The Spoon Daily news and analysis about the food tech revolution little did I know that a chance meeting in the Venetian in Las Vegas would eventually lead to the creation of Japan’s most influential and well-attended food tech event We’d just come off our second North America Smart Kitchen Summit a couple of months prior and two first-time attendees who had made the trip to Seattle we got together in Tokyo for the first-ever SKS Japan and I was wonderfully surprised at the excitement and innovation around the food system taking place in this beautiful country and it’s incredible how this show has matured into one of the most important food tech events on the calendar This year’s event spanned three days and brought in over one hundred speakers from across the globe and the food system Above: Myself and Hiro Tanaka giving the opening keynote to kick of SKS Japan 2024 (and announce SKS 2025 dates) Kevin Yu (SideChef) and Assaf Pashut (Chefee) join me for a conversation about how the consumer kitchen has evolved over the past decade and where it will go over the next ten years Above: Jasmin Hume (Shiru) joins me on stage and Adam Yee (Sobo Foods) and Tarini Naravane (Grainge AI) dial in to talk about the impact of AI on developing new food inputs and products comes to Japan from Seattle to talk about his fast-growing restaurant and how he leverages technology such as robotics to help “scale craft” Above: Author and Wired’s kitchen tech reviewer Joe Ray sits for a fireside chat on his thoughts about food tech journalism and first impressions of Japan’s food and restaurant scene Above: A talk about new approaches using upcycling and food life extension technologies with Moody Soliman (Ryp Labs) Hiroki Nakajima (University of Tokyo) during a session entitled Tokyo Regenerative Food Lab in Action Bottom right is demo table for AlgaleX’s upcycled algae protein product line Above: Exhibitors head out to the streets of Tokyo for a street fair on the final day of SKS Japan to share their innovations with a wider audience Just enter your email and we’ll take care of the rest: and website in this browser for the next time I comment Δdocument.getElementById( "ak_js_1" ).setAttribute( "value" Feed your mind! Subscribe to one of our podcasts! © 2016–2025 The Spoon Metrics details This study investigated the three-dimensional (3D) cellular interactions and tunneling nanotubes (TNTs) during fetal mouse skin regeneration on embryonic days 13 (E13) and 15 (E15) We aimed to understand spatial relationships among cell types involved in skin regeneration and assess the potential role of TNTs Full-thickness skin incisions were performed in E13 and E15 embryos processed for 3D reconstruction (1 μm thickness sections) and subjected to whole-mount immunostaining We conducted in vitro co-culture experiments with fetal macrophages and fibroblasts to observe TNT formation To assess the effect of TNTs on skin regeneration an inhibiting agent (cytochalasin B) was administered to amniotic fluid Results revealed that E13 epidermal keratinocytes interacted with dermal fibroblasts and macrophages TNT structures were observed at the E13-cell wound sites confirmed through in vitro co-culture experiments In vitro and utero cytochalasin B administration hindered those formation and inefficient skin texture regeneration at E13 wound sites This emphasizes the necessity of 3D cellular interactions between epidermal and dermal cells during skin regeneration in mouse embryos at E13 The prevalence of TNT structures indicated their involvement in achieving complete skin texture restoration we aimed to enhance the 3D cellular interactions that have been previously identified in various mouse fetal developmental stages we induced wounds in mouse fetal skin at various developmental stages and acquired thick semi-thin consecutive tissue Sects (1 μm thickness each) from the wound sites these sections were overlaid and reconstructed in 3D to analyze the spatial relationships between cells we performed whole-mount immunostaining on thicker tissue sections using confocal microscopy to examine the 3D microstructures of the cells relevant to wound healing with the formation of dense tunneling nanotubes (TNT) specifically among macrophages and between macrophages and fibroblasts We demonstrated inhibition of skin texture regeneration by administering a inhibiting agent of actin polymerization This groundbreaking discovery aligns with the results of this present study (A): Skin wounds of mouse fetuses at E15-24 h Comparison between 7 and 1 μm images; the 1 μm image offers distinctive cell boundaries (B): 3D reconstructed images in the z-axis direction of skin wounds at E13-24 (C): Comparison of epidermis-dermis interactions in consecutive section alignment images at E13-24 h and E15-24 h The epidermis at E13-24 h contracts and closes via actin cables the epidermal cells proliferate and thicken as they close This difference indicates that at E15-24 h the thickened part of the epidermis comes into contact with the deep layer dermis This interaction between the epidermis and dermis differed from that observed at E13-24 h indicating its role in disrupting skin texture regeneration In this study, macrophages accumulated in the wound area, even at E13, and were present in normal skin. When examined using a confocal microscope, these cells displayed numerous thin, elongated processes, forming a network of intercellular interactions [Fig. 2A,B]. (A): Observation of wound-site macrophages and blue (4′,6-diamidino-2-phenylindole [DAPI]) staining (b): Enlarged image showing the F4/80-positive linear structure Yellow arrows (wound ridge) and white arrows (tunneling nanotube [TNT]) (B): Macrophage-phagocytic enlarged images at the wound center This image shows F4/80-positive macrophages engulfing multiple cells (C): Macrophage phagocytosis after toluidine blue staining on E13-24 h (D): Overview of the images at each time point The number of phagocytic cells per 10 slices (1 μm/slice) around the wound center is measured (A): 3D image of whole-mount staining on E13-24 h and E15-24 h Numerous F4/80-positive tunneling nanotube (TNT) structures are observed along the wound ridge TNT structures are observed among macrophages at developmental stage In comparison to E15-24 h, E13-24 h exhibited a higher density of macrophages at the wound center. However, both conditions resulted in elongated TNT structures along the periphery of the wound. Conversely, there was no local density of TNT-like structures in normal skin areas at E13-24 h and E15-24 h (corresponding to E14 and E16, each) [Fig. 3C] The F4/80-positive macrophages were isolated from E13 fetal trunk skin using MACS and subjected to co-cultivation with dermal fibroblasts from the corresponding period The presence or absence of TNTs was observed using SEM Those structures of the macrophage were observed to be interconnected with another macrophage Scanning electron microscopy (SEM) images of co-cultured E13 macrophages and fibroblasts TNT structures in certain macrophages are indicated by white arrowheads (A): Immunohistochemistry images of E13 macrophages and adult macrophages (B): Immunostaining after administration of cytochalasin (B) to fibroblast cultures at different concentrations (Control (C): Inhibition of TNT formation in E13 macrophages with Cytochalasin B TNT structures were observed as indicated by white arrowheads (D): Observation of actin polymerization inhibition in the skin wound site under Cytochalasin B administration many cells at the wound margin exhibit a rounded morphology due to Cytochalasin B the fibers of actin polymerization can be clearly observed (A): Cytochalasin B administration to fetuses and whole-mount staining of 3D images at E13-24 h and E15-24 h (B): Comparison between the cytochalasin B-treated and control groups after 24 h The cytochalasin B-treated group shows a reduction in macrophage density within the wound and TNT formation The zoom shows that macrophage cells were detected with surface mode by Imaris (C): Comparison among the number of F4/80-positive tunneling nanotubes in the wounds Measurements are performed at three different locations (D): Microscopic images taken 72 h after cytochalasin B administration to fetal wound sites The administered group exhibits the disappearance of skin texture at E13-72 h which typically undergoes complete regeneration E: H&E staining 72 h after cytochalasin B administration depicts delayed re-epithelialization at E15-72 h compared to the control F: Whole-mount staining after cytochalasin B administration on E13-72 hand E15-72 h we aimed to further investigate the relationship between various cells and their intricate structures and observe the spatial relationships among cell populations beneath the wound site we used dual fixation and epoxy resin embedding a reliable technique that ensures consistent collection of continuous sections with minimal risk of cutting errors or missed sections We observed the aggregation of macrophages at the wound site despite the relatively low-inflammatory conditions in fetal wounds Although further analysis is required to determine their specific roles we observed that macrophages are involved in activities indicating their significance in wound healing during full skin regeneration This transition in macrophage origin corresponds to variations in their functions during wound healing and the timing of cessation of skin texture regeneration yolk sac-derived macrophages may affect skin regeneration Schematic representation of the wound healing process at E13 (characterized by complete regeneration) and E15 (characterized by skin texture loss) yellow (dermal fibroblasts in the superficial dermis) Additionally, the dynamics of actin directly regulate fibrosis16 inhibition of those formation resulted in a considerable reduction of TNTs at the 24-h time point hindering skin texture regeneration at E13-72 h indicating the involvement of those structures in skin texture regeneration the molecular mechanisms and the substances involved in this process require further investigation it is essential to elucidate the contributions of macrophages during the developmental stages differences between superficial and deep fibroblast subtypes spatial and temporal molecular interactions among inflammatory cells and the functions of TNTs associated with these processes We consulted the ARRIVE guideline (https://arriveguidelines.org) to ensure proper reporting of animal experiments The research protocol was approved by the Institutional Animal Care and Use Committee of Keio University School of Medicine (Approval Number: A2023-006) All experiments were followed with the Facility Guidelines This experiment used Pregnant Slc:ICR mice obtained from Sankyo Labo Service Corporation (Tokyo Embryos were labeled E0 when vaginal plugs were observed Fetal surgeries were performed at E13 and E15 with a minimum of four mothers undergoing surgery at each time point Pregnant mice were anesthetized using 3–5% isoflurane inhalation and their abdominal walls were incised to expose the pregnant uterus an incision was made in the uterine muscle layer covering the fetus the amniotic sac and yolk sac were exposed and a small incision was made in the amniotic sac to partially expose the fetal skin a full-thickness incision of approximately 1 mm in length was made on the fetal lateral chest only the amniotic and yolk sacs were sutured with 9–0 nylon The fetus remaining within the amniotic sac and uncovered by the uterine muscle layer was returned to the abdominal cavity before suturing the abdomen a small incision was made from the top of the uterus to the amniotic and yolk sacs only the uterine muscle layer was sutured with 9–0 nylon the uterus was returned to the abdominal cavity and sutured ritodrine hydrochloride (FUJIFILM Wako Pure Chemical Co. was intraperitoneally administered at a rate of 1 μg/g body weight The abdominal wall and skin were sutured using 5–0 nylon sutures For fetal specimens collected 48 and 72 h postoperatively the wound site was identified by labeling with DiI (0.25%) dissolved in 1% ethanol in phosphate-buffered saline (PBS) for visualization At various time points following wound creation the mothers were euthanized through cervical dislocation with a minimum of four fetuses per time point undergoing wound creation Fetal trunk skin wound regions were collected at each time point under a stereomicroscope and subjected to the fixation and embedding procedures described below The samples were pre-fixed with 2.5% glutaraldehyde (TAAB Laboratories Equipment UK) and 2% paraformaldehyde (PFA) (Nakalai Tesque Japan) in 0.1 M phosphate buffer (pH7.4) (PB) for 2 h at room temperature (RT) After washing in PB for 10 min × 3 times at RT the samples were post-fixed with 1% OsO4 (TAAB Laboratories Equipment They were washed in cold distilled water for 10 min × 3 times The samples were dehydrated with a graded series of ethanol (50 and 99.5% for 10 min each and 3 times in 100% for 10 min each) and propylene oxide (twice for 15 min each) then followed by infiltration and embedding in Epon 812 (TAAB Laboratories Equipment Samples were degassed using an aspirator at RT (1 μm thickness) (800 to 1,000 sections from each sample) were cut with a diamond knife (DiATOME Switzerland) equipped with an ultramicrotome ULTRACUT R (Leica Biosystems Germany) and collected on slide glass CREST (Matsunami Japan) (about 15 sections on each slide glass) After drying the sections for 24 h on a heater at 60 ℃ they were stained with 0.5% toluidine blue (Merck images were captured using a NanoZoomer (Hamamatsu Photonics Japan) with a 40 × objective lens and saved in a TIFF format The images were processed using a free software GNU Image Manipulation Program (Gimp 2.10.14) The following procedures were done using free-software The image set consisted of about 1000 images from each sample were arranged in order After performing color adjustment and contrast enhancing the images were converted to 8-bit grayscale images The region of interest was trimmed from each image 3D volume images were reconstructed using the medical imaging software (Cybernet, Tokyo, Japan, https://www.cybernet.co.jp/medical-imaging/products/expertintage/overview.html) The skin wound sections were fixed by immersion in PFA (4%) for 24 h fixed tissues were embedded in paraffin and cut into sections of 7 µm thickness Deparaffinization was accomplished using xylene and ethanol followed by hematoxylin and eosin (H&E) staining using hematoxylin and eosin stain (Muto Pure Chemicals Co. Frozen sections were used for immunostaining immersed in sucrose solutions (20 and 40%) in PBS embedded in optimal cutting temperature (OCT) compound (Sakura Finetek Japan Co. Sections of 10 µm thickness were obtained using a cryostat (Leica Biosystems The immunostaining procedure involved the following steps and PBST (PBS containing 0.2% Triton X-100) followed by blocking with 5% bovine serum albumin (BSA) in PBST USA; 1:200) and anti-TNFa-IP2 antibody (Santa Cruz were added to the sections and incubated at 4 °C overnight Sections were washed and mounted on glass slides using ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific For whole-mount samples measuring 20 × 15 mm Skin samples were collected from the trunks of E13 fetuses after euthanizing two Slc: ICR mice were dissected on ice using scissors and a No The dissected tissue was subjected to a reaction in a mixture of Dispase II (GIBCO Collagenase type IV (in PBST: PBS with 0.1% Triton X-100) the tissue was neutralized using RPMI 1640 with GlutaMAX supplement (GIBCO USA) and 5 mL of 10% fetal bovine serum (FBS) buffer it was strained using a cell strainer (70 μm) and the tissue was incubated in lysis buffer (BD Pharm Lyse and the cell suspension was adjusted in PBS (pH 7.2) containing 0.5% BSA and 2 mM ethylenediaminetetraacetic acid (EDTA) magnetic-activated cell sorting (MACS) buffer The tissue was further processed by incubating with Anti-F4/80 MicroBeads UltraPure (Miltenyi Biotec Germany) at a 1:10 dilution in 100 μL for 15 min at 4 °C A Miltenyi magnetic separation (MS) column was prepared in MACS MultiStand and washed thrice with MACS buffer The cell suspension was passed through a cell strainer (100 μm) into the column the column was removed from the magnetic field and MACS buffer (500 μL) was added to elute the F4/80-positive cells for further use Macrophage-tunneling nanotubes were co-cultured with fibroblasts for the same duration Fibroblasts were obtained through dissection and fine mincing of fetal skin followed by culturing in Dulbecco's modified Eagle’s medium (DMEM) with low glucose (FUJIFILM Wako Pure Chemical Co. Japan) containing FBS (10%) and penicillin/streptomycin (P/S) (1%) Fibroblasts were separated using trypsin–EDTA at 37 °C for 3 min and placed in a 10 cm culture dish Dermal fibroblasts from E13 were derived from a pool of fibroblasts and cells from passage 3 onwards were used for in vitro experiments Macrophages and fibroblasts were co-cultured in RPMI supplemented with FBS (10%) and P/S (1%) (1:1 ratio) for 24 h The following steps were employed to prepare samples for scanning electron microscope (SEM) analysis The samples were pre-fixed in a solution containing PFA (4%) and glutaraldehyde (2.5%) in 0.05 M cacodylate buffer (pH 7.4) at RT for 30 min The samples were then rinsed five times in 0.05 M cacodylate buffer for 5 min each Postfixation was performed by immersing the samples in a solution of OsO4o (1%) and cacodylate buffer (0.05 M) at RT for 2 h the samples were washed thrice with distilled water The samples were dehydrated using a series of ethanol solutions with increasing concentrations (25 The samples were immersed in an equal-volume mixture of ethanol (100%) and isoamyl acetate for 30 min followed by two 15-min immersions in isoamyl acetate at RT for substitution the samples were dried using a critical point drying apparatus (EM CPD300 The dried samples were observed using SEM (SU6600-A To observe live-cell interactions among primary macrophages 50% of the macrophages were incubated with DiI (0.25%) dissolved in ethanol (1%) for 5 min or Cell mask Deep Red Actin Tracking Stain (Invitrogen DiI-unstained macrophages were co-cultured with DiI-stained macrophages in a 1:1 ratio in RPMI containing FBS (10%) and P/S (1%) Live imaging was immediately performed using a confocal laser scanning microscope (FLUO-VIEW FV3000 with differential interference contrast and Cy5 Dermal fibroblasts were treated with cytochalasin B (1 or 10 μg/mL) (FUJIFILM Wako Pure Chemical Co. and immunostaining was performed using standard culture medium The cells were initially fixed with PFA (4%) for 10 min followed by three 5-min washes in PBS and a 5-min incubation in PBST (0.1%) After another three washes with PBS for 5 min each the cells were blocked with BSA (1%) for 20 min followed by another three 5-min PBS washes the sections were incubated with Acti-stain™ 488 phalloidin (Cytoskeleton Inc. USA; 1:400) and rabbit anti-αSMA antibody (Abcam The cells were then washed thrice with PBS followed by a 1-h incubation with secondary antibodies from the Alexa Fluor series (Invitrogen nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) staining solution (1:500) The cells were then mounted on glass slides using ProLong™ Gold Antifade Mountant and observed under a confocal microscope at an in vivo administration concentration of 10 μg/mL Similar fetal surgical procedures were performed on Slc: ICR mice on the 13th and 15th days of pregnancy Each fetus was subcutaneously injected with cytochalasin B (10 ng/mL) immediately before wounding and wounds were created immediately post-injection The wound sites were collected after 24 and 72 h 0.25% DiI in PBS was used for staining after 72 h Tissues collected after 24 and 72 h were subjected to whole-mount staining following a procedure similar to that described previously The data that support to this study are available from the corresponding author Data is provided within the manuscript or supplementary information files Fetal skin possesses the ability to regenerate completely: Complete regeneration of skin Reactions of mammalian fetal tissues to injury II Second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation Downregulation of Lhx2 markedly impairs wound healing in mouse fetus Fibroblast growth factor 7 suppresses fibrosis and promotes epithelialization during wound healing in mouse fetuses Effect of sonic hedgehog on the regeneration of epidermal texture patterns Actin cable formation and epidermis-dermis positional relationship during complete skin regeneration M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex Functional connectivity between immune cells mediated by tunneling nanotubules Dihydrocytochalasin B enhances transforming growth factor-beta-induced reexpression of the differentiated chondrocyte phenotype without stimulation of collagen synthesis Shen, A. et al. An integrative web-based software tool for multi-dimensional pathology whole-slide image analytics. Phys. Med. Biol. https://doi.org/10.1088/1361-6560/ac8fde (2022) Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages Regulation of MKL1 via actin cytoskeleton dynamics drives adipocyte differentiation Distinctive effects of cytochalasin B in chick primary myoblasts and fibroblasts Download references We would like to thank Editage (www.editage.jp) for English language editing Department of Plastic and Reconstructive Surgery Department of Tissue Repair and Regenerative Medical Science supervised the technique for electron microscopy and 3D reconstruction The authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-024-68083-6 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Cardiac neuroimaging with 123I-metaiodobenzylguanidine (123I-MIBG) has been officially used in clinical practice in Japan since 1992 The nuclear cardiology guidelines of the Japanese Circulation Society recommended cardiac 123I-MIBG imaging for the management of heart failure (HF) patients particularly for the assessment of HF severity and prognosis of HF patients Consensus in North American and European countries regarding incorporation into clinical practice This article summarizes 22 y of clinical applications in Japan of 123I-MIBG imaging in the field of cardiology; these applications are reflected in cardiology guidelines A standardized cardiac 123I-MIBG parameter is the basis for clinical decision making and enables common use of parameters beyond differences in institutions and studies Several clinical studies unanimously demonstrated its potent independent roles in prognosis evaluation and risk stratification irrespective of HF etiologies An HMR of less than 1.6−1.8 and an accelerated washout rate are recognized as high-risk indicators of pump failure death and fatal arrhythmias and have independent and incremental prognostic values together with known clinical variables such as left ventricular ejection fraction and brain natriuretic peptide Another possible use of this imaging technique is the selection of therapeutic strategy such as pharmacologic treatment and nonpharmacologic treatment with an implantable cardioverter–defibrillator or cardiac resynchronization device; however this possibility remains to be investigated Recent multiple-cohort database analyses definitively demonstrated that patients who were at low risk for lethal events and who were defined by an HMR of greater than 2.0 on 123I-MIBG studies had a good long-term prognosis Future investigations of cardiac 123I-MIBG imaging will contribute to better risk stratification of low-risk and high-risk populations to the establishment of cost-effective use of this imaging technique for the management of HF patients and to worldwide acceptance of this imaging technique in clinical cardiology practice resulting in official approval by Japanese social health insurance the use of 131I-MIBG in the field of oncology was approved The clinical indications for 123I-MIBG include neuroblastoma and pheochromocytoma This review surveys a history of cardiac 123I-MIBG imaging recent advances in standardization of this imaging technique the possible efficacies and future directions for clinical decision making in the management of HF are discussed The number of 123I-MIBG studies for HF was estimated to be approximately 10,000 per year The use of myocardial perfusion SPECT has slightly decreased in recent years in Japan but it is noteworthy that the use of 123I-MIBG imaging has been gradually increasing Number of 123I-MIBG studies performed since 2000 (A) and breakdown in 2012 (B) in Japan 123I-MIBG Prognostic Studies in Japan with Endpoint of Cardiac Death Recommendations for 123I-MIBG Sympathetic Imaging in Japanese Circulation Society’s Guidelines (4) Cardiac 123I-MIBG activity is affected by imaging conditions particularly the collimator type; the HMR obtained with a medium-energy (ME) collimator is greater than that obtained with a low-energy (LE) one our recommendation is to use background subtraction for consistency among various studies other than HF SPECT imaging can assess regional 123I-MIBG defects, which indicate viable but denervated, or injured, myocardial tissue. The Japanese Society of Nuclear Medicine (JSNM) Working Group database is the first 123I-MIBG SPECT database created for 180° and 360° rotations in each sex (Fig. 2) (29) there are several limitations of SPECT imaging probably because of physiologic changes brought about by aging when cardiac 123I-MIBG activity is globally and markedly reduced reconstruction of SPECT images and regional assessment with a scoring system are difficult to achieve nonspecific inferior wall defects are observed inferior wall defects are also observed in diabetic hearts although the regional assessment of 123I-MIBG distribution with high-quality imaging is useful for the detection of localized denervation it seems to be supplementary to the global assessment of 123I-MIBG activity in HF Normal values for HMR (A) and WR (B) and polar maps (C) based on JSNM Working Group database Italic numerals in bars indicate reference ranges BG = background; LME = low to medium energy HMR) in clinical decision making for patients with chronic HF Standardization of cardiac 123I-MIBG activity quantified as HMR by conversion of data obtained with low- to medium-energy (LME) collimator to data obtained with LE high-resolution (LEHR) collimator (A) 32-y-old male with 22% LVEF and NYHA class 3 His HMR was converted from 1.36 obtained with LME collimator to 1.23 obtained with LEHR collimator and 5-y mortality rate was reestimated to be more than 50% (10%/y) (B) 86-y-old woman with 51% LVEF and NYHA class 2 and 5-y mortality rate was recalculated to be 10% (2%/y) This method enables the calibration of data obtained for any kind of HMR (either ME or LE collimator) contributing to the universal application and comparison of HMRs in the selection of a therapeutic strategy Process of standardization of cardiac 123I-MIBG data for calculation of HMR and risk-based decision making in management of chronic HF demonstrating that cardiac 123I-MIBG imaging can be used—independently of conventional prognostic markers—to risk-stratify patients with HF (low risk vs high risk for lethal events) and predict the probability of long-term survival with pharmacologic or nonpharmacologic treatments Cumulative mortality curves comparing patients with idiopathic dilated cardiomyopathy (A) and coronary artery disease (B) when cutoff values of 1.70 for HMR and 35% for LVEF were used with Japanese pooled databases (2) cardiac 123I-MIBG imaging can be used to monitor the effects of treatment with β-blocking agents or their combinations by correlating an increase in cardiac 123I-MIBG activity (HMR) and a decrease in 123I-MIBG WR with an improvement in NYHA functional class treatment with angiotensin-converting enzyme inhibitors or β-blockers significantly reduced cardiac death prevalence and the 5-y cardiac mortality rate compared with treatment without these drugs (15% vs and the risk reduction rate at 5 y in patients with an HMR of 1.53 or more was significantly greater than that in patients with an HMR of less than 1.53 and receiving the same drug treatment (67% vs current optimal drug treatment can improve survival rate but the efficacy for clinical outcomes is likely to depend on cardiac 123I-MIBG activity strongly suggesting that cardiac 123I-MIBG activity not only can estimate drug effects but also can predict cardiac risk improvement with appropriate drug treatment In addition to the secondary prevention of SCD or lethal arrhythmic events the most accepted ICD indication for the primary prevention of SCD is based on chronic HF presenting with prior myocardial infarction cardiac 123I-MIBG imaging enables cardiologists to help identify patients who are most susceptible to lethal arrhythmias and event risks and who can actually benefit most from device therapy by overcoming the limitations of current device therapy criteria most of which consist of surrogate markers of lethal events and QRS prolongation (intraventricular electrical dyssynchrony) Assessment of the cardiac reinnervation process with cardiac 123I-MIBG imaging may be useful for the management of patients with heart transplants in an outpatient care unit by determining the appropriate exercise prescription evaluating the exercise training effect and predicting an improvement in long-term survival for differentiating high-risk patients from low-risk patients and for anticipating survival time in each patient with chronic HF Low risk of mortality in subjects with HMR of greater than 2.0. (A) All-cause mortality curves over 10 y for Japanese pooled databases (n = 1,322) (2), indicating low probability of lethal events independent of LVEF when HMR on 123I-MIBG studies was more than 2.0. (B) Cardiac mortality curves at 5 y, estimated with logistic model of HMR on 123I-MIBG studies (n = 933) (33) indicating low probability of lethal events independent of age difference when HMR on 123I-MIBG studies was more than 2.0 Current Tentative Clinical Use of Cardiac 123I-MIBG Imaging for HF This work was partly supported by the working group activity of the Japanese Society of Nuclear Medicine and by Grants-in-Aid for Scientific Research in Japan (to Kenichi Nakajima) Kenichi Nakajima has done collaborative research with Fujifilm RI Pharma Co. No other potential conflict of interest relevant to this article was reported ↵* Contributed equally to this work Thank you for your interest in spreading the word on Journal of Nuclear Medicine NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it Japanese midfielder Masaya Okugawa struck his maiden UEFA Champions League goal Tuesday for Red Bull Salzburg in their 6-2 loss to title holders Bayern Munich the 24-year-old Okugawa evened the score at 2-2 just moments after stepping onto the pitch at Stadion Salzburg But the Austrian champions collapsed under an onslaught from the German heavyweights who piled on four goals in the final 11 minutes plus injury time The loss leaves Salzburg at the bottom of Group A with one point from their three matches Japan winger Shoya Nakajima was a 75th-minute substitute for Porto in their 3-0 win against Marseille who started Japan defender Hiroki Sakai at right-back The Portuguese champions are second in Group C with two wins and one loss Football: Japan beat Ivory Coast 1-0 on late Ueda header Football: Takefusa Kubo has goal, two assists for Villarreal in Europa League To have the latest news and stories delivered to your inbox, subscribe here. 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Volume 11 - 2020 | https://doi.org/10.3389/fmicb.2020.552418 (A) Schematic diagram of hyaluronate (HA) degradation of Hyl and UGL β1,4 linkages between glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) were degraded by hyaluronate lyase (Hyl; represented in red arrows) The resultant unsaturated HA disaccharides were hydrolyzed by unsaturated glucuronyl hydrolase (UGL; represented by a red arrow) The chemical formula was drawn by MarvinSketch software (ChemAxon) (B) Genetic diversity of streptococcal hyaluronate lyases (Hyls) from S pyogenes strain 476 (locus tag: M1GAS476_0819) Streptococcus pneumoniae strain R6CIB17 (E5Q10_01570) Streptococcus agalactiae strain COH1 (GBSCOH1_1103) 1LXM) and HylD (homology modeling using 1LXM) from S Phylogenetic analysis was conducted using MEGA 5 software with neighbor joining method (Tamura et al., 2011) for streptococcal Hyls from S. pyogenes strain 476 (locus tag: M1GAS476_0819), Streptococcus pneumoniae strain R6CIB17 (E5Q10_01570), S. agalactiae strain COH1 (GBSCOH1_1103), and SDSE strain 124 (SDEG_0654). Tree was visualized by Fig Tree.1 Bacterial strains SDSE-124 (GenBank accession number AP010935.1), SDSE-C167 (AP012976.1), GAS M1 476 (AP012491.2), GBS COH1 (HG939456.1), and GBS A909 (CP000114.1) were utilized in this study. Additional bacterial strains included TPCH-F36, which was isolated in Toyama Prefecture Central Hospital (Japan); KNZ01, KNZ03, KNZ10, and KNZ16 from Kanazawa University Hospital, Japan (Matsue et al., 2020); and GAS M1 SMD from National Center of Global Health and Medicine (Japan) The bacteria utilized in this study were cultured in Todd Hewitt broth (Becton Dickinson USA) supplemented with 0.2% yeast extract (THY Becton Dickinson) or brain-heart infusion (BHI Becton Dickinson); bacterial cultures were grown overnight at 37°C in a 5% CO2 atmosphere All experiments were conducted in accordance with the WHO Laboratory Biosafety Manual and institutional safety procedures at Kanazawa University Degraded HA halo assays were performed as previously described with some modifications (Smith and Willett, 1968; Oiki et al., 2019) Agar plates consisting of 0.5% peptone (Becton Dickinson and 0.4 mg/ml hyaluronic acid (FUJIFILM Wako Pure Chemical Corporation Bacterial strains were grown in BHI medium and resuspended in fresh BHI at the indicated optical density at 600 nm (OD600) One microliter of bacterial suspensions at the indicated concentration was spotted on the peptone agar followed by overnight incubation at 37°C in a 5% CO2 atmosphere The plates were then flooded with 2 N acetic acid and allowed to stand for 10 min Clear regions demarcate zones of HA degradation The oligonucleotides were purchased from Eurofin Genomics Standard curves for quantitative analysis of hyl and mutS gene expression were prepared with the indicated copy numbers of genes inserted into plasmids pQE30/hylB All media were filtered through a 0.45 μm-pore-size filter prior to use washed with fresh CDM with no added carbohydrate [CDM (−)] A specific amount of bacterial suspension was transferred into 3 ml of CDM with or without added carbohydrate to achieve an optical density of 0.05 at 600 nm These bacterial suspensions were incubated at 37°C in a 5% CO2 atmosphere OD600 was measured with a Genesys 20 spectrophotometer (Thermo Scientific USA) and the bacterial growth in each medium was assessed in three independent tests Hyaluronate lyases from GBS-COH1 and SDSE-124 were overexpressed in Escherichia coli. We could not obtain full-length Hyl. Since a previous report showed that Hyl undergoes an autocatalytic conversion to a smaller enzymatically active form (Jedrzejas and Chantalat, 2000) we utilized truncated versions of HylB and HylD both with N-terminal 170 amino acid deletions The hyl and ugl genes were cloned into pCold I (Takara Bio) using a Gibson Assembly Kit (New England Biolabs BL21(DE3) was transformed with the resultant plasmid and homogenized for recombinant protein purification with TALON Metal Affinity Resin (Takara Bio) After buffer exchange with 5 mM HEPES-NaOH (pH 7.0) using PD-10 columns (GE Healthcare the purified recombinant protein solutions were utilized for enzymatic assays Protein concentrations were measured by BCA Protein Assay kit (ThermoFisher Scientific) Ten microliters of the enzyme (recombinant HylB or HylD) solution (0.05 mg/ml) were mixed with 500 μl of 1 mg/ml HA solution containing 0.2 M buffer (described below) and left at 37°C The increase in absorbance at 235 nm due to C=C double bond formation was monitored with a U-3210 spectrometer (Hitachi Industries Japan) at a final pH of 5.0 (in the presence of citrate buffer) Five microliters of recombinant HylD and/or UGL solution (0.1 mg/ml) was mixed with 100 μl of 1 mg/ml HA solution containing 0.1 M citrate buffer pH 6.0 and left overnight at 37°C Ten microliters of the solutions were spotted on a silicagel 70 TLC plate (FUJIFILM Wako Pure Chemical Corporation) The resulting products were separated with thin layer chromatography using a 1-butanol/acetic acid/water (4:1:1 The products were visualized after spraying them with 5% sodium phosphomolybdate n-hydrate (FUJIFILM Wako Pure Chemical Corporation) in ethanol and heating to 200°C for 5 min Skin wounds were made according to a previous report with some modifications (Mukai et al., 2016) 5 week-old C57BL/6JJ mice were anesthetized through intraperitoneal administration of a mixture of Dormicum (4 mg/kg; Astellas Pharma Vetorphale (5 mg/kg; Meiji Seika Pharma Co. and Domitor (0.3 mg/kg; Nippon Zenyaku Kogyo Co. and their skin was disinfected with ethanol A circular full-thickness wound (4 mm in diameter) including the panniculus carnosus muscle was made using a sterile biopsy punch (Kai Industries Co The wounds were covered with a transparent dressing (Tegaderm; 3 M Health Care Japan) for 24 h to maintain a moist environment a 0.1-ml aliquot containing approximately 1.4 × 106 CFUs of SDSE-124 (WT or ΔhylD) in PBS was inoculated directly onto the wounds of the mice under anesthesia which were then covered with sterile gauze and transparent dressing the infected mice were anesthetized with Sevofrane (Maruishi Pharmaceutical Co. blood was immediately collected for determination of Interleukin 6 levels (R&D Systems and tissue was excised from the skin lesion The tissue samples were stained with hematoxylin and eosin (H&E; Muto Pure Chemicals Co. Japan) and Gram stain (Nissui Pharma Medical Sales Co. Japan) according to the manufacturer’s instructions An optical microscope (Eclipse E600 with U-III Film Camera System; Nikon Corp. Japan) was used to examine the stained tissue sections and images were captured using the NIS-Elements imaging software (Nikon) All animal experiments were conducted in accordance with the Declaration of Helsinki and Act on Welfare and Management of Animal (Ministry of Environment All genetic recombination experiments were approved by the Committee for Genetic Recombination Experiments (Kanazawa University) Significant HA degradation was observed on plates spotted with SDSE strains from patients with streptococcal toxic shock syndrome (strains C167 and TPCH-F36) as well as those isolated from joint fluid (strain KNZ01) and open wounds (strains KNZ10 and KNZ16); these results indicated that all SDSE strains could degrade HA deletion of hylD gene (ΔhylD) in SDSE-124 completely eliminated the capacity for HA degradation although SDSE TPCH-F36 expresses a phage-related hyaluronate lyase gene encoding HylP (WP_003058192.1) its capacity to degrade HA was not significantly higher than that of the KNZ01 or KNZ16 strains; these results suggested that the expression or activity of HylD is more significant than that of HylP in this SDSE strain Although GBS-COH1 could degrade HA at high concentrations the area of HA degradation surrounding the GBS-COH1 spots was significantly smaller compared with those containing the SDSE strains No HA degradation was observed surrounding the spots containing GBS-A909 We then proceeded to compare expression levels of the genes encoding hyl and ugl (UGL, ENZYME entry EC 3.2.1.179). SDSE-124 and GBS-COH1 bacteria were cultured in hyaluronate- and/or glucose-containing medium without agar and collected for RT-qPCR analysis. As shown in Figure 2C expression of both hyl and ugl was detected at dramatically higher levels in SDSE-124 compared with GBS-COH1 These results suggest that SDSE can express comparatively large amounts of both Hyl and UGL that facilitate rapid HA degradation The high expression of hyl and ugl in SDE-124 was not observed in glucose-containing medium indicating that HA acts as a signal to produce more HylD and UGL in SDSE These results indicate that SDSE can grow and divide more rapidly using HA as a carbon source than the other β-hemolytic streptococci evaluated here OD600 of SDSE decreased after 48–96 h of incubation suggesting that SDSE grows rapidly but reaches a plateau sooner (24 h) UGL acted on Hyl-produced disaccharides but not on full-length HA right panel); these results suggested that SDSE-124 coordinately regulates these genes so that HA is completely eliminated The fact that we observed no ugl upregulation in the SDSE-124ΔhylD mutant indicates that either (a) disaccharides produced by HylD triggers ugl expression or (b) ugl expression is triggered by HylD itself Infection with SDSE-124 and SDSE-124ΔhylD was compared in response to intraperitoneal injection of equivalent numbers of bacteria into C57BL6/J mice. We found that 53% of the mice inoculated with SDSE-124 (eight of 15 mice) died within 1 day post-infection; 67% mortality was observed at 7 days post-inoculation (Figure 5B) only 13% of the SDSE-124ΔhylD-inoculated mice (two of 16 mice) died overall Survival was significantly higher in response to infection with the SDSE-124ΔhylD strain; these results suggest that HylD is a critical element promoting the pathogenicity of SDSE These results suggest that HylD is involved in the inflammation of wounded skin (A) H&E staining and (B) Gram staining of tissue sections taken from the wound areas (C) IL-6 concentrations of SDSE-infected mice Data represent the mean ± SD of log-transformation of IL-6 concentrations Mice were infected with SDSE strains (1.4 × 106 CFU/mouse) via the wound areas blood was immediately collected for IL-6 tests and samples of the wound areas were taken for H&E and Gram stains The value of p was calculated using Student’s t-test high concentrations of UGL may lead to a decrease in the concentration of accumulated disaccharides and thus promote inflammatory responses; this will represent a significant disadvantage from the perspective of bacterial evasion It remains unclear as to whether disaccharides generated by the actions of HylD can limit inflammation and promote SDSE-124 immune evasion GBS but not SDSE secrete hyaluronate capsules that promote immune evasion; deletion of the hylB gene in GBS may promote formation of a more substantial capsule and thereby promote lethal infection indicating that SDSE can target HA accumulated at the healing sites Further analysis will be required to elucidate the mechanism of SDSE invasion we characterized the expression of genes encoding for HylD and UGL in bacterial strain SDSE-124 These two enzymes were upregulated in response to HA This investigation suggested that HylD and UGL play important roles in nutrient acquisition from hosts followed by the bacterial pathogenicity damaging host tissues This is the first report that has identified and characterized a specific virulence factor from SDSE The original contributions presented in the study are included in the article/Supplementary Material further inquiries can be directed to the corresponding author The animal study was reviewed and approved by the Committee for Genetic Recombination Experiments (Kanazawa University) MM contributed to mice experiments and improved the quality of the manuscript NT contributed to study design for revision of the manuscript KM contributed to design of wound infection experiments YN contributed to sample preparation for histological analysis and TW contributed to histological analysis of skin tissues WH critically revised the manuscript and contributed to hyaluronate degradation assay and TLC analysis SO contributed to ethical approval of the animal experiments and progress of the study All authors contributed to the article and approved the submitted version This work was partly supported by JSPS Grant-in-Aid for Scientific Research C (number 18K07133 to KO) and Takeda Science Foundation (KO) The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest We thank Advanced Science Research Center (Kanazawa University) for Sanger sequencing and Enago (www.enago.jp) for the English language review The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.552418/full#supplementary-material 1. http://tree.bio.ed.ac.uk/software/figtree/ Molecular parameters of hyaluronic acid in normal and arthritic human fluids Group G streptococci in healthy school-children and in patients with glomerulonephritis in Trinidad PubMed Abstract | CrossRef Full Text | Google Scholar The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences Human infections due to 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virulence factors and carbohydrate metabolism during invasive diseases induced by group g streptococci Severe invasive streptococcal infection by Streptococcus pyogenes and Streptococcus dysgalactiae subsp equisimilis (SDSE) endocarditis with endogenous endophthalmitis and aortic root abscess Received: 16 April 2020; Accepted: 28 August 2020; Published: 24 September 2020 Copyright © 2020 Nguyen, Ogura, Matsue, Takemoto, Mukai, Nakajima, Hoang, Iwata, Sakai, Wada, Hashimoto, Okamoto and Ichimura. