Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site.

RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs)1,2. However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.

Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2.

Components of the Fanconi anemia and homologous recombination pathways play a vital role in protecting newly replicated DNA from uncontrolled nucleolytic degradation, safeguarding genome stability. Here we report that histone methylation by the lysine methyltransferase SETD1A is crucial for protecting stalled replication forks from deleterious resection. Depletion of SETD1A sensitizes cells to replication stress and leads to uncontrolled DNA2-dependent resection of damaged replication forks. The ability of SETD1A to prevent degradation of these structures is mediated by its ability to catalyze methylation on Lys4 of histone H3 (H3K4) at replication forks, which enhances FANCD2-dependent histone chaperone activity. Suppressing H3K4 methylation or expression of a chaperone-defective FANCD2 mutant leads to loss of RAD51 nucleofilament stability and severe nucleolytic degradation of replication forks. Our work identifies epigenetic modification and histone mobility as critical regulatory mechanisms in maintaining genome stability by restraining nucleases from irreparably damaging stalled replication forks.

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.

Validation of the cell culture monitoring using a Raman spectroscopy calibration model developed with artificially mixed samples and investigation of model learning methods using initial batch data.

The development of calibration models using Raman spectra data has long been challenged owing to the substantial time and cost required for robust data acquisition. To reduce the number of experiments involving actual incubation, a calibration model development method was investigated by measuring artificially mixed samples. In this method, calibration datasets were prepared using spectra from artificially mixed samples with adjusted concentrations based on design of experiments. The precision of these calibration models was validated using the actual cell culture sample. The results showed that when the culture conditions were unchanged, the root mean square error of prediction (RMSEP) of glucose, lactate, and antibody concentrations was 0.34, 0.33, and 0.25 g/L, respectively. Even when variables such as cell line or culture media were changed, the RMSEPs of glucose, lactate, and antibody concentrations remained within acceptable limits, demonstrating the robustness of the calibration models with artificially mixed samples. To further improve accuracy, a model training method for small datasets was also investigated. The spectral pretreatment conditions were optimized using error heat maps based on the first batch of each cell culture condition and applied these settings to the second and third batches. The RMSEPs improved for glucose, lactate, and antibody concentration, with values of 0.44, 0.19, and 0.18 g/L under constant culture conditions, 0.37, 0.12, and 0.12 g/L for different cell lines, and 0.26, 0.40, and 0.12 g/L when the culture media was changed. These results indicated the efficacy of calibration modeling with artificially mixed samples for actual incubations under various conditions.

Associations of remaining teeth number, periodontal disease, and occlusal support status with erectile dysfunction in community-dwelling men.

OBJECTIVES: Several systematic reviews and meta-analyses have reported positive relationships between erectile dysfunction (ED) and periodontal disease. However, no study has evaluated the relationships of occlusal support status and the number of remaining teeth with ED. The aim of the present study was to investigate the relationships between ED and the remaining teeth number, periodontal disease, and occlusal support status. METHODS: This study included 400 community-dwelling men. Periodontal health status and occlusal support condition were evaluated using the Community Periodontal Index (CPI) and Eichner classification. Multivariable analyses were performed to evaluate the relationships between ED and the remaining teeth number, periodontal disease, and occlusal support status. RESULTS: The median age was 53 years. Of the 400 men, 333 (83%) were classified into ED group. In univariable analyses, remaining teeth number, CPI score, and Eichner classification were significantly associated with ED. In multivariable analyses, the remaining teeth number (odds ratio [OR]: 0.907, p = 0.114) and CPI score (OR: 0.978, p = 0.864) were not significantly associated with ED, whereas the Eichner classification was independently and significantly associated with ED (OR: 3.490, p = 0.042). CONCLUSIONS: Poor occlusal support status was significantly associated with ED in community-dwelling men, as opposed to remaining teeth number and periodontal health status.

Utility of prognostic nutritional index and systemic immune-inflammation index in oral cancer treatment.

