Cross-cultural adaption, validity, and reliability of the Japanese version of the Central Aspects of Pain in the Knee (CAP-Knee-J) questionnaire in patients with knee pain: a validation study.

BACKGROUND: Knee pain is a prominent concern among older individuals, influenced by the central nervous system. This study aimed to translate the Central Aspects of Pain in the Knee (CAP-Knee) questionnaire into Japanese and investigate its reliability and validity in older Japanese individuals with knee pain. METHODS: Using a forward-backward method, CAP-Knee was translated into Japanese, and data from 110 patients at an orthopedic clinic were analyzed. The Japanese version (CAP-Knee-J) was evaluated regarding pain intensity during walking, central sensitization inventory, and pain catastrophizing scale. Statistical analyses confirmed internal validity and test-retest reliability. Concurrent validity was assessed through a single correlation analysis between CAP-Knee-J and the aforementioned measures. Exploratory factor analysis was employed on each CAP-Knee-J item to examine structural validity. RESULTS: CAP-Knee-J showed good internal consistency (Cronbach's α = 0.86) and excellent test-retest reliability (intraclass correlation coefficient = 0.77). It correlated significantly with pain intensity while walking, central sensitization inventory scores, and pain catastrophizing scale scores. Exploratory factor analysis produced a three-factor model. CONCLUSIONS: CAP-Knee-J is a reliable and valid questionnaire for assessing central pain mechanisms specific to knee pain in older Japanese individuals, with moderate correlations with the CSI and weak with the PCS, thus indicating construct validity. This study supports the development of effective knee pain treatments and prognosis predictions.

Age- and sex-related oculomotor manifestation of dopamine deficiency in Segawa disease.

OBJECTIVE: To investigate the oculomotor manifestations of Segawa disease (SD), considered to represent mild dopamine deficiency and discuss their pathophysiological basis. METHODS: We recorded visually- (VGS) and memory-guided saccade (MGS) tasks in 31 SD patients and 153 age-matched control subjects to study how basal ganglia (BG) dysfunction in SD evolves with age for male and female subjects. RESULTS: SD patients were impaired in initiating MGS, showing longer latencies with occasional failure. Patients showed impaired ability to suppress reflexive saccades; saccades to cues presented in MGS were more frequent and showed a shorter latency than in control subjects. These findings were more prominent in male patients, particularly between 13 and 25 years. Additionally, male patients showed larger delay in MGS latency in trials preceded by saccades to cue than those unpreceded. CONCLUSIONS: The findings can be explained by a dysfunction of the BG-direct pathway impinging on superior colliculus (SC) due to dopamine deficiency. The disturbed inhibitory control of saccades may be explained by increased SC responsivity to visual stimuli. SIGNIFICANCE: Oculomotor abnormalities in SD can be explained by dysfunction of the BG inhibitory pathways reaching SC, with a delayed maturation in male SD patients, consistent with previous pathological/physiological studies.

A Rare Case of Meningitis Caused by Streptococcus gallolyticus subsp. pasteurianus in an Immunocompetent Young Adult.

Bacterial meningitis is a life-threatening condition that is mainly caused by Streptococcus pneumoniae and Neisseria meningitis. Although Streptococcus gallolyticus subsp. pasteurianus (Sgp) is also known to cause meningitis, its frequency is quite low, especially in adults. We herein report the first immunocompetent Japanese adult patient (20-year-old woman) with bacterial meningitis caused by Sgp. The patient showed dramatic improvement after antibiotic treatment. Although previous reports have described an association between Sgp infection and an immunosuppressive status, bowel and hepatobiliary diseases, or strongyloidiasis, our case did not demonstrate any of these conditions, suggesting that Sgp can cause meningitis even in young immunocompetent adults.

Absolute quantification of DNA damage response proteins.