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Kohei Ogura, b2d1cmFAc3RhZmYua2FuYXphd2EtdS5hYy5qcA== Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish So translates the name awarded to Japanese pilot Hiroyoshi Nishizawa following his death in 1944 unexpected; an anticlimactic end to a remarkable tale his superiors honored him with a Zen Buddhist moniker; Bukai-in Kohan Giko Kyoshi this young man bore a terrifying reputation The Second World War encompassed all corners of the world where the Allies faced the deadly power of the Japanese air force much of the combat was defined by the rise of the aces Skilled pilots on both sides fought terrifying aerial battles carried out daring raids against the enemy and engaged with combatants in the air Yet even amongst the many outstanding Japanese aces This was a man who seemed to find no fear in death – a man whose courage and daring knew no bounds flew six consecutive loops directly above enemy positions the soldiers on the ground held their fire and by the time Nishizawa returned to his own base a letter had already arrived congratulating him on his maneuvers – and inviting him back for the “all-out welcome” he deserved The Devil of Rabaul chose to decline that particular invitation but if his enemies hadn’t quite believed his reputation before that day even amongst his own comrades he seemed like a figure out of legend Nishizawa was known as a strange and solitary character for he seemed ever more content with the status of an outsider as his celebrated status increased and even once his name became synonymous with acts of courage and valor but the only place he ever seemed truly comfortable was the sky itself elements of mystery still cling to the man who seemed to stray so close to myth Nishizawa had already been present at some of the key battles fought in that geographical theater of the war and October 1944 found him escorting the first of Japan’s major kamikaze attacks against the Allies He himself was only present to back up the five bombers The young pilot watched his comrades hurtling to certain death their planes ripping into the US warships below the explosions caused by four of the five planes triggered chain reactions throughout the vessels successfully bringing down two F6F Hellcats and raising the total number of his confirmed kills to 88 It was a clear victory for the Japanese fighters While the carnage unfolded before his eyes he saw another event take place –his own death Though accounts vary as to the exact nature of the fate he envisioned for himself he returned from the mission without a shadow of a doubt in his mind While another man might have tried to run from his fate the Devil of Rabaul wasted no time in facing his destiny head on with his premonition still at the forefront of his mind he himself requested a position on the next suicide squad kamikaze mission Hiroyoshi Nishizawa was going to do it in style It would have been madness to throw away a man of his caliber so lightly the young man was already being referred to by his enemies as the Ace of Aces – losing him in a suicide mission would have been simply unthinkable Nishizawa’s superiors sealed the fate of their finest pilot He was assigned to a different mission in the end and the following morning set out as a passenger on a transport aircraft with clear skies and low winds – the region had always been known for its gentler climate the assignment with which they had been tasked should not have posed any difficulty to any of the men on board; they were transporting replacement planes from Clark Field Nishizawa’s own Zero having been destroyed in a separate operation High in the clear October skies over Mindoro Island though even they had no idea just who they were bearing down upon As the three planes flew above the town of Calapan Newell sent the lumbering transport plane before him down in flames the Japanese Ace of Aces had earned the respect of his enemies and his comrades alike He had become a nationally recognized symbol of bravery Hiroyoshi Nishizawa walks to this day a unique line between a man and a myth with a story rivalled by few others in its mysterious and evocative nature the legacy of the man now known as Bukai-in Kohan Giko Kyoshi lives on Nishizawa is remembered as an honored Buddhist person Malcolm Higgins is one of the authors writing for WAR HISTORY ONLINE Metrics details High-pressure synthesized Pb-based perovskites exhibit diverse functional properties displays distinctive diffuse scattering and valence skipping the spatial distribution of Pb ions in the crystal remains largely unexplored we elucidate the role of Pb ions with different valences through Sr substitution using high-resolution transmission electron microscopy combined with elemental mapping This approach allows us to accurately examine the distribution of Sr and Pb ions in the same atomic columns while Pb ions exhibit positional distortions Simulations based on the experimentally determined Pb distribution reproduce the diffuse scattering observed in PbCrO3 These findings suggest that the lone pair electrons of s orbitals are responsible for the local lattice distortion Our study provides atomistic insights into the local structures of materials exhibiting valence skipping for which no viable substitutes currently exist it is exempt from the RoHS Directive (EU directive on the restriction of hazardous substances in electronic and electrical equipment) Understanding the mechanisms behind the unique properties of Pb compounds is crucial for developing effective alternatives and advancing toward a Pb-free society Pb2+ (6s2) and Pb4+ (6s0) states are stabilized whereas the intermediate Pb3+ (6s1) configuration is inherently unstable These unusual phenomena may stem from the valence flexibility of Pb ions it is unclear whether Pb2+ and/or Pb4+ are responsible for off-centering further investigation is needed to clarify the origin of the characteristic diffuse scattering observed in PbCrO3 the valence state of the former phase is considered to be Pb2+0.3Sr2+0.2Pb4+0.5Cr3+O3 they are expected to reduce the number of Pb2+ ions the role of Pb4+ in PbCrO3 can be inferred through Sr2+ substitution because Sr2+ lacks lone-pair electrons in its s orbitals An additional advantage of Sr substitution is that it allows atomic displacements of both Pb and Sr ions to be observed in the same atomic columns using transmission electron microscopy We investigated the local structure of Pb0.8Sr0.2CrO3 to clarify the origin of diffuse scattering Using high-resolution scanning transmission electron microscopy (STEM) and atomic-resolution energy-dispersive X-ray spectroscopy (EDS) Our findings reveal that Pb off-centering is strongly related to the inactive lone-pair electrons of Pb2+ this study proposes a method for estimating the distribution of Pb2+ and Pb4+ ions in the charge glass state through Sr2+ substitution using atomic-resolution EDS mapping Observation of diffuse scattering in Pb0.8Sr0.2CrO3 (a) Selected-area electron diffraction pattern of Pb0.8Sr0.2CrO3 (b) Selected-area electron diffraction pattern of PbCrO3 for comparison (c) Low-magnification HAADF–STEM image of the (001) plane of Pb0.8Sr0.2CrO3 (d) Intensity profile of the diffuse scattering at the arrow positions in panels (a) and (b) Atomic-scale configurations of each atom in Pb0.8Sr0.2CrO3 Energy dispersive X-ray spectroscopy images using (b) Cr K The yellow grid lines are added as eye guides to confirm atomic displacements The dotted grid lines in the figure indicate that Sr is typically located at the intersection points the bright spots in the Pb map appear distorted or slightly displaced from the grid Sr2+ (5s0) ions are found to occupy the lattice points suggesting that Pb4+ (6s0) ions remain undisturbed owing to their similar electronic configurations Because Pb ions can exist in either divalent or tetravalent states and Pb4+ ions are assumed to be at the lattice points the Pb ions slightly off-center from these lattice points are likely Pb2+ ions would distort the coordination environment in the crystal causing off-centering similar to what is observed in ferroelectric perovskites (a) Simulated pattern calculated using Pb atom coordinates derived from observations (b) Fast Fourier transform (FFT) pattern from a high-resolution TEM image Observation of atomic-scale configuration in other regions. (a, b) Pb and Sr ion maps in the same field of view. (c, d) Observation of a different area in the same grain. These results were obtained from a grain different from that in Fig. 2 We believe these atomistic insights could be valuable for designing Pb-free functional materials with high performance particularly for a randomly displaced configuration has proven difficult because X-ray diffraction only provides information on average structures The observation methods presented in this study will be useful for investigating local structures in materials exhibiting valence skipping using a cubic-anvil high-pressure synthesis technique A powder mixture is enclosed in a platinum capsule then pressurized and heated using a graphite heater The accelerating voltage was set to 200 kV and all measurements were performed at room temperature Because elemental maps were filtered to reduce noise discussing absolute quantities based on intensity was challenging Local structural analysis using STEM is particularly useful for specimens prepared by high-pressure synthesis owing to the ability to observe a small amount of sample and to distinguish grains of target materials from those of impurities the powder specimen is embedded in epoxy resin After mechanical polishing to a thickness of approximately 10 μm it was attached to a molybdenum single-hole mesh The mesh is further processed by Ar-ion milling while rotating until a hole is formed The cross section at the edge of the perforated sample has a wedge shape and the area near the hole represents the thin film region that can be observed by TEM This milling method offers the advantage of preserving the original structure without the damage typically caused by thin-film processing with focused ion beams the Ar-ion milling method allows for a 100 µm field-of-view making it easier to search grains with target crystal orientations An electron diffraction pattern was simulated using xHREM (HREM Research Inc.) A multislice method based on a supercell was used to calculate the diffuse scattering intensity The supercell for the simulation was constructed from EDS elemental maps Ionic positions were determined from Sr and Pb elemental maps by automatically measuring the center position of each ion Sr ion sites were assumed to be at the origin without distortion and the displacement of Pb sites was calculated as the distance from the origin 42 Pb sites were extracted from the EDS maps The data that support the findings of this study are available from the corresponding author upon reasonable request Lead exposure and the derivation of maximum allowable concentrations and threshold limit values Origin of the high piezoelectric response in PbZr1−xTixO3 A monoclinic ferroelectric phase in the Pb(Zr1−xTix)O3 solid solution The missing boundary in the phase diagram of PbZr1−xTixO3 Review of crystal and domain structures in the PbZrxTi1−xO3 solid solution Chemical structures and performance of perovskite oxides Efficient planar perovskite solar cells using halide Sr-substituted Pb perovskite Evidence for Pb-O covalency in tetragonal PbTiO3 Crystallographic features and tetragonal phase stability of PbVO3 Structural Percolation in the PbM1−xMx′O3 (M Pressure-induced transformation of 6H hexagonal to 3C perovskite structure in PbMnO3 and magnetic and electronic properties of PbNiO3 with perovskite-and LiNbO3-type structures On the structure and microstructure of “PbCrO3” Theory of charge Kondo effect on pair hopping mechanism Melting of Pb charge glass and simultaneous Pb-Cr charge transfer in PbCrO3 as the origin of volume collapse Unusual inhomogeneous microstructures in charge glass state of PbCrO3 Stereochemical expression of ns2 electron pairs in metal halide perovskites Visualizing the role of Bi 6s “lone pairs” in the off-center distortion in ferromagnetic BiMnO3 First-principles study of spontaneous polarization in multiferroic BiFeO3 Lamellar-like nanostructure in a relaxor ferroelectrics Pb(Mg1/3Nb2/3)O3 Atomic-resolution electron microscopy of nanoscale local structure in lead-based relaxor ferroelectrics Direct observation of monoclinic polar nanoregions in relaxor ferroelectric Pb(Yb1/2Nb1/2)O3-PbTiO3 Local atomic order and hierarchical polar nanoregions in a classical relaxor ferroelectric An ab-initio study of the rôle of lone pairs in the structure and insulator-metal transition in SnO and PbO Charged domain boundaries stabilized by translational symmetry breaking in the hybrid improper ferroelectric Ca3–xSrxTi2O7 Stabilization of layered perovskite structures via strontium substitution in Ca3Ti2O7 revealed via elemental mapping Download references This study was supported in part by JSPS KAKENHI (Grant Numbers JP19H05625 and the Collaborative Research Projects of Materials and Structures Laboratory Kanagawa Institute of Industrial Science and Technology (KISTEC) Research Center for Autonomous System Materialogy performed the STEM observations and analyzed the data synthesized the polycrystalline specimen via high-pressure synthesis M initiated and supervised the research project All authors discussed the results and contributed to the manuscript Download citation DOI: https://doi.org/10.1038/s41598-025-93984-5 Metrics details established by the Japanese Ministry of Health Labour and Welfare and involving 27 institutions aimed to compare postoperative outcomes between laminoplasty (LM) and posterior fusion (PF) for cervical ossification of the posterior longitudinal ligament (OPLL) in order to address the controversy surrounding the role of instrumented fusion in cases of posterior surgical decompression for OPLL 478 patients were considered for participation in the study; from among them respectively) were included and evaluated using the Japanese Orthopaedic Association (JOA) scores the JOA Cervical Myelopathy Evaluation Questionnaire (JOACMEQ) Basic demographic and radiographical data were reviewed and the propensity to choose a surgical procedure was calculated there were no significant differences among the participants in terms of patient backgrounds radiographical measurements (K-line or cervical alignment on X-ray OPLL occupation ratio on computed tomography increased signal intensity change on magnetic resonance imaging) or clinical status (JOA score and JOACMEQ) after adjustments The overall risk of perioperative complications was found to be lower with LM (odds ratio [OR] 0.40 and the rate of C5 palsy occurrence was significantly lower with LM (OR 0.11 The range of motion (20.91° ± 1.05° and 9.38° ± 1.24° p < 0.0001) in patients who had PF was significantly smaller than in those who had LM multivariable logistic regression analysis showed no significant difference among the participants in JOA score OPLL progression was greater in the LM group than in the PF group (OR 2.73 Both LM and PF for cervical myelopathy due to OPLL had resulted in comparable postoperative outcomes at 2 years after surgery the two techniques must be compared under equal conditions and with the fact that severe cases are more commonly dealt with via PF taken into account the objective of the present study was to compare postoperative outcomes between LP and PF for cervical OPLL in a propensity score-matched analysis adjusted for baseline factors and radiographical characteristics of spinal cord compression A representative case of cervical laminoplasty The thickness of OPLL was 7.1 mm (double-arrow) The neutral position and range of motion at C2-C7 were 9° and 40° (b) Preoperative CT and MRI sagittal images (c) Two years postoperative functional X-rays The thickness of OPLL was 9.4 mm (double-arrow) The neutral position and range of motion at C2–C7 were 3° and 25° A representative case of cervical posterior fusion The thickness of OPLL was 8.0 mm (double-arrow) The neutral position and range of motion at C2–C7 were 11° and 20° (c) 2 years postoperative functional X-rays The thickness of OPLL was 7.4 mm (double-arrow) The neutral position and range of motion at C2–C7 were 19° and 0° and quality of life (QOL) were also evaluated preoperatively and at 24 months postoperatively with a higher score indicating better health status The JOACMEQ also incorporates visual analog scale (VAS) scores for pain or stiffness in the neck or shoulder We evaluated improvement in each of the five JOACMEQ domains Clinically significant improvement was confirmed if (1) the patient answered all the questions necessary to calculate the functional score for a domain and an increase of ≥ 20 points was obtained for that score or (2) the functional score after treatment was > 90 points even if answers for any unanswered questions were assumed to be the worst possible answers Each attending surgeon was also required to record all adverse events throughout the study period A central panel of investigators classified each adverse event in relation to the surgery; any discrepancies among reviewers were resolved by consulting source documents Perioperative complications were defined as surgery-related events occurring within 30 days of surgery The most compressed level and the presence of a signal intensity change in the spinal cord were also investigated on mid-sagittal MRI spinal canal occupation ratio of OPLL on axial CT at the maximum cord compression level and signal intensity change on T2 weight MRI were investigated The cervical lordotic angle (C2–7 angle) and range of motion (ROM) in flexion–extension were also measured using tangential lines drawn on the posterior edge of the C2 and C7 vertebral bodies on lateral radiographs taken in a neutral position Propensity score methods were used to estimate treatment effects from the observational data in the present study A backward elimination logistic regression model was created to estimate the probability of treatment assignment including all the relevant baseline variables with p-values less than 0.25 In order to balance the distribution of baseline variables between treatment groups a pseudo sample was created by weighting standardized inverse probability of treatment in which we replaced extreme values of weight with those of the 1st and 99th percentiles The Student t-test and Chi-square test were used to compare differences in means and proportions of baseline covariates in the weighted sample The covariates that were found to be different between the two groups with p-values less than 0.05 were further adjusted in the generalized linear model to compare treatment effects 30 days and 2 years after the operation We calculated odds ratios (ORs) and the 95% confidence intervals (95% CIs) for the dichotomized indices and mean differences for the continuous variables adjusted for age A two-sided p-value less than 0.05 was considered statistically significant Statistical analysis was conducted using SAS version 9.4 (SAS Institute Table 2 shows demographic parameters and QOL in the weighed sample (post propensity score matching) of patients undergoing LP or PF There was no significant difference in age although there was no significant difference between the two groups with regard to diabetes mellitus the frequency of cerebrovascular disease was higher in the LP group than in the PF group (7.4% and 1.4% Baseline radiographical measurements were not significantly different between the groups as assessed by confirming degree of cervical lordosis spina canal occupation ratio of the OPLL > 60% Baseline functional status and QOL were also not significantly different The two exceptions were the bladder function score in JOACMEQ which was significantly higher in the PF group than in the LP group (mean (standard deviation SD) of 79.7 (33.6) compared with 72.8 (20.5); p = 0.03) and VAS pain or numbness level in the arms or hands in JOACMEQ which was significantly higher in the PF group than in the LP group (mean (SD) of 68.0 (32.8) compared with 59.9 (32.4); p = 0.048) suggesting that the choice of PF or LP might have only a limited effect on the outcome for cervical function Since this study did not investigate whether or not prophylactic foraminotomy was performed further investigation is needed to determine the usefulness of concomitant foraminotomy which might be associated with lower JOACMEQ cervical function score Although JOACMEQ has criteria for determining whether these differences are clinically meaningful multivariate analysis showed no significant differences in postoperative improvement of cervical function the effect on QOL was considered to be limited although the perioperative complication of C5 palsy was more frequent in cases when PF was performed the majority of C5 palsy cases showed improvement at two-years post-surgery it can be said that this complication was not associated with significant negative outcomes at two years post-surgery it is highly likely that micromotion after laminoplasty will promote ossification and that fixation will inhibit ossification within the area of fixation since there is a possibility that ossification may develop outside the area of fixation further study is needed to determine the usefulness of the surgical technique in long-term follow-up The findings of this study are likely to be more generalizable than findings from single-center studies since patients were prospectively enrolled at 24 multicenter sites The large number of recruitment sites allowed us to evaluate outcomes for 189 patients with OPLL who received LP or PF we evaluated outcomes using different radiographical and questionnaire tools allowing for a comprehensive assessment of surgical outcomes in patients with OPLL future large cohort studies need a separate analysis for the two procedures was performed by highly experienced surgeons but the number of years of experience of each of the surgeons is unknown We cannot deny the possibility that the difference in experience of the surgeons between the two techniques may have affected the results in considering the usefulness of surgical treatment a comparison with the natural course of cervical OPLL progression must be made the present data set was not compared with data for the natural course We are currently investigating the natural course of cervical OPLL in a multicenter study which will make it possible to report on comparative results for quality of life in the future cervical LP and PF provide almost comparable functional and QOL improvements at two years after surgery although perioperative complications were more numerous in cases where PF was performed Pathological studies on the ossification of the posterior longitudinal ligament (opll) Nouri, A., Tetreault, L., Singh, A., Karadimas, S. K. & Fehlings, M. G. Degenerative cervical myelopathy: epidemiology genetics and pathogenesis. Spine https://doi.org/10.1097/brs.0000000000000913 (2015) Fehlings, M. G. et al. Change in functional impairment, disability, and quality of life following operative treatment for degenerative cervical myelopathy: A systematic review and meta-analysis. Glob. Spine J. 7, 53s–69s. https://doi.org/10.1177/2192568217710137 (2017) Comparison of outcomes of surgical treatment for ossification of the posterior longitudinal ligament versus other forms of degenerative cervical myelopathy Kato, S., Ganau, M. & Fehlings, M. G. Surgical decision-making in degenerative cervical myelopathy: Anterior versus posterior approach. J. Clin. Neurosci. 58, 7–12. https://doi.org/10.1016/j.jocn.2018.08.046 (2018) Ma, L. et al. Comparison of laminoplasty versus laminectomy and fusion in the treatment of multilevel cervical ossification of the posterior longitudinal ligament: A systematic review and meta-analysis. Medicine 97, e11542. https://doi.org/10.1097/md.0000000000011542 (2018) Yuan, X. et al. Comparison of laminectomy and fusion vs laminoplasty in the treatment of multilevel cervical spondylotic myelopathy: A meta-analysis. Medicine 98, e14971. https://doi.org/10.1097/md.0000000000014971 (2019) Minimum 10-year followup after en bloc cervical laminoplasty Chiba, K. et al. Long-term results of expansive open-door laminoplasty for cervical myelopathy: Average 14-year follow-up study. Spine 31, 2998–3005. https://doi.org/10.1097/01.brs.0000250307.78987.6b (2006) Long-term results after expansive open-door laminoplasty for the segmental-type of ossification of the posterior longitudinal ligament of the cervical spine: A comparison with nonsegmental-type lesions Nakashima, H. et al. Prediction of outcome following surgical treatment of cervical myelopathy based on features of ossification of the posterior longitudinal ligament: A systematic review. JBJS Rev. 5, 01874474. https://doi.org/10.2106/jbjs.rvw.16.00023 (2017) A new concept for making decisions regarding the surgical approach for cervical ossification of the posterior longitudinal ligament: The K-line Long-term results of expansive laminoplasty for ossification of the posterior longitudinal ligament of the cervical spine: more than 10 years follow up Iwasaki, M. et al. Surgical strategy for cervical myelopathy due to ossification of the posterior longitudinal ligament: Part 1: Clinical results and limitations of laminoplasty. Spine 32, 647–653. https://doi.org/10.1097/01.brs.0000257560.91147.86 (2007) Reoperation for late neurological deterioration after laminoplasty in individuals with degenerative cervical myelopathy: Comparison of cases of cervical spondylosis and ossification of the posterior longitudinal ligament Multicenter study investigating the postoperative progression of ossification of the posterior longitudinal ligament in the cervical spine: A new computer-assisted measurement Fehlings, M. G. et al. Laminectomy and fusion versus laminoplasty for the treatment of degenerative cervical myelopathy: Results from the AOSpine North America and International prospective multicenter studies. Spine J. 17, 102–108. https://doi.org/10.1016/j.spinee.2016.08.019 (2017) Long-term results of cervical myelopathy due to ossification of the posterior longitudinal ligament with an occupying ratio of 60% or more What are the important predictors of postoperative functional recovery in patients with cervical OPLL Nakashima, H. et al. Comparative effectiveness of open-door laminoplasty versus French-door laminoplasty in cervical compressive myelopathy. Spine 39, 642–647. https://doi.org/10.1097/brs.0000000000000252 (2014) Duetzmann, S., Cole, T. & Ratliff, J. K. Cervical laminoplasty developments and trends, 2003–2013: A systematic review. J. Neurosurg. Spine 23, 24–34. https://doi.org/10.3171/2014.11.spine14427 (2015) Mikhail, C. M., Dowdell, J. E. 3rd. & Hecht, A. C. Posterior fusion for the subaxial cervical spine: A review of the major techniques. HSS J. 16, 188–194. https://doi.org/10.1007/s11420-019-09722-x (2020) Operative results and postoperative progression of ossification among patients with ossification of cervical posterior longitudinal ligament Kato, S. et al. Does posterior scoliosis correction improve respiratory function in adolescent idiopathic scoliosis? A systematic review and meta-analysis. Glob. Spine J. 9, 866–873. https://doi.org/10.1177/2192568218811312 (2019) Li, H. & Dai, L. Y. A systematic review of complications in cervical spine surgery for ossification of the posterior longitudinal ligament. Spine J. 11, 1049–1057. https://doi.org/10.1016/j.spinee.2011.09.008 (2011) Nakashima, H. et al. Multivariate analysis of C-5 palsy incidence after cervical posterior fusion with instrumentation. J. Neurosurg. Spine 17, 103–110. https://doi.org/10.3171/2012.4.spine11255 (2012) A late neurological complication following posterior correction surgery of severe cervical kyphosis Takemitsu, M., Cheung, K. M. C., Wong, Y. W., Cheung, W. Y. & Luk, K. D. K. C5 nerve root palsy after cervical laminoplasty and posterior fusion with instrumentation. J. Spinal Disord. Tech. 21, 267–272. https://doi.org/10.1097/BSD.0b013e31812f6f54 (2008) Nakashima, H. et al. Complications of cervical pedicle screw fixation for nontraumatic lesions: A multicenter study of 84 patients. J. Neurosurg. Spine 16, 238–247. https://doi.org/10.3171/2011.11.spine11102 (2012) Can prophylactic bilateral C4/C5 foraminotomy prevent postoperative C5 palsy after open-door laminoplasty Machino, M. et al. Cervical alignment and range of motion after laminoplasty: Radiographical data from more than 500 cases with cervical spondylotic myelopathy and a review of the literature. Spine 37, E1243-1250. https://doi.org/10.1097/BRS.0b013e3182659d3e (2012) Kim, T. H., Lee, S. Y., Kim, Y. C., Park, M. S. & Kim, S. W. T1 slope as a predictor of kyphotic alignment change after laminoplasty in patients with cervical myelopathy. Spine 38, E992-997. https://doi.org/10.1097/BRS.0b013e3182972e1b (2013) Kim, B. et al. Relationship between T1 slope and loss of lordosis after laminoplasty in patients with cervical ossification of the posterior longitudinal ligament. Spine J. 16, 219–225. https://doi.org/10.1016/j.spinee.2015.10.042 (2016) Lee, S. H. et al. Does extension dysfunction affect postoperative loss of cervical lordosis in patients who undergo laminoplasty?. Spine 44, E456–E464. https://doi.org/10.1097/brs.0000000000002887 (2019) Fujishiro, T. et al. Gap between flexion and extension ranges of motion: A novel indicator to predict the loss of cervical lordosis after laminoplasty in patients with cervical spondylotic myelopathy. J. Neurosurg. Spine 35, 8–17. https://doi.org/10.3171/2020.10.spine201723 (2021) Radiological study of cervical ossification of the posterior longitudinal ligament Ando, K. et al. Comparative study of surgical treatment and nonsurgical follow up for thoracic ossification of the posterior longitudinal ligament: radiological and clinical evaluation. Spine 42, 407–410. https://doi.org/10.1097/brs.0000000000001769 (2017) Nagoshi, N. et al. Comparison of surgical outcomes after open- and double-door laminoplasties for patients with cervical ossification of the posterior longitudinal ligament: A prospective multicenter study. Spine 46, E1238-e1245. https://doi.org/10.1097/brs.0000000000004094 (2021) Download references This work was supported by Health and Labour Science Research grants (H29-nanchi(nan)-ippan-040) and by a grant from the Japan Agency for Medical Research and Development (16ek0109136h0002) There are no other financial associations that may be relevant or seen as relevant to this work A list of authors and their affiliations appears at the end of the paper Nagoya University Graduate School of Medicine Wakayama Medical University Kihoku Hospital Hirosaki University Graduate School of Medicine Niigata University Medicine and Dental General Hospital Chiba University Graduate School of Medicine Faculty of Medicine and Graduate School of Medicine Department of Orthopaedics and Rehabilitation Medicine Dokkyo Medical University School of Medicine Shunsuke Fujibayashi & Shunsuke Fujibayashi Fujita Health University School of Medicine Department of Public Health and Health Systems Yoshiharu Kawaguchi & Yoshiharu Kawaguchi wrote the initial draft of the manuscript; H.N. All authors read and approved the final manuscript Download citation DOI: https://doi.org/10.1038/s41598-021-04727-1 Journal of Orthopaedic Surgery and Research (2024) Japan showed plenty of attacking energy but were foiled by Trinidad and Tobago keeper Marvin Phillip in Wednesday's scoreless draw Although fans at Toyota Stadium were denied a national team debut from 18-year-old wunderkind Takefusa Kubo attacked relentlessly but could not get onto the scoreboard against 93rd-ranked Trinidad With Japan completely dominant in midfield the visitors were unable to maintain possession for long throughout the first half Phillip repeatedly turned away first-half shots set up by a steady salvo of deadly accurate crosses into the penalty area Al-Duhail midfielder Shoya Nakajima sniped away at the goal while FC Groningen midfielder Ritsu Doan wreaked havoc up the middle often in concert with Marseille defender Hiroki Sakai coming up the right flank a Doan through pass unleashed Sakai down the right Werder Bremen forward Yuya Osako found space in front of Phillip's goal and stung the keeper's hands after a well-timed cross from Sakai Football: Takefusa Kubo marks 18th birthday on eve of potential Japan debut Football: Hajime Moriyasu picks squad of Olympic hopefuls for Copa America That ushered in nearly 20 minutes of non-stop pressure from the Japanese that included a Nakajima missile blocked by Phillip and a free kick from the Japanese midfielder that came off the bar "We did well until it came to shooting," Nakajima said Starved of the ball through the first half while his teammates were under pressure Trinidad forward Levi Garcia had the best chance of the first half but was blocked by Japan's Daniel Schmidt The American-born Vegalta Sendai keeper then stopped a point-blank header off the ensuing corner Phillip was once more put to the test in a 30-second rush in that saw him deflect an 85th-minute strike from Japan captain Gaku Shibasaki and then tip away a roller seconds later and then stopping a shot from Sho Genji Japan went with a three-man back line for the first time under manager Hajime Moriyasu "I think it was very hard for them in some ways but they gave it their best shot," Moriyasu said "If they can maintain a feel for this (setup) that gives us options as we go forward." they were able to see defender Yuto Nagatomo bring