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Exogenous hydrogen sulfide reduces atrial remodeling and atrial fibrillation induced by diabetes mellitus via activation of the PI3K/Akt/eNOS pathway
- Authors:
Affiliations: Center for Comprehensive Treatment of Atrial Fibrillation, Department of Cardiothoracic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China, Department of Rehabilitation, Elderly Rehabilitation Hospital, Suzhou Red Cross Society, Suzhou, Jiangsu 215009, P.R. China
- Published online on: June 30, 2020 https://doi.org/10.3892/mmr.2020.11291
- Pages: 1759-1766
Copyright: © Xue et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
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As the most common sustained arrhythmia diagnosed in the clinic, atrial fibrillation (AF) significantly increases the mortality and morbidity of patients by impairing cardiac function and causing stroke (1). Epidemiological studies have demonstrated that diabetes mellitus (DM) is one of the major risk factors leading to the pathogenesis of AF, which increases the risk by ~40% (2). It was previously reported that autonomic, electrical, electromechanical and structural remodeling, including oxidative stress, connexin remodeling and glycemic fluctuations, triggered by DM, contribute to AF pathophysiology (3). However, the underlying mechanism of the mutual relationship between AF and DM remains mostly unknown, which limits the prevention and treatment of DM-induced AF. Streptozotocin (STZ) is a natural chemical, which can be taken up by pancreatic B cells via the glucose transporter and exert toxic effects. Due to its distinctive effect, STZ is widely applied as a tool to induce experimental DM animal models (4). Hayami et al (5) investigated the impact of a DPP-4 inhibitor on AF in a rat model induced by STZ, and found that the administration of STZ greatly increased atrial fibrosis, but sulfonylurea and DPP-4 inhibitors inhibited inflammation and fibrosis of the atria.
Previous research has demonstrated that inflammation and oxidative stress play a critical role in the relationship between DM and AF (6). Therefore, agents mitigating inflammation and oxidative stress might be useful in preventing DM-induced AF. Hydrogen sulfide (H2S) has drawn considerable attention, not only due to its inherent activity in human physiology, but also because of its potential as a cardiovascular protector (7). Most studies of H2S and cardiovascular disorders have focused on myocardial ischemia/reperfusion injury rather than arrhythmia, especially AF (7,8). In addition, there is evidence that downregulated PI3K-Akt-eNOS expression is related to an increased AF inducibility in diabetic rats (9). In addition, it has been noted that H2S is associated with inflammation and oxidative-stress responses (10,11). In the present study, it was hypothesized that H2S could reverse AF and reduce the incidence of AF in diabetic rats via the PI3K-Akt-eNOS signaling pathway, so the effect of H2S on DM-induced AF and its underlying mechanisms were explored.
Materials and methods
Animal model
A total of 80 adult Sprague-Dawley (SD) rats were used in this study, which was approved by the Experimental Animal Administration Committee of the Second Military Medical University (Shanghai, China). Animal experiments were performed following the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85–23, revised 1996). The 12-week-old male rats (weight 200–220 g) were randomly divided into the control group, the DM group, the H2S group, and the DM+H2S group (each group included 20 rats). All rats were fed a routine diet and maintained in a standard room environment (18-23°C, humidity 40–60%, 12-h light/dark cycle, and free access to food and water). Animal health and behavior were monitored and evaluated every week, including general condition, appetite, sleep status and survival.
Rats of the DM group and the DM+H2S group were administered STZ (60 mg/kg; Wako Chemicals USA, Inc.) dissolved in citrate buffer (Sigma-Aldrich; Merck KGaA) intraperitoneally (i.p.) at the age of 12 weeks. After one week, the rats were tested for fasting blood glucose (FBG) level. The fasting time was 16 h. If the FBG level was >200 mg/dl, then the rats were selected for the following experiments (5). At the same time, the control group and the H2S group were administered citrate buffer only. After the injection, the H2S group and the DM+H2S group were administered 15 µmol/kg (i.p.) sodium hydrosulfide (NaHS, exogenous H2S donor; Sigma-Aldrich; Merck KGaA) every week for 8 consecutive weeks, whereas the control group and the DM group were administered normal saline only. At the age of 20 weeks, all rats were subjected to the following experiments.
Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT)
As previously reported (12), IPITT and IPGTT were conducted at the age of 20 weeks. In terms of IPGTT, the rats were fasted overnight for 15 h, and were given 1.5 gr/kg glucose through an i.p. injection. Blood was collected from the tail vein for glucose measurements at 0, 10, 20, 30, 60, 90 and 120 min after glucose injection. As for IPITT, the rats received 2.5 U/kg insulin via i.p. injection, and then blood was drawn from the tail vein for glucose measurements at 0, 20, 30, 40, 60, 90 and 120 min after insulin administration.
Intracardiac programmed electrical stimulus
As previously described, all groups were examined with electrophysiological characteristics via intracardiac programmed electrical stimulus (13). Each animal was anesthetized with 2% isoflurane (Beijing Hongxin Technology) inhalation and fixed on a heated pad at the prone position to maintain 37°C body temperature. Surface electrocardiogram (ECG; B&W TEKSystems, Inc.) and intracardiac electrograms were recorded simultaneously. P wave duration and an indicator of intra-atrial conduction time were measured. An electrode catheter (1.1 F; Millar) was placed into the right atrium through the right jugular vein. A burst of electrical stimuli for 30 sec was used to test the inducibility of atrial arrhythmias. The burst test was conducted 3 times, and >two incidents of AF observed (P wave disappeared and lasted >2 sec) was judged as the onset of AF. LabChart Software 7.0 (ADInstruments) was used to record and analyze electrocardiogram data.
Western blot analysis
As previously described (14), expression levels of key proteins of the PI3K/Akt/endothelial nitric oxide synthase (eNOS) pathway in the atrial myocardium were examined with western blotting. The membranes were incubated with the following primary antibodies overnight at 4°C, anti-PI3K (1:2,000, cat. no. ab151549; Abcam), anti-Akt (1:2,000, cat. no. ab8805; Abcam), anti-phosphorylated (p)-Akt (1:500, cat. no. 9611; Cell Signaling Technology, Inc.), anti-p-eNOS (1:500, cat. no. ab184154; Abcam), anti-eNOS (1:500, cat. no. ab76198, Abcam) and anti-β-actin (1:5,000, cat. no. ab179467; Abcam). Following the primary incubation, membranes were incubated with rabbit anti-mouse (1:5,000, cat. no. ab150113; Abcam) or goat anti-rabbit HRP-conjugated secondary antibody (1:5,000, cat. no. ab150077; Abcam). Gel Imaging system (Bio-Rad Laboratories, Inc.) and ImageJ software (Version 1.8, National Institutes of Health) were used to image and analyze the western blotting bands.
Reverse transcription-quantitative (RT-q)PCR
Total RNA from atrial myocardium samples was extracted using TRIzol (Takara Biotechnology Co., Ltd.). Total RNA (1,000 ng) was reverse transcribed into cDNA using the Prime-Script™ RT Reagent kit (Takara Biotechnology Co., Ltd.). Before performing the RT reaction, RNA (10 µl) was heated to 65°C for 5–10 min, and then quenched on ice. Then, the inversion system was prepared according to the instructions of the cDNA kit, the sample was placed at 25°C for 10 min and then incubated at 42°C for 1 h. Following which, the cDNA was denatured at 95°C and placed on ice.
RT-qPCR was performed using SYBR-Green (Takara Biotechnology Co., Ltd.) and normalized to β-actin expression. The fibroblast-to-myofibroblast transition marker α-smooth muscle actin, and atrial fibrosis markers, transforming growth factor (TGF)-β, collagen I, collagen III, were examined. A total of 30 cycles of PCR were performed: Denaturation at 95°C for 30 sec, annealing at 60°C for 45 sec, and extension at 72°C for 60 sec.
The primer sequences are listed as follows: Akt, 5′-AGCATGGAGTGTGTGGACAG-3′ (forward) and 5′-GATGATCCATGCGGGGCTT-3′ (reverse); eNOS, 5′-CGACTATCCTGTATGGCTCTGAG-3′ (forward) and 5′-GATCCCCATTGCCAAATGTGC-3′ (reverse); PI3K, 5′-GCTCTTTCCCCAGCTGAACT-3′ (forward) and 5′-ACACAGTGTCGCTGGTTTGA-3′ (reverse); β-actin: 5′-CATCCTGCGTCTGGACCGG-3′ (forward) and 5′-TAATGTCACGCACGATTTCC-3′ (reverse); TGF-β, 5′-CCCAGCATCTGCAAAGCTC-3′ (forward) and 5′-GTCAATGTACAGCTGCCGCA-3′ (reverse); collagen I, 5′-CATGTTCAGCTTTGTGGACCT-3′ (forward) and 5′-GCAGCTGACTTCAGGGATGT-3′ (reverse). The average expression of β-actin was used as an internal reference gene to normalize input cDNA. Relative gene expression was calculated via the threshold cycle value (CT) and the formula 2−∆∆Cq (15). All of the reactions were repeated at least twice, with at least 3 replicates for every sample.