BACKGROUND: DNA damage response (DDR) and repair are vital for safeguarding genetic information and ensuring the survival and accurate transmission of genetic material. DNA damage, such as DNA double-strand breaks (DSBs), triggers a response where sensor proteins recognize DSBs. Information is transmitted to kinases, initiating a sequence resulting in the activation of the DNA damage response and recruitment of other DDR and repair proteins to the DSB site in a highly orderly sequence. Research has traditionally focused on individual protein functions and their order, with limited quantitative analysis, prompting this study's attempt at absolute quantification of DNA damage response and repair proteins and capturing changes in protein chromatin affinity after DNA damage through biochemical fractionation methods. RESULTS: To assess the intracellular levels of proteins involved in DDR and repair, multiple proteins associated with different functions were quantified in EPC2-hTERT cells. H2AX had the highest intracellular abundance (1.93 × 106 molecules/cell). The components of the MRN complex were present at the comparable levels: 6.89 × 104 (MRE11), 2.17 × 104 (RAD50), and 2.35 × 104 (NBS1) molecules/cell. MDC1 was present at 1.27 × 104 molecules/cell. The intracellular levels of ATM and ATR kinases were relatively low: 555 and 4860 molecules/cell, respectively. The levels of cellular proteins involved in NHEJ (53BP1: 3.03 × 104; XRCC5: 2.62 × 104; XRCC6: 5.05 × 105 molecules/cell) were more than an order of magnitude higher than that involved in HR (RAD51: 2500 molecules/cell). Furthermore, we analyzed the dynamics of MDC1 and γH2AX proteins in response to DNA damage induced by the unstable agent neocarzinostatin (NCS). Using cell biochemical fractionation, cells were collected and analyzed at different time points after NCS exposure. Results showed that γH2AX in chromatin fraction peaked at 1 h post-exposure and gradually decreased, while MDC1 translocated from the isotonic to the hypertonic fraction, peaking at 1 hour as well. The study suggests increased MDC1 affinity for chromatin through binding to γH2AX induced by DNA damage. The γH2AX-bound MDC1 (in the hypertonic fraction) to γH2AX ratio at 1 h post-exposure was 1:56.4, with lower MDC1 levels which may attributed to competition with other proteins. CONCLUSIONS: The approach provided quantitative insights into protein dynamics in DNA damage response.

The Pathophysiology of Gilles de la Tourette Syndrome: Changes in Saccade Performance by Low-Dose L-Dopa and Dopamine Receptor Blockers.

AIM: To elucidate the pathophysiology of Gilles de la Tourette syndrome (GTS), which is associated with prior use of dopamine receptor antagonists (blockers) and treatment by L-Dopa, through saccade performance. METHOD: In 226 male GTS patients (5-14 years), we followed vocal and motor tics and obsessive-compulsive disorder (OCD) after discontinuing blockers at the first visit starting with low-dose L-Dopa. We recorded visual- (VGS) and memory-guided saccades (MGS) in 110 patients and 26 normal participants. RESULTS: At the first visit, prior blocker users exhibited more severe vocal tics and OCD, but not motor tics, which persisted during follow-up. Patients treated with L-Dopa showed greater improvement of motor tics, but not vocal tics and OCD. Patients with and without blocker use showed similarly impaired MGS performance, while patients with blocker use showed more prominently impaired inhibitory control of saccades, associated with vocal tics and OCD. DISCUSSION: Impaired MGS performance suggested a mild hypodopaminergic state causing reduced direct pathway activity in the (oculo-)motor loops of the basal ganglia-thalamocortical circuit. Blocker use may aggravate vocal tics and OCD due to disinhibition within the associative and limbic loops. The findings provide a rationale for discouraging blocker use and using low-dose L-Dopa in GTS.

Abnormal saccade profiles in hereditary spinocerebellar degeneration reveal cerebellar contribution to visually guided saccades.