his usual dynamic to the pitch in his 117th senior international game He had been tied with former goalkeeper Yoshikatsu Kawaguchi and Leicester forward Shinji Okazaki for third in career appearances for Japan Japan next take on El Salvador at Hitomebore Stadium Miyagi on Sunday before leaving for the Copa America To have the latest news and stories delivered to your inbox Simply enter your email address below and an email will be sent through which to complete your subscription Please check your inbox for a confirmation email Thank you for reaching out to us.We will get back to you as soon as possible Metrics details Positive association between ossification of the posterior longitudinal ligament of the spine (OPLL) and obesity is widely recognized; however few studies focused on the effects of obesity on treatment of cervical OPLL The effects of obesity on surgical treatment of cervical OPLL were investigated by a Japanese nationwide 478 patients with cervical myelopathy due to OPLL were prospectively enrolled To clarify the effects of obesity on the surgical treatment for cervical OPLL non-obese (< BMI 30.0 kg/m2) and obese (≥ BMI 30.0 kg/m2) groups The mean age of the obese group was significantly younger than that of non-obese group There were no significant differences between the two groups in other demographic information the obese group had a significantly higher rate of surgical site infection (SSI) than that of non-obese group Approach-specific analyses revealed that the SSI was significantly higher in the obese group than in the non-obese group A logistic regression analysis revealed that age and duration of symptoms were significant factors affecting the postoperative minimum clinically important difference success The result of this study provides useful information for future cervical OPLL treatment few studies have a comprehensive discussion focusing on the effects of obesity on the treatment of cervical OPLL The purpose of this study is to investigate the effects of obesity on surgical treatment of cervical OPLL including perioperative complications by a Japanese nationwide Flowchart of patients through the study OPLL: ossification of the posterior longitudinal ligament; JOA score: Japanese Orthopedic Association score; MCID: minimum clinically important difference Radiographical assessments revealed that type of OPLL, C2-7 angle, C2-7 range of motion (ROM), K-line condition, and the presence of an intramedullary signal intensity change on T2-weighted magnetic resonance (MR) imaging were not significantly different between non-obese and obese groups (Table 2) The canal occupying ratio (COR) of the obese group (47.8 ± 17.8%) was high enough to be almost significantly different from that of the non-obese group (43.2 ± 15.4%) (p = 0.050) there were 12 cases of anterior–posterior surgery we also investigated the incidence of SSI in non-obese and obese groups with laminoplasty group alone and confirmed that obese group had a significantly higher incidence of SSI (1.5% vs The JOA recovery rate of the non-obese and obese groups at 2 years after surgery were 42.2 ± 36.5 and 46.9 ± 32.4 showing no significant difference (p = 0.32) Overall, 184 out of 402 patients (45.8%) achieved an minimum clinically important difference (MCID) success at 2 years after the surgery. Then, the factors affecting the MCID success were evaluated by logistic regression analysis (Table 8) This analysis revealed that age (OR: 0.946 95% CI 0.989–0.998 p = 0.002) were significant factors affecting the postoperative MCID success A post hoc power analysis showed that the actual sample size of this study had 99.3% power since there was no significant difference between the non-obese and obese groups in comorbidities that were thought to affect perioperative complications such as diabetes mellitus we considered that the obesity had a significant impact on high SSI incidence we do not immediately deny the possible involvement of factors that did not differ statistically We believe that the real impact of statistically non-significant factors on SSI will need to be investigated in larger samples in the future The surgical procedure for cervical OPLL should be comprehensively determined in careful consideration of cervical spine alignment but anterior surgery may be beneficial for patients who are prone to SSI we should carefully explain that obese patients with cervical OPLL are at high risk of SSI it is advisable to consider a preoperative weight loss program aimed at reducing BMI we must keep in our mind that the results of this review by Cofano et al fewer citations for papers dealing with cervical spine surgery compared to thoracolumbar spine surgery the inclusion of cases other than cervical OPLL which is the most selected treatment for cervical OPLL we believe that no conclusions have been established regarding the actual effect of obesity on postoperative neurological improvement of cervical OPLL and further studies are needed to clarify it We also confirmed a correlation between duration of symptoms and likelihood of MCID success in the present study The longer the preoperative exacerbation of neurological symptoms the more irreversible changes can occur in the spinal cord which can exacerbate postoperative neurological improvement We believe that this is one of the main causes of the correlation between preoperative duration of symptoms and likelihood of MCID success long-term spinal cord compression can have a negative impact on postoperative improvement of the symptom when planning a weight loss program for BMI reduction before surgery we believe that consideration should be given to making it as short as possible the average age of the obese group in the present study was significantly younger than that of non-obese group obesity may be involved not only in the early onset of cervical OPLL One of the reasons for this may be the lack of evidence however further studies need to clarify the real effects of obesity on the cervical OPLL including its treatment and development Otherwise, the present study imposes several limitations. This study is a prospective study of surgically treated patients with cervical OPLL, but the surgical procedure has not been randomized. The surgical procedure was determined according to the algorithm shown in the “Patients and methods” the surgical procedure for each case was decided BMIs of the patients in this study were much lower than expected in other populations such as in North America and Europe we were able to investigate the effects of obesity on cervical OPLL treatment in short-term outcomes Longer follow-up involving much higher BMI ranges is needed to clarify these issues the favorable aspect of the present study is that this is the first Japanese nationwide prospective study comprehensively disclosing the effects of the obesity on the treatment for patients with cervical OPLL we investigated the effects of obesity by surgical approaches and clarified for the first time that special attention should be paid to SSI especially in posterior surgery this is the first well-powered Japanese nationwide multicenter prospective study that identified the effect of obesity on the treatment for the patients with cervical OPLL Special attention should be paid to SSI when planning posterior surgery for the treatment of obese patients with cervical OPLL This Japanese nationwide, multicenter, prospective study (https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000039771 UMIN000035194) involved 28 academic institutions of the Japanese Multicenter Research Organization for Ossification of the Spinal Ligament (JOSL) formed by the Japanese Ministry of Health Labour and Welfare and the Japanese Agency for Medical Research and Development (AMED) The protocol for this study has been approved by institutional review board (IRB) of Tokyo Medical and Dental University as a Central IRB and IRB of Shiga University of Medical Science and the other all participating institutions as a local participating institution This research was conducted in accordance with the "Declaration of Helsinki" and the Ministry of Health Science and Technology "Ethical Guidelines for Medical Research for Humans" mean age 64.1 ± 11.6 years) with cervical myelopathy due to OPLL were prospectively enrolled from April 2015 to July 2017 The definition of cervical myelopathy due to OPLL is that (1) clear spinal cord compression by OPLL can be confirmed and (2) spinal cord compression by OPLL can be diagnosed as the cause of the present symptoms by neurological examination Patients with (i) less than 20 years old; (ii) a history of cervical spine surgery; (iii) neurological impairments due to cervical disc herniation All patients provided written informed consent on entry into the registry The surgical procedure was determined according to the following algorithm cervical laminoplasty was selected for K-line (+) cases with the lordotic alignment in patients with K-line (−) cases and/or high spinal canal occupying ratio of OPLL and patients with kyphosis anterior decompression and fusion and/or posterior decompression and fusion were employed anterior surgery is a technical demand surgery with higher incidence of complications and was adopted after comprehensively evaluating the experience of the surgeon and the wishes of the patient to clarify the effects of obesity on this issue we divided the surgery into anterior (anterior decompression and fusion) and posterior (laminoplasty and posterior decompression and fusion) surgeries Patients who underwent anterior–posterior surgery were excluded in this approach-specific analyses The K-line is a straight line connecting the midpoints of the spinal canals of C2 and C7 in the lateral radiograph Patients were considered K-line (−) if OPLL crossed the K-line C2-7 range of motion (ROM) was measured on flexion–extension lateral standard radiographs The most compressed level and the presence of a signal intensity change in the spinal cord were also investigated on T2-weighted magnetic resonance (MR) imaging Spinal canal occupying ratio (COR) of OPLL on axial computed tomography (CT) at the maximum cord compression level was also investigated as a condition resulting in an abscess or other evidence of infection in the skin the presence of SSI was confirmed by reoperation or by histopathological or radiographical investigation The software application used for the analysis was SPSS version 26.0 (SPSS Inc. On paraplegia depending on the ligament of the spine Ossification off the Longitudinal Ligament (eds Okawa Risk factors for surgical complications in the management of ossification of the posterior longitudinal ligament Approach-related complications after decompression for cervical ossification of the posterior longitudinal ligament Cervical ossification of the posterior longitudinal ligament: A computed tomography-based epidemiological study of 2917 patients Comparison of outcomes of surgical treatment for ossification of the posterior longitudinal ligament versus other forms of degenerative cervical myelopathy: Results from the prospective multicenter AOSpine CSM-international study of 479 patients A systematic review and meta-analysis comparing anterior decompression with fusion and posterior laminoplasty for cervical ossification of the posterior longitudinal ligament 2019 clinical practice guideline for ossification of spinal ligaments working group Japanese Orthopaedic Association (JOA) clinical practice guidelines on the management of ossification of the spinal ligament and national prevalence of overweight and obesity in children and adults during 1980–2013: A systematic analysis for the Global Burden of Disease Study 2013 and metabolic syndrome with obesity: Findings from the National Health and Nutrition Examination Survey physical activity and obesity-related chronic diseases Morbid obesity increases cost and complication rates in spinal arthrodesis Economic impact of comorbidities in spine surgery Body mass index as a predictor of complications and mortality after lumbar spine surgery Obesity is associated with an increased rate of incidental durotomy in lumbar spine surgery Obesity and spine surgery: A qualitative review about outcomes and complications Is it time for new perspectives on future researches A systematic review of complications in cervical spine surgery for ossification of the posterior longitudinal ligament Prevalence and distribution of ossified lesions in the whole spine of patients with cervical ossification of the posterior longitudinal ligament a multicenter study (JOSL CT study) Incidence of surgical site infection after spine surgery: A systematic review and meta-analysis Ossification of the posterior longitudinal ligament: Surgical approaches and associated complications Perioperative complications of anterior decompression with fusion versus laminoplasty for the treatment of cervical ossification of the posterior longitudinal ligament: Propensity score matching analysis using a nation-wide inpatient database Natural history of the ossification of cervical posterior longitudinal ligament: A three dimensional analysis Prediction of outcome following surgical treatment of cervical myelopathy based on features of ossification of the posterior longitudinal ligament: A systematic review Ossification of the posterior longitudinal ligament of the spine Inter-and intra-observer reliability of the Japanese Orthopaedic Association Scoring system for evaluation of cervical compression myelopathy Minimum clinically important difference and patient acceptable symptom state of Japanese Orthopaedic Association Score in degenerative cervical myelopathy patients Guideline for prevention of surgical site infection Hospital infection control practices advisory committee Surgical site infections following spine surgery: Eliminating the controversies in the diagnosis Drain tip culture is not prognostic for surgical site infection in spinal surgery under prophylactic use of antibiotics Download references This work was supported by Japanese Agency for Medical Research and Development (AMED) and Health and Labour Science Research Grants This study was approved by each institutional review board Niigata University Medical and Dental General Hospital Japanese Multicenter Research Organization for Ossification of the Spinal Ligament contributed to planning and conduct of the present study and to reporting the present manuscript contributed to conception and design of the present study and to reporting the present study contributed to conducting the present study and to edit the present manuscript All the authors read and approved the final manuscript supervised all the statistical analyses in the present study Download citation DOI: https://doi.org/10.1038/s41598-022-12625-3 A day after Japan's impressive 4-3 friendly win over Uruguay manager Hajime Moriyasu on Wednesday lauded his new-look side for their seamless integration of overlapping generations of players The victory Tuesday in Saitama was the third in three matches under the new boss and the first combining established players from this summer's World Cup with newer members who have quickly made their mark on the international stage "They really melded together," said Moriyasu who took the reins after Akira Nishino stepped down at the end of the World Cup in Russia (Man of the moment Takumi Minamino scored twice against Uruguay to take his tally to four goals in his first three national team starts) The new wave -- including 24-year-old Shoya Nakajima 23-year-old Takumi Minamino and 20-year-old Ritsu Doan -- blended smoothly in attack with the likes of veterans Yuto Nagatomo as they continually made inroads into the Uruguayan defense With Nakajima attracting attention for his aggressive playmaking from the left side left-back Nagatomo repeatedly made attacking runs down the wing to draw defenders and open up space for the Portimonense midfielder Football: Rising stars lead way as Japan beats Uruguay 4-3 Right-back Sakai duplicated the strategy on the opposite side creating room for right midfielder Doan to operate Werder Bremen forward Osako said the movement into attacking territory by the two wingbacks had been integral to Japan's game plan "Because of the balance provided by Nagatomo and Sakai we were able to attack from both sides," Osako said Choosing to make just two late substitutions in the win over world No Moriyasu appears to have settled upon the foundation of his team for the Asian Cup kicking off in January in the United Arab Emirates (Shoya Nakajima unleashes a shot in the first half of Tuesday's win) has hinted at further experimentation with his team selections and tactics including the use of a defensive back three A somewhat controversial omission from the World Cup squad Nakajima has become the lynchpin of Japan's attack under Moriyasu while Salzburg forward Minamino has delivered a remarkable four goals in his first three starts beginning with the 3-0 victory over Costa Rica last month Groningen midfielder Doan has been equally impressive combining an incisive attacking game with a nose for winning contested balls in key areas (Ritsu Doan opens his national team account with a crisp finish) Moriyasu said he hopes to take a full-strength senior side to the Copa America starting next June in Brazil when Japan will participate in the South American continental tournament for the second time as an invited nation While it was initially thought Japan would send an under-23 squad in preparation for the 2020 Tokyo Olympic Games Moriyasu said he wanted his strongest possible side in Brazil but I've asked for discussions to take place Because it is not sanctioned by the Asian Football Confederation clubs have no obligation to release Japanese players to take part in the tournament The Japan Football Association would therefore need to negotiate the release of players with their respective J-League or overseas clubs Metrics details Although conventional polymer gels are known as mechanically weak materials their fracture toughness can be effectively improved by introducing weak and brittle bonds into soft and stretchy polymer networks denoted as the ‘sacrificial bond principle’ When force is applied to such modified gels with an initial crack brittle bonds surrounding the crack tip are widely and catastrophically ruptured prior to macroscopic crack propagation As this extensive brittle bond fracture requires significant energy input the total energy required for gel fracture is remarkably increased Since the gel toughness is increased due to sacrificing the introduced brittle bonds I describe some extremely tough gels prepared by our group using this principle double- or multiple-network gels with high water content featuring covalent sacrificial bonds self-healing polyampholyte gels containing ionic sacrificial bonds and PDGI/PAAm gels based on hydrophobic sacrificial bonds exhibiting stress-responsive structural colors (artificial) knee articular cartilage is continuously exposed to large compression and impact forces for several decades typical gels like jelly or tofu are so brittle that they can easily be fractured by applying a small amount of energy real-life applications require synthetic gels that are soft but mechanically robust posing a big challenge for material chemists The mechanical robustness of materials can be characterized by various parameters such as tensile or compressive fracture stress fracture energy and viscoelastic properties fracture energy is an index of toughness and describes resistance against crack propagation being best suited to describe mechanical robustness since material failure is a result of crack propagation J m−2) is defined as the energy required to create a unit area of fracture surface and the intrinsic fracture energy (G0) of rubbery materials is generally described by the Lake–Thomas theory as wide applications of gels require their additional toughening The properties of common hard and tough materials provide a guiding principle for toughening gels When force is applied to these materials (e.g. an obvious internal structure transition around the crack tip is detected prior to macroscopic crack propagation exemplified by dislocation of crystals in metals crazing in polycarbonate and strain-induced crystallization in natural rubbers For materials exhibiting such structural transitions (a) General structure of a tough gel relying the sacrificial bond principle consisting of a highly stretchable matrix with a high density of introduced brittle bonds c) Possible fracture processes of (b) a single-network gel and (c) a sacrificial bond gel brittle bonds are widely ruptured prior to macroscopic crack propagation around the crack tip (shadowed zone) A full color version of this figure is available at Polymer Journal online Our group has synthesized a series of tough hydrogels containing several kinds of sacrificial bonds based on the above principle with some examples and corresponding toughening mechanisms introduced in this focus review a wide fracture of the 1st brittle network occurs in a damage zone with thickness h around the crack tip prior to macroscopic crack propagation (scission of the 2nd stretchy network) If the energy required to produce a damage zone of unit volume is defined as gdamage (J m−3) tough DN gels based on neutral 1st networks have recently been synthesized by introduction of additional components The fracture stress and work of deformation at fracture of these elastomers and gels are sufficiently increased upon incorporation of the 3rd and 4th neutral networks PA gels show considerably improved fracture energies compared to conventional gels Friction and lubrication of hydrogels—its richness and complexity Polyelectrolyte hydrogels for replacement and regeneration of biological tissues Designing hydrogels for controlled drug delivery Biomedical applications of hydrogels: a review of patents and commercial products Fracture energy of polymer gels with controlled network structures Multi-scale multi-mechanism design of tough hydrogels: building dissipation into stretchy networks Double-network hydrogels with extremely high mechanical strength Determination of fracture energy of high strength double network hydrogels True chemical structure of double network hydrogels A facile method for synthesizing free-shaped and tough double network hydrogels using physically crosslinked poly(vinyl alcohol) as an internal mold Importance of entanglement between first and second components in high-strength double network gels Brittle-ductile transition of double network hydrogels: mechanical balance of two networks as the key factor Mechanically strong double network photocrosslinked hydrogels from N,N-dimethylacrylamide and glycidyl methacrylated hyaluronan Double-network strategy improves fracture properties of chondroitin sulfate networks Large strain hysteresis and mullins effect of tough double-network hydrogels Characterization of internal fracture process of double network hydrogels under uniaxial elongation paste-like behaviors and distinct necking of double network gels with enhanced heterogeneity Yielding criteria of double network hydrogels A model of the fracture of double network gels A local damage model for anomalous high toughness of double-network gels Localized yielding around crack tips of double-network gels Direct observation of damage zone around crack tips in double-network gels Direct observation on the surface fracture of ultrathin film double-network hydrogels Robust bonding and one-step facile synthesis of tough hydrogels with desirable shape by virtue of the double network structure Microgel-reinforced hydrogel films with high mechanical strength and their visible mesoscale fracture structure Structure optimization and mechanical model for microgel-reinforced hydrogels with high strength and toughness Fracture process of microgel-reinforced hydrogels under uniaxial tension A universal molecular stent method to toughen any hydrogels based on double network concept Synthesis and fracture process analysis of double network hydrogels with a well-defined first network Proteoglycans and glycosaminoglycans improve toughness of biocompatible double network hydrogels Toughening elastomers with sacrificial bonds and watching them break Nonionic double and triple network hydrogels of high mechanical strength Fabrication of tough hydrogels from chemically cross-linked multiple neutral networks Physical hydrogels composed of polyampholytes demonstrate high toughness and viscoelasticity A phase diagram of neutral polyampholyte—from solution to tough hydrogel Crack blunting and advancing behaviors of tough and self-healing polyampholyte hydrogel Self-healing behaviors of tough polyampholyte hydrogels Self-adjustable adhesion of polyampholyte hydrogels Oppositely charged polyelectrolytes form tough Free reprocessability of tough and self-healing hydrogels based on polyion complex tough and highly stretchable ionic nanocomposite physical hydrogels Anisotropic hydrogel based on bilayers: color Unidirectional alignment of lamellar bilayer in hydrogel: one-dimensional swelling and stress/strain tunable structural color Mechano-actuated ultrafast full-colour switching in layered photonic hydrogels Lamellar hydrogels with high toughness and ternary tunable photonic stop-band Lamellar bilayers as reversible sacrificial bonds to toughen hydrogel: hysteresis Phase-separation-induced anomalous stiffening Tough and self-healing hydrogels formed via hydrophobic interactions Reliable hydrogel with mechanical ‘Fuse Link’ in an aqueous environment Thermoresponsive toughening with crack bifurcation in phase-separated hydrogels under isochoric conditions Molecular mechanistic origin of the toughness of natural adhesives Bone indentation recovery time correlates with bond reforming time An amino acid ionic liquid-based tough ion gel membrane for CO2 capture Download references This work was partially supported by JSPS KAKENHI This research was also partially funded by the ImPACT Program of the Council for Science Jian Ping Gong (Hokkaido University) and other co-workers for their great contribution to this research Global Institution for Collaborative Research and Education The authors declare no conflict of interest Download citation Metrics details Influenza A and B viruses show clear differences in their host specificity and pandemic potential Recent studies have revealed that the host protease TMPRSS2 plays an essential role for proteolytic activation of H1 and H7 subtype strains of influenza A virus (IAV) in vivo IAV possessing a monobasic cleavage site in the haemagglutinin (HA) protein replicates poorly in TMPRSS2 knockout mice owing to insufficient HA cleavage human isolates of influenza B virus (IBV) strains and a mouse-adapted IBV strain were analysed The data showed that IBV successfully underwent HA cleavage in TMPRSS2 knockout mice and that the mouse-adapted strain was fully pathogenic to these mice The present data demonstrate a clear difference between IAV and IBV in their molecular mechanisms for spreading in vivo the molecular bases generating the biological differences between IAV and IBV are poorly understood the role of TMPRSS2 for the in vivo replication of IBV was examined HeLa cells constitutively expressing mTMPRSS2 or hTMPRSS2 (HeLa/mTM2 and HeLa/hTM2 respectively) and the parental HeLa cells were infected with B/Aichi/99[V] (A) or MA-B/Ibaraki/85[V] (B) at an MOI of 1.0 in the presence or absence of trypsin IBV HA in cells was detected by SDS-PAGE and immunoblotting Histopathological findings in the lungs of WT and TMPRSS2 KO mice infected with human isolates of IBV Data obtained by hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) for IBV antigens are shown The inflammation scores of individual mice (n = 3) are shown: 0 Based on the structure of B/HongKong/8/73 (PDB 2RFU), the predicted locations of the mutated amino acid residues in MA-B/Ibaraki/85[V] HA are shown. The mutated residues are shown in magenta in a sphere model. The cyan circle indicates the position of the cleavage site. (A) WT and TMPRSS2 KO mice (n = 3) were intranasally inoculated with 6.7 × 103 PFU of MA-B/Ibaraki/85[V] Lung lavage fluids and lung homogenates at 2 Filled and open bars indicate data of WT and TMPRSS2 KO mice (B) The HA and NP protein levels in lung lavage fluids at 2 dpi were analysed by SDS-PAGE and immunoblotting Each lane corresponds to data from an individual mouse The same amounts of lung lavage fluids were loaded (C) Quantification of cleavage by measuring the chemiluminescent signals for the cleaved HA subunit (HA1 and HA2) signals to total HA (HA1 identification of the protease(s) responsible for IBV HA cleavage may reveal a molecular basis for the different biological features between IAV and IBV and contribute to the development of anti-IBV drugs All experiments with animals were performed in strict accordance with the Animal Experimentation Guidelines of the National Institute of Infectious Diseases and the protocol was approved by the Institutional Animal Care and Use Committee of the institute TMPRSS2 KO mice were reported previously10 the mice were generated from TMPRSS2 gene KO C57BL/6 ES cells (KOMP Repository Knockout Mouse Project; Product ID: VG13341) and have a complete C57BL/6 genetic background WT C57BL/6 mice were purchased from Japan SLC WT and TMPRSS2 KO mice (6–7-week-old) were infected intranasally with 4.6 × 103 The body weight and clinical signs were monitored daily WT and TMPRSS2 KO mice were euthanized and autopsied at 2 days p.i The viral antigens of IBV were detected using a mouse antiserum against IBV NP (Lot This antiserum was obtained from BALB/c mice immunized intraperitoneally with inactivated IBV virions (Victoria lineage) using the Sigma adjuvant system (Sigma-Aldrich) The inflammation levels in individual mice were scored as follows: 0 The cut-off in body weight loss for euthanasia was 25% Lung lavage fluids and lung homogenates were collected at 2 WT and TMPRSS2 KO mice (6–7-week-old) were infected intranasally with 2.0 × 103 These doses of MA-B/Ibaraki/85[V] correspond to 30 and 3,000 mouse lethal dose 50 (MLD50) for WT mice similar mice were also infected intranasally with 6.7 × 103 PFU of MA-B/Ibaraki/85[V] and autopsied at 2 days p.i WT and TMPRSS2 KO mice (n = 3) were infected with 2.0 × 103 PFU of MA-B/Ibaraki/85[V] and lung lavage fluids were collected at 2 Monolayers of MDCK cells were infected with serially diluted lung lavage fluid and lung homogenate samples for 1 hour at 4 °C and incubated for 24 hours at 37 °C to allow the viruses to enter the cells Trypsin was omitted to avoid HA cleavage before virus entry the cell monolayers were additionally overlaid with MEM/1% agarose supplemented with 4.0 μg/mL of trypsin to allow plaque formation To determine the infectious titres including viruses that had not been activated in vivo but possessed an infectious potential virus samples were treated with 2.0 μg/mL of trypsin WT and TMPRSS2 KO mice were infected with 6.7 × 103 PFU of MA-B/Ibaraki/85[V] (n = 3) or mock-infected (n = 1) Lung lavage fluids were collected at 2 days p.i and parental HeLa cells were infected with B/Yamagata/88[Y] and parental B/Ibaraki/85[V] at a multiplicity of infection (MOI) of 1.0 The lung lavage fluids and cells were lysed with lysis buffer to make a final solution containing 150 mM NaCl The polypeptides were separated by SDS-PAGE in 10–20% polyacrylamide gels (e-PAGEL and blotted onto PVDF membranes (WSE-4051; ATTO) Rabbit antisera raised against IBV HA (#11053-RP01; Sino Biological Inc.) and NP (#GTX128539; GeneTex Inc.) were used for detection of HA and NP TMPRSS2 Independency for Haemagglutinin Cleavage In Vivo Differentiates Influenza B Virus from Influenza A Virus ) 1186–1243 (Lippincott Williams & Wilkins New world bats harbor diverse influenza A viruses A distinct lineage of influenza A virus from bats Evolutionary pattern of the hemagglutinin gene of influenza B viruses isolated in Japan: cocirculating lineages in the same epidemic season Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983 Domestic pigs are susceptible to infection with influenza B viruses Tmprss2 is essential for influenza H1N1 virus pathogenesis in mice TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice The host protease TMPRSS2 plays a major role in in vivo replication of emerging H7N9 and seasonal influenza viruses Hemagglutinin activating host cell proteases provide promising drug targets for the treatment of influenza A and B virus infections Host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses Journal of molecular and genetic medicine: an international journal of biomedical research 3 Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium Cleavage of influenza virus hemagglutinin by host cell protease Proteolytic activation of the 1918 influenza virus hemagglutinin The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium Cleavage activation of the human-adapted influenza virus subtypes by matriptase reveals both subtype and strain specificities DESC1 and MSPL activate influenza A viruses and emerging coronaviruses for host cell entry Influenza virus hemagglutinin with multibasic cleavage site is activated by furin Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses Endogenous protease-dependent replication of human influenza viruses in two MDCK cell lines Heterogeneity of the MDCK cell line and its applicability for influenza virus research A mutant H3N2 influenza virus uses an alternative activation mechanism in TMPRSS2 knockout mice by loss of an oligosaccharide in the hemagglutinin stalk region Crystal structure of unliganded influenza B virus hemagglutinin Cross-protection against influenza B type virus infection by intranasal inoculation of the HA vaccines combined with cholera toxin B subunit Download references and Akira Ainai for providing the IBV strains This work was supported by Grants-in-Aid from the Japan Agency for Medical Research and Development Tokyo University of Agriculture and Technology The authors declare no competing financial interests Download citation Metrics details Charged domain walls and boundaries in ferroelectric materials display distinct phenomena such as an increased conductivity due to the accumulation of bound charges we report the electron microscopy observations of atomic-scale arrangements at charged domain boundaries in the hybrid improper ferroelectric Ca2.46Sr0.54Ti2O7 Like in the prototype improper ferroelectric YMnO3 we find that charged domain boundaries in Ca2.46Sr0.54Ti2O7 correspond to out-of-phase boundaries which separate adjacent domains with a fractional translational shift of the unit cell our results show that strontium ions are located at charged domain boundaries The out-of-phase boundary structure may decrease the polarization charge at the boundary because of the ferrielectric nature of Ca2.46Sr0.54Ti2O7 thereby promoting the stabilization of the charged state By combining atomic-resolution imaging and density-functional theory calculations this study proposes an unexplored stabilization mechanism of charged domain boundaries and structural defects accompanying out-of-phase translational shifts in the improper ferroelectric Ca3–xSrxTi2O7 the existence and effect of translation domains have not been observed at the atomic scale an out-of-phase boundary is called an antiphase boundary when the displacement phase is π (i.e. a domain boundary represents a crystallographic defect that separates two ferroelectric domains with opposite polarization directions while a domain wall means a ferroelectric domain boundary without a compositional change The out-of-phase displacement allows local electric polarization vectors of adjacent domains to align in the same direction at the boundaries which stabilizes the charged domain boundaries This study proves that charged domain boundaries are correlated with crystallographic defects in Ca2.46Sr0.54Ti2O7 a Dark-field image for g = \(\bar{3}11\) in the (1\(\bar{1}\)0) plane. b Dark-field image for g = 113. The areas indicated by squares show the locations at which the EDS images in Figs. 3 and 4 were acquired The red and blue arrows show the projected polarization directions in the ferroelectric domains the area labeled 3b has the same position in panels a and b The straight lines indicate out-of-phase boundaries with translational shifts whereas the broad boundaries correspond to regions with SrTiO3 structures a HAADF-STEM image of a charged domain boundary in the area indicated in Fig. 2 The contrast level of the dark-field image determines the projected polarization direction Ps b HAADF-STEM image of the edge of the charged domain boundary c–g HAADF-STEM image and corresponding elemental maps of the edge of the boundary The labels L and K represent the absorption edges for EDS mapping The segregation of Sr atoms is shown in panel e these configurations should be more stable than boundaries without a translational shift This mechanism is possible because of the ferrielectric nature of Ca2.46Sr0.54Ti2O7 which exhibits layered polarization vectors with opposite directions this phenomenon should be characteristic of layered perovskite structures showing ferrielectricity This is significantly different from the antiphase boundary due to the transition in YMnO3 The result obtained for Ca2.46Sr0.54Ti2O7 is also supported by the presence of Sr ions at the boundaries because the phase transition will not move Sr which would take their positions after the material has become crystalline the out-of-phase boundaries should exist in the I4/mmm paraelectric phase charged boundaries are selectively formed at the out-of-phase boundary positions because of the above-explained energy gain at the ferroelectric transition These images also show that this type of boundary runs along the [1\(\bar{1}\)0] direction a, b HAADF-STEM images showing boundary structures. The square area indicated by a dashed yellow line is shown in high magnification in panel b. The inset shows the magnified dark-field image of Fig. 2 The dashed line denotes a charged domain boundary Ps indicates the direction of the electric polarization in the domains The corresponding elemental maps are shown in panels c–f The areas with the SrTiO3 perovskite structure have no polarization because of the nonpolar simple cubic structure of SrTiO3 the extended perovskite region reduces the spontaneous polarization which is defined as the electric polarization per volume the charged out-of-phase boundaries presented in this study may have different valence states of Sr and Ti ions at the boundaries These properties will be explored in future works Our results provide mechanistic insights into the microscopic structure of boundaries and the Sr substitution effect on the properties of the layered perovskite oxide Ca3–xSrxTi2O7 this study shows that atomic-resolution elemental mapping is important for understanding structural defects and macroscopic properties in ferroelectric materials The single crystal was grown via the floating zone method Thin specimens along the (1\(\bar{1}\)0) plane were prepared via focused ion beam at 30 kV up to a thickness <100 nm Subsequent Ar-ion milling at 4 kV and an incident angle of 8° was used to remove the damage from the specimen and reduce the specimen thickness Domain-wall engineering and topological defects in ferroelectric and ferroelastic materials Domain wall conduction and polarization-mediated transport in ferroelectrics