Masson's trichrome staining
According to a previous report (16), Masson's trichrome staining was conducted to evaluate the extent of atrial fibrosis. After the rats were sacrificed, atrium samples were rapidly excised, rinsed with PBS, fixed in 4% paraformaldehyde at 4°C overnight, and embedded in paraffin. Then, 5-µm-thick sections at of the atrium were cut. Masson's trichrome kit (cat. no. HT15; Sigma-Aldrich; Merck KGaA) was used according to the manufacturer's instruction. ImageJ software (Version 1.8; National Institutes of Health) was used to evaluate the area of fibrosis. Each section was examined in 5 randomly-selected high-power fields (×400) and middle-power fields (×200) under a light microscope. Collagen volume fraction (CVF)=Collagen area (blue)/Total area ×100%.
Cell culture
Cardiac fibroblasts (CFs) were harvested from the different groups, according to a previous report (17). Following attachment to uncoated culture plates, all unattached or weakly attached cells were removed. Then, the attached fibroblasts were washed and grown in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C. CFs were plated on a 96-well plate at a density of 1×104/well and cultured for 24 h.
Cell proliferation assay
As previously described (18), cell proliferation was examined using an MTT assay. Data values are expressed as fold-change compared with the control group.
Cell migration
As previously described (18), cell migration was examined with the real-time cell analyzer (RTCA) migration experiment. The CFs were seeded in RTCA Cim-16 plates in a serum-free DMEM medium with a cell density of at least 20,000/well. A chemoattractant of full growth medium (DMEM with 5–10% bovine serum albumin; cat. no. A1933; Sigma-Aldrich; Merck KGaA) was applied in the lower chamber. The RTCA device (xCELLigence RTCA; Roche Diagnostics) was used for measurements in a time-resolved manner (18). The values are expressed as fold-change compared with the control group.
Statistical analysis
GraphPad Prism 7.0 (GraphPad Software, Inc.) was used for statistical analysis. The AF incidence of four groups was compared with the Chi-square test. For comparison of normally distributed variables, one-way ANOVA and Tukey's post hoc test was performed. For comparison of AF duration, Kruskal-Wallis test and Dunn's multiple comparisons post hoc test were conducted. Data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.
DM model results
As demonstrated in Fig. 1, the FBG value was evaluated in the four groups at different points, including before the STZ injection, 1, 4 and 8 weeks after STZ injection. Before the injection, there was no difference in FBG among the four groups (P=0.5204), and the FBG varied between 90 and 110 mg/dl (Fig. 1A). However, 1 week after the STZ injection, the DM group and the DM+H2S group demonstrated a significant increase in the FBG level to 410.8±92.0 and 454.1±93.4 mg/dl, respectively, which were significantly higher than the control group and the H2S group (P<0.0001). At the same time, there was no significant difference between the DM and DM+H2S group, and between the control group and the H2S group (both P>0.05; Fig. 1B). The FBG level was examined 4 and 8 weeks after STZ injection, when NaHS had been administered. The results presented similar trends with the FBG level 1 week after STZ injection. The FBG of the DM group and the DM+H2S group was significantly higher than the control group and the H2S group (P<0.0001). At the same time, there was no difference between the DM group and the DM+H2S group, the control group and the H2S group (both P>0.05) (Fig. 1C and D).
Figure 1.
FBG level in the four groups at different time points. (A) FBG level before STZ injection. (B) FBG level at 1 week after STZ injection. (C) FBG level at 4 weeks after STZ injection. (D) FBG level at 8 weeks after STZ injection. (E) IPGTT results of the four groups. (F) IPITT results of the four groups. n=20 for each group. Data are presented as mean ± SD. ****P<0.0001 vs. the control group; ####P<0.0001 vs. the DM group; ++++P<0.0001 vs. the H2S group. FBG, fast blood glucose; STZ, streptozotocin; DM, diabetes mellitus; H2S, hydrogen sulfide; IPGTT, Intraperitoneal glucose tolerance test; IPITT, intraperitoneal insulin tolerance test.