OBJECTIVE: To study how the pathophysiology underlying hereditary spinocerebellar degeneration (spinocerebellar ataxia; SCA) with pure cerebellar manifestation evolves with disease progression using saccade recordings. METHODS: We recorded visually- (VGS) and memory-guided saccade (MGS) task performance in a homogeneous population of 20 genetically proven SCA patients (12 SCA6 and eight SCA31 patients) and 19 normal controls. RESULTS: For VGS but not MGS, saccade latency and amplitude were increased and more variable than those in normal subjects, which correlated with cerebellar symptom severity assessed using the International Cooperative Ataxia Rating Scale (ICARS). Parameters with significant correlations with cerebellar symptoms showed an aggravation after disease stage progression (ICARS > 50). The saccade velocity profile exhibited shortened acceleration and prolonged deceleration, which also correlated with disease progression. The main sequence relationship between saccade amplitude and peak velocity as well as saccade inhibitory control were preserved. CONCLUSIONS: The cerebellum may be involved in initiating VGS, which was aggravated acutely during disease stage progression. Dysfunction associated with disease progression occurs mainly in the cerebellum and brainstem interaction but may also eventually involve cortical saccade processing. SIGNIFICANCE: Saccade recording can reveal cerebellar pathophysiology underlying SCA with disease progression.

Early detection of cognitive decline in Alzheimer's disease using eye tracking.

Background: Patients with Alzheimer's disease (AD) are known to exhibit visuospatial processing impairment, as reflected in eye movements from the early stages of the disease. We investigated whether the pattern of gaze exploration during visual tasks could be useful for detecting cognitive decline at the earliest stage. Methods: Sixteen AD patients (age: 79.1 ± 7.9 years, Mini Mental State Examination [MMSE] score: 17.7 ± 5.3, mean ± standard deviation) and 16 control subjects (age: 79.4 ± 4.6, MMSE score: 26.9 ± 2.4) participated. In the visual memory task, subjects memorized presented line drawings for later recall. In the visual search tasks, they searched for a target Landolt ring of specific orientation (serial search task) or color (pop-out task) embedded among arrays of distractors. Using video-oculography, saccade parameters, patterns of gaze exploration, and pupil size change during task performance were recorded and compared between AD and control subjects. Results: In the visual memory task, the number of informative regions of interest (ROIs) fixated was significantly reduced in AD patients compared to control subjects. In the visual search task, AD patients took a significantly longer time and more saccades to detect the target in the serial but not in pop-out search. In both tasks, there was no significant difference in the saccade frequency and amplitude between groups. On-task pupil modulation during the serial search task was decreased in AD. The number of ROIs fixated in the visual memory task and search time and saccade numbers in the serial search task differentiated both groups of subjects with high sensitivity, whereas saccade parameters of pupil size modulation were effective in confirming normal cognition from cognitive decline with high specificity. Discussion: Reduced fixation on informative ROIs reflected impaired attentional allocation. Increased search time and saccade numbers in the visual search task indicated inefficient visual processing. Decreased on-task pupil size during visual search suggested decreased pupil modulation with cognitive load in AD patients, reflecting impaired function of the locus coeruleus. When patients perform the combination of these tasks to visualize multiple aspects of visuospatial processing, cognitive decline can be detected at an early stage with high sensitivity and specificity and its progression be evaluated.

Discovery of a new pyrimidine synthesis inhibitor eradicating glioblastoma-initiating cells.

BACKGROUND: Glioblastoma-initiating cells (GICs) comprise a tumorigenic subpopulation of cells that are resistant to radio- and chemotherapies and are responsible for cancer recurrence. The aim of this study was to identify novel compounds that specifically eradicate GICs using a high throughput drug screening approach. METHODS: We performed a cell proliferation/death-based drug screening using 10 560 independent compounds. We identified dihydroorotate dehydrogenase (DHODH) as a target protein of hit compound 10580 using ligand-fishing and mass spectrometry analysis. The medical efficacy of 10580 was investigated by in vitro cell proliferation/death and differentiation and in vivo tumorigenic assays. RESULTS: Among the effective compounds, we identified 10580, which induced cell cycle arrest, decreased the expression of stem cell factors in GICs, and prevented tumorigenesis upon oral administration without any visible side effects. Mechanistic studies revealed that 10580 decreased pyrimidine nucleotide levels and enhanced sex determining region Y-box 2 nuclear export by antagonizing the enzyme activity of DHODH, an essential enzyme for the de novo pyrimidine synthesis. CONCLUSION: In this study, we identified 10580 as a promising new drug against GICs. Given that normal tissue cells, in particular brain cells, tend to use the alternative salvage pathway for pyrimidine synthesis, our findings suggest that 10580 can be used for glioblastoma therapy without side effects.Key Points1. Chemical screening identified 10580 as a novel GIC-eliminating drug that targets DHODH, an essential enzyme for the de novo pyrimidine synthesis pathway. 2. Compound 10580 induced cell cycle arrest, apoptosis, and differentiation in GICs.