Ferroelectric translational antiphase boundaries in nonpolar materials Direct evidence of polar nature of ferroelastic twin boundaries in CaTiO3 obtained by second harmonic generation microscope Conduction at domain walls in oxide multiferroics Nonvolatile ferroelectric domain wall memory Free-electron gas at charged domain walls in insulating BaTiO3 Physics and applications of charged domain walls Insulating interlocked ferroelectric and structural antiphase domain walls in multiferroic YMnO3 Conduction of topologically protected charged ferroelectric domain walls Anisotropic conductance at improper ferroelectric domain walls Functional electronic inversion layers at ferroelectric domain walls Hybrid improper ferroelectricity: a mechanism for controllable polarization-magnetization coupling Experimental demonstration of hybrid improper ferroelectricity and the presence of abundant charged walls in (Ca Structural domain walls in polar hexagonal manganites Electrostatic topology of ferroelectric domains in YMnO3 Evolution of the domain topology in a ferroelectric Topology breaking of the vortex in multiferroicY0.67Lu0.33MnO3 and nucleation of out-of-phase boundaries (OPBs) in epitaxial films of layered oxides Bismuth volatility effects on the perfection of SrBi2Nb2O9 and SrBi2Ta2O9 films Domain structure of epitaxial Bi4Ti3O12 thin films grown on (001) SrTiO3 substrates Electron optical studies of barium titanate single crystal films Dynamical theory of electron diffraction in Laue-case Domain topology and domain switching kinetics in a hybrid improper ferroelectric Hidden antipolar order parameter and entangled Néel-type charged domain walls in hybrid improper ferroelectrics Domain structure of rochelle salt and KH2PO4 Polar octahedral rotations: a path to new multifunctional materials Introduction to Conventional Transmission Electron Microscopy Topological defects at octahedral tilting plethora in bi-layered perovskites Domain-wall conduction in ferroelectric BiFeO3 controlled by accumulation of charged defects Optimal noise filters in high-resolution electron microscopy Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set Generalized gradient approximation made simple Structural transitions in hybrid improper ferroelectric Ca3Ti2O7 tuned by site-selective isovalent substitutions: A first-principles study Special points for Brillouin-zone integrations Crystal structural evolution and hybrid improper ferroelectricity in Ruddlesden-Popper Ca3-xSrxTi2O7 ceramics VESTA 3 for three-dimensional visualization of crystal Download references This work was supported in part by JSPS KAKENHI Grant Numbers JP19H05814 The crystal growth at Rutgers was supported by the center for Quantum Materials Synthesis (cQMS) funded by the Gordon and Betty Moore Foundation’s EPiQS initiative through grant GBMF10104 Present address: Department of Physics and Astronomy performed the density-functional theory calculations Mori initiated and supervised the research project Peer review information Communications Materials thanks Zijian Hong and the other reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s43246-021-00215-1 Metrics details This retrospective cohort study established malnutrition’s impact on mortality and neurological recovery of older patients with cervical spinal cord injury (SCI) It included patients aged ≥ 65 years with traumatic cervical SCI treated conservatively or surgically The Geriatric Nutritional Risk Index was calculated to assess nutritional-related risk 789 patients (mean follow-up: 20.1 months) were examined and 47 had major nutritional-related risks on admission and activities of daily living (ADL) at 1 year post-injury were compared between patients with major nutrition-related risk and matched controls selected using 1:2 propensity score matching to adjust for age the median survival times were 44.9 and 76.5 months for patients with major nutrition-related risk and matched controls Matched controls had more individuals with a neurological improvement of American Spinal Injury Association Impairment Scale ≥ 1 grade (p = 0.039) and independence in ADL at 1 year post-injury than patients with major nutrition-related risk (p < 0.05) 6% of older patients with cervical SCI had major nutrition-related risks; they showed a significantly higher 1 year mortality rate and lower ADL at 1 year post-injury than matched controls the influences of malnutrition in older populations with SCI on clinical outcomes including mortality and neurological recovery this study aimed to determine nutrition-related risk and its impact on mortality and neurological recovery of older patients with cervical SCI using GNRI All study participants provided written informed consent The Institutional Review Board of the representative facility reviewed and approved this study (Approval No.: 3352-1) All methods were performed in accordance with the principle of the Declaration of Helsinki and Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan The following patients were included those aged ≥ 65 years with traumatic cervical SCI those treated conservatively or surgically between 2010 and 2020 at one’s institution and those followed up for at least 3 months post-injury The exclusion criteria were cervical metastases and missing data number of ventilator dependents due to SCI-induced respiratory dysfunction blood examination results at the first visit and survival data at 1-year post-injury were collected Blood tests included those for hemoglobin (g/dL) ADL data on admission and at 1 year post-injury were extracted from the database AIS data on admission and at 1 year post-injury were extracted from the database including conservative or surgical treatment All treatments were determined on a case-by-case basis by each attending physician The GNRI has the following grading system: > 98 = absence of nutrition-related risk; 92 to ≤ 98 = low risk; 82 to < 92 = moderate risk; and < 82 = major risk A matched control group was created using propensity score matching we fitted a logistic regression model using age The nearest-neighbor 1:2 matching procedure was used restricting the matched propensities to be within 0.01 units of each other The mortality rate within 1 year was compared between patients with major nutrition-related risk and matched controls using the chi-square test Kaplan–Meyer analysis was used to calculate the survival curve and median survival time with 95% CI post-trauma the log-rank test was used to compare the results between patients with major nutrition-related risks and matched controls and the ADL at 1 year post-injury were also compared between the groups Continuous variables are presented with a mean ± 1.0 standard deviation All analyses were performed using IBM SPSS Statistics for Windows Statistical significance was set at p values < 0.05 The institutional review board of the representative facility reviewed and approved this study After 1:2 propensity score matching, 94 patients were selected as matched controls. No significant differences were found in age, sex ratio, number of patients with diabetes, pre-traumatic ADL, AIS grade, or number of ventilator-dependent or treatment procedures between patients with major nutrition-related risk and matched controls (Table 3 Mortality rate within 1 year post-injury. Survival analysis of patients with and those without major nutrition-related risk In the subgroups of patients with AIS grades A–C on admission, the matched controls had a significantly higher proportion of patients who achieved a neurological improvement of at least one AIS grade than patients with major nutrition-related risk (57.1% vs. 34.5%, p = 0.039, Table 4) and 1 patient among individuals with major nutrition-related risk and 22 and 6 individuals among matched controls improved their neurological impairment from AIS grades from A to B no significant differences were observed between patients with major nutrition-related risk and matched controls in the subgroup of patients with AIS grade D on admission (11.1% vs the ratios of the independent patient and wheelchair/bedridden individual were significantly lower and higher in patients with major nutrition-related risk than in those of matched controls residual analysis: p < 0.05 respectively) approximately 35% of the older patients with cervical SCI had nutrition-related risk 6% of these patients had severe malnutrition which could have major nutrition-related risks Such patients showed substantially higher mortality rates within 1 year and lower levels of ADL at 1 year post-injury than matched controls these results could encourage physicians to assess the GNRI of older patients with SCI on admission our study demonstrated that malnutrition in older patients with SCI was considerably associated with increased mortality within 1 year and a poorer improvement of neurological impairment The mortality rate was not unexpected because the GNRI was designed to identify nutrition-related risk as was validated in many other pathologies described above our study results might add to the knowledge that the GNRI would be useful for predicting mortality in older patients with SCI Such reasons or other factors might be entangled with each other resulting in poor neurological improvement and lower levels of ADL at 1 year post-injury in older patients with malnutrition after SCI Our study results validated the significance of their guide and statements that a multidisciplinary approach with a long-term aspect is essential for older patients with malnutrition and SCI to decrease the mortality rate and improve neurological recovery This study had some limitations that should be addressed Although patients without missing major data were registered in our database we did not have information about the exact number of cases excluded from the database the treatment strategy was determined by each physician and may not have been consistent among patients the study population included only Japanese and was heterogeneous we did not evaluate nutritional intervention which limits us from reaching a definitive conclusion on this topic we did not consider several potentially important factors for outcomes such as the length of rehabilitation and patients’ comorbidities a prospective international multicenter study should be conducted to test the evidence-based clinical effectiveness and cost-effectiveness of nutritional intervention for older patients with SCI and malnutrition approximately 35% of the older patients with cervical SCI in our study had nutrition-related risks 6% of these patients had severe malnutrition with major nutrition-related event risks They also exhibited considerably shorter survival times assessing nutrition-related risk for older patients with SCI as well as a multidisciplinary approach with long-term follow-up for such patients with nutrition-related risk will be essential to decrease the mortality rate and improve neurological recovery and ADL Incidence of traumatic spinal cord injury worldwide: A systematic review World_Health_Organization. Spinal Cord Injury. https://www.who.int/news-room/fact-sheets/detail/spinal-cord-injury Accessed 9 March 2024 (2013) The epidemiology of traumatic spinal cord injury in British Columbia A nationwide survey on the incidence and characteristics of traumatic spinal cord injury in Japan in 2018 SPINE20 A global advocacy group promoting evidence-based spine care of value Prevention of falls and consequent injuries in elderly people Traumatic spinal cord injury in the United States Management of acute spinal cord injury: Where have we been Ministry_of_Health_Labour_and_Welfare. The Results of National Health and Nutrition Survey 2018 (written in Japanese). https://www.mhlw.go.jp/stf/newpage_14156.html Is undernutrition risk associated with an adverse clinical outcome in spinal cord-injured patients admitted to a spinal centre? Relationship between nutritional status and mortality during the first 2 weeks following treatment for cervical spinal cord injury Relationship between nutritional status and improved ADL in individuals with cervical spinal cord injury in a convalescent rehabilitation ward Geriatric Nutritional Risk Index: A new index for evaluating at-risk elderly medical patients Simplified nutritional screening tools for patients on maintenance hemodialysis is a significant predictor of mortality in chronic dialysis patients Geriatric nutrition risk index: Prognostic factor related to inflammation in elderly patients with cancer cachexia Potential association with malnutrition and allocation of combination medical therapies in hospitalized heart failure patients with reduced ejection fraction Prognostic factors for cervical spinal cord injury without major bone injury in elderly patients Differences in clinical characteristics of cervical spine injuries in older adults by external causes: A multicenter study of 1512 cases Prognostic factors for respiratory dysfunction for cervical spinal cord injury and/or cervical fractures in elderly patients: A multicenter survey International standards for neurological classification of spinal cord injury (revised 2011) The analysis of residuals in cross-classified tables Nutritional status in chronic spinal cord injury: A systematic review and meta-analysis Beliefs about inevitable decline among home-living older adults at risk of malnutrition: A qualitative study Musculoskeletal morbidity following spinal cord injury: A longitudinal cohort study of privately-insured beneficiaries A primary care provider’s guide to diet and nutrition after spinal cord injury SPINE20 recommendations 2022: Spine care-working together to recover stronger Download references Osaka Metropolitan University Graduate School of Medicine Graduate School of Comprehensive Human Sciences National Hospital Organization Murayama Medical Center Yamaguchi University Graduate School of Medicine Tohoku University Graduate School of Medicine Nagoya City University Graduate School of Medical Sciences Kobe University Graduate School of Medicine Graduate School of Medical and Dental Sciences Gunma University Graduate School of Medicine Mie University Graduate School of Medicine International University of Health and Welfare International University of Health and Welfare Narita Hospital Department of Orthopaedic Surgery and Spine and Spinal Cord Center International University of Health and Welfare Mita Hospital Osaka University Graduate School of Medicine All authors contributed to the collection of clinical data KT and HT led the drafting of this manuscript in collaboration with other authors All authors contributed to all sections of the manuscript and edited it for key intellectual content All other authors have read and provided substantive intellectual comments to the draft and have approved the final version of the paper Download citation DOI: https://doi.org/10.1038/s41598-024-56527-y Metrics details We have successfully determined the internuclear distance of I2 molecules in an alignment laser field by applying our molecular structure determination methodology to an I 2p X-ray photoelectron diffraction profile observed with femtosecond X-ray free electron laser pulses we have found that the internuclear distance of the sample I2 molecules in an alignment Nd:YAG laser field of 6 × 1011 W/cm2 is elongated by from 0.18 to 0.30 Å “in average” relatively to the equilibrium internuclear distance of 2.666 Å the present experiment constitutes a critical step towards the goal of femtosecond imaging of chemical reactions and opens a new direction for the study of ultrafast chemical reaction in the gas phase results on the transient structure of molecules during chemical reaction which will be obtainable with the UXPD methods The reported diffraction profiles for such molecules can be regarded as a snapshot of a “molecular movie” visualizing the femtosecond structural dynamics in a pump-probe experiment a fundamental question arises: whether the structure of a molecule in an intense alignment-laser field is the same as that in its ground state or not we have applied the UXPD method to a simple I2 molecule to determine its structure ‒ in other words its internuclear distance ‒ in the alignment-laser field Two laser beams propagating along the x-axis in a collinear arrangement intersect a supersonic pulsed molecular beam along the z-axis at the centre of a vacuum chamber A Nd:YAG laser is used to adiabatically align the sample I2 molecules that are probed by the XFEL XPD images of the photoelectrons are recorded by the upper VMI The degree of alignment is quantified using the 2D momentum distributions of the ionic fragments 2D momentum images of electrons and ions and their polar plots The white circles indicate the radii of 26 and 30.5 mm to distinguish the central-ring image of low-energy electrons (b) The I 2p XPD profile expressed as the polar plot The short bars denote the statistical errors of the experimental data and the solid curve is the fitted result of the Legendre polynomials of F(θe) ∝ P0(θe) + 1.49P2(θe) + 0.31P4(θe) + 0.24P6(θe) (c) Fragment-ion image indicating that the molecular axis distributions are aligned along the polarization vector of the Nd:YAG laser parallel to the z-axis The white circles correspond to radii of 5 and 10 mm (d) The molecular axis distributions expressed as the polar plot in which the background has been eliminated from the raw image and the solid curve P(θ) = cos2 θ + 1.82 cos12 θ is the result of the numerical simulation the photoelectron peak with the mean energy of εp~140 eV has a width of | ± ΔE/2| × 2~24 eV (full width at half maximum) the parameter range of ΔEa covers the possible range for the muffin-tin zero energy of V0 R-factor map as a function of parameters ΔEa and ΔRI-I (a) and relevant I 2p XPD profiles (b) In (a), a valley located in region A and a hill in region B. In (b), simulated XPD profiles at the minimum value of the R-factor in region A and at the maximum in region B are shown by red and blue curves, respectively. The experimental data are represented by the short bars, which are the same as those in Fig. 2(b). Schematic view of a one-dimensional muffin-tin potential of an I2 molecule. εp: photoelectron kinetic energy from the vacuum level, V0: energy between the vacuum level and the muffin-tin constant, and Ep: photoelectron energy in the molecular region. rI+ and rI are the muffin-tin radius of the I+ ion and that of I atom, respectively. Based on the above molecular structure determination methodology, we can conclude that the internuclear distance of the sample I2 molecules in the alignment Nd:YAG laser fields of 6 × 1011 W/cm2 is elongated by from 0.18 to 0.30 Å “in average” relatively to the equilibrium internuclear distance of 2.666 Å. XPD profile integrated over the molecular axis distributions (a) and its decompositions (b) the reference axis of the XPD profile is the polarization vector of the XFEL which is indicated by the double headed arrow the central polar plot expresses the molecular axis distribution P(θ) = cos2 θ + 1.82 cos12 θ The decomposed XPD profiles exhibit dramatic change depending on the geometries of the polarization vector of the X-rays and the molecular axis Numbers on the left and right sides of the figures stand for their linear magnifications we can investigate the correlations and couplings between electrons in the ground and excited states using the moderately intense laser fields with energy 800 mJ/pulse and duration ~10 ns were focused to ~80 μm by a spherical lens placed outside the vacuum chamber The spatial overlap of the XFEL and the Nd:YAG pulses was first examined by monitoring the images on a Ce:YAG phosphor screen in the interaction region and then confirmed by monitoring the degree of alignment of the sample molecules The temporal overlap of the pulses was monitored by a fast photodiode The Nd:YAG pulses were synchronized with the XFEL pulses by a pulse generator (Stanford Research which was triggered by a master signal that delivers the XFEL pulses with a repetition rate of 30 Hz and triggered both the pulsed solenoid valve and the cameras The valve was heated to 60 °C to provide a partial pressure of ~100 Pa for I2 The molecular beam passed through a 3-mm-diameter skimmer and was introduced into the interaction region where the Nd:YAG and XFEL laser pulses were overlapped The source and the main chamber were differentially pumped by turbo-molecular pumps and their typical pressures during the experiments were 1 × 10−4 and 2 × 10−6 Pa The pulse duration of the valve was changed from 20.5 to 22 μs by monitoring the pressure of the source chamber The pulsed valve was operated at a repetition rate of 15 Hz The images (signal + background) with and those (background) without the sample molecules were alternately measured and then the images without the noise from the residual gas and the scattered XFEL were obtained by subtracting the background from the (signal + background) images We acquired momentum-image data for 600,000 XFEL pulses we employed the intensity I of the laser pulse and the rotational temperature Trot as the fitting parameters we determined that both I = 6 × 1011 W/cm2 and Trot = 5 K reproduced fairly well the experimental data This numerical simulation of the molecular axis distribution resulted in the expectation value of cos2 θ,<cos2 θ > = 0.734 ± 0.003 we determined that the molecular axis distribution can be well represented by the simple functional form of P(θ) = cos2 θ + 1.82cos12 θ Note that the intensity I can be regarded as the effective one when the non-uniform laser intensity is averaged over the ionization volume of the XFEL pulses In the above evaluation of the degree of alignment for the sample molecules we used the equilibrium internuclear distance of 2.666 Å in the ground state To examine the internuclear distance effect on the degree of alignment we assumed that all the molecules in the ensemble were elongated by 10% of the equilibrium internuclear distance Then we estimated the degree of alignment for such molecular ensemble in which the other conditions were set to the same as the beforementioned case we obtained the degree of alignment of <cos2 θ > = 0.762 ± 0.003 which is used to construct theoretical XPD profiles expected from this value for the degree of alignment was nearly the same as that for <cos2 θ> = 0.734 ± 0.003 The experimentally prepared molecular ensemble in the Nd:YAG laser are between the two extreme cases all the molecules are either in the ground state or in the excited states the averaged degree of alignment over all the molecules in the ensemble must be between <cos2 θ > = 0.734 ± 0.003 and <cos2 θ> = 0.762 ± 0.003 we can conclude that the internuclear distance effect on the degree of alignment does not affect our molecular structure determination procedure with detectable amount The positions of the detected electrons were determined offline by calculating the centre of the intensity weighted by the density of activated pixels, which provides a sub-pixel spatial resolution. The electron image shown in Fig. 2(a) has two components; the low-energy peak which is associated with the Auger shake-off processes and the high-energy ring of the I 2p photoelectrons Although the I 2p photoelectron ring is almost separated from the intense low-energy peak parts of the two peak components overlap with each other the radial distribution of the central part with radii 3‒20 mm was approximated with a Gaussian function by a least-squared procedure for every 6-degree sector from the polarization vector of the Nd:YAG laser the extrapolated tail component was subtracted from the electron signals in the region 26‒30.5 mm where the I 2p photoelectron ring is located Finally considering the symmetry restriction for the XPD profile we averaged the I 2p photoelectron signals detected above and below the x-axis and those on the left and right of the z-axis to obtain the photoelectron polar plot depends on the molecular axis because the polarization vector is fixed in the present experimental geometry the molecular axis distributions strongly affect the profiles of the measured XPD profiles In the extreme case of a fully random alignment the XPD profiles cannot be measured but the photoelectron angular distributions can be observed relatively to the polarization vector of the X-rays This geometry was selected for a convenient illustration and there are no restrictions to the photoionization of the py orbital Structure determination of molecules in an alignment laser field by femtosecond photoelectron diffraction using an X-ray free-electron laser Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Potential for biomolecular imaging with femtosecond X-ray pulses First lasing and operation of an ångström-wavelength free-electron laser A compact X-ray free-electron laser emitting in the sub- ångstrom region Femtosecond X-ray protein nanocrystallography Single mimivirus particles intercepted and imaged with an X-ray laser Imaging live cell in micro-liquid enclosure by X-ray laser diffraction Imaging molecular motion: femtosecond X-ray scattering of an electrocyclic chemical reaction Imaging molecules from within: Ultrafast angström-scale structure determination of molecules via photoelectron holography using free-electron lasers Photoelectron diffraction from single oriented molecules: Towards ultrafast structure determination of molecules using x-ray free-electron lasers Retrieving transient conformational molecular structure information from inner-shell photoionization of laser-aligned molecules Femtosecond photoelectron diffraction on laser-aligned molecules: Towards time-resolved imaging of molecular structure Femtosecond x-ray photoelectron diffraction on gas-phase dibromobenzene molecules Imaging of molecular structure through femtosecond photoelectron diffraction on aligned and oriented gas-phase molecules Photoelectron diffraction from laser-aligned molecules with X-ray free-electron laser pulses experimental stations and photon beam diagnostics for the hard x-ray free electron laser of SACLA X-ray Data Booklet (ed. Thompson, A. C.) Rv. 3 (LBNL/PUB-490, Berkley, 2009) http://xdb.lbl.gov/ Aligning molecules with strong laser pulses Fixed-molecule photoelectron angular distributions Complete photoionization experiment in the region of the 2σg → σu shape resonance of the N2 molecule Photoelectron angular distributions from single oriented molecules: Past Photoionization cross sections and photoelectron angular distributions for X-ray line energies in the range 0.132–4.509 keV targets: 1 ≤ Z ≤ 100 Photoelectron-angular-distribution parameters for rare-gas subshells Softening of the H2+ molecular bond in intense laser fields Femtosecond dynamics of multielectron dissociative ionization by use of a picosecond laser Role of electron localization in intense-field molecular ionization Alignment of CS2 in intense nanosecond laser fields probed by pulsed gas electron diffraction Quantum interference during high-order harmonic generation from aligned molecules Accurate retrieval of structural information from laser-induced photoelectron and high-order harmonic spectra by few-cycle laser pulses Retrieving photorecombination cross sections of atoms from high-order harmonic spectra Experimental observation of revival structures in picosecond laser-induced alignment of I2 Focusing of X-ray free-electron laser pulses with reflective optics Cooling of large molecules below 1 K and He clusters formation Alignment and trapping of molecules in intense laser fields Polarization of molecules induced by intense nonresonant laser fields McGuinness, C. Robert, D. Cowan’s Atomic Structure Code. http://www.tcd.ie/Physics/people/Cormac.McGuinness/Cowan/ (2009) Theoretical study of X-ray photoelectron diffraction for fixed-in-space CO molecules C 1s photoelectron angular distributions from fixed-in-space CO molecules in the high energy continuum ≥ 50 eV Multiple-scattering calculations for 1s photoelectron angular distributions from single oriented molecules in the energy region above 50eV Download references The authors thank the operation and engineering staff members of SACLA for their support in performing the XFEL experiments which were conducted at the BL3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No This research was supported by JSPS KAKENHI Grant Number 25246041 and 16H02132 National Institutes for Quantum and Radiological Science and Technology Graduate School of Advanced Integration Science Japan Synchrotron Radiation Research Institute had the responsibility for the XFEL operation contributed to the preparation of aligned sample molecules The draft of the manuscript was written by A.Y with all discussions and improvements from all authors Download citation Japan's Samurai Blue demolished Mongolia 6-0 in the second round of Asian qualifying for the 2022 World Cup on Thursday for their second win in Group F Takumi Minamino opened the scoring in the 22nd minute at Saitama Stadium who found the Red Bull Salzburg man unmarked dead center in front of goal Southampton defender Maya Yoshida doubled the lead moments later heading home a loose ball after Hiroki Sakai had got a boot to a rebound from a shot blocked by Mongolia keeper Batsaikhan Ariunbold Yuto Nagatomo then made it 3-0 with his first goal for Japan since 2009 after Ariunbold failed to cut out a cross to the Galatasaray defender and FC Tokyo striker Kensuke Nagai headed in Japan's fourth in the 40th minute The rout continued after the break with further goals from Wataru Endo and Daichi Kamada "Scoring all those early goals made things easy for us," Yoshida said "Playing in front of a big crowd at Saitama Stadium and with so many fans watching around the country on TV the players had prepared well to show what they could do." "I'm proud of the fact that we didn't fit our game to match Mongolia's Rather it was a case of the players exceeding the demands of the moment and imposing their will on our opponents." Japan entered the game on the back of a 2-0 win at Myanmar on Sept 11 thanks to goals from Shoya Nakajima and Minamino Japan's next game is on Tuesday in Tajikistan who have beaten Mongolia and Kyrgyzstan by 1-0 scores The group's top two teams qualify for the third round of World Cup qualifying while the group winner also qualifies for the Asian Cup Metrics details this nationwide retrospective study aimed to evaluate the actual incidence and risk factors of HBVr in Japanese patients with MM This study was performed in accordance with the ethical principles of the Declaration of Helsinki and was approved by the ethics review board of each participating institution Informed consent was obtained from all patients All patients had been treated with either novel agents (bortezomib and ixazomib) or had undergone autologous stem cell transplantation (auto-SCT) and 760 (15.0%) exhibited resolved HBV infection Prophylactic antiviral agents were administered to 46 (88.5%) of the 52 HBV carriers to prevent hepatitis; one of the remaining 6 developed hepatitis These results suggest that lenalidomide may decrease AGO2 levels and inhibit HBV proliferation in patients with MM only 17 patients with MM who experienced HBVr were reported from non-Asian regions It is possible that auto-SCT is a risk factor for HBVr not only in Asia but also worldwide HBV-DNA monitoring is more economical than antiviral prophylaxis; these results suggest that prophylactic antiviral therapy might not always be recommended in MM there was no significant association between anti-HBs negativity and HBVr in the present study and serological markers could not be thoroughly assessed because each institution used different assay methods There was no significant association between post-transplant maintenance therapy and HBVr in our study (p = 0.7913) the actual prevalence of HBVr in MM patients who experienced prolonged treatment could not be determined as the incidence of HBVr gradually increased up to 5 years (260 weeks) after initiating treatment the relationship between HBVr and allogeneic SCT or recently approved novel agents apart from bortezomib The sample size was too small to evaluate the relationship nationwide retrospective study showed that HBVr in patients with MM was significantly higher especially among patients who received auto-SCT Additional prospective studies with long-term observation periods are needed to evaluate the optimal duration of HBV-DNA monitoring and to develop an effective strategy to prevent HBVr in patients with MM Download references We thank all the following physicians of the cooperating hospitals for providing the clinical data: Michiaki Koike (Juntendo University Shizuoka Hospital) Hideto Tamura and Keiichi Moriya (Nippon Medical School) Shigeki Ito and Maki Asahi (Iwate Medical University School of Medicine) Yoichi Imai and Junji Tanaka (Tokyo Women’s Medical University) Hiroshi Handa and Hiromi Koiso (Gunma University) Sakae Tanosaki (The Fraternity Memorial Hospital) Yoshihiro Hatta (Nihon University School of Medicine) Jian Hua and Masao Hagihara (Eiju General Hospital) Ilseung Choi (National Hospital Organization Kyushu Cancer Center) Naoko Harada (National Hospital Organization Kumamoto Medical Center) Kensuke Ohta (Osaka Saiseikai Nakatsu Hospital) Toshihiro Fukushima (Kanazawa Medical University Hospital) Yoshitaka Imaizumi (Nagasaki University Hospital) Hiroyuki Kuroda (Steel Memorial Muroran Hospital) Yuichi Nakamura (Saitama Medical University Hospital) Tomoharu Takeoka (Otsu Red Cross Hospital) Mitsufumi Nishio (NTT Higashinihon Sapporo Hospital) Takeshi Yamashita (Keiju Kanazawa Hospital) Msayoshi Kobune (Sapporo Medical University School of Medicine) Nobuhiko Nakamura (Gifu University Hospital) Miyuki Ookura (University of Fukui Hospital) Eriko Sato (Juntendo University Nerima Hospital) Masahiro Onozawa (Hokkaido University Graduate School of Medicine) Shikiko Ueno (Kumamoto University Graduate School of Medicine) Riko Tsumanuma (Yamagata Prefectural Central Hospital) Hiroyuki Takamatsu (Kanazawa University Graduate School of Medical Science) Nobuhiko Uoshima (Japanese Red Cross Kyoto Daini Hospital) Tsuyoshi Takahashi (Mitsui Memorial Hospital) Akio Kohno (JA Aichi Konan Kosei Hospital) Shigeru Hoshino (Saitama Red Cross Hospital) Atsushi Inagaki (Nagoya City West Medical Center) Kazuki Tanimoto (Fukuoka Red Cross Hospital) Katsumichi Fujimaki (Fujisawa City Hospital) Hirokazu Okumura (Toyama Prefectural Central Hospital) Tsuyoshi Nakamaki (Showa University School of Medicine) Norisato Hashimoto (Tokai University Hachioji Hospital) Masayuki Ohnishi (Niigata Minami Hospital) Hiroto Kaneko (Aiseikai Yamashina Hospital) Masahiro Fujiwara (Kashiwazaki General Hospital and Medical Center) Masato Shikami (Daiyukai General Hospital) Takahiro Karasuno (Rinku General Medical Center) Nobuhisa Hirase (Nakatsu Municipal Hospital) Masanobu Nakata (Seirei Hamamatsu General Hospital) Yoshihito Iwahara (National Hospital Organization Kochi National Hospital) Tatsuyuki Hayashi (Tokyo Metropolitan Police Hospital) Sachiya Takemura (Yokohama Ekisaikai Hospital) Masaru Shibano (Sakai City Medical Center) Naoki Takezako (National Hospital Organization Disaster Medical Center) This work was supported by the grant (Takeda Research Support) from Takeda Pharmaceutical Company (to M.S.) National Hospital Organization Okayama Medical Center National Hospital Organization Shibukawa Medical Center Japan Community Health Care Organization Kyushu Hospital Department of Transfusion and Cell Transplantation Tokyo Metropolitan Cancer and Infectious Diseases Center Graduate School of Sports and Health Science and received research funding from Celgene received honoraria from Chugai Pharmaceutical The remaining authors declare that they have no conflicts of interest Download citation DOI: https://doi.org/10.1038/s41408-017-0002-2 Metrics details Most plants interact with arbuscular mycorrhizal fungi which enhance disease resistance in the host plant Because the effects of resistance against bacterial pathogens are poorly understood we investigated the effects of mycorrhizal colonization on virulent and avirulent pathogens using phytopathological and molecular biology techniques Tomato plants colonized by Gigaspora margarita acquired resistance not only against the fungal pathogen but also against a virulent bacterial pathogen salicylic acid (SA)- and jasmonic acid (JA)-related defense genes were expressed more rapidly and strongly compared to those in the control plants when challenged by Pst indicating that the plant immunity system was primed by mycorrhizal colonization Gene expression analysis indicated that primed tomato plants responded to the avirulent pathogen more rapidly and strongly compared to the control plant where the effect on the JA-mediated signals was stronger than in the case with Pst We found that the resistance induced by mycorrhizal colonization was effective against both fungal and bacterial pathogens including virulent and avirulent pathogens the activation of both SA- and JA-mediated signaling pathways can be enhanced in the primed plant by mycorrhizal colonization which have been used for investigation of disease resistance mechanisms Although primed plants are able to respond more rapidly and strongly to pathogenic infection to protect themselves no or weak expression of the major defense-related genes through SA- or JA-mediated signaling pathways were observed before pathogenic infection Since these analyses have been performed only for MIR induced by R analysis of MIR by other arbuscular mycorrhizal fungi will provide further insights into the mechanism of MIR The question has been raised as to whether MIR is distinctly effective against biotrophic bacteria; however only limited information is available on MIR against foliar bacterial pathogens To better understand the effects of MIR on bacterial pathogens we investigated the effects of MIR induced by Gigaspora margarita colonization on virulent and avirulent bacterial pathogens in tomato (Solanum lycopersicum L Momotaro) and the related priming effects on defense signaling This is the first study to elucidate the priming responses against bacterial pathogens in MIR The data presented here demonstrate the potentiality of MIR for controlling various diseases To verify root-colonization by G. margarita, 14 and 28 days after inoculation (with 25 spores per plant), tomato roots were cleared and stained with trypan blue for microscopic evaluation of infection (Fig. 1A). The results revealed that the G. margarita colonization rate was only 1.5% at 14 days but increased to 10–15% at 28 days after inoculation. (A) Colonization of tomato root with G margarita (25 spores/pot) by soil drenching Roots were washed and strained with trypan blue 6 days after the inoculation with G (B) Induction of resistance against tomato leaf speck disease by G margarita (25 spores/pot) (AMF) 14 days prior to challenge inoculation with Pst (1 × 103 CFU/ml) SAR was induced by treatment with BIT (5 mg/pot) by soil-drenching method 5 days prior to challenge inoculation The growth of Pst in tomato leaflet was evaluated 2 days after the inoculation Each experiment was done with more than 4 plants Values are shown as the means ± SE (n = 8) of a single experiment Different letters indicate statistically significant differences between treatments (one-way ANOVA with Tukey’s post-test The experiment was repeated three times with similar results (C) Photograph of representative disease symptoms taken 5 days after