IPITT and IPGTT (Fig. 1E and F) was performed to test insulin tolerance and glucose tolerance. The results were consistent with the FBG test, showing that the DM group and the DM+H2S group shared similar insulin and glucose tolerance, which was notably higher than the control group and the H2S group. However, there was no difference between the control group and the H2S group, and no difference DM+H2S group and the DM group. No significant side effects of NaHS were observed in the H2S group and the DM+H2S group.
AF inducibility and persistence
As shown in Fig. 2, the intracardiac programmed electrical stimulus was applied to examine the AF inducibility of the four groups. Fig. 2A shows representative ECG figures of burst pacing and the subsequent heart rhythm. Compared with the other three groups, the DM group exhibited a much higher susceptibility of induced AF, with a significantly higher AF incidence (P=0.017). There was no statistical difference between the control group, the H2S group and the DM+H2S group in AF incidence (Fig. 2B). The AF duration of the DM group was significantly higher than the control group and the H2S group (P=0.005). Although the AF duration of the DM+H2S group was lower than the DM group, there was no statistical significance (P=0.085; Fig. 2C).
Figure 2.
Intracardiac programmed electrical stimuli results. (A) Representative burst pacing electrocardiogram wave. (B) AF incidence in the four groups. (C) AF duration in the four groups. n=20 for each group. Data are presented as mean ± SD. **P<0.01 vs. the control group; #P<0.05 vs. the DM group. DM, diabetes mellitus; AF, atrial fibrillation; H2S, hydrogen sulfide.
Atrial fibrosis
Atrial remodeling of the four groups was also evaluated using Masson's trichrome staining (Fig. 3). More blue-stained collagen was observed in the DM group, indicating a more severe atrial fibrosis than the other groups, whereas the DM+H2S group suffered less fibrosis than the DM group (Fig. 3A). Fibrosis was quantified the with ImageJ software. The CVF value was calculated for the four groups, which indicated that the CVF of the DM group was significantly higher than the other groups (P<0.0001). At the same time, there was no significant difference among the control group, the H2S group and the DM+H2S group (all P>0.05; Fig. 3B). The key markers of fibrosis, including TGF-β and Collagen I, were also determined (Fig. 3C and D), which demonstrated that TGF-β and Collagen I of the DM group was significantly higher than the other groups (P<0.0001). Fibrosis of the different groups was also examined at the cellular level by comparing the proliferation and migration of CFs. CF proliferation and migration of the DM group was significantly increased. In contrast, proliferation and migration of the DM+H2S group were lower than the DM group (P<0.0001; Fig. 3E and F).
Figure 3.
Atrial structural remodeling of fibrosis. (A) Representative Masson's trichrome staining sections of the four groups. Scale bar, 100 µm. (B) CVF (%) in the four groups. (C) TGF-β mRNA expression in the four groups. (D) Collagen I mRNA expression in the four groups. (E) CF proliferation rate of the four groups. (F) CF migration rate of the four groups. n=20 for each group. Data are presented as mean ± SD. *P<0.05 and ****P<0.0001 vs. the control group; ##P<0.01 and ####P<0.0001 vs. the DM group; ++P<0.01 vs. the H2S group. Fold-change means the value compared with the control group. CF, cardiac fibroblasts; CVF, collagen volume fraction; TGF-β, tumor growth factor β; DM, diabetes mellitus; H2S, hydrogen sulfide.
PI3K/Akt/eNOS signaling pathway
As shown in Fig. 4, the classic PI3K/Akt/eNOS signaling pathway was evaluated in the four groups with RT-qPCR (Fig. 4A-C) and western blot analysis (Fig. 4D-G). The results demonstrated that this pathway was significantly downregulated in the DM group compared with the control group, which was reversed in the DM+H2S group. The expression levels of PI3K/Akt/eNOS were significantly higher in the DM+H2S group (P<0.0001). Compared with the DM group, p-Akt/Akt and p-eNOS/eNOS ratios were significantly increased in the DM+H2S group (Fig. 4F and G).
Figure 4.
PI3k/Akt/eNOS pathway expression. (A) PI3K mRNA expression from atrium tissue normalized to β-actin. (B) Akt mRNA expression from atrium tissue normalized to β-actin. (C) eNOS mRNA expression from atrium tissue normalized to β-actin. (D) Representative western blotting bands of PI3K/Akt/eNOS pathway proteins. (E) PI3K protein expression level normalized to β-actin. (F) p-Akt/Akt protein level ratio. (G) p-eNOS/eNOS protein level ratio. n=20 for each group. Data are presented as mean ± SD. **P<0.01, ***P<0.001 and ****P<0.0001 vs. the control group; #P<0.05, ##P<0.01, ###P<0.001 and ####P<0.0001 vs. the DM group. Fold-change indicates the value compared with the control group. p-, phosphorylated; eNOS, endothelial NOS; DM, diabetes mellitus; H2S, hydrogen sulfide; STZ, streptozotocin.
