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Histone proteins, building blocks of chromatins, participate in enzymatic reactions in cells heated at around 45°C though in vitro the denaturation of histones significantly proceeds at a similar temperature. It implies that unidentified mechanisms prevent thermal denaturation of histones in vivo. However, studies on the histone structures in the heated cells have been scarce. Here, we analyzed the secondary structures of histone H2A-H2B proteins originating from the heated cells using circular dichroism spectroscopy. The secondary structure contents of the H2A-H2B extracted from the heated cells differed from those of H2A-H2B both native and denatured in vitro but reverted to the native structures by incubating the heated cells at 37°C within 2 h. Such structural flexibility may play a role in protecting genomic functions governed by chromatin structures from heat stresses.

The COVID-19 pandemic was unprecedented and forced countries to organise and combine resources and develop coordinated responses. Temporary systems were established with the support of researchers and other stakeholders. Dr Junna Oba, Assistant Professor at the Department of Extended Intelligence for Medicine at Keio University School of Medicine (KUSM) is devising methodologies for developing adjustable temporary systems that are quick and efficient to build, apply and dissemble when emergency situations have passed. KUSM helped to respond to the COVID-19 crisis by collaborating with basic science researches to reinforce the PCR testing system in hospitals. Among those involved in this effort were Professor Masako Toriya of Keio University Global Research Institute (KGRI) and Professor Koichi Matsuo from the KUSM's Collaborative Research Resources group. The researchers built from scratch a workflow for effective testing and information sharing. Since this time, the team has been working to build a cooperative network, developing a novel approach to planning, analysing and recording various processes and strategies to address emergency situations that will be shared with the wider healthcare community and policy makers. The researchers used systems engineering methods to launch an unprecedented collaborative system that offers the ability to gain an overall picture of the interrelationships between departments that are working together on the diagnosis, treatment and management of COVID-19.

(A, B) Weight of AXT cell-derived tumors formed in mice treated with saline or simvastatin administered either by intraperitoneal injection (40 mg/kg per day) (A) or by oral gavage (80 mg/kg per day) (B) Data are means {plus minus} SD. (C) Viability of AXT cells treated with the indicated concentrations of TOFA for 1 or 2 days. (D) Weight of AXT cell-derived tumors at 3 weeks after cell injection in mice fed a normal or fat-free diet. The fat-free diet was initiated 4 days before cell injection. Data are means {plus minus} SD. (E) Model for the mechanism of apoptosis induction by simvastatin.

(A) Phase-contrast microscopy of AXT cells treated with 10 μM simvastatin for 24 h in the absence or presence of mevalonate, GGPP, or FPP. (B) Representative migratory AXT cells stained with crystal violet after exposure to simvastatin for 12 h. (C, D) Flow cytometric analysis of DNA content in AXT cells treated with 5 μM simvastatin for 24 h in the absence or presence of mevalonate, GGPP, or FPP (C). The size of the sub-G1 fraction of cells was also determined (D).

Supplementary Figure Legends.

Supplementary Figure S4: (A,B) Viable AXT cells after the treatment of chemotherapeutic agents. (C) Representative flow cytometric analysis of γ-H2AX abundance in AXT cells as described in Fig.4E. (D) Immunofluorescence analysis of γ-H2AX in AXT cells.

PDF file - 155K, RNF8 suppresses RAD51 assembly in BRCA1/53BP1-depleted 293T cells

PDF file, 4242K, Induction of a constitutively active form of NFATc1 (NFATc1CA) promotes cancer cell migration.

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Supplementary Figure S2: (A) Representative flow cytometric analysis of DNA content in AXT cells. (B) Cell cycle distribution for cells treated as in (A). (C) Immunofluorescence analysis of Ki67 expression in AXT cells. (D) Representative flow cytometric analysis of Ki67 expression in AXT cells cultured in the presence of insulin. (E) Representative flow cytometric analysis of DNA content in SAOS2 cells. (F) Cell cycle distribution for cells treated as in (E). (G) Immunoblot analysis of cyclin expression in AXT cells.

PDF file, 821K, NFATc1 directly activates SNAI1 transcription.

PDF file - 121K, A schematic of the assembly of DNA damage response-related proteins at DSB sites

Supplementary Table S1. Sequences of primers and predicted product sizes for real-time RT-PCR analysis

PDF file - 522K, RAD18 is not required for RAD51 assembly at DSB sites in the absence of BRCA1 and 53BP1

PDF file - 657K, Depletion of 53BP1 rescues RAD51 assembly at DSB sites in BRCA1-depleted human cells

PDF file - 217K, Cell cycle analysis

XLS file, 25232K, List of genes.