inoculation with Pst margarita-colonized tomato against bacterial pathogens was caused by the activation of the plant immunity system corroborating that the resistance was due to colonization by G margarita and probably not to the spore-associated bacteria or endobacteria alone (A) Expression of defense-related genes in G Terminal leaflets of the 4th compound leaves were collected 14 days after the inoculation with G SAR was induced by treatment with BIT (5 mg/pot) by soil drenching for 5 days Real time PCR analysis was performed to evaluate the expression of SAR marker genes (PR1b and PR2a) and JA-related genes (Loxd Transcript levels were normalized to the expression of ACT4 measured in the same samples The means and SEs were calculated from 4 independent samples Asterisk indicates statistically significant difference between data of the control and BIT-treated plants (two-sided t-test Terminal and its neighboring leaflets of 4th compound leaves were harvested at 14 days after inoculation with G The levels of free and total SA (free SA + SA-glucoside) were quantified by HPLC Values presented are the means ± SE from 6 samples These results indicated that the JA-mediated defense signaling was not activated by G Expression of defense-related genes after infection with the virulent pathogen margarita inoculation) and the water-treated control tomato plants were inoculated with Pst Leaf disks were taken from the Pst-infiltrated part of the leaflets at the indicated time points (0 20 h post inoculation (hpi)) and used for gene expression analyses of SA-related genes (PR1b and PR2a) and JA-related genes (LOXd water-treated control plant; closed circle Asterisks indicate statistically significant difference between data of the water-treated control and G margarita-colonized plants (two-sided t-test Although the induction levels of JA-related gene expression were quite low these results indicated that the activation of the JA-mediated signaling pathway in response to pathogen infection was accelerated in mycorrhizal plants Response of tomato leaves to infection with avirulent pathogens Terminal leaflets of the 4th leaves of 3-week-old tomato plants were inoculated with Pso or Pso∆fliC (1 × 104 Photographs were taken 30 h after inoculation The experiment was repeated twice with similar results Expression of defense-related genes after infection with avirulent pathogen mutant Pso∆fliC margarita inoculation) and the water-treaterd control tomato plants were inoculated with Pso∆fliC Leaf disks were taken from the Pso∆fliC-infiltrated part of the leaflets at the indicated time points (0 16 h post inoculation (hpi)) and used for gene expression analyses of SA-related genes (PR1b and PR2a) and JA-related genes (LOXd margarita-colonized plants (two-sided t-test; ** These results indicated that the JA-mediated signaling pathway in response to infection with avirulent pathogens margarita-colonized plants compared to that in the water-treated control plants Disease resistance induced by symbiotic soilborne microbes in plants should be an important adaptative strategy to diversified environments which is also an important mechanism for crop production To understand the underlying mechanisms at the molecular level we investigated the defense responses in G margarita-colonized tomato plants to fungal and bacterial pathogens The resistance induced by this mycorrhizal symbiosis revealed protective effects against the fungal pathogen B Analyses of gene expression and SA accumulation indicated that the G margarita colonization did not activate the defense signals mediated by SA and JA; however the activation of these defense signals by infection with Pst was enhanced in G suggesting that the immune system was primed by G HR induction was not influenced by the priming of tomato plants; however the SA- and JA-mediated responses to the lower bacterial concentration were enhanced margarita-primed tomato plants exhibited accelerated induction of defense signaling upon infection with a virulent strain Both Pst and Pso had relatively strong effects on SA- and JA-signaling pathways in tomato Some of these priming mechanisms have been analyzed using bacterial pathogens whereas the priming mechanisms of MIR have been analyzed only using fungal pathogens this is the first demonstration of a priming response against a bacterial pathogen in MIR The priming effects on each of these hormonal signal transductions differed according to the type of infecting pathogens which is partly due to the antagonistic crosstalk between SA- and JA-mediated signaling pathways In analyses of defense response against avirulent pathogens infection with Pso at the bacterial concentration as Pst (1 × 105 CFU/mL) provided unstable and unreliable data on gene expression (data not shown) This result was probably due to either—or both— intense response to the avirulent factors or the initiation of cell death events at an imperceptible rate To precisely characterize cellular defense responses in mycorrhizal plants by avoiding these unfavorable conditions we searched for the appropriate bacterial concentration for infection and sampling time points for gene expression analysis We found that infection with Pso at a concentration of 1 × 104 CFU/mL resulted in a similar time course of gene expression patterns to those observed in the case of Pst our study indicated that MIR induction without the activation of SA- and JA-mediated defense was effective against both necrotrophic and biotrophic pathogens These results suggested that the effectiveness of MIR on necrotrophs or biotrophs was likely dependent on the SA-JA antagonistic crosstalk although this is not the case for all MIRs they could be a potent tool to clarify the molecular mechanism of MIR in tomato the deletion of the fliC gene from Pso had no influence on HR under our experimental conditions both defense response and its enhancement by priming especially the expression of JA-related genes after infection with this mutant were observed earlier than those with the wild-type strain This result was probably due to the lack of recognition and response to flagellin or the lack of bacterial motility would provide more information about the enhancement mechanism of defense responses in primed tomato plants the physiological state of primed plants may vary according to the mycorrhizal colonization rates analyzing the resistance against bacterial pathogens in other plant-mycorrhiza interactions would reveal the complex regulation mechanism of defense signals in MIR The operations were performed under sterile condition by utilizing XbaI and BamHI sites in the multi-cloning sites of these vectors The resulting plasmid pMCPso was introduced into E coli S17-1 by electrotransformation and then transferred to Pso by bacterial conjugation The nalidixic acid-resistant and kanamycin-sensitive Pso colonies were selected as the fliC-deficient mutant followed by PCR analysis to confirm the deletion Japan) were sown and grown in sterilized soil (Raising seedling soil Japan) in plastic pots (5 cm × 5 cm × 5 cm) in a growth chamber (16:8 h L:D One-week-old tomato seedlings were inoculated with mycorrhizal spores (25 spores per plant) by placing spores with a micropipette at 4 points (3-cm-deep) in the soil Crushed spores (25 spores in 0.1 mL sterilized water) were prepared by crushing using a pestle with a small amount of fine-ground sea sand in a 1.5 mL plastic tube plants were treated with BIT (5 mg/pot) using the soil-drenching method 5 days before pathogen inoculation margarita-colonized (14 days after inoculation) and the water-treated plants were used for pathogen inoculation assay Pst was cultured in nutrient broth containing rifampicin (100 µg/mL) at 30 °C for 24 h Bacterial suspension (1 × 103 CFU /mL) prepared using 10 mM MgCl2 were infiltrated into the terminal and its neighboring leaflets of the 4th compound leaves using a 1-mL syringe without a needle Leaf disks (4-mm diameter) were taken from the infiltrated part of the leaflet 2 days after inoculation Bacterial cells were extracted by homogenizing the leaf disks (5 disks per sample) in 10 mM MgCl2 The number of CFUs was estimated by culturing bacterial cells in nutrient broth agar plates after dilution more than 4 plants were used and 8 samples were prepared Pso and its mutant Pso∆fliC were cultured in nutrient broth at 30 °C for 24 h and 1 × 106 CFU/mL in 10 mM MgCl2) were prepared and used for inoculation into leaflets of 4th compound leaves For the gene expression analysis in leaves of the G margarita-colonized (14 days after inoculation) and the water-treated control plants terminal leaflets of the 4th compound leaves were harvested at the time of pathogen inoculation These were used for total RNA extraction using Sepasol-RNA I super reagent (Nacalai Tesque followed by cDNA synthesis using the PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio Quantitative RT-PCR was performed using a LightCycler 96 System (Roche Thermal cycling conditions consisted of 30 s at 95 °C The PCR reaction mixture contained 2 µL of tenfold diluted cDNA template 0.8 µL of primer solution (containing 5 µM each of forward and reverse primers) and 10 µL of SYBR Premix Ex Taq II (Takara Bio The gene-specific primer pairs used are as follows: for PR1b forward 5’- CTTGCGGTTCATAACGATGC-3’ and reverse 5’- TAGTTTTGTGCTCGGGATGC-3’; for PR2a reverse 5’- GGGCATTAAAGACATTTGTTTCTGG-3’; for LOXd reverse 5’-ACACTGCTTGGTTGCTTTTCTTC -3’;for OPR3 reverse 5’-AGGATTAGAGATGAAAAGACGACCA-3’; for PI2 reverse 5’-TTGCCTCCACCGAAAACC-3’; for ACT4 The culture and preparation of bacterial suspensions were performed using a method similar to that used for the pathogen-inoculation assay The bacterial concentration of Pst was 1 × 105 CFU/mL whereas those of Pso and Pso∆fliC were 1 × 104 CFU/mL to avoid the quick response leading to HR Bacterial suspensions were infiltrated into terminal leaflets of the 4th compound leaves of the G margarita-colonized (14 days after inoculation) and control plants followed by a sampling of leaf tissues from the pathogen-infiltrated parts at several time points after inoculation more than 4 plants were used and 6 RNA samples were prepared No permission is required for plant material as it was purchased from certified dealer of local area The study has been conducted without violating any ethical codes of conduct All data generated or analyzed during this study are present in this paper and the supplementary materials Cross talk between signaling pathways in pathogen defense Networking by small-molecule hormones in plant immunity Salicylic acid and disease resistance in plants Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action Induced resistance and phytoalexin accumulation in biological control of Fusarium wilt of carnation by Pseudomonas sp Differential induction of systemic resistance in Arabidopsis by biocontrol bacteria Simultaneous interaction of Arabidopsis thaliana with Bradyrhizobium sp tomato DC3000 leads to complex transcriptome changes Redox-active pyocyanin secreted by Pseudomonas aeruginosa 7NSK2 triggers systemic resistance to Magnaporthe grisea but enhances Rhizoctonia solani susceptibility in rice Effects of colonization of a bacterial endophyte The arbuscular mycorrhizal symbiosis promotes the systemic induction of regulatory defence-related genes in rice leaves and confers resistance to pathogen infection The nitrogen availability interferes with mycorrhiza-induced resistance against Botrytis cinerea in tomato The arbuscular mycorrhizal symbiosis reduces disease severity in tomato plants infected by Botrytis cinerea Enhanced tomato disease resistance primed by arbuscular mycorrhizal fungus Localized versus systemic effect of arbuscular mycorrhizal fungi on defence responses to Phytophtora infection in tomato plants Arbuscular mycorrhiza reduces susceptibility of tomato to Alternaria solani Role and mechanisms of callose priming in mycorrhiza-induced resistance Arbuscular mycorrhizal symbiosis is accompanied by local and systemic alterations in gene expression and an increase in disease resistance in the shoots Arbuscular mycorrhizal symbiosis limits foliar transcriptional responses to viral infection and favors long-term virus accumulation The arbuscular mycorrhizal symbiosis attenuates symptom severity and reduces virus concentration in tomato infected by Tomato yellow leaf curl Sardinia virus (TYLCSV) Priming of anti-herbivore defense in tomato by arbuscular mycorrhizal fungus and involvement of the jasmonate pathway Arbuscular mycorrhizal symbiosis alters plant gene expression and aphid weight in a tripartite interaction Assessment of local and systemic changes in plant gene expression and aphid responses during potato interactions with arbuscular mycorrhizal fungi and potato aphids Population and function analysis of cultivable bacteria associated with spores of arbuscular mycorrhizal fungus Gigaspora margarita Gigaspora margarita and its endobacterium modulate symbiotic marker genes in tomato roots under combined water and nutrient stress Mycorrhiza-induced resistance and priming of plant defenses Mycorrhiza-induced changes in disease severity and PR protein expression in tobacco leaves Abscisic acid modulates salicylic acid biosynthesis for systemic acquired resistance in tomato Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression The priming molecule ß-aminobutyric acid is naturally present in plants and is induced by stress Chemical priming of immunity without costs to plant growth Arbuscular mycorrhizal symbiosis: plant friend or foe in the fight against viruses? The Arabidopsis thaliana-pseudomonas syringae interaction tomato avrE1/hopM1 mutant is severely reduced in growth and lesion formation in tomato tomato DC3000: A model pathogen for probing disease susceptibility and hormone signaling in plants Plant immunity: From signaling to epigenetic control of defense Defense responses of Arabidopsis thaliana inoculated with Pseudomonas syringae pv tabaci wild type and defective mutants for flagellin (ΔfliC) and flagellin-glycosylation (Δorf1) Extensive tubular vacuole system in an arbuscular mycorrhizal fungus Gigaspora margarita Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum a simple staining technique for arbuscular-mycorrhizal fungi An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots Download references This work was partially supported by Ministry of Agriculture Forestry and Fisheries under Science and Technology Research Promotion Program for Agriculture Fisheries and Food Industry (27004A) to K.Y. by Grant-in-Aid for JSPS Fellows 19J14665 to M.Fuj and by JSPS KAKENHI Grant Numbers 18K05656 to H.N These authors contributed equally: Moeka Fujita and Miyuki Kusajima Department of Bioscience and Biotechnology Graduate School of Agricultural and Life Sciences Graduate School of Life and Environmental Sciences Center for Bioscience Research and Education M.K carried out the majority of the experiments wrote the manuscript and all authors agreed on the final manuscript Download citation DOI: https://doi.org/10.1038/s41598-022-08395-7 Metrics details Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are enzootic in poultry populations in different parts of the world and have caused numerous human infections in recent years no sustained human-to-human transmission of these viruses has yet been reported We tested nine naturally occurring Egyptian H5N1 viruses (isolated in 2014–2015) in ferrets and found that three of them transmitted via respiratory droplets causing a fatal infection in one of the exposed animals All isolates were sensitive to neuraminidase inhibitors these viruses were not transmitted via respiratory droplets in three additional transmission experiments in ferrets we do not know if the efficiency of transmission is very low or if subtle differences in experimental parameters contributed to these inconsistent results our findings heighten concern regarding the pandemic potential of recent Egyptian H5N1 influenza viruses It is unclear whether the high number of human HPAI H5N1 infections in Egypt in 2014–2015 reflects socioeconomic changes resulting in increased contact between people and infected animals or if genetic changes in the virus have increased its predilection for human infections Given that HPAI H5N1 viruses in Egypt evolve rapidly and have caused a substantial number of human infections we here characterized the respiratory droplet transmissibility of nine Egyptian HPAI H5N1 influenza viruses in ferrets Highly pathogenic HPAI H5N1 viruses are also characterized by a multibasic cleavage site in HA which allows cleavage of the HA precursor into the HA1 and HA2 subunits by ubiquitous proteases thus allowing fatal systemic viral infections in terrestrial avian species Most subclade 2.2.1 and 2.2.1.1 HA proteins possess a cleavage site of the sequence PQGERRRKKR↓G (‘↓’ denotes the cleavage site); in contrast subclade 2.2.1.2 HA proteins encode the motif PQGEKRRKKR↓G; currently it is not known if this difference affects the virulence or pathogenicity of these viruses which may facilitate receptor binding; and (iii) key amino acid residues in the viral polymerase complex for efficient avian influenza virus replication in mammals cells Respiratory droplet transmissibility of Egyptian H5N1 viruses in ferrets Ferrets were inoculated with the indicated viruses (‘Inoculated’) one naïve ferret was placed in a cage next to an inoculated ferret (‘Exposed’) Virus titers were determined on the indicated days post-inoculation or -exposure HI titers were measured pre-inoculation or -exposure and on day 26 post-inoculation or on day 25 post-exposure Horizontal lines indicate detection limits (i.e. a virus titer of 1.0 log10 PFU/ml or an HI titer of 10) one animal exposed to a ferret inoculated with A/chicken/KafrElsheikh/UT-151CAD/2015 died on day 13 post-exposure No virus was isolated and no viral antigen was detected in this animal; hence These findings suggest that the PB2-A84T and PB2-D146G mutations may enhance virus replication in the upper respiratory tract of mammals which in turn could facilitate respiratory droplet transmission To assess the receptor-binding specificity of the viruses tested here we performed a solid phase binding assay in which α2,3- or α2,6-linked sialylglycopolymers were coated onto a microtiter Receptor-binding properties of Egyptian H5N1 and control viruses (A) Binding of viruses to sialylglycopolymers containing either α2,3-linked (blue) or α2,6-linked (red) sialic acids in solid-phase binding assays and control viruses expressing the human A/Kawasaki/173/2001 (K173; H1N1) or avian A/Vietnam/1203/2004 (VN1203; H5N1) HA and NA proteins in the background of A/Puerto Rico/8/34 (H1N1) virus (B) Binding of viruses to human respiratory tissues The viruses described above were labeled with FITC and incubated with human tissue sections Tissue sections were subsequently incubated with HRP-conjugated and then with a chromogen substrate to detect virus binding These data further support the limited dual receptor-binding specificity of the Egyptian H5N1 viruses characterized in this study Whether this level of neutralizing activity would confer protection against viruses of subclade 2.2.1.2 is unclear If currently circulating HPAI H5N1 viruses acquire the ability to efficiently transmit among humans, antiviral compounds would be the first line of defense, prompting us to test the neuraminidase inhibitor sensitivity of the inoculated and transmitted viruses (Supplementary Table S16) All Egyptian isolates tested were sensitive to neuraminidase inhibitors consistent with the absence of known amino acid mutations in NA that confer oseltamivir resistance together with the alarming increase in human HPAI H5N1 virus infections in Egypt in 2014–2015 prompted us to test the respiratory droplet transmissibility of recent Egyptian HPAI H5N1 viruses in ferrets While our first study demonstrated respiratory droplet transmission among ferrets for three of nine HPAI H5N1 viruses this finding was not reproducible in three additional transmission experiments The transmission efficiency of these viruses in ferrets may be very low such that subtle differences in experimental conditions (such as the air flow and/or animal handling) may have affected the outcomes of these studies these data suggest that contemporary Egyptian HPAI H5N1 viruses may possess the ability to transmit among mammals although not at the level of seasonal human influenza viruses Human epithelial HeLa cells were maintained in MEM containing 10% FBS and antibiotics Adenocarcinomic human alveolar basal epithelial (A549) cells were maintained in a 1:1 mixture of Dulbecco’s modified essential medium (DMEM) and Ham’s F12 (DMEM/F12 Gibco) medium with 10% fetal calf serum (FCS) and antibiotics Chicken fibroblast (DF-1) cells were grown in Dulbecco’s modified essential medium (DMEM) with 10% FCS and antibiotics Cells were maintained at 37 °C or 39 °C (DF-1) in 5% CO2 Paraffin-embedded tissue sections of the human trachea and lung were purchased from US Biomax Oropharyngeal swabs were collected from household poultry as part of routine and targeted surveillance activities in different localities of Egypt (Supplementary Table S1) Swabs were placed in transport medium [phosphate-buffered saline with antibiotics] All samples were amplified in embryonated chicken eggs and virus stock titers were determined by means of plaque assays in MDCK cells The full genomic sequences of the amplified surveillance samples and viruses isolated from exposed animals were determined by Sanger sequencing The sequence data were submitted to Genbank (Accession numbers LC106039 - LC106110) Viruses (50 μl) were serially diluted two-fold with 50 μl of PBS in a U-well microtiter plate Fifty microliters of 0.5% (vol/vol) of chicken red blood cells (CRBC) or turkey red blood cells (TRBCs) were added to each well The plates were incubated at room temperature and hemagglutination was evaluated after 45 min The HA titers were calculated as the highest dilution at which complete agglutination was observed To detect hemagglutination inhibition activity serum samples were treated with receptor-destroying enzyme (RDE; Denka Seiken Co. followed by RDE inactivation at 56 °C for 30–60 min The RDE-treated sera were then serially diluted two-fold in PBS mixed with an amount of virus equivalent to eight hemagglutination units and incubated at room temperature for 30–60 min After the addition of 50 μl of 0.5% (vol/vol) CRBC or TRBC the resulting mixture was gently mixed and incubated at 4 °C or room temperature for 30–45 min HI titers were recorded as the inverse of the highest antibody dilution that inhibited 8 HA units of virus Viral neutralization assays were performed by using the methodology outlined in the WHO Manual on Animal Influenza Diagnosis and Surveillance with the following modifications sera were treated with RDE at 37 °C for 18–20 h Fifty microliters of virus (100 tissue culture infectious dose 50) was incubated with 50 μl of two-fold serial dilutions of RDE-treated sera for 30 min at 37 °C and the mixtures were added to confluent MDCK cells in 96-well microplates the cells were incubated with MEM containing 0.3% BSA and 0.75 μg/ml TPCK-trypsin at 37 °C for 48–72 h Viral cytopathic effects were observed under an inverted microscope and virus neutralization titers were calculated as described in the WHO manual Human A549 and avian DF-1 cells were transfected with plasmids for the expression of Giza virus PB2 the wild-type PB2 protein expression plasmid was substituted with plasmids expressing Dakahlia PB2-A84T or Giza PB2-D146G respectively (note that the Dakahlia and Giza PB2 genes differ by two synonymous nucleotide replacements Cells were also transfected with pPol-I-NP(0)Luc2(0) (for A549 cells) or pPol-IGG-NP(0)Fluc(0) (for DF-1 cells) which express the firefly luciferase reporter protein from a virus-like RNA transcribed by the human or avian polymerase I promoter WI) served as an internal control for the dual-luciferase assay Transfected cells were incubated for 24 h at 33 °C and 37 °C (human A549 cells) Luciferase activity was measured by using the Dual-Glo luciferase assay system (Promega) on a Glomax microplate luminometer (Promega) according to the manufacturer’s instructions The results of two experiments (both carried out in duplicate) were compared using one-way ANOVA We considered the results significant at p < 0.05 Virus-containing supernatant was centrifuged at low speed for 15 min to remove cell debris and then laid over a cushion of 30% sucrose in PBS After ultracentrifugation at 96,174 × g for 90 min at 4 °C the virus pellet was resuspended in PBS containing glycerol and stored at −80 °C The concentration of the viruses was determined by means of HA assays with 0.5% (vol/vol) chicken red blood cells we blocked the plates with 150 μl of PBS containing 4% BSA at room temperature for 1 h and then washed them four times with cold PBS Influenza virus equivalent to 32 HA units (in PBS) was then added to the plates and incubated overnight at 4 °C After washing the plates as described above we incubated them for 2 h at 4 °C with rabbit polyclonal anti-H5N1 (VN1203) or ferret polyclonal anti-H1N1 (K173) antibodies After additional washes as described above the plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Zymed) antiserum or anti-ferret IgG (Bethyl Laboratories) for 2 h at 4 °C the plates were incubated with O-phenylenediamine (Sigma) in citrate-phosphate buffer containing 0.03% H2O2 for 10 min at room temperature The reaction was stopped with 50 μl of 1 M HCl and absorbance was determined at 490 nm by using an optical plate reader (Mark microplate reader model 680; BioRad) Virus-containing supernatants were collected from infected cells and centrifuged at 1,462 × g for 15 min to remove cell debris Viruses were inactivated by incubating them with 0.1% β-propiolactone (final concentration) for at least 16 h at 4 °C Virus supernatant was laid over a cushion of 30% sucrose in PBS and ultracentrifuged at 96,174 × g for 90 min at 4 °C; virus pellet was resuspended in PBS and stored at −80 °C Virus concentrations were determined by using hemagglutination assays with 0.5% (vol/vol) TRBCs we deparaffinized and rehydrated paraffin-embedded normal human trachea and lung (US Biomax) The rehydrated sections were blocked with carbo-free blocking solution (Vector) and TNB blocking buffer (Perkin Elmer) we added the equivalent of 8 HA units of fluorescein-5-isothiocyanate (FITC)-labeled virus to the tissue sections and incubated them at 4 °C overnight The slides were washed five times with cold PBS and subsequently incubated with an HRP-conjugated rabbit anti-FITC antibody (Dako) for 30 min at room temperature the slides were incubated with AEC (3-amino-9-ethyl-carbozole) substrate-chromogen (Dako) for 15 min at room temperature counterstained with Mayer’s hematoxylin (Sigma Aldrich) for 3 min at room temperature coverslips were mounted by using Shandon Immu-Mount (Thermo Scientific) We examined all tissue sections by using a high resolution camera (AxioCam HRc) mounted on a microscope (Zeiss Six-month-old female ferrets (Triple F Farms) which were serologically negative by HI assay for currently circulating human influenza viruses six ferrets per group were intranasally inoculated with 106 PFU (0.5 ml) of A/duck/Giza/15292 S/2015 (H5N1) Three ferrets per group were euthanized on days 3 and 6 post-infection for virological and pathological examinations The virus titres in various organs were determined by use of plaque assays in MDCK cells All experiments with ferrets were performed in accordance with the Science Council of Japan’s Guidelines for Proper Conduct of Animal Experiments and guidelines set by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison The protocol was approved by the Animal Experiment Committee of the Institute of Medical Science the University of Tokyo (approval number PA15-02) and the Animal Care and Use Committee of the University of Wisconsin-Madison (protocol number V00806) Six-month-old female ferrets (Triple F Farms) were first serologically tested for exposure to currently circulating human influenza viruses by using the HI assay Two-to-six ferrets per transmission group were anaesthetized intramuscularly with ketamine and xylazine (5–30 mg and 0.2–6 mg/kg of body weight respectively) in the studies carried out at University of Tokyo (i.e. or with ketamine and dexmedetomidine (4–5 mg/kg and 0.01–0.04 mg/kg respectively) in the study carried out at the University of Madison (i.e. study 4); animals were then inoculated intranasally with 106 PFU (500 μl) of virus For ferrets anesthetized with ketamine and dexmedetomidine atipamezole was used to shorten recovery time from anesthesia The infected ferrets were housed in transmission cages that prevent direct and indirect contact between animals but allow spread of influenza virus through the air (Showa Science) one naïve ferret was placed in a cage adjacent to each inoculated ferret (exposed ferrets) All animals were assessed daily for clinical signs and symptoms and changes in body weight Nasal washes were collected from infected and exposed animals on day 1 after infection or exposure Virus titers in nasal washes were determined by means of plaque assays To determine viral titers in the organs of infected animals 6 mice/group were infected intranasally with 103 PFU of virus Three mice in each group were euthanized on days 3 and 6 post-infection and brains were collected for virus titration by use of plaque assays in MDCK cells The data shown are the mean virus titers ± standard deviation All experiments with mice were performed in accordance with the guidelines set by the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison which also approved the protocol (protocol number V00806) Excised tissues of animal organs were preserved in 10% phosphate-buffered formalin One section from each tissue sample was stained using a standard hematoxylin-and-eosin procedure; another one was stained with a mouse monoclonal antibody for type A influenza virus nucleoprotein (NP) antigen (prepared in our laboratory) that reacts comparably with all of the viruses tested in this study Specific antigen–antibody reactions were visualized with 3,3′-diaminobenzidine tetrahydrochloride staining by using the DAKO Envision detection system (DAKO Cytomation Diluted viruses were mixed with different concentrations of oseltamivir carboxylate (the active form of oseltamivir) Samples were incubated for 30 min at 37 °C followed by the addition of methylumbelliferyl-N-acetylneuraminic acid (Sigma the reaction was stopped with the addition of sodium hydroxide in 80% ethanol The fluorescence of the solution was measured at an excitation wavelength of 360 nm and an emission wavelength of 465 nm and the 50% inhibitory concentration (IC50) was calculated Because this study involved the characterization of natural influenza viruses it does not fall under the pause on gain-of-function research announced by the US Government on October 17 All experiments were approved by the respective committees at the University of Tokyo and by the University of Wisconsin-Madison’s Institutional Biosafety Committee (IBC) the University of Wisconsin’s Alternate Responsible Official (ARO) and Institutional Contact for Dual Use Research (ICDUR) was contacted and a risk assessment was performed All practices and procedures used for additional experiments followed the requirements of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules for working with mammalian-transmissible H5N1 viruses The ARO/ICDUR was kept informed of the research results This manuscript was reviewed by the University of Wisconsin-Madison Dual Use Research of Concern (DURC) Subcommittee in accordance with the United States Government September 2014 DURC Policy which concluded that the studies described herein do not constitute DURC since the natural virus isolates were not modified or sequentially passed in our laboratory the University of Wisconsin-Madison Biosecurity Task Force regularly reviews the research and practices of research conducted with pathogens of high consequence at the institution This task force has a diverse skill set and provides support in the areas of biosafety Members of the Biosecurity Task Force are in frequent contact with the principal investigator and laboratory personnel to provide oversight and assure biosecurity All experiments with HPAI H5N1 viruses were performed in enhanced biosafety level 3 laboratories at the University of Tokyo (Tokyo which are approved for such use by the Ministry of Agriculture or in biosafety level 3 agricultural (BSL-3Ag) laboratories at the University of Wisconsin-Madison approved for such use by the Centers for Disease Control and Prevention (CDC) and Animal and Plant Health Inspection Service (APHIS) Ferret transmission studies were conducted in enhanced BSL-3 containment at the University of Tokyo by PhD-level scientists who are highly experienced in such studies Mouse virulence studies were conducted in BSL-3Ag at the University of Wisconsin-Madison also by scientists who have several years of experience working with highly pathogenic influenza viruses and performing animal studies with such viruses In vitro experiments were conducted in Class II biological safety cabinets and transmission experiments were conducted in HEPA-filtered ferret isolators Staff working in enhanced BSL-3 and BSL-3Ag wear disposable overalls and powered air-purifying respirators The enhanced BSL-3 facility at the University of Tokyo includes controlled access and airtight dampers on ductwork connected to the animal cage isolators and biosafety cabinets The structure is pressure-decay tested regularly All personnel complete biosafety and BSL-3 training before participating in BSL-3-level experiments Refresher training is scheduled on a regular basis Virus inventory is submitted once a year to the Ministry of Agriculture The BSL-3Ag facility at University of Wisconsin-Madison was designed to exceed the standards outlined in Biosafety in Microbiological and Biomedical Laboratories (5th edition; http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf) HEPA-filtered supply and double-HEPA-filtered exhaust air double-gasketed watertight and airtight seals The University of Wisconsin-Madison facility has a dedicated alarm system that monitors all building controls (~500 possible alerts) Redundancies and emergency resources are built into the facility an emergency generator in case of a power failure and other physical containment measures in the facility that operate without power Biosecurity monitoring of the facility is ongoing All personnel undergo Select Agent security risk assessment by the United States Criminal Justice Information Services Division and complete rigorous biosafety and Select Agent training before participating in BSL-3-level experiments including drills and review of emergency plans The principal investigator participates in training sessions and emphasizes compliance to maintain safe operations and a responsible research environment The laboratory occupational health plan is in compliance with the University of Wisconsin-Madison Occupational Health Program is checked monthly and documentation is submitted to the University of Wisconsin-Madison Select Agent Program Manager Virus inventory is submitted 1–2 times per year to the file holder in the Division of Select Agents and Toxins at the CDC and facilities are reviewed annually by the University of Wisconsin-Madison Responsible Official and at regular intervals by the CDC and the APHIS as part of the University of Wisconsin-Madison Select Agent Program Risk assessment of recent Egyptian H5N1 influenza viruses Human cases of influenza at the human-animal interface World Health Organization/World Organisation for Animal Nomenclature updates resulting from the evolution of avian influenza A(H5) virus clades 2.1.3.2a Molecular characterization of avian influenza H5N1 virus in Egypt and the emergence of a novel endemic subclade Diversifying evolution of highly pathogenic H5N1 avian influenza virus in Egypt from 2006 to 2011 Evolution of highly pathogenic avian influenza H5N1 viruses in Egypt indicating progressive adaptation Antigenic diversity and cross-reactivity of avian influenza H5N1 viruses in Egypt between 2006 and 2011 Active surveillance for avian influenza virus Antigenic analysis of H5N1 highly pathogenic avian influenza viruses circulating in Egypt (2006–2012) Emergence of a novel cluster of influenza A(H5N1) virus clade 2.2.1.2 with putative human health impact in Egypt Recognition of N-glycolylneuraminic acid linked to galactose by the alpha2,3 linkage is associated with intestinal replication of influenza A virus in ducks Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets Airborne transmission of influenza A/H5N1 virus between ferrets In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity H5N1 hybrid viruses bearing 2009/H1N1 virus genes transmit in guinea pigs by respiratory droplet Receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the H3 hemagglutinin based on species of origin A Recommended Numbering Scheme for Influenza A HA Subtypes Antigenic analysis of highly pathogenic avian influenza virus H5N1 sublineages co-circulating in Egypt Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt Characterization of H5N1 influenza virus variants with hemagglutinin mutations isolated from patients Enhanced human receptor binding by H5 haemagglutinins Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses A single amino acid in the PB2 gene of influenza A virus is a determinant of host range Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model doi: 10.1128/JVI.79.18.12058-12064.2005 (2005) The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host 1151–1185 (Lippincott Williams & Wilkins 1186–1243 (Lippincott Williams & Wilkins Vaccines for pandemic influenza: summary of recent clinical trials Improving pandemic H5N1 influenza vaccines by combining different vaccine platforms Transmission of influenza A/H5N1 viruses in mammals doi: 10.1016/j.virusres.2013.07.017 (2013) Egyptian H5N1 influenza viruses-cause for concern The potential for respiratory droplet-transmissible A/H5N1 influenza virus to evolve in a mammalian host Enhanced expression of an alpha2,6-linked sialic acid on MDCK cells improves isolation of human influenza viruses and evaluation of their sensitivity to a neuraminidase inhibitor doi: 10.1128/JCM.43.8.4139-4146.2005 (2005) Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses A simple method of estimating fifty per cent endpoints Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(L-glutamic acid) backbone for inhibition of infection by influenza viruses RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models “Creating the CIPRES Science Gateway for inference of large phylogenetic trees” in Proceedings of the Gateway Computing Environments Workshop (GCE) phyloXML: XML for evolutionary biology and comparative genomics Download references We thank Susan Watson for scientific editing This work was supported through discretionary funds and by the Center for Research on Influenza Pathogenesis (CRIP) funded by NIAID Contract HHSN272201400008C This research was also supported by Leading Advanced Projects for medical innovation (LEAP) by the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) by the e-ASIA Joint Research Program from the Japan Agency for Medical Research and Development (AMED) and by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education The surveillance activities in Egypt were supported by the United States Agency for International Development (USAID) [grant number AID-263-IO-11-00001 Mod.