To investigate the effects and immunological mechanisms of the traditional Chinese medicine Xinjiaxiangruyin on controlling influenza virus (FM1 strain) infection in mice housed in a hygrothermal environment.Mice were housed in normal and hygrothermal environments, and intranasally infected with influenza virus (FM1). A high-performance liquid chromatography fingerprint of Xinjiaxiangruyin was used to provide an analytical method for quality control. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure messenger RNA expression of Toll-like receptor 7 (TLR7), myeloid differentiation primary response 88 (MyD88), and nuclear factor-kappa B (NF-κB) p65 in the TLR7 signaling pathway and virus replication in the lungs. Western blotting was used to measure the expression levels of TLR7, MyD88, and NF-κB p65 proteins. Flow cytometry was used to detect the proportion of Th17/T-regulatory cells.Xinjiaxiangruyin effectively alleviated lung inflammation in C57BL/6 mice in hot and humid environments. Guizhimahuanggebantang significantly reduced lung inflammation in C57BL/6 mice. The expression of TLR7, MyD88, and NF-κB p65 mRNA in lung tissue of WT mice in the normal environment, GZMHGBT group was significantly lower than that in the model group (P < 0.05). In WT mice exposed to the hot and humid environment, the expression levels of TLR7, MyD88, and NF-κB p65 mRNA in the XJXRY group were significantly different from those in the virus group. The expression levels of TLR7, MyD88, and NF-κB p65 protein in lung tissue of WT mice exposed to the normal environment, GZMHGBT group was significantly lower than those in the model group. In WT mice exposed to hot and humid environments, the expression levels of TLR7, MyD88, and NF-κB p65 protein in XJXRY group were significantly different from those in the virus group.Guizhimahuanggebantang demonstrated a satisfactory therapeutic effect on mice infected with the influenza A virus (FM1 strain) in a normal environment, and Xinjiaxiangruyin demonstrated a clear therapeutic effect in damp and hot environments and may play a protective role against influenza through downregulation of the TLR7 signal pathway. Influenza virus infection, whether seasonal or pandemic, can cause severe health problems, often leading to pneumonia . The deaFor thousands of years, Chinese medicine has accumulated a wealth of valuable experience in combating the plague. In the fight against severe acute respiratory syndrome, Chinese medicine practitioners actively participated in clinical treatment and research at various levels, and in various aspects such as prevention, treatment, and late recovery. The effectiveness and safety of an integrated traditional Chinese and western medicine method has been fully recognized by the World Health Organization. The successful prevention and treatment of influenza using Chinese medicine has also been recognized worldwide.Yinqiaosan (YQS) is a representative medicine of the cool acrid exterior-resolving method. This medicine has the effect of pungent cooling and detoxification. It is used to treat infectious diseases such as influenza, acute tonsillitis, angina, herpes, mumps, and other viral infections . XinjiaxThe present study explored the effects of YQS, GZMHGBT and XJXRY on the TLR7 pathway and immune balance of T-helper (Th) 17 and T-regulatory (Treg) cells in mice infected with H1N1 (FM1 strain) in hot and humid, and normal environments. The possible mechanism of action of Chinese medicine against influenza in these environments was investigated.The information regarding the experimental design, statistics, and resources used in this study are attached in the minimum standards of reporting checklist.High-performance liquid chromatograph ; electronic balance ;Desktop High Power CNC Ultrasonic Cleaner ; Vortex Mixer ; Small Vertical Electrophoresis Cell (Mini-Protean Tetra 1658001), Basic Electrophoresis System (PowerPac Basic164-5050) and Small-scale transfer system core (Mini Trans-Blot 1703935) were purchased from Bio-Rad, USA; optical microscope ; PCR Thermal Cycler ; Real-Time PCR System ; flow cytometer .Yinqiaosan  ; Xinjiaxiangruyin  . Guizhimahuanggebantang  contained equal part of Mahuang Tang  and Guizhi Tang , which were dissolved in water and combined. These herbs were all Chinese patent medicine granules, which were purchased from China Resources Sanjiu Medical & Pharmaceutical Co., Ltd. Oseltamivir Phosphate Capsules  was obtained from Yichang Yangtze River East Sunshine Pharmaceutical Ltd. (Lot H20065415).Chromatographically pure acetonitrile and methanol, ultrapure water, and other analytically pure reagents. Chlorogenic acid ; Forsythin ; Thymol ; Magnolol , which were purchased from Shengshi Kangpu Chemical Technology Research Institute . PrimeScript RT reagent kits , SYBR Premix EX Taq II . DEPC Water, ; TLR7 Rabbit mAb (Lot: 2633S), MyD88 Rabbit mAb (Lot:4283S), NF-kappa B p65 Rabbit mAb (Lot: 8242S), GAPDH Rabbit mAb (Lot: 2118S) were purchased from CST, USA; The mouse lymphocyte separation solution, Goat anti-rabbit secondary antibody, BCA Protein Quantitative Kit, and potent ECL kits were all products of Multi Sciences Co., Ltd. in Hangzhou, China. CD4-PE (Phycoerythrin) (Lot: 4343463); CD25-APC (Allophycocyanin) (Lot: E07106-1634); IL-17-FITC (Lot: 4291420); Foxp3-PE-cyanine 5.5 (Lot: E16149-104), eBioscience™ Foxp3/Transcription Factor Staining Buffer Set Kit (Lot: 4343791) were purchased from eBioscience Inc., USA.Virus strain: Influenza virus A , type A influenza virus FM1 murine lung adapted strain (FM1), − 80 °C cryopreserved, provided by the Department of Microbiology and Immunology, School of Basic Medical Sciences, Jinan University. After two passages of resuscitation, the hemagglutination titer was 1:640. Mortality was assessed at 14 days after statistical viral infection at different concentrations using a multiple dilution method; the virus concentration at 20% of the mouse hemagglutination titer (1:40), with 50 μL used to infect the mice.−/− mice were purchased from The Jackson Laboratory, USA. All knockout mice were backcrossed to the germline of C57BL/6 mice over 10 generations. Reproduction and breeding of TLR7−/− mice were performed in the SPF environment at the Animal Experimental Center of Jinan University. Six to eight-week-old female experimental animals were placed in artificial climate chambers: normal environment ; and hot and humid environment . The illuminance was 3000 Lx and 0 Lx each 12 h cycle. Mice were reared on a cluster basis. Thirty-Six C57BL/6 mice  and TLR7−/− mice  were housed in a hot and humid environment and randomized to six animals per group. After infection with the virus, the mice were placed in the artificial climate chambers for 12 h each day and housed in normal climate chambers for the rest of the day until the end of the experiment. The animals had ad libitum access to food and water. There were six mice in each of the following groups: blank (control); model (virus); virus + oseltamivir; virus + XJXRY; virus + GZMHGBT; and virus + YQS. Thirty-six  C57BL/6 mice of the same age were housed in a normal environment similar to the control group.Animal experiments were performed with the approval and supervision of the Experimental Animal Ethics Committee of Jinan University . SPF-grade normal C57BL/6 wild-type mice were purchased from the Experimental Animal Center of Guangdong Province (production license SCXK [Guangdong] 2013-0002). The TLR7−/− and WT C57BL/6J mice were infected with the influenza virus (FM1). The mice were first anesthetized with 6% chloral hydrate solution, and were infected intranasally with 50 μL of the FM1 strain of the influenza virus. The normal control group was given an equal volume of sterile 0.9% NaCl solution by intranasal instillation. None of the nasally infected mice died. General living conditions and body weight of the mice were observed and recorded daily.On day 11 of modeling in a hot and humid environment, both TLR7On day 11 day of modeling, the mice were infected with FM1 virus. Twenty-four hours after the mice were infected, each group of animals was treated with the corresponding drug, with dose calculated according to body weight. The dose used in mice is equivalent to 9.1 times that of humans. The treatment group was given 0.4 mL/day, with continuous intragastric administration for 5 days (12–16 days): YQS, 560 mg/mL; GZMHGBT, 350 mg/mL; XJXRY, 230 mg/mL; and oseltamivir 1.5 mg/mL. An equal amount of double distilled water was given to the normal group and the virus group. The body weight of all mice was recorded on day 17 after infection, and lung tissues and spleen were immediately obtained after the mice were euthanized.3PO4 (B); Flow rate: 1.0 mL/min; column temperature: 30 °C; detection wavelength: 280 nm; injection volume: 10 μL, the gradient elution program is shown in Table Chromatographic conditions. Columns: Cosmosil 5C18-MS-II (4.6 ID × 250 mm) Manf. No. K63663. Mobile phase: Acetonitrile (A)–0.1% H3 mg of chlorogenic acid, forsythia, thymol, and magnolol were transferred to a brown volumetric flask and methanol was added to prepare a 0.5 mg/mL standard solution.According to the ancient decocting method, lentil flower soup was obtained, cold to room temperature, and a precise quantity 12.5 mL) was transferred to a 50 mL tapered bottle. Fine particles of honeysuckle, forsythia, Magnolia, and Elsholtzia were mixed in a conical bottle and mixed with the lentil flower soup. Add the 12.5 mL methanol to the cone bottle again, weighing and ultrasonic cleaning 30 min . When it is cooled to room temperature, and then weigh it; 50% methanol was used to fill the lost weight, shake well, filter, and remove the filtrate through 0.45 µm porous filter membrane to obtain the test product solution  was used for fluorescence quantification experiments. The Ct value of each determination was recorded and GAPDH was used as the internal reference. The relative mRNA expression of the sample was calculated according to the 2−∆∆Ct method. ∆Ct = Cttarget gene − Ctreference gene, ∆∆Ct = ∆Ctexperimental group − Ctcontrol group. Each sample was measured three times and averaged, and qPCR results represented three independent experiments.The PCR reaction (20 μL): PCR forward primer (10 μM) 0.8 μL, PCR reverse primer (10 μM) 0.8 μL, SyBr Premix Ex Taq II(2*1) 10 μL, complementary DNA template 2 μL, dd HHomogenates (40 mg) were collected from lung tissue stored at − 80 °C, thawed for 15 min at 4 °C and centrifuged at 12,000 rpm, and the supernatant was collected. Bovine serum albumin (BSA) was used as standard, and a commercially available BCA protein quantitative kit was used to determine protein content. An equal amount (35 μg) of the protein contained in each sample was decomposed in a 10% Tris–glycine SDS polyacrylamide gel. After electrophoresis, the separated protein bands were wet transferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding membranes were blocked with 5% skim milk at room temperature for 1 h. The PVDF membrane was incubated with primary antibody (1:1000) and incubated overnight at 4 °C. Diluted secondary antibody was added at a ratio of 1:5000 and incubated for 1 h at room temperature. The immunodetection protein was visualized using an electroluminescence kit and the Alliance gel imaging system, and analyzed using image J software. The ratio of the target protein optical density value and the internal reference protein GAPDH optical density value was considered to represent the relative expression of the target protein.6 cells/mL. For flow cytometry, the cells were labeled with a monoclonal antibody conjugated to fluorescein  on a FACSVerse flow cytometer  and were tested and analyzed using FlowJo version 10 .The ratio of subcellular Th17/Treg (CD4+IL-17+/CD4+CD25+Foxp3+) of T lymphocytes in mice was determined using flow cytometry. Peripheral blood mononuclear cells were isolated from mouse spleens using a lymphocyte separation solution according to manufacturer’s instructions. The regulatory cell concentration was approximately 1 × 10Experimental data were processed and analyzed using SPSS version 13.0 . All indicators were expressed as mean ± standard deviation. Multiple comparisons were carried out by one-way ANOVA followed by Bonferroni’s post hoc test or one-way ANOVA with repeated measures and Bonferroni test for multiple comparisons. All tests were two-tailed and P values less than 0.05 were considered statistically significant. Differences with P < 0.05 were considered to be statistically