NFATc1 is a transcription factor that regulates T-cell development, osteoclastogenesis, and macrophage function. Given that T cells, osteoclasts, and macrophages in the tumor microenvironment are thought to modulate tumor progression, tumor cells may acquire NFATc1 expression through fusion with these NFATc1-expressing normal cells. We here revealed that a small proportion of tumor cells in human carcinoma specimens expressed NFATc1. To investigate the consequences of NFATc1 acquisition by tumor cells, we established A549 and MCF7 cell lines expressing a constitutively active form of NFATc1 (NFATc1CA) in an inducible manner. The expression of NFATc1CA promoted cancer cell invasion in association with changes in cell morphology. Analysis of gene expression and RNA interference experiments revealed that NFATc1CA suppressed E-cadherin expression by upregulating the transcriptional repressors Snail and Zeb1 in a manner independent of TGF-β signaling. Induced expression of NFATc1CA also downregulated E-cadherin expression and increased invasive activity in tumor xenografts in vivo. Our results thus suggest that the acquisition of NFATc1 expression contributes to tumor progression. Cancer Res; 73(16); 5100–9. ©2013 AACR.

PDF file - 666K, RAD51 assembles at DSB sites in RNF8-/- MEFs

PDF file, 7478K, Identification of NFATc1-positive tumor cells in human breast tumor specimens.

Supplementary Information. Supplementary Materials and Methods and Supplementary Figure Legends

PDF file - 108K, The effective knockdown of UBC13, RAP80 or RNF168 was confirmed using western blotting

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Supplementary Figure S3: (A) Immunoblot analysis of p-Akt and Akt expression in AXT cells with the supplement of Akt inhibitor IV or X. Cell viability after the treatment of Akt inhibitors. (B) Phosphorylation status of IGF1R in AXT cells.

Supplementary Figure S1: (A) Real-time PCR analysis of Igf1 expression in AXT cell-derived subcutaneous tumors. (B) Serum IGF2 concentration of control and tumor bearing mice with or without chemotherapy. (C) U2OS cells cultured under serum free or in the presence of IGF2. (D,E) Schemes of assays for cell viability (D) and RNA interference (E).

XLS file, 30K, List of a primer pair used for ChIP assay.

Supplementary Figure S5: (A,B) Viability of AXT cells was assessed after the treatment of aminooxyacetate or bafilomycin A. (C) Cell viability for AXT cells was evaluated after exposure to the indicated agents.

Supplementary movie S1

While the functions of carbon materials with precisely controlled nanostructures have been reported in many studies, their chiral discriminating abilities have not been reported yet. Herein, chiral discrimination is achieved using helical carbon materials devoid of chiral attachments. A Fe

ASO Visual Abstract

The chemokine fractalkine (FKN) is produced by various cell types, including osteoblasts and endothelial cells in bone tissue, and signals through a sole receptor, CX3CR1, which is expressed on monocytes/macrophages, including osteoclast precursors (OCPs). However, the direct effects of FKN signaling on osteoclast lineage cells under homeostatic noninflammatory conditions remain unclear. Here, we report that FKN regulates mouse OCP survival and primes OCPs for subsequent osteoclast differentiation. Wild-type but not CX3CR1-deficient OCPs grown on immobilized FKN showed enhanced osteoclast formation following receptor activator of NF-κB ligand (RANKL) stimulation, with increased expression of osteoclast differentiation markers. Interestingly, the growth of OCPs on immobilized FKN increased the expression of

Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing the toxicity. Recently, we identified a TA system termed TsbA/TsbT in the Staphylococcus aureus genome. The induction of the tsbT gene in Escherichia coli halted both DNA and RNA synthesis, reduced supercoiled plasmid and resulted in increasingly relaxed DNA. These results suggested that DNA gyrase was the target of TsbT. In addition, TsbT inhibited both E. coli and S. aureus DNA gyrase activity and induced linearization of plasmid DNA in vitro. Taken together, these results demonstrate that the TsbT toxin targets DNA gyrase in vivo. Site-directed mutagenesis experiments showed that the E27 and D37 residues in TsbT are critical for toxicity. Secondary structure prediction combining the analysis of vacuum-ultraviolet circular-dichroism spectroscopy and neural network method demonstrated that the 22nd-32nd residues of TsbT form an α-helix structure, and that the E27 residue is located around the centre of the α-helix segment. These findings give new insights not only into S. aureus TA systems, but also into bacterial toxins targeting DNA topoisomerases.