Detection of DNA damage formation by natural organic matter using EGFP-fused MDC1-expressing cells.

Studies have been conducted on the genotoxicity and carcinogenicity of disinfection by-products formed from natural organic matter (NOM) and mitigation effect for mutagens and clastogens by NOM. Whereas, reportedly, synthetic humic acid in high concentration has induced genotoxicity in human cells, and NOM samples have provoked mild oxidative and other physiological responses in aquatic organisms. Our group developed a novel detection method for DNA damage formation, namely enhanced green fluorescent protein (EGFP)-fused mediator of DNA damage checkpoint 1 (MDC1)-expressing human cells as simple and high-sensitive system. By using this method, a significant increase in the foci area was observed after 3 h, but not 24 h for 130 mgC L-1 Suwannee River fulvic acid (SRFA), 38 mgC L-1 humic acid (SRHA), and 19 mgC L-1 NOM (SRNOM). The SRNOM concentration is the original environmental one; therefore, it was suggested that the formation and repair of DNA damage associated with γ-H2AX, a biomarker for DNA double-strand breaks by mild oxidative stress, in Suwannee River (SR) were detected for the first time. The increase in the foci area was not observed for 18 mgC L-1 Lake Biwa fulvic acid (LBFA) and 50 mg L-1 catechin after both 3 h and 24 h. The difference between the SR and Lake Biwa (LB) samples may result from the differences in their electron-accepting capacity. The application of this methodology is expected to elucidate oxidative stress and toxicological effects shortly and in detail for many water samples.

Quantitative analysis of γH2AX reveals distinct responses in multiple mouse organs after administration of mitomycin C or ethyl methanesulfonate.

Ser139-phosphorylated H2AX (γH2AX) is a functional biomarker of DNA double-strand breaks. However, its conventional detection for in vivo samples relies on immunological methods using anti-γH2AX antibodies, making quantitative analysis difficult. Here, we established an absolute γH2AX quantification in vivo method for multiple organs in mice using liquid chromatography-triple quadrupole tandem mass spectrometry. When applying the method to male Institute of Cancer Research (ICR) mice (8 weeks old), the testes showed the highest γH2AX level (2.3% of total H2AX), followed by the bone marrow (0.51%), stomach (0.28%), kidney (0.20%), spleen (0.20%), liver (0.15%) and lung, which had the lowest overall level (0.10%). After intraperitoneal administration of 2 mg/kg mitomycin C in mice, γH2AX levels increased until 2-4 h, followed by a monotonical decrease to the control level in the bone marrow and spleen, and increased moderately until 24 h, followed by a slight decrease by 48 h in the liver, stomach, lung and kidney. After oral administration of 400 mg/kg ethyl methanesulphonate, γH2AX levels increased until 8 h and then decreased to the control level by 24-48 h in the spleen and kidney, increased until 24 h and then slightly decreased until 48 h in the bone marrow and lung, increased until 8 h and plateaued by 48 h in the liver, and decreased until 8 h and then increased to the control level in the stomach. Both the genotoxic chemicals did not alter γH2AX levels in the testes. These results indicate that our novel method could reveal variation in the γH2AX state in mouse organs and allows monitoring of the in vivo dynamics induced by genotoxic chemicals.

Effect of subthalamic nucleus deep brain stimulation on visual scanning.

