Investigation of Medical Accidents for the Development of Alert System to Reduce Alert Fatigue.

Alert fatigue, a decrease in sensitivity to alerts, is a problem in the medical field. In this study, a survey was conducted on medical accidents in order to develop an alert that could be expected to reduce alert fatigue. As a result, medical accidents related to drugs are common worldwide, and the need for an alert system that can detect the implementation of medical treatment was found.

RAD51 bypasses the CMG helicase to promote replication fork reversal.

Replication fork reversal safeguards genome integrity as a replication stress response. DNA translocases and the RAD51 recombinase catalyze reversal. However, it remains unknown why RAD51 is required and what happens to the replication machinery during reversal. We find that RAD51 uses its strand exchange activity to circumvent the replicative helicase, which remains bound to the stalled fork. RAD51 is not required for fork reversal if the helicase is unloaded. Thus, we propose that RAD51 creates a parental DNA duplex behind the helicase that is used as a substrate by the DNA translocases for branch migration to create a reversed fork structure. Our data explain how fork reversal happens while maintaining the helicase in a position poised to restart DNA synthesis and complete genome duplication.

An Myh11 single lysine deletion causes aortic dissection by reducing aortic structural integrity and contractility.

Pathogenic variants in myosin heavy chain (Myh11) cause familial thoracic aortic aneurysms and dissections (FTAAD). However, the underlying pathological mechanisms remain unclear because of a lack of animal models. In this study, we established a mouse model with Myh11 K1256del, the pathogenic variant we found previously in two FTAAD families. The Myh11∆K/∆K aorta showed increased wall thickness and ultrastructural abnormalities, including weakened cell adhesion. Notably, the Myh11∆K/+ mice developed aortic dissections and intramural haematomas when stimulated with angiotensin II. Mechanistically, integrin subunit alpha2 (Itga2) was downregulated in the Myh11∆K/∆K aortas, and the smooth muscle cell lineage cells that differentiated from Myh11∆K/∆K induced pluripotent stem cells. The contractility of the Myh11∆K/∆K aortas in response to phenylephrine was also reduced. These results imply that the suboptimal cell adhesion indicated by Itga2 downregulation causes a defect in the contraction of the aorta. Consequently, the defective contraction may increase the haemodynamic stress underlying the aortic dissections.

MCMBP promotes the assembly of the MCM2-7 hetero-hexamer to ensure robust DNA replication in human cells.

The MCM2-7 hetero-hexamer is the replicative DNA helicase that plays a central role in eukaryotic DNA replication. In proliferating cells, the expression level of the MCM2-7 hexamer is kept high, which safeguards the integrity of the genome. However, how the MCM2-7 hexamer is assembled in living cells remains unknown. Here, we revealed that the MCM-binding protein (MCMBP) plays a critical role in the assembly of this hexamer in human cells. MCMBP associates with MCM3 which is essential for maintaining the level of the MCM2-7 hexamer. Acute depletion of MCMBP demonstrated that it contributes to MCM2-7 assembly using nascent MCM3. Cells depleted of MCMBP gradually ceased to proliferate because of reduced replication licensing. Under this condition, p53-positive cells exhibited arrest in the G1 phase, whereas p53-null cells entered the S phase and lost their viability because of the accumulation of DNA damage, suggesting that MCMBP is a potential target for killing p53-deficient cancers.

Targeted Protein Depletion Using the Auxin-Inducible Degron 2 (AID2) System.

Targeted protein depletion using a conditional degron is a powerful method to probe the role of proteins in living cells because of the speed with which depletion can be induced and its reversibility. The auxin-inducible degron (AID) is one of the most common degron-based technologies used in cell biology. We recently established an improved system, called AID2, which involves expressing a mutant E3 ligase subunit, OsTIR1(F74G), and fusing a protein of interest to the mini-AID (mAID) tag, and that employs a new and more potent ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA). The AID2 system overcomes some of the drawbacks associated with the original AID system, i.e., leaky degradation without auxin and the requirement of high auxin doses. With AID2 it is, therefore, now possible to control a degron-fused protein more precisely, enabling target proteins to be degraded with a half-life of 10 to 45 min via the addition of a low dose of 5-Ph-IAA. Importantly, in AID2, it is not necessary to control the expression of OsTIR1(F74G) for suppressing leaky degradation and a parental cell line constitutively expressing OsTIR1(F74G) can be used for the generation of multiple mAID-tagged proteins. Here, we describe a protocol for the tagging of endogenous proteins with mAID in diploid HCT116 cells. Our protocol can be applied to other mammalian cell lines and will enhance the utility of AID2 for studying protein functions in living cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Generation of a parental HCT116 cell line expressing OsTIR1(F74G) Basic Protocol 2: Construction of CRISPR and donor plasmids for tagging endogenous genes Basic Protocol 3: Generation of cell lines expressing a protein of interest fused with mAID.