Objectives The aim of this study is to investigate the underlying mechanism of the recovery of periodontal ligament cells (PDLCs) sequentially exposed to inflammation and mechanical loading. Materials and methods We divided PDLCs into four groups: control; compressive force (CF) alone (2.0 g/cm2 ); lipopolysaccharides (LPS) pre-treatment (0.1 μg/ml) followed by simultaneous LPS and CF stimulation, simulating uncontrolled periodontitis; and LPS pre-treatment followed by CF exposure, simulating controlled periodontitis. The expression of EphB4-ephrinB2 and EphA2-ephrinA2, and the level of osteoclastogenesis and osteogenesis were evaluated. Results Simultaneous stimulation by LPS and CF, compared with CF alone and sequential LPS and CF exposure, significantly suppressed EphB4 and enhanced ephrinA2 expression. Similarly, the most intense osteoclastic differentiation was observed under simultaneous LPS and CF stimulation, whilst sequential exposure to LPS and CF only slightly increased osteoclastic cell numbers. Both the activation of EphB4 signaling and ephrinA2 silencing lowered osteoclastic differentiation, which had previously been upregulated by simultaneous LPS and CF stimulation. These treatments also increased osteogenic differentiation. Conclusions Simultaneous LPS and CF stimulation critically enhances osteoclastogenesis in PDLCs through the suppression of EphB4 and the induction of ephrinA2 signaling. Sequential LPS and CF exposure partially abolishes the osteolytic effects of simultaneous stimulation.

Dexamethasone (DEX)-sparing strategies (one-day DEX) with palonosetron as doublet antiemetic prophylaxis have previously been studied. However, DEX-sparing regimens with 5-hydroxytryptamine-3 receptor antagonist (5-HT3RA) and aprepitant (APR), as triplet antiemetic prophylaxis, have not been evaluated. This study aimed to evaluate the efficacy of a combination of 5-HT3RA, APR, and DEX on day 1 of carboplatin (CBDCA)-based chemotherapy in patients with lung cancer. Data were pooled from a nationwide, multicenter, prospective observational study using propensity score–matched analysis to compare the incidence of chemotherapy-induced nausea and vomiting (CINV) between one- and multiple-day DEX regimens in combination with 5-HT3RA plus APR. Incidence of delayed nausea was significantly higher in the one-day than in the multiple-day DEX group. Incidence of nausea was also significantly higher in the one-day than in the multiple-day DEX group on days 3–5. Kaplan–Meier curves for nausea showed a significant difference between the two groups; however, there was no significant difference in the occurrence of vomiting or the Kaplan–Meier curves of time to vomiting. To the best of our knowledge, this study is the first to evaluate the efficacy of a DEX-sparing regimen by comparing one- and multiple-day DEX combined with 5-HT3RA and APR concerning CINV incidence in lung cancer patients receiving CBDCA-based chemotherapy. Antiemetic regimens of one-day DEX result in poor control of delayed nausea; therefore, we recommend the application of the DEX-sparing strategy only after careful patient selection while considering the development of nausea.

357 Background: Remnant gastric cancer (RGC) is defined as a cancer arising in the remnant stomach after initial gastrectomy, regardless of the previous disease or surgical procedure. Despite growing evidence of the effectiveness of minimally invasive surgery (MIS) for primary gastric cancer, MIS for RGC remains controversial due to the rarity of the disease. This study aimed to evaluate the surgical and oncological outcomes of MIS for radical resection of RGC. Methods: We collected the data of patients with RGC who underwent surgery with curative intent between 2005 and 2020 from 17 institutions in Japan, and performed a propensity score (PS) matching analysis to compare the short- and long-term outcomes of MIS with open surgery. Results: A total of 327 patients were included in this study and 186 patients were analyzed after matching. The risk ratios for overall and severe complications were 0.76 (95% confidence interval [CI]: 0.45–1.27) and 0.65 (95% CI: 0.32–1.29), respectively. The MIS group had significantly less blood loss (50 ml vs. 361 ml, P

Delayed chemotherapy-induced nausea and vomiting (CINV) is not well controlled in colorectal cancer (CRC) patients undergoing oxaliplatin (L-OHP)-based chemotherapy. Whether neurokinin-1 receptor antagonist addition to a first-generation 5HT3 antagonist (1st 5-HT3 RA) and dexamethasone (DEX) is beneficial to these patients remains controversial. Furthermore, whether palonosetron (PALO) or aprepitant (APR) is more effective in controlling delayed CINV is unclear. We, therefore, investigated whether PALO+DEX or 1st 5-HT3 RA+DEX+APR was more effective in controlling delayed CINV, and the risk factors for delayed CINV, in CRC patients undergoing L-OHP-based chemotherapy. Data were pooled from two prospective observational Japanese studies and a phase III trial to compare CINV incidence between the PALO + DEX (PALO) and 5-HT3 RA+DEX+APR (APR) groups by propensity score-matched analysis. CINV risk factors were identified using logistic regression models. The CINV incidence was higher in the PALO group than in the APR group. Logistic regression analysis revealed alcohol consumption, motion sickness, and the PALO+DEX regimen as independent risk factors for delayed nausea, and female sex and the PALO+DEX regimen as those for delayed vomiting. Compared with prophylactic PALO + DEX, 1st 5-HT3 RA+DEX+APR was more effective in controlling delayed CINV. Thus, CRC patients receiving L-OHP-based chemotherapy should be treated with three antiemetics, including APR.