PURPOSE: This study aimed to evaluate the utility of inflammation-based prognostic scores (IBPS) and systemic immune-inflammation index (SII) in the treatment of oral cancer patients. METHODS: For the 183 patients enrolled in this study, IBPS and SII were calculated from peripheral blood samples obtained before and after treatment and at the time of relapse. We examined overall survival (OS) and disease-free survival (DFS) using previously reported cut-off values for IBPS. Cut-off values of neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), platelet-to-lymphocyte ratio (PLR), and prognostic nutritional index (PNI) were analyzed as NLR 1.79, PLR 114.97, LMR 5, and PNI 52.44. The cut-off value for SII was set at 569. OS and DFS were analyzed by Kaplan-Meier methods using the cutoff of each IBPS and SII. Univariate analysis and multivariate analysis using Cox proportional hazards were performed for OS and DFS. RESULTS: Kaplan-Meier methods showed the high-PNI group showed good prognosis including OS and DFS, while the high-SII group displayed poor DFS. Univariate analysis showed that pre-treatment high PNI and low SII were significantly associated with better prognosis. Multivariate analysis identified pre-treatment PNI as independently associated with OS. For DFS, univariate analysis using Cox proportional hazards modeling showed that pre-treatment high NLR and high SII were significantly associated with worse prognosis, while high PNI was significantly associated with better prognosis. Multivariate analysis identified pre-treatment PNI and SII as independently associated with DFS. Parameters of PNI and SII components were compared between pre-treatment, post-treatment and at relapse in the high- and low-PNI groups. PNI was predominantly decreased in both high- and low-PNI groups at post-treatment and at relapse compared to pre-treatment. This trend was also observed for albumin. CONCLUSIONS: Higher pre-treatment PNI was associated with better OS, while lower pre-treatment PNI and higher treatment SII were associated with poorer DFS in oral cancer patients. Our data indicated that PNI and SII might offer useful biomarkers for gauging prognosis and the efficacy of conventional therapies.

Multicenter retrospective study of nivolumab for recurrent/metastatic oral squamous cell carcinoma.

OBJECTIVES: Immunotherapy with nivolumab for patients with recurrent/metastatic oral squamous cell carcinoma has not been evaluated. Here, we aimed to examine the efficacy, safety, and prognostic factors of nivolumab in these patients. MATERIALS AND METHODS: This multicenter retrospective observational study involved patients who received nivolumab between April 2017 and June 2019. The patient characteristics were evaluated for association with progression-free and overall survival. Progression-free and overall survival rates were calculated; parameters that were significant in the univariate analysis were used as explanatory variables. Independent factors for progression-free and overall survival were identified using multivariate analysis. RESULTS: Totally, 143 patients were included. The overall response and disease control rates were 27.3% and 46.2%, respectively. The median, 1- and 2-year progression-free survival rates were 2.7 months, 25.4%, and 19.2%, respectively; those for overall survival were 11.2 months, 47.3%, and 33.6%, respectively. The independent factors affecting progression-free survival were performance status and immune-related adverse event occurrence, whereas those affecting overall survival were performance status, target disease, and number of previous lines of systemic cancer therapy. Eight patients reported grade ≥3 immune-related adverse events. CONCLUSION: Nivolumab was effective for recurrent/metastatic oral squamous cell carcinoma treatment and was well tolerated by patients.

Urinary mulberry bodies as a potential biomarker for early diagnosis and efficacy assessment of enzyme replacement therapy in Fabry nephropathy.

BACKGROUND: The inability of enzyme replacement therapy (ERT) to prevent progression of Fabry nephropathy (FN) in the presence of >1 g/day proteinuria underscores the necessity of identifying effective biomarkers for early diagnosis of FN preceding proteinuria. Here we attempted to identify biomarkers for early detection of FN. METHODS: Fifty-one Fabry disease (FD) patients were enrolled. Urinary mulberry bodies (uMBs) were immunostained for globotriaosylceramide (Gb3) and renal cell markers to determine their origin. The association between semiquantitative uMB excretion and the histological severity of podocyte vacuolation was investigated in seven patients using the vacuolated podocyte:glomerular average area ratio. The association between the semiquantitative estimate of uMB excretion and duration of ERT was analyzed. A longitudinal study was conducted to assess the effect of ERT on uMB excretion. RESULTS: Thirty-two patients (63%) had uMBs, while only 31% showed proteinuria. The uMBs were positive for Gb3, lysosomal-associated membrane protein 1 and podocalyxin, suggesting they were derived from lysosomes with Gb3 accumulation in podocytes. We observed more severe podocyte vacuolation with increased uMB excretion (P = 0.03 for trend); however, the same was not observed with increased proteinuria. The percentage of patients with substantial uMB excretion increased with shorter ERT duration (P = 0.018). Eighteen-month-long ERT reduced uMB excretion (P = 0.03) without affecting proteinuria. CONCLUSIONS: uMB excretion, implying ongoing podocyte injury, preceded proteinuria in most patients. Semiquantitative uMB estimates can serve as novel biomarkers for early FN diagnosis and for monitoring the efficacy of FD-specific therapies.

Companion Diagnosis for Retinal Neuroprotective Treatment by Real-Time Imaging of Calpain Activation Using a Novel Fluorescent Probe.