Transitional nystagmus in a Bow Hunter's Syndrome case report.

BACKGROUND: Bow Hunter's Syndrome (BHS) is known as one of cervical diseases which causes vertigo, but the details of its vertigo, especially nystagmus and eye movement, are still incompletely understood. This time, we reported the first case of BHS with a nystagmus chart with video record of transitional nystagmus. CASE PRESENTATION: The patient, a 47-year-old female, complained of vertigo caused by head rotation. When she turned her head leftward, leftward nystagmus appeared, and this was followed by dullness of the right arm. After her head was returned to the central position, downbeat nystagmus appeared, which changed to rightward nystagmus. She was diagnosed with BHS by her symptoms and images. We recorded a nystagmus video and nystagmus chart of this transitional nystagmus including downbeat nystagmus. Her vertigo was cured by the modification of a prescription for her past medical history: hypertension. CONCLUSION: The vertigo of BHS accompanies nystagmus. In this present case, the transitional nystagmus was observed, and it occurred toward the healthy side. Then the nystagmus direction was changed to the affected side via downbeat nystagmus. This is the first report with both a nystagmus chart with video of BHS. Nowadays, various kinds of vertigo induced by neck movement are known. BHS is a rare disease among vertigo diseases, but we should consider it as a different diagnosis of vertigo patients. A precise interview and proper examination are required to make the final diagnosis.

The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice.

Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.

The RIF1-long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress.

Human cells lacking RIF1 are highly sensitive to replication inhibitors, but the reasons for this sensitivity have been enigmatic. Here, we show that RIF1 must be present both during replication stress and in the ensuing recovery period to promote cell survival. Of two isoforms produced by alternative splicing, we find that RIF1-Long alone can protect cells against replication inhibition, but RIF1-Short is incapable of mediating protection. Consistent with this isoform-specific role, RIF1-Long is required to promote the formation of the 53BP1 nuclear bodies that protect unrepaired damage sites in the G1 phase following replication stress. Overall, our observations show that RIF1 is needed at several cell cycle stages after replication insult, with the RIF1-Long isoform playing a specific role during the ensuing G1 phase in damage site protection.

CDCA7 and HELLS suppress DNA:RNA hybrid-associated DNA damage at pericentromeric repeats.

Immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disorder that is caused by mutations in either DNMT3B, ZBTB24, CDCA7, HELLS, or yet unidentified gene(s). Previously, we reported that the CDCA7/HELLS chromatin remodeling complex facilitates non-homologous end-joining. Here, we show that the same complex is required for the accumulation of proteins on nascent DNA, including the DNMT1/UHRF1 maintenance DNA methylation complex as well as proteins involved in the resolution or prevention of R-loops composed of DNA:RNA hybrids and ssDNA. Consistent with the hypomethylation state of pericentromeric repeats, the transcription and formation of aberrant DNA:RNA hybrids at the repeats were increased in ICF mutant cells. Furthermore, the ectopic expression of RNASEH1 reduced the accumulation of DNA damage at a broad range of genomic regions including pericentromeric repeats in these cells. Hence, we propose that hypomethylation due to inefficient DNMT1/UHRF1 recruitment at pericentromeric repeats by defects in the CDCA7/HELLS complex could induce pericentromeric instability, which may explain a part of the molecular pathogenesis of ICF syndrome.

RIF1 controls replication initiation and homologous recombination repair in a radiation dose-dependent manner.

RIF1 controls both DNA replication timing and the DNA double-strand break (DSB) repair pathway to maintain genome integrity. However, it remains unclear how RIF1 links these two processes following exposure to ionizing radiation (IR). Here, we show that inhibition of homologous recombination repair (HRR) by RIF1 occurs in a dose-dependent manner and is controlled via DNA replication. RIF1 inhibits both DNA end resection and RAD51 accumulation after exposure to high doses of IR. Contrastingly, HRR inhibition by RIF1 is antagonized by BRCA1 after a low-dose IR exposure. At high IR doses, RIF1 suppresses replication initiation by dephosphorylating MCM helicase. Notably, the dephosphorylation of MCM helicase inhibits both DNA end resection and HRR, even without RIF1. Thus, our data show the importance of active DNA replication for HRR and suggest a common suppression mechanism for DNA replication and HRR at high IR doses, both of which are controlled by RIF1.This article has an associated First Person interview with the first author of the paper.

Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector.