α1-Acid glycoprotein (AGP) interacts with lipid membranes as a peripheral membrane protein so as to decrease the drug-binding capacity accompanying the β→α conformational change that is considered a protein-mediated uptake mechanism for releasing drugs into membranes or cells. This study characterized the mechanism of interaction between AGP and lipid membranes by measuring the vacuum-ultraviolet circular-dichroism (VUVCD) spectra of AGP down to 170 nm using synchrotron radiation in the presence of five types of liposomes whose constituent phospholipid molecules have different molecular characteristics in the head groups (e.g., different net charges). The VUVCD analysis showed that the α-helix and β-strand contents and the numbers of segments of AGP varied with the constituent phospholipid molecules of liposomes, while combining VUVCD data with a neural-network method predicted that these membrane-bound conformations comprised several common long helix and small strand segments. The amino-acid composition of each helical segment of the conformations indicated that amphiphilic and positively charged helices formed at the N- and C-terminal regions of AGP, respectively, were candidate sites for the membrane interaction. The addition of 1 M sodium chloride shortened the C-terminal helix while having no effect on the length of the N-terminal one. These results suggest that the N- and C-terminal helices can interact with the membrane via hydrophobic and electrostatic interactions, respectively, demonstrating that the liposome-dependent conformations of AGP analyzed using VUVCD spectroscopy provide useful information for characterizing the mechanism of interaction between AGP and lipid membranes., This work was financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (numbers 19K06587 and 15K07028)., [This article is part of the Special Issue: In honor and memory of Prof. Koji Nakanishi. See the first articles for this special issue previously published in Volumes 31:12, 32:3, and 32:4. More special articles will be found in this issue as well as in those to come.]

The locomotor system is highly bilateral at the macroscopic level. Homochirality of biological molecules is fully compatible with the bilateral body. However, whether and how single-handed cells contribute to the bilateral locomotor system is obscure. Here, exploiting the small number of cells in the swimming tadpole larva of the ascidian Ciona, we analyzed morphology of the tail at cellular and subcellular scales. Quantitative phase-contrast X-ray tomographic microscopy revealed a high-density midline structure ventral to the notochord in the tail. Muscle cell nuclei on each side of the notochord were roughly bilaterally aligned. However, fluorescence microscopy detected left-right asymmetry of myofibril inclination relative to the longitudinal axis of the tail. Zernike phase-contrast X-ray tomographic microscopy revealed the presence of left-handed helices of myofibrils in muscle cells on both sides. Therefore, the locomotor system of ascidian larvae harbors symmetry-breaking left-handed helical cells, while maintaining bilaterally symmetrical cell alignment. These results suggest that bilateral animals can override cellular homochirality to generate the bilateral locomotor systems at the supracellular scale.

A rapid membrane enzyme immunoassays (EIA) are frequently used to diagnose Clostridioides difficile infection (CDI). If EIA does not provide a definitive CDI diagnosis, whether treatment with anti-CD agents is to be performed depends on the pathogenesis and severity of the disease. In Japan, "MN criteria" have been proposed for the classification of disease severity. In this study, we investigated the association between disease severity and CDI prognosis when MN criteria are used.This study included 102 patients diagnosed with CDI between April 2015 and March 2020. The disease serverity classification accorditng to MN criteria was divided into two groups: non-severely ill (mild to moderate) and severely ill (severe to critical) group.Mortality was significantly higher in severely ill patients than non-severely ill patients (46.7% vs. 13.8%, p = 0.0025). Multivariable analysis showed that the mortality of patients with CDI was significantly associated with advanced age (odds ratio [OR] = 1.1; 95% confidence interval [CI] = 1.0-1.2; p = 0.019) and disease severity (OR = 4.2; 95% CI = 1.2-14.8; p = 0.023).The classification of disease severity according to the MN criteria would be particularly useful in predicting the patients' prognoses.