significant.Two days after influenza virus infection (day 13), the clinical symptoms of mice in each treatment group and virus model group became apparent, with varying degrees of weight loss, cough, fur loss, and contractures. On day 15, mice had typical flu symptoms, including hair discoloring, towering hair, curled up, arch, loss of appetite, faint. The control group exhibited a good mental state, agile movement, and natural weight gain. In the normal environment, mice in the oseltamivir, GZMHGBT were in good condition. However, in the hot and humid environment, the mice in the XJXRY and oseltamivir groups were in a better state than the virus group. = 1.892, P = 0.061). However, the weight of wild type  = 10.624, P < 0.0001) and TLR7−/− mice  = 12.108, P < 0.0001) had obvious fluctuation in hot and humid environment. TLR7−/− mice lost weight, but returned to the original weight on the 11th day.The mice were modeled in normal environment and hot and humid environment for 11 days Fig. a. We can = 34.78, P < 0.0001), the body weight of the mice on day 17 of the GZMHGBT, YQS, and oseltamivir groups decreased to 86.78%, 80.93%, 90.96% of the body weight on day 11, respectively  = 12.35, P < 0.0001), the body weight of mice on day 17 in the XJXRY, and oseltamivir decreased to 92.34%, 96.61% of the body weight on day 11, respectively  = 27.35, P < 0.0001), except for the control group, there was no difference between the other groups and the virus group  = 13.99, P = 0.0001), the WT mice XJXRY and the model group were different (P < 0.05). In the TLR7−/− mice  = 6.25, P = 0.002), there was no statistical difference between the XJXRY and the virus group . Statistical analysis revealed that in the hot and humid environment  = 23.45, P < 0.0001), compared with the model group, the dose of oseltamivir group, GZMHGBT decreased significantly (P < 0.05). In the hot and humid environment  = 8.91, P < 0.0001), there was a significant difference between the oseltamivir and XJXRY groups with the virus group (P < 0.05). In TLR7−/− mice  = 11.37, P = 0.0003), the viral load in the model group was significantly higher than that in the control group (P < 0.001) . Under normal circumstances  = 11.86, P < 0.0001), compared with the virus group, the GZMHGBT group in WT mice the ratio of Th17 cells and Treg cells in the spleen was significantly decreased (P < 0.05). In the hot and humid  = 10.34, P = 0.0005), XJXRY group was significantly lower than that of the virus group (P < 0.05). In TLR7−/− mice  = 9.23, P < 0.0001), compared with the virus group, only the positive control drug oseltamivir group Th17/Treg cell ratio was reduced  = 14.06, P = 0.0001), MyD88  = 10.07, P < 0.0001), and NF-κB p65  = 10.71, P = 0.0004) mRNA in lung tissue of WT mice in the normal environment, GZMHGBT group was significantly lower than that in the model group (P < 0.05). In WT mice exposed to the hot and humid environment, the expression levels of TLR7  = 4.78, P = 0.012), MyD88  = 9.32, P = 0.0009), and NF-κB p65  = 7.93, P = 0.0014) mRNA in the XJXRY group were significantly different from those in the virus group (P < 0.05), but not in the TLR7 knockout mice. There was no statistical difference between the three treatment groups and the virus group in the TLR7 knockout mice . Whether in normal or hot and humid environments, the mRNA levels of TLR7, MyD88, and NF-κB p65 in the lungs of oseltamivir mice were lower than those in the virus group (P < 0.05). The expression of TLR7  = 39.38, P < 0.0001), MyD88  = 7.83, P = 0.0017), and NF-κB p65  = 9.49, P = 0.0007) protein in lung tissue of WT mice exposed to the normal environment, GZMHGBT group was significantly lower than those in the model group. In WT mice exposed to hot and humid environments, the expression levels of TLR7  = 11.43, P = 0.0003), MyD88  = 28.38, P < 0.0001), and NF-κB p65  = 36.05, P < 0.0001) protein in XJXRY group were significantly different from those in the virus group (P < 0.05). There was no statistical difference among the three groups treated with Chinese medicine compared with the virus group in the TLR7 knockout mice . Under different circumstances, the expression of TLR7, MyD88, and NF-κB p65 protein in the lung tissue of oseltamivir-treated mice was lower than that of the virus group (P < 0.05). The expression levels of TLR7  of the TLR7 signaling pathway.Experimental results revealed that in the humid and hot environment model, the weight of the mice was not significantly affected. In the hot and humid environment, XJXRY clearly exhibited therapeutic effects. Under normal circumstances, GZMHGBT can effectively reduce the lung index of mice with influenza pneumonia and extend the survival time of the animals. XJXRY originated from Wu Jutong’s “Wen Bing Tiao Bian.” The prescription is composed of five herbs including honeysuckle, forsythia, Magnolia, Elsholtzia, and lentil flower. It has the effect of dispelling heat and relieving dampness, and has a significant effect on the symptoms of cold and high humidity in the summer. In pharmacology, the heat-clearing actions of tivities . Experimtivities . Honeysutivities , which itivities . Honokiolication , 21. In Recent studies have shown that the balance between Th17 cells and FOXP3+ regulatory T cells (Tregs) is of great significance. Interleukin (IL)-17 secreted by Th17 cells can induce IL-6, IL-8, granulocyte colony-stimulating factor and prostaglandin E2 in epithelial cells, endothelial cells, fibroblasts, and stromal cells. IL-17 also enhances the function of IL-1 and tumor necrosis factor (TNF) on the cell surface and the expression of intercellular adhesion molecule-1 . TherefoThe positive control drug in this experiment is oseltamivir, a neuraminidase inhibitor (NAI), which is the mainstream drug for the treatment of influenza. It inhibits NA activity and thus effectively inhibits the replication and spread of various influenza virus strains. For example, the activity of glycoside hydrolase is to cleave the glycosidic linkage of neuraminic acid to reduce viral transmission and reinfection. Existing scholars have found that oseltamivir is effective in early antiviral therapy , 32. HowIn conclusion, this study confirmed that in normal environments, GZMHGBT demonstrated significant inhibitory effects on influenza virus A (FM1) infection in mice. In hot and humid environments, XJXRY exhibited a good therapeutic effect on mice infected with the influenza A virus (FM1 strain). It can significantly reduce lung inflammation in mice, and exhibit anti-influenza and anti-inflammatory properties by down-regulating the expression of key factors in the TLR7 signaling pathway.


Although anticoagulation therapy could reduce the risk of strokes in patients with atrial fibrillation (AF), large‐scale investigations in the direct oral anticoagulant (DOAC) and AF catheter ablation (CA) era are lacking.This study was designed as a prospective, multicenter, observational study and a total of 2113 patients from 22 institutions were enrolled in the Hyogo area.2 score were 70.1 ± 10.8 years old and 1.5 ± 1.1, respectively. The follow‐up period was 355 ± 43 days. CA was performed in 614 (29%) and DOACs were prescribed in 1118 (53%) patients. Ischemic strokes/systemic embolisms (SEs) and major bleeding occurred in 13 (0.6%) and 17 (0.8%) patients, respectively. New onset dementia, hospitalizations for cardiac events, and all‐cause death occurred in eight (0.4%), 60 (2.8%), and 29 (1.4%) patients, respectively. A multivariate analysis demonstrated that persistent AF and the body weight (BW) were associated with ischemic strokes/SEs and major bleeding, respectively . AFCA history was associated with the cardiac events . Age was associated with new onset dementia .The mean age and CHADSIn the DOAC and CA era, the incidence of ischemic strokes/SEs, major bleeding and cardiac events could be dramatically reduced in patients with AF. However, some unsolved issues of AF management still remain especially in elderly patients with persistent AF and a low BW. AFatrial fibrillationCAcatheter ablationDOACdirect oral anticoagulants12 score/CHA2DS2‐VASc score number.The number of patients with atrial fibrillation (AF) is increasing at a rapid rate and is expected to reach beyond one million patients in Japan as the population ages.AF catheter ablation (AFCA) is widely performed, and some investigations have reported that it is more effective for preventing AF recurrences than medical therapy.AF patients without strokes have been followed by cardiologists in Japan. However, once cerebral vascular events occur, those patients are followed by brain surgeons or neurologists. Therefore, it is difficult for primary care doctors to share the events. To share those events, we established the HAF‐NET (HYOGO ATRIAL FIBRILLATION NETWORK) registry, which pooled the data from primary care doctors, brain surgeons, and neurologists. The primary end point was the composite of symptomatic cerebral infarctions including transient ischemic attacks (TIAs), systemic embolisms (SEs), and fatal bleeding complications requiring hospitalization including an intracranial hemorrhage. Secondary end points included a composite of a composite of new onset dementia, cardiac events requiring hospitalization, and all‐cause death. Our goal of this study was to examine the incidence of both primary and secondary endpoints and to identify the predictors for the composite primary end point, secondary end point, and each event including stroke/SE, major bleeding, new onset dementia, hospitalization for cardiac event and all‐cause mortality and to clarify the reality of AF management in Japan by using the data form the HAF‐NET Registry.22.1The HAF‐NET Registry is multicenter, prospective, observational study of Japanese patients with AF. The patients were enrolled from April 2015 to August 2016. Inclusion criteria were those aged 20 or older in whom AF was diagnosed by a 12‐lead or Holter electrocardiogram. There were no exclusion criteria. A total of 22 institutions, all of which were located in Hyogo Prefecture, participated in this registry. They consisted of eight cardiovascular centers, two affiliated or community hospitals, and 12 primary care clinics. All patients were followed through a review of the inpatient and outpatient medical records, and additional information was obtained through contact with the patients, relatives, and/or referring physicians by mail or telephone. The data were checked by clinical research coordinators. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, CONSORT 2010 guidelines and was approved by the ethical committees of Kobe University . This study will be registered with the University Hospital Medical Information Network Clinical Trial Registry (UMIN‐CTR) (UMIN000036784), and the posted information will be updated as needed to reflect the protocol amendments and study progress.2.2All patients had a certification of attendance, which contained information including the anticoagulation therapy regimen and contact information of the primary care doctor Figure . Even thThe baseline clinical background data were as follows: patient clinical characteristics including the date of birth, age, gender, body weight, serum creatinine level, date when AF was diagnosed, history of treatment including CA, cardiac surgery, percutaneous coronary intervention, or coronary arterial bypass grafting, type of AF, comorbidities, and risk factors including heart failure, hypertension, diabetes mellitus, strokes/TIAs, vascular disease, valvular disease, ischemic heart disease, cardiomyopathy, dementia, whether patients smoked or consumed alcohol at the time of enrollment, a reduced left ventricular function (%FS < 25% or EF < 35%), current medications including anticoagulant drugs (DOACs or warfarin) and antiplatelet drugs, and subjective symptoms including palpitations, dyspnea and dizziness. Paroxysmal AF was defined as AF that terminated spontaneously within 7 days, while persistent AF was defined as AF that lasted for > 7 days but could be terminated with medication or electrical cardioversion. Long‐lasting persistent AF was defined as AF that lasted > 1 year.2 score and CHA2DS2‐VASc score.The risk of a stroke was evaluated by the CHADS2.3The primary endpoints of this registry were symptomatic cerebral infarctions including TIAs, SEs, and fatal bleeding complications requiring hospitalization including an intracranial hemorrhage. A TIA was defined as a sudden onset of focal neurologic symptoms and/or a sign lasting less than 24 hours, brought