H2S, an endogenous active molecule, has been demonstrated to be effective in organ protection for ischemia/reperfusion injury (8). However, data concerning its effect on AF incidence, especially on DM-induced AF, is minimal. In the present study, SD rats were used as the research subject and a DM model was successfully established using STZ. It was demonstrated that DM rats had higher AF incidence and duration, which is associated with worse atrial fibrosis. It was also found that H2S could reduce AF incidence and mitigate the atrial fibrosis, which was achieved via the PI3K/Akt/eNOS pathway. Although there was no statistical difference between the DM group and DM+H2S group in AF duration, which could be attributed to the sample size, we propose that H2S could also shorten AF duration.
There are several ways to build DM animal models, including Zucker Diabetic Fatty rats, BioBreeding rats, LEW.1AR1-iddm rats, Goto-Kakizaki rats, chemically induced diabetic models, and non-obese diabetic mouse and Akita mouse models (19). Hayami et al used STZ to damage pancreatic islet B cells and significantly increase blood glucose, and found that diabetic rats suffered severe inflammation and atrial fibrosis (5). The present study used the same models as Hayami, and found that diabetic rats had not only worse atrial fibrosis, but also higher AF incidence and duration. Notably, the application of STZ for the DM model causes islet B cell injury and a shortage of insulin, which is similar to type 1 diabetes without insulin resistance. Saito et al (4) used another diabetic rat model by administrating a high-fat diet and low-doses of STZ. In this way, the rats had the characteristics of insulin resistance and high blood glucose, which simulates type 2 diabetes.
Research on DM and AF has demonstrated that autonomic, electrical, electromechanical and structural remodeling, including oxidative stress, connexin remodeling and glycemic fluctuations, could be important reasons why patients with DM are at higher risk of AF (20). Among all the mechanisms, oxidative stress and related structural remodeling lay the foundation of abnormal electrical activity (6). Chang et al (21) led a large-scale cohort study and found that metformin use was associated with a decreased risk of AF in patients with type 2 DM who were not using other anti-diabetic medication, most likely via attenuation of atrial cell tachycardia-induced myolysis and oxidative stress. Another study used the xanthine oxidase inhibitor allopurinol to prevent oxidative stress-mediated atrial remodeling in alloxan-induced DM rabbits (22).
Activation of the PI3K/Akt/eNOS signaling pathway is associated with reduced reactive oxygen species production (23,24). Chong et al (25) found that resveratrol could decrease left atrial fibrosis and regulate variation in ion channels to reduce AF through the PI3K/Akt/eNOS signaling pathway. Hydrogen sulfide is closely associated with the PI3K/Akt pathway. Xiao et al (26) investigated the effects of hydrogen sulfide on myocardial fibrosis and found that the protective effect of H2S against diabetes-induced myocardial fibrosis might be associated with the attenuation of autophagy via the upregulation of the PI3K/Akt1 signaling pathway. The present study found that H2S could significantly upregulate the PI3K/Akt/eNOS signaling pathway and reduce atrial fibrosis induced by DM. However, Xiao et al (26) demonstrated decreased autophagy resulted from PI3K/Akt1 activation, which was not found in this study. Another study led by Liu et al (27) also confirmed that H2S could ameliorate rat myocardial fibrosis induced by thyroxine, which may be associated with autophagy activated by upregulation of the PI3K/Akt signaling pathway and downregulation of miR-21, miR-34a and miR-214.
Several limitations of the present study must be noted. Firstly, despite the protective effect of H2S on DM-induced AF and atrial fibrosis, further mechanisms, such as inflammation, apoptosis or autophagy, were not investigated in this study due to limited time and funds. Secondly, this study only used the STZ-induced DM model, which is similar to type 1 DM, so conclusions are limited. Finally, electrical remodeling and cardiac structural function could be altered by DM status, which was also not investigated. However, these limitations can be resolved in further studies.
In conclusion, DM can lead to the structural remodeling of atrial fibrosis, increasing AF incidence and persistence. Administration of H2S does not affect glucose metabolism, but can significantly mitigate atrial fibrosis and reduce the incidence of AF, probably via the activation of the PI3K/Akt/eNOS pathway.
Not applicable.