Mid-infrared free-electron laser (MIR-FEL) is potentially applicable for tissue ablation and dissociation of pathological peptide aggregates in medicine. However, it is still poorly understood how the MIR-FEL irradiation influences on functional proteins such as enzymes. In the current study, the effect of MIR-FEL on both aggregate and non-aggregate (= native) states of hen egg-white lysozyme (HEWL) as a representative enzyme has been investigated. Absorption intensity at terahertz region (0.3–1.2 THz, 10–40 cm−1) of the aggregate of HEWL was lower than that of the native HEWL, but the former was increased nearly to the same level with the latter after the MIR-FEL irradiation tuned to 6 μm that corresponds to carbonyl stretching vibrational mode of amide bonds (amide I). This indicates that the aggregate of HEWL was converted to the native state by the irradiation. On the other hand, synchrotron-radiation vacuum-ultraviolet circular-dichroism spectroscopy showed that protein conformation of the native HEWL, which was rich in α-helix, was little changed after the MIR-FEL irradiation under the same condition with the case of the aggregate of HEWL. Furthermore, the enzymatic hydrolysis activity of the native HEWL against bacterial glycan was not remarkably decreased by the irradiation. Therefore, it can be estimated that the native structure of HEWL is little damaged although the aggregate state can be easily dissociated by the resonant excitation at amide bonds.

Intermittent parathyroid hormone (iPTH) treatment induces bone anabolic effects that result in the recovery of osteoporotic bone loss. Human PTH is usually given to osteoporotic patients because it induces osteoblastogenesis. However, the mechanism by which PTH stimulates the expansion of stromal cell populations and their maturation toward the osteoblastic cell lineage has not be elucidated. Mouse genetic lineage tracing revealed that iPTH treatment induced osteoblastic differentiation of bone marrow (BM) mesenchymal stem and progenitor cells (MSPCs), which carried the leptin receptor (LepR)-Cre. Although these findings suggested that part of the PTH-induced bone anabolic action is exerted because of osteoblastic commitment of MSPCs, little is known about the in vivo mechanistic details of these processes. Here, we showed that LepR+ MSPCs differentiated into type I collagen (Col1)+ mature osteoblasts in response to iPTH treatment. Along with osteoblastogenesis, the number of Col1+ mature osteoblasts increased around the bone surface, although most of them were characterized as quiescent cells. However, the number of LepR-Cre-marked lineage cells in a proliferative state also increased in the vicinity of bone tissue after iPTH treatment. The expression levels of SP7/osterix (Osx) and Col1, which are markers for osteoblasts, were also increased in the LepR+ MSPCs population in response to iPTH treatment. In contrast, the expression levels of Cebpb, Pparg, and Zfp467, which are adipocyte markers, decreased in this population. Consistent with these results, iPTH treatment inhibited 5-fluorouracil- or ovariectomy (OVX)-induced LepR+ MSPC-derived adipogenesis in BM and increased LepR+ MSPC-derived osteoblasts, even under the adipocyte-induced conditions. Treatment of OVX rats with iPTH significantly affected the osteoporotic bone tissue and expansion of the BM adipose tissue. These results indicated that iPTH treatment induced transient proliferation of the LepR+ MSPCs and skewed their lineage differentiation from adipocytes toward osteoblasts, resulting in an expanded, quiescent, and mature osteoblast population. © 2019 American Society for Bone and Mineral Research.

Δ9-Tetrahydrocannabinol (THC) is known to have various pharmacological effects mediated through activation of cannabinoid CB1 and CB2 receptors in rodents. In adult rats, 22- and 50-kHz ultrasonic vocalizations (USVs) serve as an effective communication system and as indicators of negative and positive states, respectively. The present study was performed to determine whether THC affects USVs in adult rats, and to determine the roles of cannabinoid receptors in these effects. THC (1, 3 mg/kg) was administered intraperitoneally to adult male Wistar rats 60 min before measurement of USVs. The CB1 antagonist, SR141716 (3, 6 mg/kg), or CB2 antagonist, AM630 (1, 10 mg/kg), was administered intraperitoneally 10 min before THC. USVs were measured during a 5-minute period without air puff stimulus or with air puff stimulus. THC did not affect 22- or 50-kHz USVs without air puff stimulus. On the other hand, THC significantly increased the number of 22-kHz USVs, but not 50-kHz USVs, after air puff stimulus. Moreover, SR141716 at 6 mg/kg, but not AM630 at either dose, inhibited the increase in number of 22-kHz USVs induced by THC after air puff stimulus. These results suggest that THC induced changes in sensitivity to aversive air puff stimuli through CB1 receptors, and as a result increased emission of 22-kHz USVs in rats.