on by a transient decrease in the blood flow, which rendered the brain ischemic in the area producing the symptom. Fatal bleeding complications were defined as a reduction in the hemoglobin level by ≥2 g/dL, a transfusion of ≥ 2 units of blood, or symptomatic bleeding in a critical area or organ, following the International Society on Thrombosis and Hemostasis definition. The secondary end points were a composite of new onset dementia, cardiac events requiring hospitalization, and all‐cause death. The diagnosis of dementia was based on the Mini‐Mental State Examination and/or Hasegawa dementia rating scale.2.4P < .05 using an unadjusted Cox proportional hazard regression analysis. To compare the clinical events between the warfarin and DOAC users, 667 age, BW, and CHADS2 score‐matched DOAC and warfarin users were tested. The cumulative incidence of a stroke or SE was determined by the Kaplan‐Meier method. The survival analysis between warfarin and DOACs was performed using a log‐rank test. A value of P < .05 was considered statistically significant. All statistical analyses were performed using SPSS, Release 25 software (SPSS).Continuous data were presented as the mean ± SD for normally distributed variables. Medians and quartiles were given for nonnormally distributed variables. If these data followed a normal distribution, they were tested with an unpaired t‐test or Welch test. If not, they were tested with a Mann‐Whitney test. Categorical variables were analyzed with the Fisher's exact test. Cox proportional hazards regression models were used to estimate the hazard ratios and 95% confidence intervals for each event. Previously reported variables including age, gender, BW, AF type, AFCA history, valvular disease, ischemic heart disease, cardiomyopathy, EF less than 35%, heart failure, hypertension, age > 75 years, diabetes mellitus, stroke/TIA, vascular disease, antiplatelet drug, DOAC use, and HAS‐BLED were also selected as cofounders. The multivariable Cox proportional hazards regression model included variables with a 3A total of 2113 patients were enrolled from 38 institutions in Hyogo prefecture between April 2015 and August 2016. Of those, 1343 (64%) were enrolled from cardiovascular centers, 66 (3%) from affiliated or community hospitals, and 704 (33%) from private clinics. Two thousand and seventy (98%) of the 2113 patients were followed for 1 year after the enrollment and the mean follow‐up period was 355 ± 43 days.3.12 and CHA2DS2‐VASc scores were 1.5 ± 1.1 and 2.6 ± 1.6, respectively. Figure 2 score and CHA2DS2‐VASc score. A CHADS2 score = 1 and CHA2DS2‐VASc score = 3 were the most common subpopulations. Table The baseline characteristics of the registered patients are summarized in Table 2 and CHA2DS2‐VASc scores, and lower HAS‐BLED score , while a stroke/TIA history was more frequently observed in those with an AFCA history . Of note, fewer anticoagulant drugs or antiplatelet drugs were used in the patients with an AFCA history .Compared to the patients without a CA history, those with a history of CA had a younger age, mainly paroxysmal AF, fewer underlying diseases, lower CHADS3.2Figure P < .001; BW: 62.5 ± 12.6 vs. 65.0 ± 12.7, P < .001; serum creatinine level: 1.07 ± 0.75 vs. 0.88 ± 0.36, P < .001). The AF types including paroxysmal, persistent, and long‐lasting AF were equally distributed in the warfarin users, while paroxysmal AF was dominantly found in the DOAC users. The symptoms were less in the warfarin users . Of note, a CA history was more frequently observed in patients without anticoagulant therapy than in those who did not undergo CA .No anticoagulant therapy was performed in 270 (13%) patients who were significantly younger and their CHADS3.3P = .03; BW: HR: 0.94, 95% CI: 0.90‐0.99, P = .03; HAS‐BLED score: HR: 1.82, 95% CI: 1.12‐2.96, P = .02; Table P = .03; diabetes mellitus: HR: 3.51, 95% CI: 1.17‐10.54, P = .03), that of hospitalization for cardiac events consisted of AFCA history as well as serum creatinine, heart failure , and that of all‐cause mortality consisted of the age and EF less than 35%  and 99 (4.4%) patients, respectively. Of those, ischemic strokes/SEs and major bleeding occurred in 13 (0.6%) and 17 (0.8%) patients, respectively. New onset dementia, hospitalization for cardiac events, and all‐cause death occurred in eight (0.4%), 60 (2.8%), and 29 (1.4%) patients, respectively. According to a multivariable Cox proportional hazard regression analysis, the most predictive model of a stroke/SE consisted of the AF type (persistent) and that of major bleeding consisted of the BW as well as the HAS‐BLED score  and 11 (4.1%) of 270 patients, respectively. Of those, ischemic strokes/SEs and major bleeding occurred in one (0.4%) and one (0.4%) patients, respectively. New onset of dementia, hospitalization for cardiac event and all‐cause death occurred in one (0.4%), six (2.2%), and five (1.9%) patients, respectively. Notably, 174 (64%) of 270 patients without anticoagulation therapy had CA history. Although hospitalization for cardiac event was found in four (2%) of 174 patients, no other events including stroke/SE, major bleeding, new onset of dementia and all‐cause death were found in the 174 patients with CA. Furthermore, 614 (29%) of 2113 patients had CA history regardless of anticoagulation therapy, primary and secondary endpoints were found in 4(0.7%) and 18(3%), respectively. Of those, stroke/SE, major bleeding, new onset of dementia, hospitalization for cardiac event, and all‐cause death were found in one (0.2%), three (0.5%), two (0.3%), 15 (2%) and one (0.2%) patients, respectively.2 score‐matched DOAC and warfarin users were compared . A composite of the primary and secondary endpoints was found in nine (1.3%) and 24 (3.6%) DOAC users, and in 15 (2.2%) and 36 (5.4%) of the 667 warfarin users, respectively. Of those, the incidence of the ischemic strokes/SEs was significant less in DOAC users .To assess the clinical impact of the DOACs, 667 age, BW, CHADS44.1The data from the HAF‐NET registry demonstrated a higher DOAC use and AFCA history as compared to the previous studies, which resulted in excellent outcomes after 1 year of follow‐up among Japanese patients with AF in the DOAC and AF ablation era. Persistent AF and a lower BW were strongly associated with stokes/SEs and major bleeding, respectively. AFCA history as well as age and heart failure were associated with the composite secondary endpoints including new onset dementia, hospitalization for cardiac events, and all‐cause mortality.4.2One‐third (800 patients) of the patients in this registry were from Chuo‐Ku, which is located in the southern region of Kobe city. The population of Chuo‐Ku is approximately 135 000 people. Based on the epidemiological prevalence of AF in the Japanese population of 0.6%, the number of AF patients in Chuo‐Ku was estimated to be approximately 810. As the number in our registry was almost equal to the estimated AF patients in Chuo‐Ku, the AF patents in this registry were assumed to fully reflect a typical ward in Kobe city.2 scores were enrolled in our registry. The mean age of the HAF‐NET, SAKURA, and FUSHIMI registries was 70, 72, and 74 years old, respectively. The mean BW was 64, 64, and 58 kg and persistent AF 49%, 63% and 54% in the enrolled patients, respectively. The prevalence of a stroke/TIA history was 9%, 11%, and 19 %, respectively. The mean CHADS2 scores were 1.5, 1.8, and 2.1, respectively.In Japan, real‐world data of the anticoagulation therapy in patients with AF have been published from two major AF registry such as FUSHIMI and SAKURA registry.The FUSHIMI registry recruited patients mainly from private clinics, while the HAF‐NET and SAKURA registries recruited those from cardiovascular centers, affiliated hospitals, community hospitals, and private clinics. Sixty‐four and 48 private clinics participated in the FUSHIMI and SAKURA registries, respectively, while there were only 15 in the HAF‐NET registry. The proportion of patients from private clinics completely differed. This might be the reason why the patient characteristics differed among the three registries.4.3Warfarin was prescribed in only around 30% of the patients in the HAF‐NET registry. As compared to the FUSHIMI and SAKURA registries, the proportion of warfarin users was significantly smaller in the HAF‐NET registry . However, the proportion of DOAC users was significantly greater . Several studies from the J‐RHYTHM and SHINKEN databases reported the increase in DOAC users, from 6.1% in 2012 to 20.4 % in 2014, and from none in 2007‐2009 to 25.5% in 2010‐2012.4.42 score was lower and a CA history was found in almost 30% of the patients who mainly had a stroke history or no anticoagulation therapy. This might have reduced the AF burden, which resulted in an excellent outcome. Iguchi et al. demonstrated that patients with preexisting heart failure had higher unadjusted rates of strokes/SEs  as well as higher incidences of all‐cause death and a composite of all‐cause death or strokes/SEs.The two major postmarket surveillance (PMS) studies  showed the incidence rates of major bleeding and thromboembolic events, suggesting that dabigatran and rivaroxaban were safe and effective in the Japanese clinical practice. The J‐Dabigatran surveillance demonstrated that major bleeding, strokes/SEs, and all‐cause death occurred in 1.1%, 1.2%, and 1.3%/year, respectively.4.52 and CHA2DS2‐VASc scores and the incidence of clinical events was extremely low. This indicated that CA could be considered as an early intervention for AF management in the real world. Based on those clinical results, the Japanese guidelines have been updated and the latest one (2018 JCS/JHRS Guideline on Non‐Pharmacotherapy of Cardiac Arrhythmias) was published in May 2019. In the latest guidelines, CA was recommended as a class IIa indication for drug refractory, recurrent paroxysmal and persistent AF regardless of heart failure. This will facilitate the CA in patients with heart failure, which might strengthen the impact of CA on AF management in the next decade.Recently, several studies have reported the impact of CA on the mortality and cardiovascular hospitalization in patients with AF. Especially in patients with heart failure, the impact has been greater. The CATSLE‐AF study clearly demonstrated that CA was associated with a significantly lower rate of the composite end point of death from any cause or hospitalization for worsening heart failure as compared to medical therapy. Although no statistical significance could be found, cerebrovascular accidents were dramatically reduced by CA as compared to medical therapy.4.6Previous RCTs have revealed better clinical outcomes especially for fatal bleeding under DOAC therapy than under warfarin therapy. However, fewer Japanese patients could be enrolled in those RCTs.4.7P = .004). The population attributable risk for dementia resulting from AF was 13%. They concluded that patients with AF should be screened for cognitive symptoms.A meta‐analysis reported that AF was independently associated with an increased risk of all forms of dementia.4.82 score‐matched DOAC, and warfarin users were compared because of the small number in each medical therapy group. Fourth, this study involved AF patients recruited from a small region of Japan, and therefore, the results might not be generalizable to the overall population.This study had several limitations. First, this study was designed as a prospective observational study, therefore, only associations were shown, not causality. The possibility of unmeasured or residual confounding factors was not ruled out. Second, anticoagulant therapy was assessed at the time of the enrollment, but the changes in the medical therapy could not be assessed. Third, to assess the impact of the DOAC therapy, age, BW, and CHADS52 scores. In the DOAC and CA era, the incidence of ischemic strokes/SEs, major bleeding and hospitalization for the cardiac events could be strongly reduced in patients with AF. However, some unsolved issues of AF management still remain especially in elderly patients with persistent AF and a low BW.The HAF‐NET registry was characterized as (a) having a high incidence of DOAC prescriptions and a CA history and (b) including relatively younger patients with lower CHADSThe Section of Arrhythmia is supported by an endowment from Medtronic JAPAN and Abbott JAPAN. The authors have reported that they have no relationship relevant to the contents of this paper to disclose. Click here for additional data file.