OBJECTIVE: Deep brain stimulation (DBS) can provide insights into the workings of the basal ganglia (BG) by interfering with their function. In patients with Parkinson's disease (PD) treated with DBS of the subthalamic nucleus, we studied the effect of DBS on scanning eye movements. METHODS: In the visual memory task, subjects viewed images of various complexities for later recall. In visual search tasks, subjects looked for and fixated one odd target ring, embedded among 48 Landolt rings, which either stood out or not from the distractors. We compared the parameters of scanning saccades when DBS was on and off. RESULTS: In the visual memory task, DBS increased the amplitude of saccades scanning simple but not complex drawings. In the visual search tasks, DBS showed no effect on saccade amplitude or frequency. CONCLUSIONS: Saccades when viewing simple images were affected by DBS since they are internally guided saccades, for which the involvement of BG is large. In contrast, saccades when viewing complex images or during visual search, made with the help of visual cues in the images (externally guided saccades) and less dependent on BG, were resistant to the effect of DBS. SIGNIFICANCE: DBS affects saccades differentially depending on the task.

Simultaneous and absolute quantification of nucleoside triphosphates using liquid chromatography-triple quadrupole tandem mass spectrometry.

BACKGROUND: Nucleoside triphosphates participate in fundamental cellular processes as building blocks of DNA and RNA, energy carriers, and cofactors in enzymatic reactions, and their balance is tightly regulated. Here, we established a simultaneous and absolute quantification method for eight nucleoside triphosphates using liquid chromatography-triple quadrupole tandem mass spectrometry and hydrophilic interaction chromatography. Our method was successfully applied to the extract of human acute myeloid leukemia Molm-13 cells. RESULTS: Levels of ribonucleoside triphosphates (2.07 × 108-2.29 × 109 molecules/cell) in Molm-13 cells were two orders of magnitude higher than those of deoxyribonucleoside triphosphates (1.72 × 106-1.40 × 107 molecules/cell). Exposure of Molm-13 cells for 24 h to thymidine, a nucleotide imbalance inducer, increased the levels of cellular dTTP, dGTP, and dATP and decreased only dCTP, resulting in significant inhibition of cell proliferation. CONCLUSION: Our quantification method for nucleoside triphosphates revealed the quantitative relationship between the arrest of cell proliferation and the imbalance of nucleoside triphosphates in thymidine-treated Molm-13 cells. Owing to the short run time (15 min/run), broad adaptability, and throughput performance, we believe that our method is a powerful tool for not only genetic and molecular biology research but also for studying the mechanism of genotoxic compounds and anti-cancer or anti-virus drugs, drug screening, clinical studies, and other fields.

High-content image analysis (HCIA) assay has the highest correlation with direct counting cell suspension compared to the ATP, WST-8 and Alamar blue assays for measurement of cytotoxicity.

INTRODUCTION: Various cytotoxicity assays measuring indicators such as enzyme activity, dye uptake, or cellular ATP content are often performed using 96-well microplates. However, recent reports show that cytotoxicity assays such as the ATP assay and MTS assay underestimate cytotoxicity when compounds such as anti-cancer drugs or mutagens induce cell hypertrophy whilst increasing intracellular ATP content. Therefore, we attempted to evaluate the reliability of a high-content image analysis (HCIA) assay to count cell number in a 96-well microplate automatically without using a cell-number indicator. METHODS: We compared cytotoxicity results of 25 compounds obtained from ATP, WST-8, Alamar blue, and HCIA assays with those directly measured using an automatic cell counter, and repeating individual experiments thrice. RESULTS: The number of compounds showing low correlation in cell viability measured using cytotoxicity assays compared to automatic cell counting (r2<0.8, at least 2 of 3 experiments) were follows: ATP assay; 7; WST-8 assay, 2; Alamar blue assay, 3; HCIA cytotoxicity assay, 0. Compounds for which correlation was poor in 3 assays, except the HCIA assay, induced an increase in nuclear and cell size. However, correlation between cell viability measured by automatic cell counter and the HCIA assay was strong regardless of nuclear and cell size. Additionally, correlation coefficients between IC50 values obtained from automatic cell counter and from cytotoxicity assays were as follows: ATP assay, 0.80; WST-8 assay, 0.84; Alamar blue assay, 0.84; and HCIA assay, 0.98. DISCUSSION: From the above, we showed that the HCIA cytotoxicity assay produces similar data to the automatic cell counter and is highly accurate in measuring cytotoxicity.