Conditional degron-based methods are powerful for studying protein function because a degron-fused protein can be rapidly and efficiently depleted by adding a defined ligand. Auxin-inducible degron (AID) is a popular technology by which a degron-fused protein can be degraded by adding an auxin. However, compared with other technologies such as dTAG and HaloPROTAC, AID is complicated because of its two protein components: OsTIR1 and mAID (degron). To simplify the use of AID in mammalian cells, we constructed bicistronic all-in-one plasmids that express OsTIR1 and a mAID-fused protein using a P2A self-cleavage sequence. To generate a HeLa mutant line for the essential replication factor MCM10, we transfected a CRISPR-knockout plasmid together with a bicistronic plasmid containing mAID-fused MCM10 cDNA. After drug selection and colony isolation, we successfully isolated HeLa mutant lines, in which mAID-MCM10 was depleted by the addition of indole-3-acetic acid, a natural auxin. The bicistronic all-in-one plasmids described in this report are useful for controlling degradation of a transgene-derived protein fused with mAID. These plasmids can be used for the construction of conditional mutants by combining them with a CRISPR-based gene knockout.

Control of homologous recombination by the HROB-MCM8-MCM9 pathway.

DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8-MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB-MCM8-MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8-MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.

Analysis of the Stay Time of Patients in Gunma University Heavy Ion Medical Center (GHMC) Using RFID Technology.

We observed the stay time of patients and staff in Gunma University Heavy Ion Medical Center. The stay time of patients with the prostatic cancer and the facing time with radiotherapy technicians in treatment rooms were significantly reduced as times goes by. This decreasing in time has an implication in scheduling algorithm development: for patients. RFID technology can be a potential method to track both staff and patients and thereby to assess the resource utilization efficiency.

Association of Uric Acid with Incident Metabolic Syndrome in a Japanese General Population.

Uric acid is associated with cardiovascular disease (CVD) and its risk factors. Here, we examined the association between the serum uric acid level and incident metabolic syndrome in a Japanese general population. This retrospective, observational study was based on data obtained from an annual health checkup program in Gunma Prefecture, Japan. We evaluated 14,793 participants who did not use antihypertensive or antidiabetic medications and did not present with CVD or metabolic syndrome at the study baseline in 2009. Metabolic syndrome was defined as per the Japanese diagnostic criteria. A discrete proportional hazards regression model was used to evaluate the association between the serum uric acid level at baseline and the incident metabolic syndrome through 2012 and was adjusted for age, gender, waist circumference, systolic and diastolic blood pressure, fasting blood glucose, high-density lipoprotein cholesterol, and triglyceride. At baseline, the average age of the participants was 48.9 years, who were comprised of 40% women. The mean serum uric acid level at baseline was 5.3 ± 1.4 mg/dL. During the three-year follow-up, 7% of the cohort (n = 1,031) developed metabolic syndrome. The uric acid level was strongly associated with incident metabolic syndrome in the multivariable model (adjusted hazard ratio: 1.10; 95% confidence interval, 1.04-1.17; P < 0.01 per 1 mg/dL increase for uric acid). Higher uric acid levels were independently associated with a greater risk of incident metabolic syndrome in a Japanese general population.

Quantitative Analysis of Iodine Image of Dual-energy Computed Tomography at Rest: Comparison With 99mTc-Tetrofosmin Stress-rest Single-photon Emission Computed Tomography Myocardial Perfusion Imaging as the Reference Standard.

PURPOSE: Dual-energy computed tomography (DECT) can be used for visual determination of iodine distribution in the myocardium (iodine image); however, the accuracy and reproducibility of the process remains debatable. Because of the low contrast-to-noise ratio of CT, we hypothesized that quantitative measurement may be more accurate for detecting myocardial ischemia. In this study, we evaluated our quantitative method by comparing it with visual analysis using Tc-tetrofosmin (TF) stress-rest single-photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) as the reference standard. MATERIALS AND METHODS: Forty-three patients who had a significant stenosis on cardiac rest DECT and had received Tc-TF stress-rest SPECT MPI within 1 month were retrospectively analyzed. The regions of interest were set on iodine images in accordance with the American Heart Association (AHA) 17-segment model (a total of 731 segments). The regions of interest values were divided by the amount of iodine (mg) per unit weight (kg) and defined as perfusion value (perfusion value analysis). All segments were also visually analyzed and receiver operating characteristic curve analysis performed to identify the superior analysis. RESULTS: The receiver operating characteristic curve analysis showed that perfusion value analysis is significantly superior to visual analysis [the area under the curve: 0.921 (95% confidence interval, 0.860-0.981) versus 0.685 (95% confidence interval, 0.580-0.791), respectively, P<0.05], with 93.8% sensitivity, 99.1% specificity, 98.9% accuracy, 83.3% positive predictive value, and 99.7% negative predictive value (P<0.01). CONCLUSIONS: Quantitative analysis of the iodine image of rest DECT, called perfusion value analysis, is more accurate than visual analysis when compared with Tc-TF SPECT MPI as the reference standard.