Cirrhinus mrigala, Cirrhinus reba, Catla catla, Lebio rohita, Crossocheilus latius, Clupisoma garua, and Mystus tengara) collected from local markets of Varanasi, Allahabad, Mirzapur, and Kanpur of Uttar Pradesh, India. The Cu concentration was found at Varanasi (4.58 mg/l), Allahabad (2.54 mg/l), and Mirzapur (2.54 mg/l). Pb was recorded 0.54, 0.62, 0.85, and 0.24 mg/l at Kanpur, Allahabad, Mirzapur, and Varanasi, respectively. The Cd concentration was recorded 0.54, 0.68, 0.78, and 0.85 mg/l at Kanpur, Allahabad, Mirzapur, and Varanasi, respectively. The Cr, Cd, and Pb concentrations in the river water were observed over the prescribed safe limits at all sampling sites, while Cu concentration was higher than the standards at all sites except Kanpur. However, Zn was observed under the permissible limits (15 mg/l) at all sampling sites. In case of fish tissues, WHO reported the concentration of Pb, Cd, and Cr higher than the prescribed safe limits. The results determined that the highest heavy metals accumulation was found settled in the liver of all selected fish species. Zn ranked the highest quantity, which was found in fish tissues with the concentration of 32.41 ± 2.55 μg/g in the gill of C. catla and 4.77 ± 0.34 μg/g in the gill C. Reba. The metals followed the magnitude order of Zn > Pb > Cu > Cd > Cr in selected fish tissues.This paper assesses the potential human health risks posed by five heavy metals  found in seven most consumable fish species ( With an average annual running water potential of 525.02 Bm3 yr−1 (Billion cubic meters per year), which comes from all major Indian river basins 55], the 3.2C. mrigala and C. garua fish species are major sources of protein and consumed throughout India. Thus, selected species were taken for in this study and also analyzed for different metals. The highest load of Zn was found in all the studied fish species followed by L. rohita, M. tengara, C. garua, C. latius, C. mrigala, C. catla and C. reba. The heavy metal concentration trend was Zn > Cu > Pb > Cd > Cr in almost all fish species. Findings of the present study also confirmed the results reported in C. reba a species .3.2.3C. reba and 3.15 ± 1.22 μg/g in liver for C. catla, while the length and weight of both fish species were higher than the other selected fish species. The FAO and WHO proposed a limit of 0.5 μg/g for Pb in food, while FEPA set this value to 2.0 μg/g. The larger fish  tend to accumulate more heavy metals due to extensive column feeding nature [C. reba, C. garua, and M. tengara, which was due to their smaller body size [L. rohita was investigated similar to P. sophore studied in by other researchers in other rivers [The lead (Pb) concentration ranged from 1.12 ± 0.03 to 4.77 ± 0.34 μg/g among the fish selected from the study area. The highest Pb concentration was detected 4.77 ± 0.34 μg/g in gill, for g nature ,66,76. T3.2.4C. mrigala and also 0.53 ± 0.13 μg/g to 1.42 ± 0.23 μg/g in muscles.Cadmium (Cd) is a serious contaminant and a highly toxic element, which is transported in the water and air and found in different sources. The Cd concentration ranged from 0.32 ± 0.07 to 2.54 ± 0.33 in the selected fish tissues. The high load of Cd in the Ganga River is due to different industrial and domestic channels induced in the Ganga River. The maximum concentration of Cd (2.54 ± 0.05) was detected in the liver of C. striatus. Vannoort and Thomson observed a lower Cd concentration (compared to the present study) varied from 0.003-0.036 mg/kg with a mean of 0.01367 mg/kg in vacuum packaged smoked fish species  [The highest volume of Cd was recorded by the authors in  as 1.42  mykiss) . For ins mykiss) . In anot mykiss) .3.2.5C. reba, 0.35 ± 0.11 μg/g in C. mrigala, and 0.44 ± 0.03 μg/g in C. garua, respectively. European Union Commission, suggested the daily tolerable chromium concentration to be 1 μg/g, while the FEPA suggested 0.15 μg/g and WHO suggested 0.15 μg/g. Earlier reports in regard to the Cr concentrations from the southeast coast of India indicated the range of 0.41–1.56 μg/g and 0.65–0.92 μg/g [The chromium (Cr) concentration among the selected fish tissue ranged from 0.28 ± 0.03 to 1.74 ± 0.31. The lowest levels of the chromium concentration in muscle were recorded as 0.28 ± 0.03 μg/g in .92 μg/g ,82.E. suratensis in muscle [The Cr concentration in the present study was almost similar to n muscle . The soun muscle ,85. In an muscle ,87. In cn muscle .3.3C. catla and C. mrigala at all selected fish organs including other heavy metals. The accumulation of Cd and Cu was reported to occur due to the waste of electroplating, petrochemical, production, and chemical-intensive industries [C. catla and C. mrigala had already been observed in other studies [dustries ,91. Accu studies . The neg3.4Bio-concentration factors (BCFs) of heavy metals in fish tissues are the ratio of the heavy metals in tissue to surrounded water . In the 3.5The accumulation of heavy metals in the fish could affect directly the health conditions of the consumers living both in and outside the fishing site and consuming the fish on a daily basis. Therefore, the health risk assessment is essentially needed for fishes coming from contaminated resources. The health risk assessments, which are conducted based on the assumption of the most chemicals with noncancerous effects, exhibit a threshold response . The TarThe intake of heavy metals-contaminated freshwater fish has a high concern for human health ,96. The 4The finding of the present study was compared with national and international standards (BIS and WHO) for drinking water, and it was found that the Ganga River water is not suitable for consumption without proper treatment at the selected sites. The Cu concentration was 4.58 mg/l at Varanasi, while it was 2.54 mg/l at the Allahabad and Mirzapur sites. Pb was 0.54 mg/l at the Kanpur site, 0.62 mg/l at Allahabad, 0.85 mg/l at Mirzapur, and 0.24 mg/l at Varanasi. The Cd concentration was observed 0.54, 0.68, 0.78, and 0.85 mg/l at Kanpur, Allahabad, Mirzapur, and Varanasi, respectively. The Cr, Cd, and Pb were observed over the prescribed safe limits at all sampling sites. Cu was higher at all sites except Kanpur. Zn was observed under the permissible limits (15 mg/l) at all sampling sites. The toxic metals were found accumulated in muscle, gill, and liver, where the highest concentration found in the liver. The high carcinogenic risk for consumers related to Cd, Cr and Pb were found out of permissible limits. Although Estimation of daily intake (EDI) calculation of heavy metals concentration is less than the recommended daily allowance. The heavy metals concentrations in the fish living in the Ganga River were considerably higher than the safe limits suggested by WHO and FAO.According to BAFs of Pb, Cd and Zn are most readily absorbed and bioaccumulation heavy metals in the River Ganga fishes. The THQ was not more than 1 for in all fish species. The bioaccumulation of heavy metals in edible fish species may be considered as a warning for the negative impacts of fish consumption on human health. The present study shows that effective precautionary measures need to be taken in order to prevent future metal contaminants in the Ganga River water. Heavy metals contamination in the fish stock of the Ganga River must motivate imperative, urgent, and corrective actions from all the responsible parties to not only prevent and mitigate the situation, but also protect the well-being of local inhabitants significantly.The authors declare that they have no conflict of interest.