Chemically induced strong cellular hypertrophy often reduces the accuracy of cytotoxicity measurements obtained using the ATP assay.

The ATP assay is a highly sensitive and versatile method for measuring cytotoxicity. However, the correlation between the cell viability results obtained using the ATP assay and those obtained using direct cell counting has not been widely reported. Therefore, to evaluate the reliability and limitations of the ATP assay, we compared the results of ATP assay with those of automatic cell counter, which can measure the number and diameter of cells directly, by using 24 compounds and repeating individual experiments thrice. The correlation between the data was low for 7 of the 24 compounds (r2 < 0.8, at least 2 out of 3 experiments). These were the top 7 of the 11 compounds that induced cell hypertrophy. These 7 compounds were also observed to increase the area of mitochondria. However, the last 4 of the 11 compounds increased the cell size but did not increase the mitochondrial area. For the remaining 13 compounds, which had no effect on cell size, a good correlation was observed between the results of the two methods (r2 > 0.8, at least 2 out of 3 experiments), and the cell size was effectively the same as that of the controls. We concluded that the poor correlation between the two methods was attributable to an increase in the content of intracellular ATP because of the chemically induced cell and mitochondrial hypertrophy. We showed that the ATP assay is unsuitable for assessing the cytotoxicity of compounds that induce cell hypertrophy with increase in the mitochondrial area and ATP content.

Disruption of DNA Damage-Response by Propyl Gallate and 9-Aminoacridine.

The DNA-damage response (DDR) protects the genome from various types of endogenous and exogenous DNA damage, and can itself be a target of certain chemicals that give rise to chromosomal aberrations. Here, we developed a screening method to detect inhibition of Mediator of DNA damage Checkpoint 1 (MDC1) foci formation (the Enhanced Green Fluorescent Protein (EGFP)-MDC1 foci formation-inhibition assay) using EGFP-MDC1-expressing human cells. The assay identified propyl gallate (PG) and 9-aminoacridine (9-AA) as inhibitors of camptothecin (CPT)-induced MDC1 foci formation. We demonstrated that the inhibition of CPT-induced MDC1 foci formation by PG was caused by the direct suppression of histone H2AX phosphorylation at Ser139 (γH2AX), which is required for MDC1 foci formation, by quantifying γH2AX in cells and in vitro 9-AA also directly suppressed H2AX Ser139-phosphorylation in vitro but the concentration was much higher than that required to suppress CPT-induced MDC1 foci formation in cells. Consistent with these findings, PG and 9-AA both suppressed CPT-induced G2/M cell-cycle arrest and increased the number of abnormal nuclei. Our results suggest that early DDR-inhibitory effects of PG and 9-AA contribute to their chromosome-damaging potential, and that the EGFP-MDC1 foci formation-inhibition assay is useful for detection of and screening for H2AX Ser139-phosphorylation-inhibitory effects of chemicals.

Nuclear pyruvate kinase M2 complex serves as a transcriptional coactivator of arylhydrocarbon receptor.

Pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase complex (PDC) regulate production of acetyl-CoA, which functions as an acetyl donor in diverse enzymatic reactions, including histone acetylation. However, the mechanism by which the acetyl-CoA required for histone acetylation is ensured in a gene context-dependent manner is not clear. Here we show that PKM2, the E2 subunit of PDC and histone acetyltransferase p300 constitute a complex on chromatin with arylhydrocarbon receptor (AhR), a transcription factor associated with xenobiotic metabolism. All of these factors are recruited to the enhancer of AhR-target genes, in an AhR-dependent manner. PKM2 contributes to enhancement of transcription of cytochrome P450 1A1 (CYP1A1), an AhR-target gene, acetylation at lysine 9 of histone H3 at the CYP1A1 enhancer. Site-directed mutagenesis of PKM2 indicates that this enhancement of histone acetylation requires the pyruvate kinase activity of the enzyme. Furthermore, we reveal that PDC activity is present in nuclei. Based on these findings, we propose a local acetyl-CoA production system in which PKM2 and PDC locally supply acetyl-CoA to p300 from abundant PEP for histone acetylation at the gene enhancer, and our data suggest that PKM2 sensitizes AhR-mediated detoxification in actively proliferating cells such as cancer and fetal cells.