#3] in the framework of the OSRO/EGY/101/USA project jointly implemented by the FAO General Organization for Veterinary Services (GoVS) and National Laboratory for Veterinary Quality Control of Poultry Production (NLQP) The following reagents were obtained from the NIH Biodefense and Emerging Infections Research Resources Repository NIH: Polyclonal anti-monovalent influenza subvirion vaccine rgA/Vietnam/1203/2004 (H5N1) NR-4109; and human reference antiserum to influenza A/Indonesia/05/2005 (H5N1) National Laboratory for Veterinary Quality Control on Poultry Production General Organization for Veterinary Services International Research Center for Infectious Diseases planned the experiments and/or analyzed the data has received speaker’s honoraria from Toyama Chemical and Astellas Inc. has received grant support from Chugai Pharmaceuticals Reprints and permissions Download citation Metrics details and the lack of pre-existing immunity among humans to viruses of this subtype Here we characterize two early human A(H7N9) isolates A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1 Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04) Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates Anhui/1 did not replicate well in miniature pigs after intranasal inoculation Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors a property that may be critical for virus transmissibility in ferrets Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential the PB2-627K or PB2-701N markers have been detected in almost all human suggesting ready adaptation of A(H7N9) viruses to humans these findings demonstrate that Anhui/1 is as pathogenic as CA04 and more pathogenic than Dk/GM466 in mice Viral-antigen-positive cells were detected in the tracheal glandular and alveolar epithelial cells of all ferrets Anhui/1- and CA04-infected ferrets displayed numerous viral-antigen-positive cells whereas Dk/GM466-infected ferrets presented far fewer antigen-positive cells Viral antigens were also detected in pneumocytes in localized lung lesions of each ferret viral antigen was detected in Anhui/1- and CA04-infected ferrets only Anhui/1 and Shanghai/1 thus established a robust infection in the upper respiratory organs of ferrets that was unlike most avian H5N1 influenza virus infections in ferrets which consistently cause severe symptoms including profound weight loss Shown are pathological findings in the trachea (a–d u–x) of macaques infected with Anhui/1 (a–l) or Dk/GM466 (m–x) at 3 d.p.i w) or immunohistochemistry for influenza viral antigen (b Haematoxylin and eosin staining of the lung of an uninfected macaque is shown Ferrets were infected with 5 × 105 p.f.u three naive ferrets (contact ferrets) were each placed in a cage adjacent to an infected ferret Nasal washes were collected from infected ferrets on day 1 after inoculation and from contact ferrets on day 1 after co-housing and then every other day (up to 9 days) for virus titration preliminary analysis of the specificity of the recombinant Anhui/1 H7 HA revealed preferential binding to α2,3 sialosides (R.P.d.V. the ‘human-type’ receptor specificity of A(H7N9) viruses assessed by glycan array seems to reflect the combined activities of HA and NA determined by plaque-reduction assays in MDCK cells were low for Anhui/1 and the control CA04 virus (1.4 μg ml−1 and 1.2 μg ml−1 suggesting that this compound could be a treatment option against A(H7N9) viruses resistant to NA inhibitors Mice were intranasally inoculated with 103 or 104 p.f.u e) or recombinant Anhui/1 virus possessing Shanghai/1-NA-294K (c mice were treated with oseltamivir phosphate PBS served as a control for intranasal administration; distilled water served as a control for oral administration Three mice per group were euthanized at 3 and 6 d.p.i. and the virus titres in lungs were determined by plaque assays in MDCK cells (d–f) Statistically significant differences between virus titres of control mice and those of mice treated with antiviral drugs were determined by using Welch’s t-test or Student’s t-test on the result of the F-test The resulting P values were corrected by using Holm’s method (*P < 0.05).**Virus was not recovered from all three animals infected with CA04 virus (for the 104 p.f.u the virus titres for the individual animals were 102.0 p.f.u the virus titres for the individual animals were 102.2 and 101.8 p.f.u on the basis of their sequences and phylogenetic relationships Anhui/1 and Shanghai/1 originated from an avian host but possess several characteristic features of human influenza viruses such as efficient binding to human-type receptors efficient replication in mammalian cells (probably conferred by PB2-627K) and respiratory droplet transmission in ferrets (Anhui/1) together with the low efficacy of NA inhibitors and the lack of human immunity (we tested 500 human sera collected from various age groups in Japan and found no antibodies that recognized Anhui/1) make A(H7N9) viruses a formidable threat to public health A(H7N9) viruses were propagated in embryonated chicken eggs to produce viral stocks Control viruses were propagated as described in Methods All experiments with A(H7N9) viruses were carried out in approved biosafety level (BSL3) containment laboratories All animals were used according to approved protocols for their care and use Detailed procedures are provided in Methods Six-week-old female BALB/c mice (Japan SLC; groups of six) were intranasally inoculated with 103 or 104 p.f.u CA04 or a recombinant virus possessing the Shanghai/1 NA gene encoding NA-294K and the remaining genes from Anhui/1 mice were administered antiviral compounds as described in detail in Methods Three mice per group were euthanized at 3 or 6 d.p.i and the virus titres in lungs were determined by plaque assays in MDCK cells and Dk/GM466 were propagated in embryonated chicken eggs For antigenic characterization by haemagglutination inhibition assays (see below for detailed procedures) Kawasaki/173 and Vietnam/1203 were propagated in MDCK cells All experiments with H7N9 viruses were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo and the National Institute of Infectious Diseases Japan; or in enhanced BSL3 containment laboratories at the University of Wisconsin-Madison which are approved for such use by the Centers for Disease Control and Prevention and by the US Department of Agriculture All cells were incubated at 37 °C with 5% CO2 Laninamivir and laninamivir octanoate were kindly provided by Daiichi Sankyo Co Favipiravir was kindly provided by Toyama Chemical Co and oseltamivir carboxylate was provided by F Zanamivir was kindly provided by GlaxoSmithKline Peramivir was kindly provided by Shionogi & Co were transfected into 293T cells with the help of a transfection reagent culture supernatants were collected and inoculated to embryonated chicken eggs for virus propagation MDCK cells were infected in triplicate in 12-well plates with Anhui/1 Dk/GM466 or CA04 at a multiplicity of infection (m.o.i.) of 0.01 the viral inoculum was replaced with MEM containing 0.3% bovine serum albumin (with 0.75 μg ml−1 trypsin treated with l-1-tosylamide-2- phenylethyl chloromethyl ketone) Culture supernatants collected at the indicated time points were subjected to virus titration by using plaque assays in MDCK cells Cultures of differentiated NHBE cells grown on semipermeable membrane supports were washed extensively with DMEM to remove accumulated mucus and infected in triplicate with virus at a m.o.i The inoculum was removed after 30 min of incubation at 33 °C or 37 °C and cells were further incubated at 33 °C or 37 °C 72 and 96 h post-infection from the apical surface Apical collection was performed by adding 500 μl of medium to the apical surface followed by incubation for 30 min at 33 °C or 37 °C and removal of the medium from the apical surface The titres of viruses released into the cell culture supernatant were determined by plaque assay in MDCK cells Six-week-old female BALB/c mice (Japan SLC) were used in this study Baseline body weights were measured before infection four mice per group were intranasally inoculated with 101 Body weight and survival were monitored daily for 14 days For virological and pathological examinations six mice per group were intranasally infected with 104 or 106 p.f.u (50 μl) of the viruses and three mice per group were euthanized at 3 and 6 d.p.i The virus titres in various organs were determined by plaque assays in MDCK cells All experiments with mice were performed in accordance with the University of Tokyo’s Regulations for Animal Care and Use and approved by the Animal Experiment Committee of the Institute of Medical Science Five- to eight-month-old female ferrets (Triple F Farms) which were serologically negative by haemagglutination inhibition assay for currently circulating human influenza viruses six ferrets per group were intranasally inoculated with 106 p.f.u Three ferrets per group were euthanized at 3 and 6 d.p.i for virological and pathological examinations All experiments with ferrets were performed in accordance with the University of Tokyo’s Regulations for Animal Care and Use and approved by the Animal Experiment Committee of the Institute of Medical Science Pairs of ferrets were individually housed in adjacent wireframe cages that prevented direct and indirect contact between animals but allowed spread of influenza virus by respiratory droplets three ferrets per group were intranasally inoculated with 0.5 ml of 106 p.f.u three naive ferrets (contact ferrets) were each placed in a cage adjacent to an infected ferret (in these cages infected and contact ferrets are separated by ∼5 cm) Body weight and temperature were monitored every other day Nasal washes were collected from infected ferrets on day 1 after inoculation and from contact ferrets on day 1 after co-housing and then every other day (for up to 9 days) for virological examinations The virus titres in nasal washes were determined by plaque assays in MDCK cells Approximately 2-year-old male cynomolgus macaques (Macaca fascicularis) from Cambodia (obtained from Japan Laboratory Animals) weighing 2.2–2.8 kg and serologically negative by AniGen AIV antibody ELISA which detects infection of all influenza A virus subtypes (Animal Genetics) and neutralization against A/Osaka/1365/2009 (H1N1pdm09) B/Tokyo/UT-E2/2008 (type B) and A/duck/Hong Kong/301/78 (H7N2) viruses six and four macaques were inoculated with Anhui/1 or Dk/GM466 (107 p.f.u through a combination of intratracheal (4.5 ml) ocular (0.1 ml per eye) and oral (1 ml) routes (resulting in a total infectious dose of 6.7 × 107 p.f.u.) Nasal and tracheal swabs were collected at 1 Three Anhui/1- and two Dk/GM466-infected macaques per group were euthanized at 3 and 6 d.p.i Virus titres were determined by plaque assays in MDCK cells All experiments with macaques were performed in accordance with the Regulation on Animal Experimentation Guidelines at Kyoto University (5 February 2007) and were approved by the Committee for Experimental Use of Nonhuman Primates in the Institute for Virus Research Two- to three-month-old female specific-pathogen-free miniature pigs (NIBS line; Nippon Institute for Biological Science) which were serologically negative by neutralization assay for currently circulating human and swine influenza viruses Baseline body temperatures were measured before infection Four and two pigs were intranasally inoculated with 107 p.f.u Nasal swabs were collected every day for virological examinations Two pigs per group were euthanized at 3 d.p.i for virological and pathological examinations; the remaining two Anhui/1-inoculated pigs were euthanized at 6 d.p.i All experiments with miniature pigs were performed in accordance with guidelines established by the Animal Experiment Committee of the Graduate School of Veterinary Medicine and were approved by the Institutional Animal Care and Use Committee of Hokkaido University Four-week-old female specific-pathogen-free chickens (Nisseiken Co.) were used in this study Six chickens per group were intranasally inoculated with 2 × 106 p.f.u Tracheal and cloacal swabs were collected every day for virological examinations Three chickens per group were euthanized at 3 and 6 d.p.i The virus titres in various organs and swabs were determined by plaque assays in MDCK cells All experiments with chickens were performed in accordance with the Animal Experimentation Guidelines of the National Institute of Infectious Disease (NIID) and were approved by the Animal Care and Use Committee of the NIID Four-month-old female quails (Yamanaka Koucho En) were used in this study Six quails per group were intranasally inoculated with 2 × 106 p.f.u Three quails per group were euthanized at 3 and 6 d.p.i All experiments with quails were performed in accordance with the University of Tokyo's Regulations for Animal Care and Use and were approved by the Animal Experiment Committee of the Institute of Medical Science Excised tissues of animal organs preserved in 10% phosphate-buffered formalin were processed for paraffin embedding and cut into 3-μm-thick sections One section from each tissue sample was stained using a standard haematoxylin and eosin procedure whereas another one was processed for immunohistological staining with a rabbit polyclonal antibody for type A influenza nucleoprotein antigen (prepared in our laboratory) that reacts comparably with all of the viruses tested in this study Specific antigen–antibody reactions were visualized with 3,3′-diaminobenzidine tetrahydrochloride staining by using the DAKO LSAB2 system (DAKO Cytomation) Macaque lung homogenates and serum samples were processed with the MILLIPLEX MAP Non-human Primate Cytokine/Chemokine Panel–Premixed 23-Plex (Merck Millipore) Array analysis was performed by using the Bio-Plex Protein Array system (Bio-Rad Laboratories) In vitro NA activity of viruses was determined by using the commercially available NA-Fluor Influenza Neuraminidase Assay Kit (Applied Biosystems) diluted viruses were mixed with the indicated amounts of oseltamivir carboxylate laninamivir or peramivir and incubated at 37 °C for 30 min Methylumbelliferyl-N-acetylneuraminic acid (MUNANA) was then added as a fluorescent substrate and the mixture was incubated at 37 °C for 1 h The reaction was stopped by adding 0.12 M Na2CO3 in 40% ethanol The fluorescence of the solution was measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm 8 × 105 MDCK cells were infected with approximately 50 p.f.u the viral inoculum was replaced with agarose medium containing various concentrations of favipiravir After the cells were incubated at 37 °C for 2 days plaques were visualized by crystal violet staining and counted six mice per group were intranasally inoculated with 103 or 104 p.f.u mice were treated with the following antiviral compounds: (1) oseltamivir phosphate: 4 or 40 mg per kg per 200 μl administered orally twice a day for 5 days; (2) zanamivir: 0.8 or 8 mg per kg per 50 μl administered intranasally once daily for 5 days; (3) laninamivir: 0.75 mg per kg per 50 μl administered intranasally once during the entire experimental course; (4) favipiravir: 30 or 150 mg per kg per 200 μl administered orally twice a day for 5 days; (5) or PBS intranasally (50 μl) and distilled water orally administered three mice per group were euthanized at 3 and 6 d.p.i The virus titres in lungs were determined by plaque assays in MDCK cells These data were used to select antibodies for glycan arrays collected in Japan in November 2012 from 200 donors ranging in age from 20 to 63 years were treated with receptor-destroying enzyme (Denka Seiken Co) Twofold serial dilutions of the treated sera were mixed with 100 p.f.u MDCK cells were inoculated with the virus-serum mixtures and cultured for 3 days The neutralizing activity of the sera was determined based on the cytopathic effects in inoculated cells All experiments with human sera were approved by the Research Ethics Review Committee of the Institute of Medical Science the University of Tokyo (approval number: 21-38-1117) A total of 300 serum samples were also obtained from the serum bank of the National Institute of Infectious Diseases in Japan Samples were collected from different regions of Japan during 2010–2011 and analysed for antibodies against Anhui/1 by use of the haemagglutination inhibition assay with 0.5% turkey red blood cells All statistical analyses were performed using JMP Pro 9.0.2 (SAS Institute) Statistically significant differences between the virus titres of Dk/GM466-infected mice and those of other mice were determined by using Welch’s t-test with Bonferroni’s correction Comparisons of virus titres in antiviral sensitivity assays in mice were also done using Welch’s t-test or Student’s t-test on the result of the F-test The resulting P values were corrected by using the Holm’s method All recombinant DNA protocols were approved by the University of Wisconsin-Madison’s Institutional Biosafety Committee after risk assessments were conducted by the Office of Biological Safety and by the University of Tokyo’s Subcommittee on Living Modified Organisms the University of Wisconsin-Madison Biosecurity Task Force regularly reviews the research program and ongoing activities of the laboratory The task force has a diverse skill set and provides support in the areas of biosafety All experiments with H7N9 viruses were performed in enhanced BSL3 containment laboratories Ferret transmission studies were conducted by two scientists with DVM and/or PhD degrees that each had more than 5 years of experience working with highly pathogenic influenza viruses and performing animal studies with such viruses chickens and quails wear disposable overalls and powered air-purifying respirators that filter the air Biosecurity monitoring of the facilities is ongoing BSL3 and Select Agent (for the US laboratory) training before participating in BSL3-level experiments The laboratory occupational health plans are in compliance with policies at the respective institutions (University of Wisconsin-Madison and the University of Tokyo Occupational Health Programs) Human infection with a novel avian-origin influenza A (H7N9) virus Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China Genomic signature and protein sequence analysis of a novel influenza A (H7N9) virus that causes an outbreak in humans in China Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity Rescue of an influenza A virus wild-type PB2 gene and a mutant derivative bearing a site-specific temperature-sensitive and attenuating mutation Severity of pneumonia due to new H1N1 influenza virus in ferrets is intermediate between that due to seasonal H1N1 virus and highly pathogenic avian influenza H5N1 virus Classical swine H1N1 influenza viruses confer cross protection from swine-origin 2009 pandemic H1N1 influenza virus infection in mice and ferrets Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets Reassortment between avian H5N1 and human H3N2 influenza viruses in ferrets: a public health risk assessment Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity Quantitative characterization of glycan-receptor binding of H9N2 influenza A virus hemagglutinin Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin Contemporary North American influenza H7 viruses possess human receptor specificity: implications for virus transmissibility Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2en-derived inhibitors Centers for Disease Control & Prevention Emergence of avian influenza A(H7N9) virus causing severe human illness - China Evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network Poor responses to oseltamivir treatment in a patient with influenza A (H7N9) virus infection Basal cells of differentiated bronchial epithelium are more susceptible to rhinovirus infection Generation of influenza A viruses entirely from cloned cDNAs Efficient selection for high-expression transfectants with a novel eukaryotic vector Replication and transmission of influenza viruses in Japanese quail Highly pathogenic H5N1 influenza virus infection in migratory birds Zhu, H. et al. Infectivity, transmission, and pathology of human H7N9 influenza in ferrets and pigs. Science http://dx.doi.org/10.1126/science.1239844 (2013) A recurring motif for antibody recognition of the receptor-binding site of influenza hemagglutinin Architecture of ribonucleoprotein complexes in influenza A virus particles Download references Shu for A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9) viruses We thank the IMSUT serum bank for providing human sera Webster for providing monoclonal antibody to A/seal/Massachusetts/1/80 (H7N7) was obtained through the National Institutes of Health (NIH) Biodefense and Emerging Infections Research Resources Repository National Institute of Allergy and Infectious Diseases (NIAID) Hoffmann-La Roche for providing oseltamivir carboxylate GlaxoSmithKline for providing zanamivir and Shionogi & Co This work was supported by the Japan Initiative for Global Research Network on Infectious Diseases from the Ministry of Education Japan; by grants-in-aid from the Ministry of Health Japan; by ERATO (Japan Science and Technology Agency); by NIAID Public Health Service research grants AI099274 and AI058113 to J.C.P. and by an NIAID-funded Center for Research on Influenza Pathogenesis (CRIP Noriko Nakajima and Masaki Imai: These authors contributed equally to this work has received speaker’s honoraria from Chugai Pharmaceuticals GlaxoSmithKline and Astellas; grant support from Chugai Pharmaceuticals Toyama Chemical and Otsuka Pharmaceutical Co.; is a consultant for Crucell; and is a founder of FluGen Supplementary Tables S1-S18 and a Supplementary Reference Reprints and permissions Download citation Environmental Science and Pollution Research (2021) there had been 134 laboratory-confirmed human cases of infection with avian influenza A H7N9 virus infection Yoshihiro Kawaoka and colleagues characterize the biology of two recent isolates of the virus They provide a wealth of data from infections in mice H7N9 virus is shown to be less sensitive to neuraminidase inhibitors than pandemic H1N1 virus but equally susceptible to an experimental polymerase inhibitor Terrence Tumpey and colleagues determine the capacity of two clinical H7N9 isolates to cause disease and transmit between mammals They show that the virus can replicate in human airway cells and in the respiratory tract of ferrets to a higher level than can seasonal H3N2 virus and show higher lethality in mice than genetically related H7N9 and H9N2 viruses the H7N9 virus showed limited transmission in ferrets by respiratory droplets Ron Fouchier and colleagues investigate the transmissibility of H7N9 virus between ferrets They show that airborne transmission can occur virus variants that have higher avian receptor binding higher pH of fusion and lower thermostability are selected and they suggest that these characteristics may result in reduced transmissibility a comprehensive magazine aimed at IT professionals announced its "IT Japan Award 2023" winners The awards have been granted annually by Nikkei Computer since 2007 They recognize companies and organizations demonstrating outstanding construction and application of IT systems and sharing of their successful knowhow with the broader public A third-party screening committee selects winners from among bodies covered in Nikkei Computer and the digital Nikkei xTECH which delivers information on the latest developments in technology and business Prizewinners are celebrated for their innovative use of IT systems during the past year The screening committee for the 17th IT Japan Awards met on Friday May 19 to judge nominees from the three perspectives of "contribution to management innovation and business reformation," "uniqueness in system construction and application," and "innovative implementation of technology and method." This year's Grand Prize went to Panasonic Holdings Corporation which was recognized for its groupwide initiative to promote digital transformation the company has implemented the "PX (Panasonic Transformation)" digital transformation project to promote reformation across the group to support digital transformation of the supply chain business at its group company Panasonic Connect and to enhance traditional manufacturing by introducing artificial intelligence and other powerful types of software A total of six companies were selected by the screening committee as below: one Grand Prize Panasonic Holdings CorporationDigital transformation at Panasonic TORIDOLL Holdings CorporationShifting every business system to SaaS to accelerate overseas expansion through its "don't own" strategy Ltd.AI forecasting of tomato yields up to five weeks ahead to secure stable supply KamakurayaImplementation of "salaried worker agriculture" utilizing IT; auto dealer president becoming one of the largest Japanese soba farmers in Nagano Prefecture Ltd.Achieving an electric power reduction effect with on-train IoT allowing Tobu Railway to create "train formations with one fewer car" for the Noda Line Ltd.Visualization of salespeople's online customer service to open a new approach to practices in the apparel industry Information Systems Society of JapanMasayoshi Sakai Executive Director of Japan Users Association of Information SystemsIn cooperation with: Nikkei xTECH Editorial Team For further details, please contact:Nikkei Business Publications, Inc. Unlock the insight" is the expression that describes our mission we meet diverse business community needs in the three key categories of "management," "technology" and "lifestyle." announced the launch of a new digital media focused on the gaming industry Communication techniques from the perspective of cognitive science" by Mutsumi Imai published by Nikkei Busi Participants of the 17th final round held in March 2024 Nikkei Inc will host the final of the annual Japanese Speech Contest for University Students A joint seminar was held by Endpoints News and Nikkei Biotechnology & Business to provide industry leaders with insights on how developing new me Metrics details We report on the measurement of deep inner-shell 2p X-ray photoelectron diffraction (XPD) patterns from laser-aligned I2 molecules using X-ray free-electron laser (XFEL) pulses aligned parallel to the polarization vector of the XFEL were well matched with our theoretical calculations we propose a criterion for applying our molecular-structure-determination methodology to the experimental XPD data we have demonstrated that this approach is a significant step toward the time-resolved imaging of molecular structures these measurements suffered from very low X-ray elastic-scattering cross sections making it difficult to acquire good signal-to-noise ratios these measurements suffered from cluster contamination in sample molecules making it difficult to obtain XPD data of the laser-aligned molecules the differences between the XPD data with and without alignment laser have been examined The difference data manifesting interference between photoelectron partial-waves have been well reproduced by theoretical calculations XPD data of the laser-aligned target molecules are desirable for their structure determination Illustration of X-ray photoelectron diffraction of a single aligned molecule a 2p photoelectron wave emitted from the left I atom (a) and a scattered wave by the right I atom (b) cause a fringe pattern due to interference between the two waves (c) The fringe pattern depends on an internuclear distance and photoelectron energy although this simulation was done under the condition of an equilibrium internuclear distance and photoelectron energy of 140 eV Schematic drawing of the experimental set-up Two laser beams propagating in a collinear arrangement intersect a supersonic pulsed molecular beam at the center of a vacuum chamber A Nd:YAG laser is used to align the sample molecules probed by the XFEL XPD images of photoelectrons are recorded by the upper VMI The degree of alignment is quantified using 2D momentum distributions of ionic fragments We neglect the interaction between the spin and the orbital motion of each electron hereafter the experimental details are described in the Methods section 2p photoelectron momentum images and fragment-ion momentum images of the I2 molecules under different experimental conditions are shown in Fig. 3, along with illustrations of relevant polarization geometries. Each image was obtained from alternating measurements of the images with and without the I2 molecular beams, which was operated at half of the XFEL repetition rate. 2D momentum images of electrons and ions Outer rings in (a–c) are due to I 2p photoelectrons Rings in (d–f) are due to fragment ions Im+ Centers of the rings shift toward the down stream direction (b,e) and (c,f) were measured simultaneously Insets in the middle row indicate polarization geometries Polarization vectors of the Nd:YAG laser and XFEL are indicated by double-headed arrows the photoelectron image bounded by white circles with the radii of 27.5 and 35 mm is distingushed from the central-ring image of low-energy electrons the fragment-ion image bounded by white circles with the radii of 7.5 and 10.5 mm indicates that molecular axis distributions are aligned along the polarization vector of the Nd:YAG laser because the degree of alignment in this experiment is not sufficient, which means that about 60% of all the molecules have their axis located within a cone of 40° Polar plots of I 2p photoelectron angular distributions (a–c) were constructed from the photoelectron images in Fig. 3(a–c) Filled circles with error bars represent the experimental data and solid curves are fitted results The errors did not have a normal distribution To see the small differences in the figures the characteristic features of the azimuthal-angle distribution are restricted by conservation of angular momentum component on the molecular axis so that the XPD pattern observed in this perpendicular polarization geometry provides less information about the molecular geometry than that in the parallel polarization (a) Blue curve: full multiple-scattering calculation; red curve: single-scattering calculation (b) The green bold and dotted curves are the 2pz XPD patterns from left-side and right-side I atoms under the single-scattering approximation The red curve is an incoherent superposition of the two XPD patterns (c) Same as (b) but for the 2px XPD pattern (d) Purple curve: primary photoemission amplitude from the 2pz in the left-side I atom and light-blue curve: interference term of (with positive values expressed by the bold curve and negative values by the dotted curve) In (e) the black curve for and the light-blue curve for are barely visible in which a double-headed arrow indicates the polarization vector of the XFEL Comparison of computational and experimental I 2p XPD patterns Bold solid curve: full multiple-scattering calculation taking the molecular axis distribution into account. Filled circles with error bars: experimental data. Thin solid curve: fitted result. The experimental data are the same as in Fig. 4(b) Each result was normalized by the area of the polar plots I 2p XPD patterns depending on internuclear distances (a) Patterns for and (b) for . Red curves: equilibrium internuclear distance of 2.666 Å; blue curves: internuclear distance of (2.666 + 0.5) Å; and green curves: internuclear distance of (2.666–0.5) Å. The red curve in (a) is the same as the bold solid curve in Fig. 6 we have successfully measured 2p XPD patterns from laser-aligned I2 molecules using XFEL pulses which were in strong agreement with the multiple-scattering XPD theory calculations we have proposed the criterion for applying our molecular-structure-determination methodology to experimental XPD data the present work is a step toward ultra-fast photoelectron diffraction which may enable the capture of ultra-fast molecular movies A spatial overlap between the XFEL and Nd:YAG laser was confirmed by monitoring their spot images on a Ce:YAG screen installed at the interaction region A temporal overlap between the XFEL and YAG laser pulses was introduced by adjusting the time delay of the Nd:YAG laser pulses to the XFEL pulses which were measured by a pin photo-diode for the former and a home-made photo-diode for the latter and placed equidistant from the interaction point which was then collimated by a skimmer with a diameter of 3 mm The valve was operated with a duration of 20.7 μs for a driving pulse The stagnation pressure of He was 35 bar and the sample pressure of I2 gas was ~0.006 bar which was evaporated from solid I2 by heating the valve containing it to 60 °C Operating the pulse valve at the half of the XFEL repetition rate results in alternating measurements of electron and ion images with and without the sample gas The static electric fields in the extraction regions and the drift regions of the VMI were adjusted to optimize the velocity focusing to I 2p photoelectrons with a kinetic energy of 140 eV The VMI was equipped with a micro-channel plate (MCP) backed by a phosphor screen with an active diameter of 75 mm Electron images were recorded with a scientific complementary metal-oxide-semiconductor (sCMOS) camera mounted to the upper VMI while ion images were recorded with a CCD camera mounted to the lower VMI We accumulated momentum-image data for 740,000 XFEL pulses without Nd:YAG laser pulses for 1,150,000 XFEL pulses with horizontally polarized Nd:YAG laser pulses and for 550,000 XFEL pulses with vertically polarized Nd:YAG pulses Spots on the diagonal line correspond to fragment-ion pairs with equal charges Off-diagonal spots correspond to fragment-ion pairs with unequal charges A small portion of I+ ion signal is created by the Nd:YAG laser Three-dimensional representation of the electron image shown in Fig Compared to the outer-ring intensity for 2p photoelectrons the intensity in the central region is extremely strong The tail of the central region extends to the outer ring the sidebands may be included in our photoelectron images these are not visible since they are smeared out by the photon-energy shot-by-shot fluctuation of The photoelectron angular distribution from a gas of isolated denotes the Legendre polynomial of the second order If, instead, the molecules have a definite orientation, then the angular distribution is described by an alternative form from Eq. (1). For example, when the molecular axis is parallel to the electric vector of the XFEL (see Fig. 3) the angular distribution in the xz plane can be expressed by the angular distribution in the xz plane is written as the azimuthal-angle distribution is restricted by the conservation of the angular momentum component on the molecular axis for linear molecules the polar-angle distribution is determined by intramolecular photoelectron diffraction which imply molecular symmetry restrictions The XPD patterns can be reproduced by both the photoionization and XPD theories although they are based on different approximations Irrespective of the degree of alignment, the polar-angle distribution of the XPD pattern for linear molecules has the same form as Eq. (2) although values of the AL coefficients depend on the degree of alignment the degree of alignment has been incorporated to reproduce our photoelectron diffraction data A useful formula for X-ray photoelectron diffraction (XPD) amplitude M(k) for measuring photoelectron momentum k is written as the amplitude can be written as multiple scattering series: describes the direct photoemission amplitude without scattering from surrounding atoms is then the single-scattering amplitude and the third term is the double-scattering amplitude and so on describes the interference effect between the direct and single-scattering waves Structure determination through correlated fluctuations in X-ray scattering Extending the methodology of X-ray crystallography to allow imaging of micrometer-sized non-crystalline specimens Solution of the crystallographic phase problem by iterated projections A unified evaluation of iterative projection algorithms for phase retrieval Application of a real-space three-dimensional image reconstruction method in the structural analysis of noncrystalline biological macromolecules enveloped by water in coherent X-ray diffraction microscopy X-ray diffraction from isolated and strongly aligned gas-phase molecules with a free-electron laser Photoelectron diffraction from single oriented molecules: towards ultrafast structure determination of molecules using X-ray free-electron lasers Velocity map imaging of ions and electrons using electrostatic lenses: application in photoelectron and photofragment ion imaging of molecular oxygen Controlling the alignment of neutral molecules by a strong laser field Laser-induced alignment and orientation of quantum-state-selected large molecules Diffraction and holography with photoelectrons and Auger electrons: some new directions Adsorbate structure determination on surfaces using photoelectron diffraction Diffraction and holography with photoelectrons and fluorescent X-rays Adsorbate structure determination using photoelectron diffraction: methods and applications Femtosecond photoelectron diffraction on laser-aligned molecules: towards time-resolved imaging of molecular structure Femtosecond X-ray photoelectron diffraction on gas-phase dibromobenzene molecules C 1s photoelectron angular distributions from fixed-in-space CO molecules in the high-energy continuum ≥50eV A compact X-ray free-electron laser emitting in the sub-ångström region alignment and orientation of large molecules using static electric and laser fields State- and conformer-selected beams of aligned and oriented molecules for ultrafast diffraction studies Laser-field-free orientation of state-selected asymmetric top molecules Even, U. & Lavie, N. Even Lavie Valve. Available at: https://sites.google.com/site/evenlavievalve/home (Date of access: 22/12/2014) Scientific Instrument Services, Inc. SIMION. Available at: http://simion.com (Date of access: 15/01/2015) Free-Free Transitions Following Six-Photon Ionization of Xenon Atoms Two-colour IR + XUV spectroscopies: the “soft-photon approximation” On the angular distribution in nuclear reactions and coincidence measurements Complete photoionization experiment in the region of the 2σg → σu shape resonance of the N2 molecule Theory of photoionization cross sections by dynamical theory in the X-ray region Depth distribution function calculated by quantum scattering theory Download references The authors thank the operation and engineering staff of SACLA for their support in performing the XFEL experiments This research was supported by JSPS KAKENHI Grant Number 25246041 Present address: Japan Synchrotron Radiation Research Institute The draft of the manuscript was written by K.N discussed with all the authors and then improved by them Download citation This plane made its maiden flight on April Fool’s Day 1939 and entered into operational service with the Imperial Japanese Navy (IJN) Air Service in July of the following year this was undoubtedly the deadliest fighter of the Pacific Theater with speed – maxing out at 331 miles per hour – and maneuverability unmatched by