Salmonella enterica by using a compact disc (CD)-shaped device that can reduce reagent consumption in conventional PCR. The detection method, which combines culture and PCR, is more rapid than the conventional culture method and is more sensitive and cheaper than PCR. In this study, we also examined a sample preparation method that involved collecting bacterial cells from food. The bacteria collected from chicken meat spiked with S. enterica were mixed with PCR reagents, and PCR was performed on the device. At a low concentration of S. enterica, the collected S. enterica was cultured before PCR for sensitive detection. After cultivation for 4 h, S. enterica at 1.7 × 104 colony-forming units (CFUs)·g−1 was detected within 8 h, which included the time needed for sample preparation and detection. Furthermore, the detection of 30 CFUs·g−1 of S. enterica was possible within 12 h including 8 h for cultivation.Rapid detection of food-borne pathogens is essential to public health and the food industry. Although the conventional culture method is highly sensitive, it takes at least a few days to detect food-borne pathogens. Even though polymerase chain reaction (PCR) can detect food-borne pathogens in a few hours, it is more expensive and unsatisfactorily sensitive relative to the culture method. We have developed a method to rapidly detect  Salmonella spp., Escherichia coli or Staphylococcus. Especially, Salmonellaenterica is the causative agent of human salmonellosis and one of the most notorious food-borne pathogens worldwide [S. enterica is often caused by ingesting chicken meat or eggs. This is because chickens sometimes carry S. enterica in their intestines. Even if chickens or eggs are not infected by S. enterica when they are produced, after production of these materials, there are various opportunities for infection by S. enterica, before they are consumed, including during processing, transportation, packaging, sales, cooking and serving food. Therefore, rapid detection of S. enterica at all stages from production to eating is necessary to avoid an outbreak. There is a need for faster and simpler screening tests to detect S. enterica in not only foods but also food manufacturing plants.Rapid detection of food-borne pathogens is of great importance to public health and the food industry. Food-borne illnesses are often caused by bacteria, viruses, chemical agents or natural toxins. Half of all food-borne illnesses are caused by bacteria such as orldwide . InfectiS. enterica requires multiple subculture steps followed by biochemical tests. Although the conventional culture method is highly sensitive and can detect a few colony-forming units (CFUs)·mL−1 of S. enterica, it takes at least three days to confirm that food is not contaminated by any Salmonella species, and five to six days to clarify whether food is contaminated by S. enterica [Salmonella genes such as the 16S rRNA gene [invA gene [phoP gene [phoE gene [fimA gene [et al. reported that the limit of detection (LOD) of real-time PCR-based detection of S. enterica was 103 CFUs·mL−1 [The conventional method to detect enterica . Separatenterica ,5,6,7,8.RNA gene , the invFUs·mL−1 , which iet al. reported digital PCR on Lab-on-a-Disk in 2010 [et al. also reported digital droplet PCR on Lab-on-a-disk in 2015 [The microfluidic devices are useful to reduce the reagents because the handling of a small amount of reagent is enabled in the devices. Especially, the development of the centrifugally driven microfluidic platforms, which are generally referred to as “Lab-on-a-Disk”, is of great interest . The adv in 2010 . The PCR in 2015 . The PCR−1 or lower, almost all bacterial cells were isolated as a single cell into each microchambers on the device. Moreover, we successfully achieved sequential PCR of the invA gene of isolated S. enterica by hot cell-direct PCR without the need to extract DNA [i.e., 25 μL. Furthermore, in the conventional PCR method, 1 μL of PCR reagent in a tube is too little to perform PCR normally, because the reagent is evaporated. In our device, the LOD of S. enterica is 5 × 104 cells·mL−1 [S. enterica in food. Furthermore, since food may be contaminated by various bacteria, one objective of this study was to be able to detect S. enterica from other bacterial cells using the device. Therefore, at first, we examined the specific detection of S. enterica from a mixture of S. enterica and E. coli, which is the most common bacterium. The food sample selected for this study was ground chicken because S. enterica infection is often caused by consuming chicken. Moreover, we evaluated the speed of detection of S. enterica in food using a combination of culture and PCR to improve the LOD of S. enterica in the device. By combining the culture method and PCR on the CD-shaped device, our detection method was more rapid than the conventional culture method, and more sensitive than real-time PCR based method.We have developed a compact disc (CD)-shaped device for single cell isolation to detect isolated cells easily and rapidly without the need for a micro-pumping system . By spinract DNA . In thislls·mL−1 . TherefoS. enterica and E. coli were purchased from RIKEN BRC . Nutrient broth was purchased from Sanko Junyaku Co., Ltd. . Nutrient broth with 0.5% NaCl medium (NB medium) (pH 7.2) and buffered peptone water (BPW) (pH 7.2) was used to grow S. enterica while Lysogeny Broth (LB) medium (pH 7.0) was used as the growth medium for E. coli. Bacterial cells were cultivated in liquid media overnight at 37 °C. Cell concentration was confirmed under an optical microscope and diluted to the desired concentration with 10 mM phosphate buffer solution (PBS). Furthermore, the number of CFUs was confirmed by cultivation on agar medium at 37 °C for 24 h.Salmonella Detection Kit Ver. 2.0 . This kit contains a 2× Cycleave reaction mixture and a 5× SIN primer/probe mix. The Cycleave reaction mixture contains Taq polymerase, RNaseH, buffer, dNTP mixture and internal control DNA. The internal control DNA is a sequence non-related to the target gene and works to detect false negatives. When the target gene is not detected, and the internal control DNA shows a positive signal, this indicates the absence of PCR inhibition and that the concentration of the target gene in the sample is below the detection limit. When both target and internal control are not detected, this indicates that PCR does not occur properly and that a reaction inhibitory factor exists in the sample. The SIN primer/probe mix is a mixture of primer and probe to detect the invA gene and internal control DNA. The probe for the invA gene was labeled with 6-carboxyfluorescein (FAM) and a quencher. The probe for the internal control DNA was labeled with X-Rhodamine (ROX) and a quencher. The increase in fluorescence from FAM and ROX occurred after an amplification of the invA gene and internal control DNA, respectively. After the probe is hybridized to the PCR product, it is cut by RNaseH. Then fluorescence intensity is increased by uncoupling the quencher from the probe. The reaction mixture consisted of a 1× Cycleave reaction mixture, a 1× SIN primer/probe mix, 0.5 U·μL−1 Taq polymerase (TaKaRa EX Taq hot start version) and the desired concentration of the suspension of S. enterica. The reaction mixture without S. enterica was used as the negative control.PCR was performed using a CycleavePCR S. enterica recovered by Percoll®  was used as a template in real-time PCR, which was performed with a 7500 Real-Time PCR System . Thermal cycling was initiated at 95 °C for 2 min to lyse S. enterica, followed by 40 cycles of 95 °C for 5 s, 55 °C for 10 s, and 72 °C for 30 s.The number of washing treatments necessary to remove the inhibitor from ground chicken in PCR was also evaluated. In this experiment, 5 μL of The CD-shaped device was fabricated according to a previously described method . In brieS. enterica using the CD-shaped device was performed by the detection system as shown in S. enterica, followed by 40 cycles of 95 °C for 5 s, 55 °C for 10 s, and 72 °C for 10 s. After PCR, the fluorescence intensity of each microchamber was measured by the detection system. The relative fluorescence intensity (RFI) is expressed as: We previously developed a detection system that could detect the fluorescence image of all microchambers in a microchannel of the CD-shaped device before and after PCR . As showS. enterica in ground chicken, 500 μL of sediment after Percoll®  liquid medium and cultured at 37 °C for 4 or 8 h. After cultivation and washing with PBS, this S. enterica sample was detected on the CD-shaped device.As shown in S. enterica and E. coli.Bacterial samples from chicken meat as the food sample were prepared as follows. Ground chicken was obtained from a local grocery and used as the food sample. To recover bacteria from chicken meat, 5 g of ground chicken and 45 mL of BPW were mixed in a stomacher bag . After stirring for 1 min, 100 μL of filtrate, which was passed through the filter of the stomacher bag, was cultivated on NB agar medium and LB medium at 37 °C to detect the existence of S. enterica cells was spiked to 5 g of ground chicken and mixed with 45 mL of BPW in the stomacher bag. After stirring for 1 min, 1 mL of filtrate was mixed with 0.7 mL of Percoll®. After the mixture was centrifuged at 14000 rpm for 15 min at 4 °C, 0.5 mL of sediment was collected as the S. enterica sample. After washing the sample with 10 mM PBS one to three times, part of it served for hot cell-direct PCR while another part was cultivated on NB agar medium at 37 °C.Samples were prepared for PCR as follows b. The deS. enterica contamination in food would require discrimination from among various bacteria. Therefore, we examined whether S. enterica was selectively detected by our detection system from a sample containing many general bacteria. In this study, 103 CFUs·μL−1 of E. coli was used as the general bacteria, because the standard value of the number of the general bacteria for non-heated food was less than 106 CFUs·g−1. In this experiment, 50, 100 and 400 cells·μL−1 of S. enterica with 1000 cells·μL−1 of E. coli were examined. −1 of S. enterica with 1000 cells·μL−1 of E. coli. To determine the detection threshold value of S. enterica, RFI of the negative control was measured on the CD-shaped device and was lower than 1.4. Therefore, microchambers displaying an RFI value greater than 1.4 were identified as those containing S. enterica in this study. The chamber with RFI value greater than 1.4 was observed around the turn of microchannel. We observe such localization on the isolation of rod-like bacteria such as salmonella sp. but no localization of ball-shaped cell and particle was observed [−1 of S. enterica, respectively. Furthermore, these numbers depended on the concentration of S. enterica. Therefore, we could selectively detect S. enterica in a sample containing large number of E. coli cells. In our previous report [−1 of S. enterica in our CD-shaped device was 90 microchambers among 200 microchambers. The observed number of microchambers with RFI value greater than 1.4 was larger than expected number. In this experiment, cell concentration was confirmed under an optical microscope but not by colony formation, so that we might detect dead S. enterica cells as well as living cells.The detection of observed . Such los report , the numS. enterica-spiked chicken, the number of bacteria contaminated in chicken purchased from a grocery store was confirmed by the colony counting method. The concentration of general bacteria contaminated in chicken was 200 CFUs·g−1, but no S. enterica cells were found. Generally, the infection by S. enterica is often caused by ingesting food contaminated by more than 105 to 106 CFUs [S. enterica-spiked chicken was examined. At first, 2.3 × 107 CFUs of S. enterica was added to 5 g of ground chicken and 45 mL of BPW in a stomacher bag. After treatment with Percoll®, the number of S. enterica cells, which were collected from S. enterica-spiked chicken, was confirmed by colony counting. The concentration of S. enterica was 2.7 × 106 CFUs·g−1. Therefore, the recovery rate for S. enterica was 59% after Percoll® treatment. The remaining 41% of S. enterica would likely be adsorbed to the surface of the ground chicken or the stomacher bag.Generally speaking, ground chicken, which is purchased from a grocery store, has