Acetylation of Histone H2AX at Lys 5 by the TIP60 Histone Acetyltransferase Complex Is Essential for the Dynamic Binding of NBS1 to Damaged Chromatin.

The association and dissociation of DNA damage response (DDR) factors with damaged chromatin occurs dynamically, which is crucial for the activation of DDR signaling in a spatiotemporal manner. We previously showed that the TIP60 histone acetyltransferase complex acetylates histone H2AX, to facilitate H2AX exchange at sites of DNA damage. However, it remained unclear how the acetylation of histone H2AX by TIP60 is related to the DDR signaling. We found that the acetylation but not the phosphorylation of H2AX is essential for the turnover of NBS1 on damaged chromatin. The loss of H2AX acetylation at Lys 5 by TIP60 in cells disturbed the accumulation of NBS1 at sites of DNA damage. Although the phosphorylation of H2AX is also reportedly required for the retention of NBS1 at damage sites, our data indicated that the acetylation-dependent NBS1 turnover by TIP60 on damaged chromatin restricts the dispersal of NBS1 foci from the sites of DNA damage. These findings indicate the importance of the acetylation-dependent dynamic binding of NBS1 to damaged chromatin, created by histone H2AX exchange, for the proper accumulation of NBS1 at DNA damage sites.

Protective role of ALDH2 against acetaldehyde-derived DNA damage in oesophageal squamous epithelium.

Acetaldehyde is an ethanol-derived definite carcinogen that causes oesophageal squamous cell carcinoma (ESCC). Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme that eliminates acetaldehyde, and impairment of ALDH2 increases the risk of ESCC. ALDH2 is produced in various tissues including the liver, heart, and kidney, but the generation and functional roles of ALDH2 in the oesophagus remain elusive. Here, we report that ethanol drinking increased ALDH2 production in the oesophagus of wild-type mice. Notably, levels of acetaldehyde-derived DNA damage represented by N(2)-ethylidene-2'-deoxyguanosine were higher in the oesophagus of Aldh2-knockout mice than in wild-type mice upon ethanol consumption. In vitro experiments revealed that acetaldehyde induced ALDH2 production in both mouse and human oesophageal keratinocytes. Furthermore, the N(2)-ethylidene-2'-deoxyguanosine levels increased in both Aldh2-knockout mouse keratinocytes and ALDH2-knockdown human keratinocytes treated with acetaldehyde. Conversely, forced production of ALDH2 sharply diminished the N(2)-ethylidene-2'-deoxyguanosine levels. Our findings provide new insight into the preventive role of oesophageal ALDH2 against acetaldehyde-derived DNA damage.

hCAS/CSE1L regulates RAD51 distribution and focus formation for homologous recombinational repair.

Homologous recombinational repair (HR) is one of the major repair systems for DNA double-strand breaks. RAD51 is a key molecule in HR, and the RAD51 concentration in the cell nucleus increases after DNA damage induction. However, the mechanism that regulates the intracellular distribution of RAD51 is still unclear. Here, we show that hCAS/CSE1L associates with RAD51 in human cells. We found that hCAS/CSE1L negatively regulates the nuclear protein level of RAD51 under normal conditions. hCAS/CSE1L is also required to repress the DNA damage-induced focus formation of RAD51. Moreover, we show that hCAS/CSE1L plays roles in the regulation of the HR activity and in chromosome stability. These findings suggest that hCAS/CSE1L is responsible for controlling the HR activity by directly interacting with RAD51.