Calpain activation induces retinal ganglion cell (RGC) death, while calpain inhibition suppresses RGC death, in animal studies. However, the role of calpain in human retinal disease is unclear. This study investigated a new strategy to study the role of calpain based on real-time imaging. We synthesized a novel fluorescent probe for calpain, acetyl-l-leucyl-l-methionine-hydroxymethyl rhodamine green (Ac-LM-HMRG) and used it for real-time imaging of calpain activation. The toxicity of Ac-LM-HMRG was evaluated with a lactate dehydrogenase cytotoxicity assay, retinal sections, and electroretinograms. Here, we performed real-time imaging of calpain activation in a rat model. First, we administered N-methyl-d-aspartate (NMDA) to induce retinal injury. Twenty minutes later, we administered an intravitreal injection of Ac-LM-HMRG. Real-time imaging was then completed with a noninvasive confocal scanning laser ophthalmoscope. The inhibitory effect of SNJ-1945 against calpain activation was also examined with the same real-time imaging method. Ac-LM-HMRG had no toxic effects. The number of Ac-LM-HMRG-positive cells in real-time imaging significantly increased after NMDA injury, and SNJ-1945 significantly lowered the number of Ac-LM-HMRG-positive cells. Real-time imaging with Ac-LM-HMRG was able to quickly quantify the NMDA-induced activation of calpain and the inhibitory effect of SNJ-1945. This technique, used as a companion diagnostic system, may aid research into the development of new neuroprotective therapies.

Histone H2A variants confer specific properties to nucleosomes and impact on chromatin accessibility.

In eukaryotes, variants of core histone H2A are selectively incorporated in distinct functional domains of chromatin and are distinguished by conserved sequences of their C-terminal tail, the L1 loop and the docking domain, suggesting that each variant confers specific properties to the nucleosome. Chromatin of flowering plants contains four types of H2A variants, which biochemical properties have not been characterized. We report that in contrast with animals, in Arabidopsis thaliana H2A variants define only four major types of homotypic nucleosomes containing exclusively H2A, H2A.Z, H2A.X or H2A.W. In vitro assays show that the L1 loop and the docking domain confer distinct stability of the nucleosome. In vivo and in vitro assays suggest that the L1 loop and the docking domain cooperate with the C-terminal tail to regulate chromatin accessibility. Based on these findings we conclude that the type of H2A variant in the nucleosome impacts on its interaction with DNA and propose that H2A variants regulate the dynamics of chromatin accessibility. In plants, the predominance of homotypic nucleosomes with specific physical properties and their specific localization to distinct domains suggest that H2A variants play a dominant role in chromatin dynamics and function.

Cancer-associated mutations of histones H2B, H3.1 and H2A.Z.1 affect the structure and stability of the nucleosome.

Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may provide important information toward understanding how the mutations promote cancer progression.

Replication stress induces accumulation of FANCD2 at central region of large fragile genes.

During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.

Falls Due to Loss of Consciousness are Associated With Maxillofacial Fracture Severity.

PURPOSE: Falls are a common cause of the maxillofacial fractures, and falls associated with loss of consciousness might have special characteristics. The purpose of the present study was to measure the association between the types of falls and maxillofacial injury severity. PATIENTS AND METHODS: The present retrospective cross-sectional study focused on patients with maxillofacial fractures resulting from falls who had been treated at the Hirosaki University Hospital from 1990 to 2016. The falls were divided into 2 categories according to the reason for their occurrence: 1) falls from slipping, tripping, or stumbling (STSFs); and 2) falls from loss of consciousness (LOCFs). The primary outcome measure of the present study was the severity of the maxillofacial fractures. The secondary outcomes were the pattern of maxillofacial fractures, pattern of concomitant injuries, and treatment modality. Multiple linear regression analysis was performed to evaluate the independent predictors for fracture severity. RESULTS: A total of 148 patients had been admitted for maxillofacial fractures resulting from falls. The sample included 107 STSFs (72.3%) and 41 LOCFs (27.7%). The cause of the LOCFs was orthostatic-hypotension syncope in 13 patients, neurally mediated syncope in 10, cardiogenic syncope in 9, epilepsy in 5, and other in 4 patients. The proportion of mandibular fractures and the mean facial injury severity scale score were significantly greater in the LOCF group (2.20 ± 1.19) than in the STSF group (1.65 ± 1.15; P = .0067). The incidence of concomitant injuries was significantly greater in the STSF group than in the LOCF group (P = .023), and the distribution of sites was significantly different between the 2 groups (P = .039). CONCLUSIONS: Our results have shown that maxillofacial fractures secondary to LOCFs tend to be more severe and to have a lower incidence of concomitant injuries compared with STSFs. We believe these features originate from the absence of protective reflexes resulting from the loss of consciousness.

Amplifierless PAM-4/PAM-8 transmissions in O-band using a directly modulated laser for optical data-center interconnects.