a small amount of bacteria. Therefore, before examining the recovery rate for 106 CFUs . Then, t® treatment was examined. In this experiment, 1.5 × 109 CFUs·g−1 of S. enterica was spiked to ground chicken. As a control sample, the same concentration of S. enterica in PBS was examined. As shown in S. enterica was not spiked to chicken (control), the RFI of FAM for the invA gene and the RFI of ROX for the internal control DNA were 2.7 and 10, respectively. After Percoll® treatment and washing one to three times, the RFI of FAM was 2.6, 2.7 and 2.8, respectively while the RFI of ROX was 6.3, 9.8 and 11, respectively. The low RFI of ROX observed after washing once indicated that the inhibitor in the food sample was not sufficiently removed. In contrast, the inhibitor in food samples was sufficiently removed when samples were washed at least twice. Therefore, the number of washes was set to two to sufficiently remove the inhibitor.To sufficiently remove the inhibitor to PCR in the food sample, the number of washes after PercollS. enterica sample spiked to ground chicken was examined on the CD-shaped device. As the threshold value of RFI for the detection of S. enterica was 1.4 on the device, a microchamber with an RFI value greater than 1.4 was identified as one containing S. enterica. Spiked concentrations of 4.6 × 107, 4.6 × 106, 4.6 × 105 and 4.6 × 104 CFUs·g−1 were examined immediately after washing. As shown in 7, 4.6 × 106, 4.6 × 105 and 4.6 × 104 CFUs·g−1, 170, 24, 6 and 0 microchambers, respectively exceeded the threshold. Furthermore, for every concentration, microchambers with an RFI value lower than the threshold were observed: the average RFI of those microchambers was approximately 1.2, almost equal to that of the negative control. In the case of 4.6 × 105 CFUs·g−1, the concentration of S. enterica in the reaction mixture was expected to be 9 CFUs·μL−1. Taking the recovery into consideration, the number of microchambers that exceeded the threshold was expected to be three or four, while the observed number was six in this experiment. This result might suggest that dead S. enterica were detected by PCR. Furthermore, cultivation before PCR is necessary to detect S. enterica at concentrations lower than 4.6 × 104 CFUs·g−1.After the number of washes was optimized, the S. enterica collected from ground chicken following filtration and Percoll® treatment was cultivated before PCR and detection on the CD-shaped device. 6, 1.7 × 105 and 1.7 × 104 CFUs·g−1 was 184, 124 and 10, respectively. In contrast, without cultivation, six microchambers exceeded the threshold when S. enterica concentration was 1.7 × 106 CFUs·g−1. Furthermore, when S. enterica concentration was 1.7 × 105 and 1.7 × 104 CFUs·g−1, no microchambers exceeded the threshold. Therefore, the detection of S. enterica by PCR when S. enterica concentration was 1.7 × 104 CFUs·g−1 was confirmed within 8 h between sampling and detection. The number of microchambers that exceeded the threshold after cultivation for 8 h is shown in −1, the number of microchambers that exceeded the threshold was 105, 37, 30 and 20, respectively. The sensitive detection of 30 CFUs·g−1 of S. enterica was possible on the CD-shaped device in 12 h, which included 8 h of cultivation. These results demonstrated that this method is more than 300 times more sensitive than the real-time PCR method and more than 10 times as rapid as the conventional culture method. Furthermore, detection of the number of only viable bacterial cells is also important in food-borne illness. By cultivating S. enterica before PCR in the CD-shaped device, we could detect only viable S. enterica in a sample carrying a low concentration of this bacterium because not only viable S. enterica but also dead bacterial cells were not detected at less than 4.6 × 104 CFUs·g−1 of S. enterica without cultivation.To achieve highly sensitive detection, S. enterica after sample preparation was 59%. The inhibitor in the food sample was sufficiently removed after two washes with 10 mM PBS. The detection of S. enterica at 4.6 × 106 CFUs·g−1 was confirmed after 4 h without cultivation before PCR. After cultivation for 4 h, S. enterica at 1.7 × 104 CFUs·g−1 was detected after 8 h. Furthermore, the sensitive detection of 30 CFUs·g−1 of S. enterica was possible on the CD-shaped device within 12 h, which included 8 h for cultivation. Our detection method is more than 10 times as rapid as the conventional culture method and more than 300 times more sensitive than the real-time PCR method. Cultivating S. enterica before PCR allows for the rapid and sensitive detection of viable S. enterica in a sample of S. enterica in food.The recovery rate for


In this work, muscovite single crystal was irradiated with Co-60 γ ray in air at a dose rate of 54 Gy min−1 with doses of 0–1000 kGy. Then, structure transformation and mechanism were explored by Raman spectrum, Fourier-transform infrared spectrum, X-ray diffraction, thermogravimetric analysis, CA, scanning electron microscope and atomic force microscopy. The main results show that variations in the chemical/crystalline structure are dose-dependent. Low-dose irradiation sufficiently destroyed the structure, removing Si–OH, thus declining hydrophilicity. With dose increase up to 100 kGy, CA increased from 20° to 40°. Except for hydrophilicity variation, shrink occurred in the (004) lattice plane which later recovered; the variation range at 500 kGy irradiation was 0.5% close to 0.02 Å. The main mechanisms involved were framework break and H2O radiolysis. Framework break results in Si–OH removal and H2O radiolysis results in extra OH introduction. The extra introduced OH probably results in Si–OH bond regeneration, lattice plane shrink and recovered surface hydrophilicity. The importance of framework break and H2O radiolysis on structure transformation is dose-dependence. At low doses, framework break seems more important while at high doses H2O radiolysis is important. Generally, variations in the chemical structure and surface property are nonlinear and less at high doses. This indicates using the chemical structure or surface property variation to describe irradiation is correct at low doses but not at high doses. This finding is meaningful for realizing whether muscovite is suitable for detecting high-dose irradiation or not, and mechanism exploration is efficient for identifying the procedure for defect formation within the matrix of clay used in disposal HLRW in practice.Intensive study on structure transformation of muscovite single crystal under high-dose  In addition to being a source of power, benefiting economy and defence, two features are crucial for sustainable development of this energy. One is sufficient shielding or detecting irradiation as irradiation is accompanied by nuclear energy  and is very dangerous . The oth2) [2 generates toxic gas under irradiation [In the field of irradiation detection, for good stability and low cost, muscovite is proposed as a detector –9. Its s2) . However2) –14, the 2) , the comadiation . In thisadiation  and tranadiation ,16. It madiation . Thus, a2O radiolysis [+, UO2+ diffusion [3+ reduction [2O radiolysis [For disposal of HLRW, deep geological disposal is recommended . In thisdiolysis ,26, thusdiolysis . In thisdiolysis ,29. Natuiffusion ,31, Fe3+eduction ,32, H2O diolysis –35). In diolysis –41, sewadiolysis –44, drugdiolysis , etc.2O normally exists in the material itself or in the environment. On exposure to irradiation, matrix, impurity and H2O would generate numerous radicals. They might react with each other leading to a complex product. Thus, mechanism exploration and stability evaluation are difficult [To date, the main mechanism and stability are not clearly understood for the disposal of HLRW due to the complexity of material composition and environmental conditions. Normally, clay is a composite containing numerous impurities like oxides, organics, which account for as much as 40% . Additioifficult .In reality, the main property of clay is that of a matrix. Having a clear understanding of stability and mechanisms for defect formation within the clay matrix is useful. The clay matrix is a phyllosilicate and defects in phyllosilicate are similar . Taking 2(AlSi3O10)(OH)2-single crystal is a pure phyllosilicate crystal showing a ‘2 : 1’ layered structure (T–O–T). Two SiO4 tetrahedron sheets are linked together by an AlO6 octahedron sheet forming a tri-layer structure. One-fourth tetrahedral Si are substituted by Al and one-third octahedron sites show vacancies [+ ion via weak ionic bonds [2O in this material is low, thus reducing the complexity of the material component and making the system simple, benefiting understanding. In this case, the obtained variation could be mainly ascribed to the matrix, benefiting the speculation for defect formation. Normally, H2O is difficult to be removed completely [γ-ray irradiation seems more important due to its strong penetration. For strong penetration, γ ray can even penetrate packing material and is widely used for radiation modification [γ-ray irradiation on muscovite seems more meaningful.Muscovite-KAlacancies ,50. Vacaic bonds . For strmpletely ,34,54, afication –57. Explγ ray in air at a dose rate of 54 Gy min−1 with doses up to 1000 kGy. Then, the variation in the structure and intrinsic mechanism was investigated. The main objectives of this work were to (1) explore the sensibility of muscovite under high-dose irradiation to ensure whether it is suitable for detecting high-dose irradiation or not, (2) understand the mechanism for structure transformation, and (3) better understand the mechanism for defect formation within the matrix of clay used in the disposal of HLRW. The main results show muscovite to be sensitive to low-dose irradiation but not to high dose. Using this material to detect high-dose irradiation is improper. H2O radiolysis is essential especially at high doses.Therefore, in this work, muscovite single crystal was irradiated with Co-60 2.2.1.2(AlSi3O10)(OH)2-single crystal film  was bought from the University of Cambridge, U.K. Its accurate composition has been analysed and reported as MUS2 in the literature [Muscovite-KAlterature ,59.2.2.γ ray in air at the Institute of Nuclear Physics and Chemistry, China Academy of Engineering Physics  at room temperature, with a dose rate of 54 Gy min−1 with doses up to 1000 kGy. Then, the samples were stored in air at room temperature before characterization.Prior to irradiation, a film with a thickness less than 200 µm was dried at 65°C for 5 h to remove the absorbed water. Then, the film was wrapped with aluminium foil and irradiated with Co-60 2.3.2.3.1.−1 with a spectrum resolution of 0.9 cm−1, a 532 nm laser source and a power of 4.5 mW.Raman spectrum (RS) experiments were carried out on a Nicolet ALMEGA XR Instrument from 90 to 1300 cm2.3.2.−1 with a spectrum resolution of 4 cm−1, 32 scans per spectrum. Transmission-mode experiments were performed on a Thermo Fisher Nicolet iS50 spectrometer from 2400 to 4000 cm−1 with a spectrum resolution of 2 cm−1, 32 scans per spectrum. The sample prepared for the transmission-mode experiment was cut into a square with a size of 16 mm × 18 mm, and the spectrum was normalized by mass as 10 mg.Reflection-model experiments were performed on a Bruker Tensor 27 spectrometer from 400 to 4000 cm2.3.3.α irradiation (λ = 0.15418 nm) with a voltage of 40 kV and a current of 40 mA. Step size and scanning 2-theta (2θ) were set to be 0.02° and 5–90°, respectively, and all patterns were analysed by Jade 5 software.X-ray diffraction (XRD) experiments were carried out on a D8 Advances X-ray powder diffractometer by using Cu k2.3.4.−1, and an argon flow of 50 ml min−1.Thermogravimetric analysis (TGA) experiments were carried out on a Netzsch STA 449 F3 instrument from 50 to 500°C with a heating rate of 10°C min2.3.5.Static contact angle (CA) experiments were performed on an XG-CAM contact Angle Meter. A drop of 2 µl purified water was dropped onto the sample surface and a picture was recorded by a camera immediately . Then, C2.3.6.Scanning electron microscope (SEM) measurements were carried out on a Zeiss MERLIN Compact 14184 instrument with an acceleration voltage of 8 kV. Prior to the measurement, a thin layer of gold was coated on the sample surface to increase electrical conductivity .2.3.7.Atomic force microscopy (AFM) experiments were performed on an NTEGRA Prima instrument (NT-MDT Co.). Prior to the measurement, a thin layer of double-faced adhesive tape was fixed on a glass slide, then the film was fixed on the tape. The tapping mode of scanning was adopted and datum was analysed by Nova-px software.3.3.1.