSIV genes revealed that the virus did not belong to any of the four known genera within the family Iridoviridae. Thus a new, fifth genus was established and designated Megalocytivirus to reflect the characteristic presence of enlarged basophilic cells within infected organs. Indirect immunofluorescence tests employing recently generated monoclonal antibodies and PCR assays are currently used in the rapid diagnosis of RSIVD. For disease control, a formalin-killed vaccine was developed and is now commercially available in Japan for several fish species. Following the identification of RSIV, markedly similar viruses such as infectious spleen and kidney necrosis virus (ISKNV), dwarf gourami iridovirus (DGIV), turbot reddish body iridovirus (TRBIV), Taiwan grouper iridovirus (TGIV), and rock bream iridovirus (RBIV) were isolated in East and Southeast Asia. Phylogenetic analyses of the major capsid protein (MCP) and ATPase genes indicated that although these viruses shared considerable sequence identity, they could be divided into three tentative species, represented by RSIV, ISKNV and TRBIV, respectively. Whole genome analyses have been reported for several of these viruses. Sequence analysis detected a characteristic difference in the genetic composition of megalocytiviruses and other members of the family in reference to the large and small subunits of ribonucleotide reductase (RR-1, RR­2). Megalocytiviruses contain only the RR-2 gene, which is of eukaryotic origin; whereas the other genera encode both the RR-1 and RR-2 genes which are thought to originate from Rickettsia-like α-proteobacteria.

Is a large fibroid a high-risk factor for uterine artery embolization?

OBJECTIVE: The objective of our study was to determine whether tumor size, specifically uterine fibroids of 10 cm or larger, predisposes a patient to an unacceptably high risk at uterine artery embolization. MATERIALS AND METHODS: One hundred fifty-two consecutive women underwent embolization for uterine fibroids. Complications and outcomes were analyzed using questionnaires and serial MRI between women with one or more uterine fibroids of 10 cm or larger diameter (mean, 12.4 cm; range, 10-19 cm) (n = 47, group 1) and women with each uterine fibroid of less than 10 cm diameter (mean, 6.8 cm; range, 2-9.5 cm) (n = 105, group 2). RESULTS: Thirty complications (19.7%, 30/152), which occurred in 27 women (17.8%, 27/152), were noted. However, 25 of 30 complications were minor, requiring no or nominal therapy. They occurred in 19.1% (9/47) of group 1 and in 15.2% (16/105) of group 2 women (p = 0.637). Major complications requiring major therapy, unplanned increased level of care, or unanticipated prolonged hospitalization (> 48 hr) or including permanent adverse sequelae were noted in 6.4% (3/47) of group 1 and in 1.9% (2/105) of group 2 women (p = 0.172). Of these five women, four underwent surgery because of sloughing fibroids. Permanent adverse sequelae were observed in one woman of group 1, who has had sexual dysfunction after embolization. No deaths occurred in either group. There was no significant difference in most outcomes or in intervals until the complete disappearance of postprocedural pain and full recovery between the two groups. CONCLUSION: We found no increased risk to patients undergoing uterine artery embolization for fibroids on the basis of tumor size. Successful outcomes can be obtained for such lesions.

Uterine artery embolization using gelatin sponge particles alone for symptomatic uterine fibroids: midterm results.

OBJECTIVE: The purpose of this study was to assess the safety and effectiveness of uterine artery embolization using gelatin sponge particles alone for women with symptomatic uterine fibroids. SUBJECTS AND METHODS: During 38 months, 60 patients (age range, 32-52 years; mean age, 42.5 years) with symptomatic uterine fibroids underwent uterine artery embolization. Only gelatin sponge particles, approximately 500-1000 microm in diameter, were used in all patients. The improvement of clinical symptoms was assessed by questionnaire. Reduction of the largest tumor and uterine volume reductions were assessed using MR imaging. The follow-up period ranged from 1 to 38 months (mean, 10.6 months). RESULTS: Menorrhagia improved markedly or moderately in 41 (98%) of 42 of patients 4 months after embolization and in 20 (100%) of 20 patients 1 year after embolization. Bulk-related symptoms improved markedly or moderately in 31 (97%) of 32 of patients 4 months after embolization and in 19 (100%) of 19 of patients 1 year after embolization. MR imaging revealed that the mean largest tumor volume reduction rates were 55% at 4 months and 70% at 1 year after embolization, and the mean uterine volume reduction rates were 40% at 4 months and 56% at 1 year after embolization. Follow-up MR imaging showed no new fibroids and no regrowth of existing fibroids. No major complications were observed in any women. CONCLUSION: We suggest that uterine artery embolization with gelatin sponge particles alone is a safe and effective treatment for symptomatic fibroids. The outcomes bear comparison with those of uterine artery embolization using polyvinyl alcohol particles, which have been reported in the literature.

Uterine leiomyoma after embolization by means of gelatin sponge particles alone: report of a case with histopathologic features.

We describe the histopathologic features of uterine leiomyoma after uterine artery embolization (UAE) in a 42-year-old woman. This patient, who was taking antiplatelet drugs for the treatment of cerebral disease, successfully underwent UAE using only gelatin sponge particles for a symptomatic uterine leiomyoma. Although menorrhagia improved moderately after the procedure, she underwent abdominal hysterectomy 11 months later because of recurrent uterine bleeding. Histopathology revealed that most of the area of the uterine leiomyoma was characterized by extensive coagulation necrosis, which support the positive result of the procedure. No significant abnormalities were noted in either the myometrium or endometrium, which also suggested that UAE using only gelatin sponge particles is an appropriate procedure to preserve the uterus. The histologic and radiologic features of this case are discussed. To the best of our knowledge, this is the first reported case of uterine leiomyoma after UAE using only gelatin sponge particles as a primary embolic agent.