any of the early Allied fighter planes consisting of two 7.7 mm Type 97 machine guns in the engine cowling and two 20 mm Type 99-1 Mk 3 cannon in the wings Ohio — Mitsubishi A62M Zero at the National Museum of the United States Air Force This plane was ofttimes for a Zero because of the two warbirds’ highly similar appearance whilst the “Oscar” belonged to the Imperial Japanese Army Air Force (JAAF) Making its maiden flight in January 1939 and being officially introduced into JAAF service in October 1941 the Ki-43 was well-liked by its pilots for its excellent maneuverability and leisurely flying characteristics Nearly all of the JAAF’s aces nabbed at least some of their kills in the Hayabusa; then-Sgt Col.) Satoru Anabuki was the top dog amongst them with armament consisting of two 7.7mm machine guns in the first variant and two 20mm cannon in the final variant  ‘Take Him Off The Ballot’: Donald Trump Gets More Bad News and private military contractor (with assignments worked in Iraq Every Pillar of Honor on Iki IslandSentinel's Peak Pillar of HonorSentinel's Peak Pillar of Honor - Immortal Hope Today's print edition Home Delivery two things are certain: You're never far from the remains of dead people and you're never far from a knife Historically an important seaport with a current population of around 830,000 Sakai is conspicuous from the air owing to its massive keyhole-shaped burial mounds ringed by moats and a wall of trees and hemmed in by urban sprawl.googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); }); which were built over hundreds of years beginning in the third century helped put Sakai on the map — literally — attracting legions of laborers and skilled craftspeople In a time of both misinformation and too much information quality journalism is more crucial than ever.By subscribing Your subscription plan doesn't allow commenting. To learn more see our FAQ Sponsored contents planned and edited by JT Media Enterprise Division While we wouldn’t suggest you try this at home that’s exactly what this video tries to answer Uploaded by CarWow, the clip pits three older models – including a Ford Focus a Honda Civic and a Peugeot 206 – against one another in a literal death match the cars were drained of all their oil and coolant while a rock held down the accelerator keeping them revving until the very end Also Watch: Woman Allegedly Filled Her Engine With Water Instead Of Oil To Cool It Down this torture test didn’t take long before it claimed its first victim The Focus was the first to surrender as its engine waved the white flag after just 20 seconds The Peugeot wasn’t far behind as smoke was seen coming from the engine shortly after the Focus called it quits the 206’s engine managed to make it 47 seconds before the inevitable happened With the three-way test down to just one, the natural question was how long would the Civic survive The model kept going strong for over a minute the engine started smoking and then dropped to around 4,000 rpm the car was still drivable and it managed to make several laps around the camera crew The engine’s situation continued to worsen but it wasn’t ready to give up until 6 minutes and 22 seconds after the test began While you’d assume the video would end there, it doesn’t. As a joke, they put Mentos and Coke into the engine the engine restarted and ran for a little bit before finally surrendering Metrics details Technologies related to hydrophobic coatings have been applied to various industrial items for droplet formation and removal from solid surfaces excellent water-shedding properties and droplet control in a desired direction are not easily attainable merely by reducing their surface energies Success requires the design and control of a nanolevel surface structure with appropriate chemical composition Various hydrophobic surfaces have been developed to date and their static hydrophobicity has been investigated carefully studies of dynamic hydrophobicity of droplets have increased recently because of the rapid advance and availability of high-speed cameras facilitating direct evaluation of droplet motion This paper presents a review of recent studies investigating the design of hydrophobic surfaces for liquid droplet control specifically focussing on recent studies of dynamic hydrophobicity small water droplets can be observed to roll off hydrophobic umbrella surfaces at different speeds A droplet on a round hydrophobic surface does not always slide down on the surface easily at a small tilt angle large moving acceleration is not always observed What determines the motion of droplets on solid surfaces Differences in droplet shape on solid surfaces are often explained by variations in wettability The wetting of a solid surface has persisted through the ages as a research subject at the border between physics and chemistry Wettability control of a solid against a liquid has been widely explored in industry for application in daily life because it can engender various physical and chemical phenomena related to solids and liquids Wettability is an important property of solid surfaces from both fundamental and practical aspects The degree of adhesion of liquid droplets onto a solid surface is commonly described in terms of the contact angle (θ) and γlv represent the interfacial free energies per unit area of the solid–gas Although the definition and measurement of the contact angle are simple and easy the nature of wettability is complex because it is related to three phases: solid various factors affect the surface wettability of a solid When one factor of a solid surface such as roughness is altered other factors such as the surface topography or its chemical composition often change simultaneously it is usually difficult to discuss the results of surface wettability obtained from experiments while retaining academic rigor The main research interest for industrial materials is the behavior of millimeter-size droplets on a solid surface of more than a few centimeters in area the discussion of wettability among samples with great differences in surface character is sometimes conducted with ignorance of the influence of other characteristics Hydrophobic coatings are extremely important for wettability control The coatings are anticipated for various industrial uses such as anti-wetting anti-rust and reduced friction resistance by reducing solid–liquid interaction a hydrophobic surface is one on which water forms a round droplet that is easily removed Recent advances in probe microscopy and surface spectroscopy and the wider availability of such techniques have revealed that macroscopic surface hydrophobicity is affected by nanolevel characteristics such as structure chemical composition and their alignment and homogeneity The expected properties for a hydrophobic surface cannot always be obtained unless precise design and control of the solid surface are applied This report presents the results of recent studies on the design of hydrophobic surfaces for droplet removal or control from the viewpoint of materials science When roughness is imparted to a solid surface, its wettability is changed. Wenzel3 modified Young's equation and described the contact angle θ′ on a rough surface as follows: θ denotes the contact angle on a smooth surface which is defined as the ratio of the actual area of a rough surface to the geometrically projected area This r differs from the value of the arithmetic mean deviation of the profile (Ra) which is commonly employed in the engineering field surface roughness enhances the hydrophobicity of a hydrophobic surface and Cassie models for hydrophobicity of solid surfaces with and without roughness Transparent superhydrophobic polymer films with brightness enhancement for backlighting The surface shape is prepared using brightness-enhancement film (BEF) (a) Droplet shape on the transparent superhydrophobic film prepared from BEF with indium tin oxide (ITO) top coating (b) scanning electron micrograph of the same film and (c) scanning electron micrograph of a BEF (d) Brightness enhancement effect in a superhydrophobic film prepared from a BEF with ITO (tilt: 30°) there are differences in the degree of contribution of the alignment of roughness or chemical composition in the hydrophobic surfaces to sliding angle and dynamic sliding behaviors such as sliding acceleration (described later) precise control of the surface composition and its structure is necessary to attain static and dynamic hydrophobicity simultaneously With proper design of the surface structure and chemical composition control of directional transportation and sliding of a liquid droplet becomes possible to determine whether a transition from the Cassie to Wenzel case is possible Results showed that the more stable configuration is always that with the smallest contact angle Superhydrophobic surfaces do not maintain a state once the energy balance is broken by some means this state is attainable under a specific condition and sustained under small external field fluctuation Both the contact angle and sliding angle of the superhydrophobic state depend on the relationship between droplet size and roughness level in many cases the surface roughness does not enhance hydrophobicity because the droplet does not reflect the surface area increase realized by imparting surface roughness when a droplet is large compared to the surface roughness and the contribution of Wenzel's mode increases The sliding angle under this state depends on the droplet mass and the resistance to droplet sliding due to surface roughness the effects of these approaches differ greatly from those required for practical use Natural lotus leaves possess self-renewal capability; clean wax is supplied to the surface by their own metabolism These two functions maintain the superhydrophobicity of the plant surface for its entire lifetime studies to overcome these weak points for artificial superhydrophobic surfaces are not often reported To make superhydrophobic surfaces a key technology in the field of surface functional materials the development of technologies for the improvement of mechanical strength and long-term outdoor durability is indispensable a superhydrophobic surface is merely an object to satisfy the curiosity of materials chemists Breakthrough studies are necessary in this area Furmidge47 derived an equation that describes the relationship between the sliding angle and the receding and advancing contact angles as follows: (ii) increasing the load and time allowed for surfaces to remain in contact and (iii) increasing the rate of separation or retraction the respective and combined contributions of these factors to practical contact angle hysteresis remain unclear their contribution is expected to differ among materials Solid Surface Wettability and its Control (in Japanese) (© 2007 Uchida Rokakuho Publishing Although the sliding angle relates the motion or deformation of droplets it is not an index of dynamic hydrophobicity The sliding angle is not a function of time; its meaning is the limitation of interface energy balance at the three-phase contact line Both the contact angle and sliding angle are essentially thermodynamic properties The surface shape of industrial items is generally determined by their size For the design of surfaces with excellent water-shedding performance information about how fast the droplet can be removed from the surface at a certain tilt angle is becoming more important than information regarding the lowest tilt angle at which the droplet begins to slide dynamic hydrophobicity relates directly to energy saving and environmentally friendly technologies by decreasing the friction drag between liquids and solids a fundamental understanding of the factors (hydrophobic solid surface characteristics) contributing to the dynamic hydrophobicity remains elusive Analyses or modeling of the fluid mechanics of droplet sliding are often conducted with the assumption of constant sliding velocity liquid droplets commonly show sliding acceleration in the early stage of sliding on hydrophobic surfaces with excellent water-shedding properties Measurement of the sliding behavior is generally feasible only for a limited range of samples corresponding research between sliding behavior and solid surface characteristics has not been well conducted studies of dynamic wettability have been stimulated by the availability of high-speed photography the advancement of motion-image sequence analysis These technologies facilitate the advancement of studies not only of sliding behavior but also of other droplet behaviors such as horizontal motion the advance of nanoscale materials characterization has enabled studies of surface characteristics that govern the dynamic hydrophobicity of solids from the perspective of nanomaterials science It is noteworthy that the dynamic hydrophobicity described here is not determined by water-spreading kinetics but by the water-shedding properties of the surface The evaluation of dynamic hydrophobicity on a hydrophilic surface requires special care revealing that their model explains experimentally obtained results for decane drops moving on polydimethylsiloxane-coated glass and demonstrated that the threshold voltage needed to move the droplet (2 μL) can be reduced to as low as 3 V They reported that gravity enhances the first resonant mode and weakens the second mode even though the positions of the resonance peaks do not differ substantially from those observed to have horizontal vibrations When a drop is vibrated on a horizontal surface with asymmetric vibration different modes are amplified asymmetrically during the forward and reverse strokes of a vibration cycle They demonstrated droplet motion on the surface and reported that the water drop movement was regulated by the degree of gradation a Laplacian pressure difference is generated between advancing and receding sides of the liquid; the liquid droplets move spontaneously (a) Field-emission scanning electron micrograph of the fluoroalkylsilane (FAS17) surface periodically line-patterned with octadecyltrimethoxysilane (ODS) at a line/space dimension of 500/500 μm (b) Evaluation of water droplet sliding behavior on these surfaces (c) Sliding behavior of droplets with various masses on a 500 μm line-surface rotated at Φ = 13° on a slope tilted at 35° The displacement angle β depends on the droplet mass Studies of hydrophobic surface have to date mainly examined the relationship between the structure or chemical composition of a solid surface and the contact angle or sliding angle of water droplets These are fundamentally examples of static hydrophobicity such studies provide no information related to dynamic hydrophobicity Detailed investigations of the effects of solid composition and their homogeneity on dynamic hydrophobicity are necessary for the design of surfaces with excellent water-shedding properties the behaviors of liquids were treated mainly on the basis of fluid mechanics and the physical chemistry of the solid–liquid interface and were investigated mainly from the point of view of surface science or colloid chemistry The processing and properties of solid materials are handled mainly in the field of materials science To achieve a better understanding of the relationship between solid surface characteristics and dynamic hydrophobicity interdisciplinary studies in these three academic fields are necessary In addition to discussions from the surface chemistry or materials science perspectives descriptions and discussions from the viewpoint of fluid mechanics and internal fluidity are indispensable Collaborative studies for dynamic hydrophobicity might establish a new academic field situated on the border separating these three sciences Liquids and gels possess greater deformation flexibility than solids The control of liquids is a key technology for new functional surfaces and devices and such technology will also contribute to the reduction of greenhouse gas emissions through a reduction in friction drag leading to higher energy conservation Basic research related to the control of liquid droplets on solid materials remains insufficient As this is a vital technology for our future further investigations are anticipated in this field Download references Department of Metallurgy and Ceramic Science Graduate School of Science and Engineering Reprints and permissions Download citation DOI: https://doi.org/10.1038/asiamat.2011.55 Metrics details A Corrigendum to this article was published on 01 March 2012 Twenty autopsy cases with 2009 pandemic influenza A (2009 H1N1) virus infection performed between August 2009 and February 2010 immunohistochemistry for type A influenza nucleoprotein antigen and real-time reverse transcription-PCR assay for viral RNA were performed on formalin-fixed and paraffin-embedded specimens the D222G amino acid substitution in influenza virus hemagglutinin was analyzed in formalin-fixed and paraffin-embedded trachea and lung sections by direct sequencing of PCR-amplified products There were several histopathological patterns in the lung according to the most remarkable findings in each case: acute diffuse alveolar damage (DAD) with a hyaline membrane (four cases) acute massive intra-alveolar edema with variable degrees of hemorrhage (three cases) neutrophilic bronchopneumonia (five cases) and tracheobronchitis with limited histopathological changes in alveoli (four cases) the main findings were due to preexisting disease Influenza virus antigen was only detected in the respiratory tract in 10 cases by immunohistochemistry The antigen was detected in type II pneumocytes (three cases) in the epithelial cells of the trachea and in the epithelial cells in both of the above (one case) The four cases with acute DAD presented with antigen-positive type II pneumocytes the D222G substitution was detected in the lung as a major sequence although 222D was prominent in the trachea suggesting that selection of the viral clones occurred in the respiratory tract the pathogenesis of 2009 H1N1 was confirmed to be viral infection in pneumocytes which caused severe alveolar damage and fatal viral pneumonia Further studies on both host and viral factors in autopsy or biopsy materials will be essential to elucidate the other pathogenic factors involved in influenza virus infection Influenza virus causes respiratory infections with the potential for high morbidity and mortality To control the seasonal epidemic of influenza virus infection Influenza virus also causes periodic pandemics The emergence of avian H5N1 influenza virus infection in humans in 1997 alerted us the future outbreaks of new pandemic influenza virus infection A total of 20 fatal cases with 2009 H1N1 infection were examined Infection was confirmed by detecting viral RNA in nasopharyngeal swab specimens using the reverse-transcriptase PCR (RT-PCR) method at the first medical examination at each hospital the swabs were collected post mortem at the medical examiner's office The autopsy tissue samples of the 20 fatal cases were sent from 15 hospitals to our laboratory for further pathological investigation between August 2009 and February 2010 Some tissue samples were formalin-fixed and others were formalin-fixed and paraffin-embedded The tissue sections included the trachea (n=15) This study was approved by the institutional medical ethical committee of the National Institute of Infectious Diseases except for rabbit polyclonal antibody for SP-D Anti-SP-D antibody was purchased from Chemicon (Temecula Alexa Fluor 568-conjugated anti-mouse or anti-rabbit IgG (Molecular Probes and Alexa Fluor 488-conjugated anti-rabbit or anti-mouse IgG (Molecular Probes) were used as secondary antibodies The clinical characteristics of the 20 patients are presented in Table 1 The median age of the patients was 39.8 years (range Thirteen (65%) patients were aged between 15 and 59 years Thirteen patients (65%) died within 3 days after the onset of influenza symptoms The mean duration of illness was 5.7 days (range The most frequent medical history or underlying disease was cardiovascular disease Five patients were obese with a body mass index exceeding 30 kg/m2 Antiviral treatment was administered in 13 cases (oseltamivir in 11 cases and zanamivir in 2 cases) within 48 h after disease onset Case 1 received oseltamivir from day 7 after disease onset 19 and 20) from the medical examiner's office and cases 2 and 9 were found in the state of cardiac pulmonary arrest Case 8 was dead on arrival without hospitalization Eleven cases were hospitalized and seven of them were intubated because of hypoxia Three cases (cases 15–17) were suspected of having fulminant myocarditis associated with influenza virus infection because they suddenly developed ventricular fibrillation and cardiac arrest 2 or 3 days after disease onset lung specimens were taken aseptically during autopsy and Streptococcus pneumoniae was cultured in one case and Pseudomonas aeruginosa in two cases pneumoniae was cultured from the sputa on admission The bacterial cultures were negative for the other seven cases Hematoxylin–eosin (H&E) staining (a and k) and immunohistochemistry for type A influenza nucleoprotein antigen (InfA-NP) (b Histopathological findings in the alveoli (a–d InfA-NP antigens were detected in the alveoli presenting with an exudative stage of diffuse alveolar damage (DAD) with hyaline membrane formation and hyperplasia of type II pneumocytes (a and b InfA-NP antigens were detected in both submucosal glands (e and f) and desquamated alveolar epithelial cells in case 4 (g and h) InfA-NP antigens were detected in the nuclei of epithelial cells of trachea with mucosal mononuclear cell infiltration (i and j) mononuclear cell infiltration around the bronchial submucosal glands (k) and viral antigens in submucosal glands (l) are shown We excluded the pathological findings in case 17 from the pathological assessment of influenza infection because they were extensively modified by complications caused by the long duration of percutaneous pulmonary support Four cases had more severe lesions in the upper airway with limited intra-alveolar edema and hemorrhage (cases 9 and 12) or no pathological changes in the lower airway (cases 15 and 16) The main histopathological findings in cases 10 and 14 included preexisting emphysema with congestion and edema The pathological findings of heart tissue specimens in three cases which were suspected of having myocarditis as described above did not show inflammatory cell infiltration or myocyte necrosis Cases 5 and 12 died of acute influenza virus-associated encephalopathy case 5 showed histopathological changes in the brain which included leakage of plasma proteins from vessels and clasmatodendrosis of astrocytes (data not shown) whereas case 12 had gross brain edema and high plasma interleukin (IL)-6 (16197 pg/ml) and IL-10 (1156 pg/ml) concentrations without remarkable histopathological changes in the brain because neither viral antigens nor viral RNA were detected in the formalin-fixed and paraffin-embedded tissue sections Type A influenza nucleoprotein antigen (InfA-NP; red) and cell-type-specific marker protein (green) are colocalized in the same cells TO-PRO-3 nucleic acid staining (blue) and differential interference contrast (DIC) images are also shown (a) InfA-NP (red) and epithelial membrane antigen (EMA) are shown in epithelial cells (green) (b) InfA-NP (red) and surfactant apoprotein D (SP-D) are shown in type II pneumocytes (green) (c) InfA-NP (red) and CD68 (PG-M1) are expressed in alveolar macrophages (green) The Inf-NP (red)-positive cell on the right is a detached type II pneumocyte (d) InfA-NP (red) and cytokeratin AE1/AE3 are shown in epithelial cells in the upper airways (green) Amino acid residue at position 222 (225 in H3) in hemagglutinin of the 2009 pandemic influenza A (2009 H1N1) virus in formalin-fixed and paraffin-embedded trachea and lung tissue sections was determined by direct sequencing The nucleotide sequence around the amino acid residue at position 222 in the hemagglutinin gene of the 2009 H1N1 virus in case 1 is shown glycine) was the minor sequence in the trachea glycine) was the major sequence and AAT (N asparagine) was the minor sequence in the lung although the pathogenesis of these diseases and their relationship to influenza virus infection have not yet been elucidated there were several reasons why the antigen was not detected clearance of viral antigens after a long duration of illness and the condition of formalin-fixed and paraffin-embedded samples Emergence of a novel swine-origin influenza A (H1N1) virus in humans Cerebrospinal fluid and serum levels of cytokines and soluble tumor necrosis factor receptor in influenza virus associated encephalopathy Influenza-associated acute encephalopathy in Japanese children in 1994–2002 Acute encephalopathy and pandemic (H1N1) 2009 Fulminant myocarditis associated with pandemic H1N1 influenza A virus in children A national survey on myocarditis associated with the 2009 influenza A (H1N1) pandemic in Japan Pneumonia and respiratory failure from avian-origin influenza A (H1N1) in Mexico Fatalities associated with the 2009 H1N1 influenza A virus in New York city Centers for Disease Control and Prevention. CDC estimates of 2009 H1N1 influenza cases, hospitalizations and deaths in the United States, April 2009-March 13, 2010. Available from http://www.cdc.gov/h1n1flu/estimates/April_March_13.htm Deaths and hospitalizations related to 2009 pandemic influenza A (H1N1)-Greece (MMWR) Morb Mortal Wkly Rep 2010;59:682–686 Pathological changes associated with 2009 H1N1 virus Lung pathology in fatal novel human influenza A (H1N1) infection Pulmonary pathologic findings of fatal 2009 pandemic influenza A /H1N1 viral infections 2009 pandemic influenza A (H1N1): pathology and pathogenesis of 100 fatal cases in the United States Postmortem findings in eight cases of influenza A/H1N1 The first case of pandemic influenza (A/H1N1) virus infection in Japan: detection of a high copy number of the virus in type II alveolar epithelial cells by pathological and virological examination Sudden death of a patient with pandemic influenza (A/H1N1pdm) virus infection by acute respiratory distress syndrome Quasispecies of the D225G substitution in the hemagglutinin of pandemic influenza A(H1N1) 2009 virus from patients with severe disease in Hong Kong Preliminary review of D222G amino acid substitution in the haemagglutinin of pandemic influenza A (H1N1) 2009 viruses Virulence-associated substitution D222G in hemagglutinin of 2009 pandemic influenza A (H1N1) virus affects receptor binding Comparison of the ability of viral protein- expressing plasmid DNAs to protect against influenza H5N1-infected cells in lung with diffuse alveolar damage in exudative phase from a fatal case in Vietnam Vascular endothelial growth factor messenger RNA expression level is preserved in liver metastases compared with corresponding primary colorectal cancer Histopathologic and immunohistochemical features of fatal influenza virus infection in children during the 2003-2004 season Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs The pathology of influenza virus infections and pathogenesis of avian influenza A (H5N1) infection in humans Characterization of quasispecies of pandemic 2009 influenza A virus (A/H1N1/2009) by de novo sequencing using a next-generation DNA sequencer Avian flu: influenza receptors in the human airway Receptor-binding specificity of pandemic influenza A (H1N1)2009 virus determined by carbohydrate microarray Altered receptor specificity and cell tropism of D222G haemoagglutinin mutants from fatal cases of pandemic A (H1N1) 2009 influenza Download references and K Nakanishi for clinical and pathological information on the patients This work was supported by a Health and Labour Sciences Research Grant on Emerging and Re-emerging Infectious Diseases (H22 Shinko-Ippan-014) Department of Clinical Laboratory Sciences Hokkaido University Graduate School of Medicine Department of Cardiovascular Internal Medicine National Center for Global Health and Medicine Okinawa Prefectural Nanbu Medical Center and Children's Medical Center Department of Pathology and Laboratory Medicine Department of Emergency and Intensive Care Download citation DOI: https://doi.org/10.1038/modpathol.2011.125 Nakajima and Minamino lifts SAMURAI BLUE past Myanmar 2-0 at the FIFA World Cup Qatar 2022 Asian Qualification Round 2 the SAMURAI BLUE (Japan National Team) played their first match of the FIFA World Cup Qatar 2022 Asian Qualification Round 2 against the Myanmar National Team at Thuwunna Stadium in Yangon With the help of goals scored by NAKAJIMA Shoya (FC Porto) and MINAMINO Takumi (FC Red Bull Salzburg) Japan got off to a great start with a 2-0 victory As coach MORIYASU Hajime mentioned prior to the match that he will “start players who are in good form,” the Japanese coach elected to start the same line-up from their match against the Paraguay National Team at the KIRIN CHALLENGE CUP 2019 played on Thursday 5 With strong hopes to make their seventh consecutive appearance at the World Cup Japan entered the Asian qualifiers under the rainy skies of Yangon NAGATOMO Yuto (Galatasaray S.K.) and SAKAI Hiroki (Olympique de Marseille) at a high position to take the initiative of the match when DOAN Ritsu (PSV Eindhoven) intercepted the ball at the left side allowing TOMIYASU Takehiro (Bologna FC 1909) to send a quick pass to Nakajima who made a cut towards the box before curling a right footed shot over the head of the opposing goalkeeper to give Japan the early lead Doan created another opportunity when he delivered a cross towards the goal but Minamino’s header couldn’t capture the target YOSHIDA Maya (Southampton FC) connected with a corner kick to allow Tomiyasu to take a shot at goal but his attempt was saved by the goalkeeper Japan’s second goal was scored in the 26th minute when OSAKO Yuya (Werder Bremen) posted up to allow Doan to take a shot inside the box The shot was once blocked by the goalkeeper but Doan collected the loose ball and lifted a pinpoint cross to Minamino who struck a header into the goal to make it 2-0 Japan continued to threaten the opposing goal as Yoshida struck a close shot in the 29th minute followed by Nakajima’s shot in the 43rd minute Tokyo) long range effort in the 45th minute Japan continued to show their dominance in the second half but with the home crowd of 25,500 spectators supporting their back AUNG THU responded to a freekick delivered from the right side and struck a powerful shot GONDA Shuichi (Portimonense S.C.) made the save to deny their opportunity Japan continued to create chances in the second half Hashimoto struck another fine shot from mid-range followed by Osako’s header off a corner kick but both attempts were saved by the opposing goalkeeper Another close opportunity was created in the 64th minute when Osako’s header found SHIBASAKI Gaku (Deportivo de La Coruna) unmarked in front of the goal but Shibaski’s volley struck the cross bar In efforts to bring in fresh legs to their offence Japan brought in ITO Junya (KRC Genk) in the 66th minute followed by SUZUKI Musashi (Hokkaido Consadole Sapporo) in the 77th minute KUBO Takefusa (RCD Mallorca) in the 81st minute The three substitutes added more layers to the Japanese offence as they helped create more opportunities for Japan but their efforts fell short from adding further goals to their tally as the match ended with a final score of 2-0 While Japan earned three points from their first match of this round Tajikistan earned their second win with a 1-0 victory over Mongolia to take the lead in Group F while Kyrgyz Republic and Myanmar are yet to earn a point At the second round of the Asian qualifiers the group leaders will automatically advance to the final round while the top four teams among the runners-up will also advance Japan will now face Mongolia at Saitama Stadium on Thursday 10 October followed by two away matches against Tajikistan and Kyrgyz Republic on Tuesday 15 October and Thursday 14 November respectively The second half of the round will take place next year starting with a home fixture against Myanmar on Thursday 26 March followed by an away match against Mongolia on Tuesday 31 March and two home fixtures against Kyrgyz Republic and Tajikistan on Thursday 4 and Tuesday 9 June respectively Coach of SAMURAI BLUE (Japan National Team)In any given tournament the first match is always going to be a tough match our time on the pitch had been limited due to torrential rain but the players gave their best efforts within the given situation to earn this result I would like to credit our players for this clean sheet victory It was too bad that we couldn’t score further goals but the players showed great aggression throughout the match We did show signs of fatigue during the second half as we almost allowed Myanmar to gain momentum but the players managed to keep their composure to hold onto the lead I hope we can continue to showcase a positive performance I would also like to mention that our players did a great job to make the necessary adjustments with the pitch condition and played with plenty of flexibility We were all on the same page and fought hard to earn the best results Myanmar had many great players and they are a team that can fight as a team Their performance made us realise that the competition at the Asian qualifiers will not be easy We will continue to prepare ourselves for each of the upcoming matches and give our best efforts to reach a higher level DF #5 NAGATOMO Yuto (Galatasaray S.K.)Compared to the past matches at the Asian qualifiers I feel like this was one of the better matches we’ve played despite winning the match with a clean sheet DF #16 TOMIYASU Takehiro (Bologna FC 1909)With our opponent seeking to create their chances through long balls and counterattacks we made sure to challenge the ball while keeping coverage at the back The second round of this qualifiers has a unique atmosphere especially compared to matches played in Japan and Europe DF #22 YOSHIDA Maya (Southampton FC)Our goal was to earn three points in our first match so I am glad we were able to achieve that goal Our performance in the second half saw plenty of room for improvements We must continue to strive for higher standards throughout these qualifiers MF #9 MINAMINO Takumi (FC Red Bull Salzburg)The opening goal really allowed us to settle down and take control over the match Doan delivered a great cross to allow me to score the second goal The pitch condition was not as bad as we thought Earning a win was the most important thing today so I am glad we were able to achieve this result MF #10 NAKAJIMA Shoya (FC Porto)In today’s game I was committed to take shots when I was given the opportunity I still had other opportunities I could have scored so I will strive to become a better player who can convert those chances We still have plenty of room for improvements as a team so we hope to heighten the quality of our attacking combinations I am always focused on giving my best performance under any given circumstance so the rain and pitch condition didn’t really bother me MF #21 DOAN Ritsu (PSV Eindhoven)We knew our opponent was seeking for their opportunity to counter us so I was focused on forcing turnovers in the midfield I was influenced by the heavy pitch condition and started to get tired in the second half MF #17 KUBO Takefusa (RCD Mallorca)I am glad that I was given the opportunity to play in the match I hope to get myself more involved in our future matches While I was watching the game from the bench I was able to see the supporters’ enthusiasm to cheer for their country and it gave me a taste of what this competition is all about Coach of Myanmar National TeamI cannot say that I am happy with this result but we were able to find many positive aspects from this match It has been only four months since I was appointed as the coach and it is a very difficult task to face a top tier team like Japan in such short time our team was able to learn many things from this 0-2 loss We hope to seek our chances through the matches against other teams besides Japan Squad & Schedule of SAMURAI BLUE (Japan National Team) Myanmar National Team vs SAMURAI BLUE (Japan National Team)Date: Tue 10 September 2019 18:50Venue: Thuwunna Stadium (Yangon Tournament Information SAMURAI BLUE wins over Bolivia with Nakajima’s goal in the second half - KIRIN CHALLENGE CUP 2019 the SAMURAI BLUE (Japan National Team) faced the Bolivia National Team at the KIRIN CHALLENGE CUP 2019 and won the match 1-0 with the help of the goal scored by midfielder NAKAJIMA Shoya (Al Duhail SC) in the second half to record their first win in three matches As mentioned at the pre-game press conference Coach MORIYASU Hajime rotated the entire line-up from their match against the Colombia National Team on Friday 22 The fresh line-up featured eight players making their first ever start under the current regime SCHMIDT Daniel (Vegalta Sendai) made his return in goal since the AFC Asian Cup UAE 2019 while HATANAKA Shinnosuke (Yokohama F･Marinos) and MIURA Genta (Gamba Osaka) were positioned as the centre halves NISHI Daigo (Vissel Kobe) made his return to the national team since June 2011 while ANZAI Koki (Kashima Antlers) covered the left side Tokyo) and KOBAYASHI Yuki (SC Heerenveen) playing as the defensive midfielders with KAGAWA Shinji (Besiktas J.K.) filling in the number 10 role USAMI Takashi (Fortuna Dusseldorf) and INUI Takashi (Deportivo Alaves) were placed in the flanks while KAMADA Daichi (Sint-Truidense V.V.) played as the lone striker upfront Kagawa was given the armband for the first time in his national team career With Hashimoto and Hatanaka making their national team debut and three other players playing in only their second match to represent their country While Anzai and Inui actively attacked the left side Usami penetrated the right flank to create opportunities but the players couldn’t quite connect with each other to break down the Bolivian defence Inui made an effort in the 23rd minute when he dribbled in from the left side before striking a shot but his attempt was denied by the opposing goalkeeper this was their third match under the new coach and they are in the phase to prepare for the upcoming Copa America scheduled in June While the South American side dropped deep in their territory to defend their goal their young striker Leonardo VACA orchestrated their attacks from the right side A close opportunity came in the 42nd minute when Rodrigo RAMALLO connected with a cross but his header couldn’t capture the target and SHIBASAKI Gaku (Getafe C.F.) in the 60th minute the young attacking trio that has featured the team since the start of the Moriyasu regime showcased their abilities while Shibasaki played a vital role in the team’s build-up plays to create opportunities for Japan The golden moment came in the 76th minute when Doan forced a turnover in the midfield and sent a pass to Minamino The midfielder then delivered the ball to Nakajima who dribbled into the penalty area before striking a right footed shot that shook the net Nakajima came within inches to score his second goal just moments later when his mid-range shot struck the cross bar Minamino responded to the defection and fired a shot to threaten the Bolivian goal but failed to capitalise on the opportunity Other substituted players also made their attempts as SUZUKI Musashi (Hokkaido Consadole