−1 corresponding to vibrations of isosceles triangle O–H–O [6 octahedron [4 tetrahedron [−1 are assigned in −1 can be assigned to breathing vibration of the tetrahedron sheet [2–OH [Raman and Fourier-transform infrared (FT-IR) spectra are widely used to characterize the chemical structure. le O–H–O , AlO6 octahedron  and SiO4rahedron , respecton sheet , or Si–Oon sheet  or libraet [2–OH ,50. In t4 might be stable. This is because the broken Si, Al or O atoms cannot leave from their positions because of the linkage of adjacent atoms. In this case, the broken bonds (Si–O or Al–O bonds) can even be regenerated. Finally, the species of the chemical bond within the tetrahedron sheet seems to vary slightly after irradiation. For the AlO6 octahedron, OH vibration was observed, while the affiliation is uncertain. For instance, the band at 409 cm−1 can be assigned to the Al2–OH libration [4 bending [6 octahedron seems less. Normally, γ-ray irradiation leads to obvious variation in the chemical structure when partial atoms are removed or there is extra species participation [No internal band disappeared or extra band appeared after irradiation in the Raman spectra, indicating a slight variation in the framework. This can be explained as follows. Normally, RS is sensitive to nonpolar vibration such as vibration of the C–C bond . In thisibration  or SiO4  bending . Additiocipation ,62,65,66γ-ray irradiation at 0–1000 kGy. Several bands at 685, 744, 803, 895 and 967 cm−1 were observed mainly related to vibrations of the tetrahedron sheet  observed in that spectrum (z-direction is strong (the included angle between the z-direction and OH is less than 30°) [−1 was integrated and is shown in figure 3b. Generally, the band area increased with the increase in dose. For the 1000 kGy-irradiated sample, the band area was larger than the pristine sample by nearly 20%, showing that numerous Al–OH were introduced, meaning dehydroxylation was not the dominant reaction during the irradiation process. As the sample did not contact other species except air during the irradiation process, the extra OH pull-in was probably due to H2O radiolysis as partial H2O was lying on the surface, interlayer or edges [2O radiolysis rate could decline.tahedron . This isspectrum . This ishan 30°) , and Si–or edges . In realNormally, quantitatively analysing FT-IR spectrum should assign an internal standard band. Using mass to quantitatively analyse the FT-IR spectrum is unusual. In reality, we have adopted that strategy. Nevertheless, the band at a low wavenumber is easy to saturate and the vibration for OH is weak. In this case, when the signal for OH is visual, the signal at low wavenumber is saturated when the signal at the low wavenumber is proper, and the signal for OH is weak. It is difficult to use an internal standard band to quantitatively analyse the OH amount in the FT-IR spectrum obtained by the transmission-mode experiment. Thus, the spectrum was normalized by mass. We think this strategy is also effective.2O radiolysis. The extra Al–OH group's introduction indicates dehydroxylation was not dominant.Generally, Raman and FT-IR analysis indicate irradiation had no intense effect on the matrix while having an obvious effect on the microstructure. Upon irradiation, the Si–OH bond was destroyed at a low dose then regenerated at a high dose. Simultaneously, an extra Al–OH bond was introduced, probably ascribed to H3.2.a shows XRD patterns for muscovite under γ-ray irradiation at 0–1000 kGy. The main lattice planes were assigned by Jade 5 software according to standard PDF cards. For the pristine sample, there were five main lattice planes assigned as (002), (004), (006), (008) and (224) with the corresponding 2θs as 8.88°, 17.76°, 26.81°, 36.00° and 45.43°, respectively. For irradiated samples, patterns were similar to the pristine sample. For all samples, several peaks with 2θs as 55.19°, 64.43° and 76.28° were observed, probably due to lattice plane repetition as the method used was powder diffraction while sampling in film. Generally, XRD patterns did not show an obvious change in peak shape and position for the sample after irradiation, implying no obvious variation in species. If species were altered, patterns would vary obviously as different materials or phases have distinct lattice plane parameters. A similar pattern indicates no serious decomposition or phase transformation has occurred during the irradiation process. Although no serious transformation occurred, partial variation in micro probably occurred.The extra introduced OH might alter the crystalline structure, probably resulting in crystal shrink or expansion. Correspondingly, the variation in the crystalline structure could reflect the variation in the chemical structure, benefiting mechanism exploration. Figure 4b shows refined XRD patterns for (002), (004), (006) and (008) lattice planes. All patterns are similar while generally shifting to a higher angle at low dose than recovered at a dose higher than 500 kGy. Taking the (004) lattice plane as a representative, pristine, 500 kGy-irradiated and 1000 kGy-irradiated samples had 2θs as 17.76°, 17.85° and 17.77°, respectively. According to Bragg's formula (nλ = 2dsinθ), for the refined lattice plane and measurement condition, n and λ are constant. In this case, increased θ means reduced d [Figure 4educed d . In othed for the (004) lattice plane was investigated. For the aforementioned 2θs, the ratio of the interlayer space for 500 kGy-irradiated and pristine sample (d500/d0) is 99.4% (d500/d0 = sinθ0/sinθ500 = sin8.88°(17.76/2)/sin8.93°(17.85/2) = 0.9944 × 100% = 99.44%), meaning 0.5% (100–99.44% ≈ 0.5%) shrink under 500 kGy irradiation. The ratio of the interlayer space for 1000 kGy-irradiated and pristine samples (d1000/d0) is 99.9% (d1000/d0 = sinθ0/sinθ1000 = sin8.88°(17.76/2)/sin8.89°(17.77/2) = 0.9989 × 100% = 99.89%), meaning interlayer space d was recovered by extra irradiation. To understand variation more accurately, interlayer space d for this lattice plane was calculated by Jade 5 software on the aforementioned 2θs and λ = 0.15418 nm. d0, d500 and d1000 were 4.985 ± 0.002, 4.962 ± 0.002 and 4.981 ± 0.004 Å, respectively, and d500/d0 = 4.962/4.985 ≈ 0.9954 ≈ 99.54%, d1000/d0 = 4.981/4.985 ≈ 0.9991 ≈ 99.91%. The result is close to that compared by sin θ. Generally, under 500 kGy irradiation, the (004) lattice plane shrinks nearly by 0.5%, closing to 0.02 Å (4.985 (± 0.002) − 4.962 (± 0.002) = 0.023 (± 0.002) Å).To quantitatively describe this variation, interlayer space z-direction. The formation of hydrogen bond strengthens the interlayer force within tetrahedron and octahedron sheets [d of the (004) lattice plane declined, showing shrinking. This is interesting. This is probably mainly due to framework break and hydrogen bond formation. The broken bonds are probably the linkage between tetrahedron and octahedron sheets as Al–O–Si or Al–O–Al bonds as the break of the Al–OH bond would destroy hydrogen bond resulting in expansion for muscovite [z-direction. Naturally, it is the scale of the Si(Al)–O–Al–O–Si(Al) bond length in the z-direction. Except for chemical bond break, an extra Al–OH bond was introduced  effect easily inducing the displacement of atoms in a lattice [γ-ray irradiation as LET effect for γ ray is weak. For low LET effect, it is difficult to deposit huge energy in lattice. Thus, the temperature elevation in lattice and atom vibration enhanced by temperature elevation can be ignored. In this case, γ-ray irradiation cannot result in the displacement of atoms efficiently, only via random ionization or motivation can the chemical bond be broken to affect the crystalline structure. Thus, variation in lattice induced by γ-ray irradiation can be tiny. Normally, for solid material, a tiny variation in lattice could alter the macroscopic property obviously. Thus, the effect of variation range near 0.5% cannot be ignored. In reality, the refined pattern for the (006) lattice plane with 2θ near 26.81° varied obviously, especially for the 200 kGy-irradiated sample. The pattern for this sample also showed split (b).The variation range  may be small compared to variation induced by ionizing irradiation such as Au lattice ,76. Finaed split b.Generally, XRD experiments show that the (004) lattice plane shrunk near 0.5% upon 500 kGy irradiation. The main reasons probably derive from framework break and hydrogen bond formation. Lattice plane shrink also indicates dehydroxylation was not dominant, confirming the FT-IR results.3.3.2O radiolysis. The shrink of the (004) lattice was probably due to extra hydrogen bond formation. In this case, the H2O amount should decline. It is essential to certify this process. In early reports [2O that originally existed. Thus, TGA could characterize H2O amount variation.The extra introduced Al–OH bond certified by the FT-IR spectrum was probably due to H reports ,77–85, f2O, the slight decline could be ascribed to H2O volatilization as H2O generally exists in clay [ in clay ,86 and c in clay ,87,88.F2O is linear with its content and H2O amount within the sample before irradiation. In this case, variation in mass reduction could be related to the irradiation process. The pristine sample has the largest mass reduction at 4.3% (100 − 95.7% = 4.3%). Five hundred and 1000 kGy-irradiated samples have mass reductions of 2.2 (100 − 97.8% = 2.2%) and 3.9% (100 − 96.1% = 3.9%), respectively. In other words, the H2O amount in pristine, 500 kGy-irradiated and 1000 kGy-irradiated samples can be considered to be 4.3%, 2.2% and 3.9%, respectively, showing a declined H2O amount in the irradiated sample.Simultaneously, we hypothesize that the volatilization of H2O amount declined, which is interesting. To our knowledge, γ-ray irradiation is a cold irradiation model compared to ionizing irradiation like electron-beam irradiation [2O induced by temperature elevation related to irradiation during the irradiation process can be ignored. Because of this, the reason for the decline in amount of H2O could be ascribed to its radiolysis as H2O is easy to radiolyse [After irradiation, the Hadiation , and canadiolyse ,90 and r2O amount in the irradiated sample also indicates dehydroxylation was not the dominant reaction during the irradiation process as that reaction would raise the H2O amount. If that procedure was dominant, numerous Al–OH bonds would be destroyed, hydrogen bond (triangle O–H–O) [2O amount observed indicates H2O radiolysis is essential. Although dehydroxylation was not dominant, we cannot be sure whether this procedure occurred or not as the dynamic procedure is difficult to observe in situ.The decline of He O–H–O)  would bee O–H–O) . This wo2O amount in the 500 kGy-irradiated sample is lower than that in the 1000 kGy-irradiated sample and the H2O amount in the 1000 kGy-irradiated sample is close to that in the pristine sample. These results can support FT-IR analysis commendably as the 500 kGy-irradiated sample has the most intense signal for OH vibration  [d. Additionally, partially introduced OH might form hydrogen bonds. Finally, lattice plane shrink was observed. These assumptions could probably explain the conflict.Normally, FT-IR and TGA analyses indicate Hfor mica ,91. As se O–H–O) , its str2O amount in irradiated samples, indicating dehydroxylation was not the dominant reaction during the irradiation process. The main reason was probably ascribed to H2O radiolysis. In this case, the TGA result can certify extra Al–OH bond introduction and lattice plane shrink indirectly.Generally, TGA measurements show a declined H3.4.a shows optical images of water droplets on the sample surface. For the pristine sample, the droplet of water almost spreads out completely, showing excellent hydrophilicity. For irradiated samples, the sprawl of droplet is similar to the pristine sample except for the 100 kGy-irradiated sample, also showing nice hydrophilicity. It seems that hydrophilicity declined at a lower dose than recovered by extra irradiation.Chemical structure variation would alter surface properties such as wettability . Static Figure 6b. The pristine sample has the smallest CA by approximately 20°. Irradiated samples have varied CAs. For instance, CA increased to approximately 40° with dose increases to 100 kGy then decreased to approximately 23° with dose increases to 200 kGy. Then, CA seems to be constant within 23–26° with dose continuously increasing to 1000 kGy. This indicates that low-dose irradiation sufficiently declined hydrophilicity. In other words, the effect of irradiation on surface hydrophilicity is dose dependent. This can be explained as follows. According to FT-IR analysis, extra OH was introduced, promoting hydrophilicity. Except for OH introduction, surface Si–OH was removed, declining hydrophilicity. During the irradiation process, the aforementioned procedures occurred synchronously and had offset effects on the obtained CA. Finally, the mechanism for CA variation is complex. In reality, Si–O, Si–OH and Al–OH bonds are hydrophilic [+). In this case, variation in in-plane probably does not affect hydrophilicity. During the irradiation process, surface destruction cannot be avoided [b). In this case, surface hydrophilicity declined at low doses. With dose increases, H2O radiolysis became serious. Partial H• or HO• radicals might react with the tetrahedron sheet, regenerating Si–OH or Al–OH bonds, promoting hydrophilicity. Additionally, the extra introduced OH or destruction might increase micro-roughness, probably enlarging the specific surface area, strengthening the interfacial force between the tetrahedron sheet and H2O, promoting hydrophilicity. In this case, wettability can be recovered by extra irradiation and a spinodal was observed in CA. These assumptions could explain CA decrease with dose increase from 100 to 200 kGy.To quantitatively describe variation within hydrophilicity, CA was calculated and shown in figure 6rophilic ,94. In t avoided ,33,95 anb). Normally, an increase in the OH amount could promote hydrophilicity. Nevertheless, the material is hydrophilic, even with a high increase in the OH amount, CA would not decrease obviously. This phenomenon is normal. For instance, radiation grafting polyacrylic acid to increase surface hydrophilicity of a polymer or CNTs, after a threshold, the CA would increase or be constant with increase of degree of grafting. Additionally, the destruction within the tetrahedron sheet can be serious. In this case, CA seems to be constant or have slight variation.With dose continuously increasing from 200 to 1000 kGy, the extra irradiation seems to have no effect on CA. The result is expected and consistent with the FT-IR spectrum as the increased OH amount in this region is close. Simultaneously, the Si–OH bond was regenerated slightly b. Normalb, CA for the 200 kGy-irradiated sample is smaller compared to the 100 kGy-irradiated sample and a spinodal exists at 100 kGy. Whether the decrease of CA for the 200 kGy-irradiated sample is due to the artificial factor or not needs confirmation. To make a comparison, pristine and 1000 kGy-irradiated samples were also observed.Apart for the chemical structure, macro morphology also has a great effect on the obtained CA. For the hydrophilic material according to the Wenzel model, the larger the roughness, the smaller the CA. In this case, if there were grooves or cracks on the sample surface, CA would decrease sufficiently. Its effect might exceed the effect induced by irradiation. In this case, all aforementioned explanations for chemical structure variation would be improper. It is essential to eliminate the artificial factor. In figure 6γ-ray irradiation had no obvious effect on the macroscopic morphology of muscovite. This phenomenon is expected. In reality, γ ray is a high energy photon which almost has no mass compared to the framework atom. In this case, it is difficult to induce displacement of atom directly just by random ionization or motivation. As the density of the photon is incompact and crash between the photon and framework atom is random, the LET effect is slight. In this case, it is difficult to deposit huge energy in the microcell. Thus, it is difficult to induce obvious variation in macro morphology in theory. Even under 197Au (11.4 MeV n−1) ion irradiation for several minutes, which has an intense LET effect, variation in the z-direction is several nanometre for muscovite [γ-ray irradiation, for a weak LET effect, the variation would be smaller. In this condition, the variation could be in Å scale. This tiny scale probably exceeds resolution of SEM or AFM technology.z-direction is tiny as in the nanometre scale, implying smooth. To have a clear realization of the difference in the z-direction, an area is chosen randomly as shown in green colour in the image , the partial section shows large variation range. This is probably due to an existing defect as the sample is natural and in a sheet form. In this case, the surface might show bumps or depressions induced by mining. Additionally, the film is peeled randomly. If the sample contains defects internally, partial sections can be broken during peeling. In this case, partial bumps or depressions can be observed. Except for that region, the sample is generally smooth with a variation range close to ±2 nm in the z-direction in the chosen part. For 100 kGy-irradiated, 200 kGy-irradiated and 500 kGy-irradiated samples, the variation ranges are close to ±0.7, ±0.2 and ±0.6 nm. They are very tiny, showing smooth. In reality, the size of the T–O–T structure in the z-direction is close to 1.2–1.5 nm, the chemical bond length for the K–O ionic bond or the Si–O/Al–O bond is close to 0.2 nm. Seeing the value especially for the 200 kGy-irradiated sample, the variation range is close to ±0.2 nm, which is close to the length of the chemical bond as the K–O ionic bond or Si–O bond. Normally, the distribution of the interlayer ion (e.g. K+ ion) is random; in this case, the variation in the z-direction at the atom level may be close to 0.2 nm. In this work, the variation range is close to this scale. That means the surface is very smooth.To observe morphology more clearly, AFM experiments were performed. he image . Then, tz-direction after cropping is less than ±2 nm. The range is tiny. Normally, the variation range induced by the artificial factor is large, which can be in µm scale or larger. Trying to induce variation in the nanometre scale by artificial technology is difficult. In reality, this tiny variation needs special instruments or technology, which should be processed precisely such as by laser etching. For samples peeled manually, this tiny variation can be endured. Generally, this tiny variation indicates the sample is smooth, which further confirms SEM results.Generally, for all samples, the variation range in the z-direction. This can be ascribed to sample preparation or difference. For irradiated samples, they seem smoother compared to the pristine sample especially for the 200 kGy-irradiated sample. That cannot be ascribed to irradiation as it is difficult to prepare the sample manually with a constant surface structure. Simultaneously, we cannot assure that the defect in the sheet is uniform. Additionally, variation induced by irradiation in the z-direction would be very tiny as seen from the aforementioned explanations (SEM part). Its variation may be covered by sample preparation or difference. In this case, apparent roughness difference cannot be ascribed to the irradiation process. If we consider the pristine sample has the largest roughness and all samples have similar chemical surface structure, in this case, irradiated samples should have larger CAs especially for the 200 kGy-irradiated sample as this sample seems to have the smallest roughness. Nevertheless, the obtained CA is contrary to this expectation. They have close CAs  and the linkage between tetrahedron and octahedron sheets. Simultaneously, partial H2O occurred in radiolysis, generating H• or HO• radicals. These radicals are reaction-active, reacting with the framework (e.g. the linkage between tetrahedron and octahedron sheets or the broken Si–O bonds), leading to extra OH introduction. During the irradiation process, these procedures occurred synchronously. At low doses, destruction seems predominant. In this case, the Si–OH bond was removed and surface hydrophilicity declined. At high doses, H2O radiolysis seems predominant. In this case, the H2O amount declined, an extra Al–OH bond was introduced, Si–OH bond was regenerated, and surface hydrophilicity recovered. Additionally, partial OH might form hydrogen bonds, resulting in lattice plane shrink. To describe this procedure more clearly, several equations will be used as given below and a scheme will be displayed in z-direction as interlayer space d mainly reflects the scale of the Si(Al)–O–Al–O–Si(Al) bond in the z-direction, ≡Si(Al)–O–Si(Al)≡ represents the TO4 tetrahedron sheet, ≡Si–OH represents the surface Si–OH structure and ≡Si(Al)–O–Al–OH represents the Al–OH bond in the octahedron sheet. The reason for the Al element written in tetrahedron is due to the one-fourth tetrahedral Si substituted by Al. In reality, we cannot be sure whether breaks occurred in Si–O–Si or Si–O–Al bonds in the tetrahedron sheet.From the aforementioned analysis, it seems that variations in the chemical/crystalline structure and surface hydrophilicity are dose-dependent, and muscovite is sensitive to low-dose irradiation and not to high dose. The main reasons were explained generally. To have a clear cognition, the main mechanisms will be illustrated below. It seems that the main mechanisms involve framework breaks and Hγ-ray irradiation.Equations (3.1)–(3.6) are reactions probably originally induced by 2O radiolysis. Equations (3.7)–(3.10) describe OH introduction, where Equations (3.7) and (3.8) describe Si–OH bond regeneration, probably illustrating hydrophilicity recovery by extra irradiation; Equations (3.9) and (3.10) describe OH introduction in the octahedron sheet, probably illustrating lattice plane shrink. Although equation (3.5) is secondary, this reaction probably illustrates lattice plane recovery at 1000 kGy for OH destruction.and4.γ-ray irradiation at 0–1000 kGy was studied with regard to chemical/crystalline structure and surface properties. After irradiation, the chemical/crystalline structure varied obviously, the H2O amount declined, and surface hydrophilicity declined at low doses. Nevertheless, surface morphology varied slightly. The main results show that the Si–OH bond was removed at low doses, then the Al–OH bond was regenerated at high doses. Simultaneously, crystal shrink occurred at doses lower than 500 kGy. For the 500 kGy-irradiated sample, the (004) lattice plane shrunk by nearly 0.5%. Additionally, CA increased from 20° to 40° with dose increases up to 100 kGy and then became constant with the dose continuously increasing. From the chemical/crystalline structure and surface hydrophilicity analysis, it can be seen that structure transformation is obvious at low doses, while at high doses it is uncertain. That means muscovite is sensitive to low-dose irradiation but not to high-dose (e.g. 500 kGy). Additionally, the main mechanisms were speculated. It seems the main mechanisms involve framework break and H2O radiolysis. These procedures occurred synchronously and framework break seems dominant at low doses while at high doses H2O radiolysis is dominant. For framework break, the Si–OH bond was removed and surface hydrophilicity declined. For H2O radiolysis, extra OH was introduced, H2O amount declined and lattice plane shrink occurred. The finding-framework break seems dominant at low doses while at high doses H2O radiolysis is meaningful for realizing the procedure for defect formation within the matrix of clay used for HLRW disposal in practice.Structure transformation of muscovite single crystal under Some drawbacks of this work are that partial intermediate products were not certified and the main mechanisms were speculated, so further research is needed. However, through this work, we could conclude that muscovite is insensitive to high-dose irradiation. This finding is meaningful for detector development especially for detecting high-dose irradiation. Simultaneously, the mechanism is beneficial for realizing the procedure for defect formation within the matrix of clay used for disposal of HLRW in practice, which is of great significance.