Chromatin modification and NBS1: their relationship in DNA double-strand break repair.

The importance of chromatin modification, including histone modification and chromatin remodeling, for DNA double-strand break (DSB) repair, as well as transcription and replication, has been elucidated. Phosphorylation of H2AX to γ-H2AX is one of the first responses following DSB detection, and this histone modification is important for the DSB damage response by triggering several events, including the accumulation of DNA damage response-related proteins and subsequent homologous recombination (HR) repair. The roles of other histone modifications such as acetylation, methylation and ubiquitination have also been recently clarified, particularly in the context of HR repair. NBS1 is a multifunctional protein that is involved in various DNA damage responses. Its recently identified binding partner RNF20 is an E3 ubiquitin ligase that facilitates the monoubiquitination of histone H2B, a process that is crucial for recruitment of the chromatin remodeler SNF2h to DSB damage sites. Evidence suggests that SNF2h functions in HR repair, probably through regulation of end-resection. Moreover, several recent reports have indicated that SNF2h can function in HR repair pathways as a histone remodeler and that other known histone remodelers can also participate in DSB damage responses. On the other hand, information about the roles of such chromatin modifications and NBS1 in non-homologous end joining (NHEJ) repair of DSBs and stalled fork-related damage responses is very limited; therefore, these aspects and processes need to be further studied to advance our understanding of the mechanisms and molecular players involved.

Corrigendum: Chromatin modification and NBS1: their relationship in DNA double-strand break repair [Genes Genet. Syst. (2015) 90, p. 195-208].

Table 1 on p. 203 should be replaced with the corrected Table 1 shown bellow. .

Functional Role of NBS1 in Radiation Damage Response and Translesion DNA Synthesis.

Nijmegen breakage syndrome (NBS) is a recessive genetic disorder characterized by increased sensitivity to ionizing radiation (IR) and a high frequency of malignancies. NBS1, a product of the mutated gene in NBS, contains several protein interaction domains in the N-terminus and C-terminus. The C-terminus of NBS1 is essential for interactions with MRE11, a homologous recombination repair nuclease, and ATM, a key player in signal transduction after the generation of DNA double-strand breaks (DSBs), which is induced by IR. Moreover, NBS1 regulates chromatin remodeling during DSB repair by histone H2B ubiquitination through binding to RNF20 at the C-terminus. Thus, NBS1 is considered as the first protein to be recruited to DSB sites, wherein it acts as a sensor or mediator of DSB damage responses. In addition to DSB response, we showed that NBS1 initiates Polη-dependent translesion DNA synthesis by recruiting RAD18 through its binding at the NBS1 C-terminus after UV exposure, and it also functions after the generation of interstrand crosslink DNA damage. Thus, NBS1 has multifunctional roles in response to DNA damage from a variety of genotoxic agents, including IR.

Increased oxidative stress in AOA3 cells disturbs ATM-dependent DNA damage responses.

Ataxia telangiectasia (AT) is caused by a mutation in the ataxia-telangiectasia-mutated (ATM) gene; the condition is associated with hyper-radiosensitivity, abnormal cell-cycle checkpoints, and genomic instability. AT patients also show cerebellar ataxia, possibly due to reactive oxygen species (ROS) sensitivity in neural cells. The ATM protein is a key regulator of the DNA damage response. Recently, several AT-like disorders have been reported. The genes responsible for them are predicted to encode proteins that interact with ATM in the DNA-damage response. Ataxia with oculomotor apraxia types 1-3 (AOA1, 2, and 3) result in a neurodegenerative and cellular phenotype similar to AT; however, the basis of this phenotypic similarity is unclear. Here, we show that the cells of AOA3 patients display aberrant ATM-dependent phosphorylation and apoptosis following γ-irradiation. The ATM-dependent response to H2O2 treatment was abrogated in AOA3 cells. Furthermore, AOA3 cells had reduced ATM activity. Our results suggest that the attenuated ATM-related response is caused by an increase in endogenous ROS in AOA3 cells. Pretreatment of cells with pyocyanin, which induces endogenous ROS production, abolished the ATM-dependent response. Moreover, AOA3 cells had decreased homologous recombination (HR) activity, and pyocyanin pretreatment reduced HR activity in HeLa cells. These results indicate that excess endogenous ROS represses the ATM-dependent cellular response and HR repair in AOA3 cells. Since the ATM-dependent cell-cycle checkpoint is an important block to carcinogenesis, such inactivation of ATM may lead to tumorigenesis as well as neurodegeneration.