Sapporo) connected with a cross delivered by SASAKI Sho (Sanfrecce Hiroshima) in the 85th minute but his header came short from hitting the target Doan received a pass from Nakajima and struck a shot from close range but his shot was saved by the opposing goalkeeper Japan finished the match 1-0 to register a win in their final match of the Heisei era Coach of SAMURAI BLUE (Japan National Team)We rotated the entire line-up from our match against Colombia and through the two matches I am now feeling confident about the growth of our young players they still need to get better in order to have enough impact to change the dynamics of the match While we had the younger players with less experience play with the veterans to share their time and feeling of the match we made the substitutions in the second half to win the match Although the players who started the match came short from scoring a goal they utilised the width of the field to keep the ball moving and our opponents were visually worn down from chasing the ball Our players executed the game plan well against a team with solid defence and the players who came in as substitutes did their job to score the goal It’s great that we managed to finish off the two matches by conceding just one goal We were able to learn from our match against Colombia and showcased a more consistent performance by preventing the opponents from creating major opportunities Our philosophy is certainly getting rooted into our players’ minds and after seeing how smoothly the new players adapted to the team it tells me that the message is getting spread among the players and through the matches we have previously played and I will keep on demanding high standards from my players I am hoping to see a player stepping up in offence to get the job done in critical moments MF #7 SHIBASAKI Gaku (Getafe C.F.)We managed to keep the ball moving in the first half and once the opponents started to get tired in the second half This win was earned by a total team effort We played with great pace in the second half but I personally feel like we need to balance ourselves and perform at a high level throughout the entire match We need to think of more ways to carry the ball upfront If we can come up with one or two patterns we can create more opportunities to score goals MF #8 NAKAJIMA Shoya (Al Duhail SC)The entire team contributed to carry the ball upfront so I was glad I was able to score the goal especially because I missed a lot of shots in our previous match It’s true that we see more plays that leads to a goal (when playing with Minamino and Doan) but every player has their own strong points MF #10 KAGAWA Shinji (Besiktas J.K.)It’s great that the team won the match and to see the substitutes make the difference in the match I knew it was going to be a difficult match but we still saw many more chances created in the second half so we can’t be happy with just one goal I told them to stay patient and be aggressive when we are attacking Our opponents played more defensively than we expected and it was hard to crate anything at first but our persistency got paid off in the second half MF #25 KOBAYASHI Yuki (SC Heerenveen)With the opponents dropping deep in their territory we had to take the risk and challenge them so I focused on preventing the opponents from initiating counter attacks and the build-up plays to get the ball upfront in a timely fashion we had to learn about each other’s character and play style Coach of Bolivia National TeamThe match was played with great intensity and it was a great tactical battle between the two teams Our players struggled to deal with the opponents’ pressure and although we created some chances from our substitutions From our experience playing against Korea Republic we focused on marking the player on the other side This match has given our young players a great experience SAMURAI BLUE (Japan National Team) Squad & Schedule SAMURAI BLUE (Japan National Team) vs Bolivia National TeamMatch Date: Tue 26 March 2019 Kick-off time 19:30Match Venue: Noevir Stadium Kobe Tournament Information the time for making a list and checking it twice is upon us The Japan Times asked translators of Japanese literature for their biggest book wish: Which Japanese work or author do you most want to see translated into English Read on for our holiday wish list for book lovers Polly Barton: One of the first Japanese books I really fell in love with is Nao-Cola Yamazaki's debut novella “Hito no Sekkusu o Warau Na” (which translates to “Don't laugh at other people's sex lives”) It tells the story of a relationship between a young student and his art teacher twice his age that manages to feel both startlingly new and achingly true and personal.googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); }); Sam Bett: My pick is Atsushi Sato's “Arechi no Kazoku” (“Family of the wasteland”) this Akutagawa Prize-winning novel is a reckoning with disaster: what it takes away what it leaves behind and how we struggle to make sense of it funnels his pain into pruning trees in his hometown north of Fukushima where a new seawall serves as a "monument to horror." The descriptions of tree care have an ASMR quality but the only thing Yuji can stop from changing is himself It’s a view of Japan the world needs to see SAMURAI BLUE earns win over Paraguay with the help of Osako and Minamino’s goals at the KIRIN CHALLENGE CUP 2019 the SAMURAI BLUE (Japan National Team) faced the Paraguay National Team in the KIRIN CHALLENGE CUP 2019 taking place at Kashima Soccer Stadium and won the match 2-0 with the help of goals scored by OSAKO Yuya (Werder Bremen) and MINAMINO Takumi (FC Red Bull Salzburg) while positioning DOAN Ritsu (PSV Eindhoven) and NAKAJIMA Shoya (FC Porto) in the flanks It was the first time these four players shared the field since they faced Venezuela last November midfielder SHIBASAKI Gaku (Deportivo de La Coruna) carried the ball up the right flank before sending a cross towards the middle while Nakajima dribbled passed the opposing defenders to create opportunities as Japan tested the waters to get themselves on the same page when the ball was connected through HASHIMOTO Kento (F.C before the ball was delivered to NAGATOMO Yuto (Galatasaray S.K.) The veteran fullback sent a cross from the left side that allowed Osako to convert with his left foot so I just focused on connecting with the ball.” Doan came close to scoring a goal when he broke through from a counterattack who found SAKAI Hiroki (Olympique de Marseille) running up the right flank Sakai delivered a pin-point cross towards the centre where Minamino awaited to score the team’s second goal of the match After advancing to the quarterfinals at the CONMEBOL Copa America Brazil 2019 in June Paraguay changed up almost half of their roster before arriving to Japan the team had only one day to train with their entire squad prior to this match but still showcased great intensity to pressure the ball and built their attacks through great ball movements Braian SAMUDIO struck a sharp shot after receiving the ball from Derlis GONZALES where Richard SANCHEZ fired a powerful volley to threaten the Japanese goal goalkeeper GONDA Shuichi (Portimonense S.C.) made a superb save to keep the clean sheet intact After finishing the first half with a 2-0 lead and UEDA Naomichi (Cercle Brugge K.S.V.) were brought in to replace Doan while TOMIYASU Takehiro (Bologna FC 1909) was shifted to the right fullback position Kubo and Tomiyasu led the attacks from the right side Kubo made attempts from freekicks earned by himself in the 50th and 63rd minute while connecting with Haraguchi’s cut back in the 58th minute to threaten the Paraguayan goal Tokyo) and ANZAI Koki (Portimonense S.C.) were introduced to the match Kubo penetrated the right side of the penalty area and fired a shot that struck the cross bar Paraguay tirelessly made their efforts to get on the scoreboard as their final opportunity came when Samudio delivered a cross that found Oscar ROMERO unmarked at the centre but the striker's attempt at goal was denied by Gonda The second half ended with neither side scoring goals as Japan dominated the match by firing 19 shots opposed to Paraguay’s 5 shots and earned their first victory since winning over El Salvador in June The SAMURAI BLUE will depart Japan on Friday 6 to make their ways to Yangon ahead of their first match of the Asian qualifiers for the FIFA World Cup Qatar 2022 as they are set to face the Myanmar National Team on Tuesday 10 Japanese defender YOSHIDA Maya (Southampton FC) and Paraguayan defender Fabian BALBUENA both gave a speech as part of the respect campaign carried out by the JFA and the SAMURAI BLUE wore jerseys with the new logo of the campaign Coach of SAMURAI BLUE (Japan National Team)I am glad that we were able to win against a strong team like Paraguay and deliver a victory to our supporters We had a difficult time conditioning ourselves ahead of this match but each player did a great job to prepare themselves within the limited time frame while sharing a mutual understanding on how to approach the match as a team The four players in the attacking third communicated well with each other while showcasing a great performance to break down the opposing defence Their ability to maximise their strengths on the pitch helped them create great opportunities while the rest of the team also did well to utilise their strong points It was also nice to see our attacking players getting involved in defence This win was a result of our players showcasing what we have been working on we weren’t able to convert our chances to score the third goal and had some shaky moments during this match We must not sit on our laurels and continue to strive to reach higher goals There will be many challenges we must face at the World Cup qualifiers and the first match will always be a tough one when we drew against Singapore in our first match of the qualifiers We will prepare ourselves in the best way possible in order to earn the full three points against Myanmar and enter the match with a strong sense of commitment DF #2 UEDA Naomichi (Cercle Brugge K.S.V.)I am glad that I was able to showcase an improved version of myself here in Kashima it was great that we were able to keep a clean sheet but we weren’t able to score the third goal Our team seemed to have slowed down in the second half and I feel like we had to communicate more with each other in order to change the dynamics DF #16 TOMIYASU Takehiro (Bologna FC 1909)I feel like I did alright to make the adjustments to play as a fullback as well It is expected that we will be spending a lot of time in offence at the competitions in Asia so it is important that the defenders are fully prepared against counterattacks we were not perfect in that aspect in today’s game We need to be more focused and communicate more with each other in order to improve our overall performance DF #22 YOSHIDA Maya (Southampton FC)We showed good transitions between offence and defence in the first half but we made too many mistakes in the second half We saw ourselves losing the ball after forcing turnovers MF #7 SHIBASAKI Gaku (Deportivo de La Coruna)The goals came from very good combination plays and we were able to showcase great creativity throughout the match so that’s something we can be proud of as we kept our focus to deny our opponent from creating opportunities from counterattacks we saw many positives that we need to continue on in the future matches MF #10 NAKAJIMA Shoya (FC Porto)I feel like we were able to play a very good game We are now aware of each other’s characters so we were able to draw out the strong points of our teammates MF #17 KUBO Takefusa (RCD Mallorca)The match was about to turn into a see-saw game and I wanted to capitalise on this given opportunity so I entered the match with a strong intention to score a goal we just need to reset our minds towards our next match and start fresh from tomorrow FW #15 OSAKO Yuya (Werder Bremen)Being able to score at this stadium will give me a positive momentum towards our next match We managed to initiate many counterattacks and created many opportunities We were also able to keep a good distance between each other as well there were chances to score the third goal so that’s an aspect I must improve on ahead of our next match Coach of Paraguay National TeamWe weren’t able to put up an equal bout in terms of the physical aspect as we struggled to deal with Japan’s speed at one-on-one situations We were also troubled by the plays of their two forwards and side halves Although we managed to showcase an organised defence in the second half Japan had the upper hand overall and they deserved the win This match could have gone beyond 2-0 and Japan showed great defence I must say that we were deeply affected by the long travel and the short preparation time SAMURAI BLUE (Japan National Team) vs Paraguay National TeamDate: Thu 5 September 2019 19:20Venue: Kashima Soccer Stadium Tournament Information Please view the main text area of the page by skipping the main menu. The page may not be displayed properly if the JavaScript is deactivated on your browser Japanese version Japan Women's Futsal National Team get through group stage with back-to-back wins ～ The 5th Asian Indoor and Martial Arts Games Ashgabat 2017 The 5th Asian Indoor and Martial Arts GamesGroup Stage 2nd Match vs Hong Kong Women's Futsal National Team17 September 2017 (Sun.) Kick-off 10:00(Local Time) Playing Time 40min.(20min.×2）Multifunctional Sport Venue Hall-1 (Ashgabat Hong Kong Women's Futsal National Team 2-3 (0-2 Scores8'　SEKINADA Minako（Japan Women's Futsal National Team）16'　NAKAJIMA Shiori（Japan Women's Futsal National Team）23'　TAKEMURA Junko（Japan Women's Futsal National Team）30'　goal against （Hong Kong Women's Futsal National Team）38'　goal against（Hong Kong Women's Futsal National Team） SubstitutionsGK：MAEHARA RingoFP：KATO Masami Japan Women's Futsal National Team had their second match of the tournament on the next day of their opener Their opponents were Hong Kong who routed hosts Turkmenistan 6-1 in their first game Japanese coach ITO Masanori commented prior to the match ‘We are facing Hong Kong who grabbed a win in their first match just like us Let’s clinch a spot in the playoffs with a win today we must enter the game with the right competitive mind-set.’ Just as advised Japan found their early chance in the very first minute of the match as KITAGAWA Kana came close to opening the scoring but all of their efforts failed to get past the solid Hong Kong defence who fortified in blocks in front of their goal It was then in the 8th minute when the Japanese women capitalised on the scoring chance from a free kick Placed in the centre and right in front of the goal NAKAJIMA Shiori sent in a neat pass to SEKINADA Minako who slotted home an important opener for her team Japan further took advantage of a right corner on 16 minutes when Nakajima reacted to the delivery from the far side to double the lead before halftime The Japanese squad got off to an ideal start in the second half as TAKEMURA Junko’s shot was deflected off an opposing defender and into the net to stretch the lead to three goals Hong Kong did not stay quiet either, as they struck back in the 30th and 38th minutes and made it a one-point game with two minutes remaining their strong challenge was fend off by the resilient Japanese defence and Japan secured a back-to-back wins in the group stage China grabbed a win over hosts Turkmenistan This was the birthday for goalkeeper MAEHARA Ringo She celebrated her birthday along with her teammates the players of the Iran women’s squad came and sang ‘happy birthday’ for her as well The final group-stage match against hosts Turkmenistan will take place on Wednesday 20 I think it can be understood how difficult today’s match was Yet I believe that we have been able to show the play of a winner in the match We positively take the fact that we have gone through to the next stage as a top team with a win today This was our advantage since we scored from set-plays in futsal I personally think that Japan’s futsal cannot effectively use set-plays in our matches we do not take into account the defensive formation of our opponents Our strengths as Japanese players are hard-working attitude and high skills I think set-plays can be our strengths as a Japanese team it was very important that we could score the goals from set-plays in today’s match Hong Kong kept strong defence and threatened us with quick offense utilising their strengths in their forward line 7 attacker were posing constant threats to us I still see that our players responded well to them but there were some plays which we could not break through we could not deny the talented moves of the players as well as making few errors of our own there are talented players who can break through well with their own individual plays The team is required to respond to these individual talents This is our next issue to sort out and it will definitely be important for us to improve it for the semi-final and final matches The stadium was filled with supporters of the hosting country and it was very powerful I am excited to play the next match in such a wonderful atmosphere FP ＃9 SEKINADA Minako (arco-iris KOBE)First of all I am happy that we have won the second match which was against Hong Kong it was the indirect free-kick from a back-pass and it was not so far from the goal So I just smashed the dropped ball to the net I was happy since it was the opening goal that we could score finally after continuous attacks so we will check what to correct and improve FP #10 NAKAJIMA Shiori (FSF Mostoles/Spain)While we were stilled tired from playing yesterday’s match the corner-kick was good and our player just applied the final touch to the goal freely it was very good that we could score from it In the 2nd half we conceded two consecutive goals and it was the difficult time for us but the fact that we could overcome the challenge in the end will give us strengths for the future It was good that we could win the three points and advance to the semi-finals we will play against the hosting country Turkmenistan I know that there will be facing a huge crowd of Turkmenistan supporters but we definitely want to win this one and gain some momentum going into the semi-final FP #11 KITAGAWA Kana (Maruoka Ruck)We have won two of the first group stage matches I could not score any goals in these two matches but it was good that I could make two assists in the previous match against China Even though we could get the two-consecutive wins so we will correct them during the next two days before the final match of the group stage against Turkmenistan We want to finish the group stage with the victory It is very clear that we will have to play in a completely away-atmosphere so we will make sure that we can communicate amongst ourselves very well despite having difficulties in hearing each other and play well observing closely their game plan To finish the competition with the fourth-consecutive victory *The schedule is subject to change due to the team condition **Indonesia and Vietnam have withdrawn their participation in the 5th Asian Indoor and Martial Arts Games Ashgabat 2017 and the tournament fixtures and schedule were changed accordingly Japan Women's Futsal National Team advances to Semi-Final with win over Uzbekistan at the AFC Women's Futsal Championship Thailand 2018 AFC Women's Futsal Championship Thailand 2018 Quarterfinal vs Uzbekistan Women's Futsal National TeamWed (20mins.x2)Indoor Stadium Hua Mak (Bangkok Japan Women's Futsal National Team 5-1 (1-0 4-1) Uzbekistan Women's Futsal National Team Scores15' AMISHIRO Anna (Japan Women's Futsal National Team)21' EGAWA Ryo (Japan Women's Futsal National Team)26' TAKEMURA Junko (Japan Women's Futsal National Team)28' NAKAJIMA Shiori (Japan Women's Futsal National Team)33' YOTSUI Saki (Japan Women's Futsal National Team)36' Goal against (Uzbekistan Women's Futsal National Team) Starting Line-upsGK: YAMAMOTO AyakaFP: AMISHIRO Anna ReservesGK: SUGIYAMA AikoFP: KOMURA Misato the Japan Women's Futsal National Team faced Uzbekistan Women's Futsal National Team at Indoor Stadium Hua Mak in their quarterfinal match of the AFC Women's Futsal Championship Thailand 2018 The match started off with Japan taking the early initiative but their early chance were denied by the brilliant saves made by the Uzbek goalkeeper but the opening goal came in the 15th minute who has been named the match MVP in the previous two matches shook the net with her shot from the left side Japan gained more momentum in the second half Amishiro delivered a pass to EGAWA Ryo who made a brilliant turn before striking a shot After TAKEMURA Junko and YOTSUI Saki scored their first goal of the competition Japan secured their spot into the semi-final with a 5-1 victory Japan will now face Thailand in the semi-final “we are looking forward to play under an away game atmosphere.” Coach (Japan Women's Futsal National Team)We struggled facing superb saves made by the opposing goalkeeper in the first half but we still managed to create many goal scoring opportunities so I believed in our team to score goals in the second half The important thing is that we managed to win in a large competition like this We entered the tournament with very limited preparation time but I can see my players improving through each matches We would like to win against Thailand in our next match FP #7 TAKEMURA Junko (FUGADOR SUMIDA LADIES)The knockout stage has begun today we seemed to have entered the match on the wrong foot Our opponents overwhelmed us with their strength and speed We could not create a good rhythm for our side A tense atmosphere continued into the second half as the match progressed 1-0 three goals consecutively helped us gain composure I am glad I was able to score in this match so we would like to win this time and advance to the final FP #10 NAKAJIMA Shiori (FSF Mostoles/Spain)It took us a while to find our rhythm in the first half There were stretches where we struggled against the opposing power-play I would like to point out that all of the field players have scored a goal in this competition thus far We have been striving as a team to materialise Coach Kogure’s attacking style futsal and I think that has contributed to this result We have lost against Thailand when we faced them last time We will like to show an even greater intention in our next match and win the match playing our style of futsal *Local Time*The schedule is subject to change due to the team condition Tournament Period: 2 May 2018 (Wed.) - 12 May 2018 (Sat.)For more information U-16 Japan National Team match report: Match 3 against U-16 France National Team International Dream Cup 2015 JAPAN  U-16 France National Team2015-6-28 (Sun.)  - 15:00  Playing time: 90min Osaka)U-16 Japan National Team  3-1 (1st 0-0、2nd 3-1)   U-16 France National Team Score51'   NAKAJIMA Motohiko (Japan)64'   NAKAMURA Shunta (Japan)71'   Goal against (France)90+4'   SAITO Mitsuki (Japan) 1st Half MembersGK: OSAKO KeisukeDF: YOSHIDA Ayumi, HASHIOKA Daiki, NISHIYAMA Taiga, TANAKA KosukeMF: SHINADA Manato, SAITO Mitsuki, ITO Hiroki, FUJIMOTO KanyaFW: NAKAMURA Shunta, KATO Takumi 2nd Half MembersGK: OKI YuyaDF: MIZUTA Kazuma、NAKAYA Yuki、SHIOZAKI YujiMF: HORI Kenta、ARAI HikaruFW: NAKAJIMA Motohiko SubstitutesHT   SHINADA Manato → NAKAJIMA Motohiko65'   KATO Takumi → HORI Kenta75'   FUJIMOTO Kanya → ARAI Hikaru83'   ITO Hiroki → SHIOZAKI Yuji The last of three match series for U-16 Japan National Team in the U-16 International Dream Cup 2015 JAPAN Presented by JFA was against France Japan struggled against France’s physicality and quick offence-defence transition and hardly created their offensive rhythm Despite a good shooting attempt by KATO Takumi both teams were scoreless until the end of the first half took it up and struck a beautiful shot to give his side the much-awaited opener Japan went on to another scoring in the 64th minute when Nakajima sent a free kick to the near side and out of the scramble NAKAMURA Shunta volleyed a loose ball with his left foot into the back of the net France also tried to fight back as their bench made a few substitutions they converted a penalty kick to cut the deficit to a single goal they kept taking advantage of their superior physicality and creating opportunities was led by HASHIOKA Daiki’s impressive clearance right on the goal line and denied French attacks NISHIYAMA Taiga explosively dribbled up the field and made a through ball to SAITO Mitsuki The match ended with the score of 3-1 in Japan’s favour who as the result won the inaugural tournament title The Most Valuable Award and the scoring title were awarded to Hashioka and Nakamura respectively Head CoachI am very happy that we won this historic inaugural U-16 International Dream Cup 2015 JAPAN tournament this tournament was meaningful in terms of having a chance to play seriously against some of the best countries in the world and also giving ourselves extra confidence by making sure that our football can match up well against those opponents.  When I sent our players onto the pitch before today’s match I said to them “We are so fortunate to play against these countries in front of these supporters and on this great pitch Show them what you can do and enjoy yourself.” areas such as moving the ball to build up and receiving passes need further improvement we played tenaciously and physically until the end harder than anybody on their respective club to advance to the older category or higher level I appreciate all the support we received.  NAKAJIMA Motohiko (Cerezo Osaka U-18)I could score a goal in three matches in a row and more than anything I am extremely happy that we could win the title with this team Being able to play at Kincho Stadium also made me happy and all the supports and cheers got this team going to get this result.  Our coach Moriyama said to us during the halftime “I give you the green light to attack aggressively right from the start of the second half,” so I am glad that I could utilise the opportunity I go back to my club and from tomorrow I will work hard to improve my weakness day in and day out.  Match Day 1Wednesday 24 June at J-GREEN Sakai Main Field [S1]U-16 Japan National Team   7-0   U-16 Costa Rica National TeamU-16 France National Team   1-1 (PK 6-7)   U-16 Chile National Team Match Day 2Friday 26 June at J-GREEN Sakai Main Field [S1]U-16 Japan National Team   2-0   U-16 Chile National Team　　U-16 Costa Rica National Team   1-5   U-16 France National Team Match Day 3Sunday 28 June at Kincho StadiumU-16 Chile National Team   2-1   U-16 Costa Rica National TeamU-16 Japan National Team   3-1   U-16 France National Team The Cup information Japan Women's Futsal National Team win 3rd group stage match against China advance to semi-final in AFC Women's Futsal Championship Malaysia 2015 AFC Women's Futsal Championship Malaysia 2015 vs China Women's Futsal National Team2015-9-23 (Wed.) - 14:00　Playing Time 20min.×2Nilai Indoor Stadium (Negri Sembilan Japan Women's Futsal National Team   7-1 (5-0 2-1)   China Women's Futsal National Team Line-upsGK: YAMAMOTO AyakaFP: HOTTA Eriko, SEKINADA Minako, NAKAJIMA Shiori, UEMOTO Yuna SubstitutesGK: YAMASHITA Miyuki, YAMAMOTO AyakaFP: KATO Saori, AMISHIRO Anna, HIGASHIYAMA Maiko, KICHIBAYASHI Chikage, FUJITA Yasuka, TAKAO Akari, SEKI Kaori All players except for GK YAMASHITA Miyuki played in the match The third opponents for Japan Women’s Futsal National Team were China in the group stage of the AFC Women’s Futsal Championship Malaysia 2015 Japan’s coach ARIHARA said to the players in the locker room before the match “This match is as important as yesterday’s match Don’t worry about the knock-out stage now We do our best against the opponents in front of us and show our best performance.” Japan dominated the match from the beginning with solid defence and organised team plays the ball from SEKINADA Minako went over the China’s defence line and NAKAJIMA Shiori beautifully met it for the opening goal KATO Saori shot a thrown-in ball from the left with her first touch to the back of the net China’s defence started to fall apart Kato reacted to the goalkeeper’s clearance of Nakajima’s shot Then Sekinada took the ball up in the middle through China’s defence received it back from her and found the back of the net Meanwhile Japan’s defence did not relax even with the early four-goal lead AMISHIRO Anna dribbled up from the midfield and broke through the opponents’ defence with the combination with SEKI Kaori on the right and put it into the goal just before the first half finished with 5-0 in Japan’s favour Although Japan attempted to keep their defensive focus in the second half to lock down China’s attacking a Chinese player received the ball unchecked for their first goal in the 33rd minute Conceding a goal in the second half reminded of what took place in the second halves in the two previous matches but this time Japan successfully braced up their guard Japan’s beautiful passing play broke China’s formation in front of their goal and HIGASHIYAMA Maiko in the end calmly kicked the ball into the goalmouth to give the sixth goal for her team With an own goal given to Japan when Nakajima’s feed from the left to the right hit an opposing defender into the goal in the 40th minute Japan finished the match with a safe 7-1 lead Japan advanced out of the group stage a top to play the semi-final against Malaysia second-placed team in Group A on Friday 25 September Amishiro Anna received the Player of the Match recognition the important outcome was resting some of the players considering yellow cards and fatigue and at the same time making sure to achieve our goal which was winning the match Looking back the three matches we just played we started off very well and in the second half we couldn’t score as many goals as we did in the first half I rather see it that we could try different options in the second half because of the leads we gained in the first half we got good results that can help us in the upcoming semi-final and final we reset our mindset and prepare well for the semi-final FP ＃6   HIGASHIYAMA Maiko (Bardral Urayasu Las Bonitas)We were able to play this last match in the qualifying round well as a team we wanted to hold the opponents scoreless after we got the opening goal and controlled the tempo of the match in our way we will correct the mistakes we had in the qualifying round and do good preparations as a team and as a player in order to put out the best performance in the next round Also we owe the extra energy we got to finish out the matches to the people who supported us GK ＃16   YAMAMOTO Ayaka (LETIZIA)Before today’s match we were in the situation that a draw or a better result will give us first place in the pool and move us to the next round But we were strongly determined to win the match but we still think we have many things to correct as it was shown in the goals we allowed in every match We think our team are capable of facing those assignments and improving them one by one we will deepen our shared understanding and get us in the best shape possible for the semi-final For all the people who support our national team in Japan and also the supporters who came all the way to Malaysia to cheer us we will make sure to win the semi-final and go on to the final The knockout stage semi-final match of the “AFC Women's Futsal Championship Malaysia 2015” between the Japan Women's Futsal National Team and the Malaysia Women's Futsal National Team which will kick-off at 19:00 on Friday 25 September (at 20:00 on Friday 25 September Japan Time) will be broadcast live on the Internet by the Asian Football Confederation (AFC).*The broadcasting image cannot be guaranteed and depends on the condition of the Internet or the viewing environment *Local Time*The schedule is subject to change due to the team condtion AFC Women's Futsal Championship Malaysia 2015 Group RostersGroupＡ Iran, Malaysia, Uzbekistan, Hong KongGroupＢ Japan, China PR, Thailand, Vietnam Nadeshiko Japan falls to European Champions Netherland 6-2 in ALGARVE Cup 2018 ALGARVE CUP 2018Group League 1st Match vs NetherlandsWed 28 February Kick-off 15:40 (Local Time) Playing Time 90min.(45min.×2)Estadio Municipal da Bela Vista (Parchal/Portugal) Scores4' Goal against (Netherlands)8' Goal against (Netherlands)31' Goal against (Netherlands)35' Goal against (Netherlands)38' NAKAJIMA Emi (Nadeshiko Japan)44' Goal against (Netherlands)52' Goal against (Netherlands)82' IWABUCHI Mana (Nadeshiko Japan) Starting Line-upsGK: IKEDA SakikoDF: SAMESHIMA Aya SubstitutionsHT ARIYOSHI Saori → SHIMIZU RisaHT MIYAKE Shiori → TAKAGI HikariHT HAJI Madoka → YOKOYAMA Kumi70' TANAKA Mina → IWABUCHI Mana70' NAKAJIMA Emi → MASUYA Rika The opening match of the Algarve Cup 2018 kicked off just after the sky cleared wiping away the concerns of the match being called off due to heavy rain The Netherlands and Japan are no strangers as they faced each other in the last Algarve Cup as well as in other friendlies Having won the recent UEFA Women’s EURO the Netherlands are one of the best teams in the world with great momentum on their side the Netherlands sent a long ball behind the Japanese defence line The Dutch side were able to get to the end of this ball sending a cross to the centre and scoring the opening goal the Netherlands penetrated the left flank to give themselves an early two goal lead as both teams utilised their strengths in offence and defence Japan kept possession by connecting short passes while the Netherlands utilised their speed and long balls to move forward while moving the ball in their back line TANAKA Mina and NAKAJIMA Emi created chances but failed to convert while goalkeeper IKEDA Sakiko made fine saves The two teams showed an well balanced fight for a while The Netherlands scored their third goal to break the silence in the 30th minute when they fired a long range shot off of a clearance following a corner kick The European Champions added another goal in the 34th minute to make it 4-0 Japan added themselves onto the scoring sheet as HASEGAWA Yui and SUMIDA Rin exchanged passes in the centre before slotting a through ball to Nakajima who scored their first goal in the 37th minute It was thought that Nadeshiko Japan were about to begin their rally back but conversely allowed another goal off of a corner kick in the closing minutes of the first half entering half-time with a four goal deficit "Play with more power and score a goal first Take more risks and press up front." Coach TAKAKURA Asako encouraged her players during half-time before sending them off to the pitch Takakura subbed in three players from the start of the second half as SHIMIZU Risa made her Nadeshiko Japan debut After giving up a massive lead to their opponent the Japanese side were desperate to score the next goal the Dutch side scored their sixth goal in the 52nd minute Japan took control of the match for a brief stretch when Hasegawa broke through with her dribbling and SAMESHIMA Aya penetrated the flank with her overlaps YOKOYAMA Kumi also contributed with here plays up front Japan could not find the net until the 70th minute substitute IWABUCHI Mana’s individual effort Iwabuchi took on several defenders before scoring her side's second goal But that would be it for the Japanese side After conceding five goals in the first half Japan suffered a lopsided 6-2 defeat in the end Nadeshiko Japan's second group-league match against Iceland will be held on Friday 2 March after a one-day interval Coach of Nadeshiko Japan (Japan Women’s National Team)It was beyond our expectation how we were crushed so easily My players tried to manage the game by following our game plan We were able to play our game in the second half as it was evident that the players were making adjustments by talking to each other We will clarify what we managed to do and we couldn't do share them with everybody and sort them out DF #17 TAKAGI Hikari (NOJIMA STELLA KANAGAWA SAGAMIHARA)With this match being the first match of the tournament so we all kept a strong attitude heading into the match I wanted to play aggressively and change the flow of the match Although we showed more possession in the second half I personally wasn’t able to effectively change the momentum of the game as we conceded another goal early in the second half I want to play with more aggression to win the ball against bigger players With the one-day break before our match against Iceland we will try to improve the issues we found today to win our next match FW #11 TANAKA Mina (NIPPON TV BELEZA)The score speaks for itself our plays and the result weren’t good today We don’t have anything to be satisfied from this match We will tackle on the issues as a team and face our next match Tournament Period: 28 February 2018 (Wed.) - 7 March 2018 (Wed.) Nadeshiko Japan's (Japan Women's National Team) Match1st Match 28 February (Wed.) kick-off 15:40 (Japan Time : 24:40) vs Netherlands2nd Match 2 March (Fri.) kick-off 15:25 (Japan Time : 24:25) vs Iceland3rd Match 5 March (Mon.) kick-off 15:40 (Japan Time : 24:40) vs Denmark All matches to be broadcasted live across Japan by Fuji TV Group For more information US EditionUK EditionScottish SunIrish SunSearchMy Account Manchester City will begin their first FIFA Club World Cup campaign this evening as they take on Japanese side Urawa Red Diamonds Pep Guardiola’s side qualified for the competition after winning the Champions League as part of their historic treble last season They also went on to win the UEFA Super Cup leaving this title as the final trophy needed to complete their collection Should City beat the Reds in the semi-final this week, they will take on the winner of Fluminense vs Al Alhy in the final on Friday Man City have the chance to join an exclusive group of Premier League sides who have won the Club World Cup Only three other English teams have won the competition with Manchester United, Liverpool and Chelsea getting their hands on the title Jurgen Klopp’s side were the next to win the Club World Cup 11 years later, battling through extra-time to beat Brazilian outfit Flamengo 1-0 And most recently, Chelsea claimed the crown in 2021 as they were also taken to extra-time by Palmeiras, with Romelu Lukaku securing a 2-1 win in the 117th minute.  So it remains a rare occasion that an English side win this competition, but Guardiola will be eyeing another piece of European glory this week.  City boss Guardiola has been speaking about his side playing in the Club World Cup "To go there you have to win the Champions League," he said "I’m very pleased and excited to go there and try to win it the results are what they are and you have to accept it.” He continued: "To play this tournament that we have never played before – you need to be there "And we are going to fly there and see the environment and play against Urawa the best as possible to deserve to get to the final Years ago we could not imagine to be there and we are there.” This Club World Cup semi-final will take place on Tuesday It will be played at the King Abdullah Sports City Stadium in Jeddah The game is scheduled to kick-off at 6pm UK time And the action will be broadcast live on TNT Sports in the UK A moderate magnitude 4.5 earthquake hit 40 km (25 mi) away from Imaichi, Tochigi,  Japan The depth of the quake could not be determined but is assumed to be shallow.The quake was not felt (or at least not reported so).