Toward the realization of low-cost, long-, and extended-reach 400GbE data-center applications, the performance of pulse amplitude modulated (PAM) signals is studied using a state-of-the-art, high-performance 1.3-µm distributed feedback directly modulated laser, without any optical amplification or complex digital processing. Amplifierless PAM-4 transmissions of up to 64-Gb/s are achieved over 40 km of standard single-mode fiber (SSMF) for standard KP4-FEC, while 84-Gb/s PAM-8 signals are evaluated over 10 km SSMF.

Spatiotemporal dynamics of turbulent coaxial jet analyzed by symbolic information-theory quantifiers and complex-network approach.

We numerically study the spatiotemporal dynamics of a turbulent coaxial jet in a model rocket engine combustor from the viewpoints of symbolic information-theory quantifiers and complex networks. The dynamic behavior of flow velocity undergoes a significant transition from a stochastic to chaotic state as the turbulent jet moves downstream. The small-world nature exists in the near field forming a stochastic state, whereas it disappears by the formation of a chaotic state in the far field. The dynamic behavior of hydrogen and oxygen concentrations in the far field also represents deterministic chaos. The simultaneous dynamic behavior with chaotic mixing forms the phase-synchronization state.

SYCP3 regulates strand invasion activities of RAD51 and DMC1.

The synaptonemal complex is a higher-ordered proteinaceous architecture formed between homologous chromosomes. SYCP3 is a major component of the lateral/axial elements in the synaptonemal complex and is essential for meiotic recombination. Previous genetic studies showed that SYCP3 functions in meiotic homologous recombination biased to interhomologous chromosomes, by regulating the strand invasion activities of the RAD51 and DMC1 recombinases. However, the mechanism by which SYCP3 regulates RAD51- and DMC1-mediated strand invasion remains elusive. In this study, we found that SYCP3 significantly suppresses the RAD51-mediated, but not the DMC1-mediated, strand invasion reaction by competing with HOP2-MND1, which is an activator for both RAD51 and DMC1. A SYCP3 mutant with defective RAD51 binding does not inhibit the RAD51-mediated homologous recombination in human cells. Therefore, SYCP3 may promote the DMC1-driven homologous recombination by attenuating the RAD51 activity during meiosis.

Xeroderma pigmentosum group C protein interacts with histones: regulation by acetylated states of histone H3.

In the mammalian global genome nucleotide excision repair pathway, two damage recognition factors, XPC and UV-DDB, play pivotal roles in the initiation of the repair reaction. However, the molecular mechanisms underlying regulation of the lesion recognition process in the context of chromatin structures remain to be understood. Here, we show evidence that damage recognition factors tend to associate with chromatin regions devoid of certain types of acetylated histones. Treatment of cells with histone deacetylase inhibitors retarded recruitment of XPC to sites of UV-induced DNA damage and the subsequent repair process. Biochemical studies showed novel multifaceted interactions of XPC with histone H3, which were profoundly impaired by deletion of the N-terminal tail of histone H3. In addition, histone H1 also interacted with XPC. Importantly, acetylation of histone H3 markedly attenuated the interaction with XPC in vitro, and local UV irradiation of cells decreased the level of H3K27ac in the damaged areas. Our results suggest that histone deacetylation plays a significant role in the process of DNA damage recognition for nucleotide excision repair and that the localization and functions of XPC can be regulated by acetylated states of histones.

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.

Methotrexate-Related Lymphoproliferative Disorder in Patients With Osteonecrosis of the Jaw: A 3-Case Report and Literature Review.

Patients with immunodeficiency or immunosuppression are at risk of developing a lymphoproliferative disorder (LPD). Methotrexate (MTX) is an iatrogenic cause of LPD, which in up to 50% cases occurs in extranodal sites. The occurrence of MTX-related LPD with osteonecrosis of the jaw (ONJ) has rarely been reported. Moreover, there are no clear diagnostic criteria and treatment strategies for management of these lesions. In the present cases, discontinuing MTX and debridement of the necrotic bone were effective. This report describes 3 cases of MTX-related LPD in patients with longstanding rheumatoid arthritis (RA) who presented with ONJ. The first patient was a 74-year-old man with RA who had received treatment with MTX for 7 years before presenting with ONJ and submental lymphadenopathy. The second patient was a 79-year-old woman who had been treated for 21 years with MTX and who presented with ONJ. The third patient was a 67-year-old man who had been treated with MTX for more than 15 years. In all 3 cases, biopsy, histology, and immunohistochemistry using a panel of lymphoid markers (Epstein-Barr virus [EBV], CD79a, CD20, PAX-5, CD3, and CD30) resulted in the diagnosis of EBV-driven T-cell, B-cell, and Hodgkin-like LPD. All 3 patients recovered after cessation of MTX and surgical debridement. Biopsy examination, diagnostic immunohistochemistry using lymphoid immune markers, and imaging studies using computed tomography, magnetic resonance imaging, and positron-emission tomographic computed tomography were useful for the correct diagnosis of this condition.