This study was supported by the Outstanding Young Physician Program of Changzheng Hospital, Young Physician Startup Fund of Changzheng Hospital, and Project of Shanghai Science and Technology Commission (grant no. 17ZR1439100).
Availability of data and materials
The datasets used and or/analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
XX and JX designed the study. XX, QY and XL acquired and interpreted the data. WX, PW, and JS conducted the animal experiments. QY and JX prepared the manuscript and supervised the study protocol. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Ethics approval and consent to participate
The present study was approved by the Experimental Animal Administration Committee of the Second Military Medical University (Shanghai, China). Animal experiments were performed following the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85–23, revised 1996).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Portable Small Rock Crushers For Sale
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Personal Emergency Preparedness Training
Wednesday, August 26, 2020
Location: Keith Spalding B117
This course details how to build personal and family emergency plans and kits. We discuss what essential supplies you should have on hand for all members of your family, including pets. We also discuss what needs to be included in a family plan and what financial documents you need to secure. What you do now will determine your quality of life after our next big earthquake. Are you prepared to survive and recover quickly?
Visit our Training Calendar Tab for course dates and time.
To enroll in this course:
- Log onto access.caltech.
- Under SELF SERVICE, click the MyLearn link.
- Click the Course Catalog icon.
- Search for class, click on the desired date and class hyperlink.
- Click on the Enroll button at top right of pop-up screen.
For more information, please phone 626-395-6727 or email firstname.lastname@example.com or visit https://www.safety.caltech.edu/training/Personal_Emergency_Preparedness


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Etihad Airways has joined forces with travel risk management company Sitata to launch an interactive map detailing COVID-19 information in each country.
Quarantining and other COVID-19 protection measures have been a major hurdle to the aviation industry's recovery. In a recent survey from the International Air Transport Association (IATA) upwards of 80% of travellers said they would not fly to countries with quarantining measures in place.
Etihad's travel map will elevate some of these issues, continuously updating itself to show the latest global travel information. The information is vetted by Sitata's team of editors and updated as and when new information is made available.
Did you like this story?
Click here for more
Etihad Aviation Group chief digital officer Frank Meyer explained: "With travel restrictions beginning to ease in many destinations, we are keen to provide our guests with current information on COVID-19 related guidelines from the countries they plan to travel to. The current situation is still very volatile and dynamic, and with new guidelines being implemented on a daily basis – and sometimes even more frequently – it is important for us as an airline to support our guests with as much information as possible".
Information includes border restrictions, curfew timings, mandatory precautionary measures, quarantine rules, airport and flight operations and even the number of coronavirus cases in each destination.
Meyer continued: "Collaborating with Sitata to implement their travel alert map on etihad.com is yet another initiative, amongst several others, that is being implemented to ensure the continuous safety and wellbeing of our guests throughout their travels."
The airline has also recently launched a COVID-19 self-assessment tool on etihad.com.


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Bird of Paradise Flower Seeds (Strelitzia Reginae) 5+Seeds
$ 5.99
Bird of Paradise (Strelitzia Reginae) - These Strelitzia Reginae seeds produce a very unusual yet prized flower. This seed grows into a long-lasting 4 foot plant, and the Bird of Paradise flower is similar in appearance to a bird's head. The center bloom stalk is 3 feet long and the multicolored bloom is 6 - 8 inches in length. Colors include orange, copper, blue and red. The Bird of Paradise plant likes temperatures above 50 degrees F, so in most places it is better to grow it in a pot and keep the plant indoors during the winter and move outside in the summer. Move the pot outdoors slowly for brief periods of time each day, and let the Bird of Paradise acclimate or the plant can get sunburned.
Bird of Paradise flower seeds are slow to germinate, taking up to 8 or more weeks to germinate, and it will take 4 - 7 years for the plant to start blooming. When planting, soak the hard Strelitzia Reginae flower seeds for 24 - 48 hours and remove the orange tuft of hairs. Plant the flower seeds an inch deep in moist potting soil. Plants bloom best when pot-bound. If you need to re-pot a plant it might take a couple of years or more for the plant to start blooming again.
Season: Perennial
USDA Zones: 9 - 11
Height: 24 - 48 inches
Bloom Season: Fall through spring
Bloom Color: Mix
Environment: Full sun to partial shade
Soil Type: Moist, well-drained


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Hot sale good quality SV-999 Structural Glazing Silicone Sealant for Southampton Factories
Short Description:
Description SV – 999 silicone structural sealant is a one-component, neutral curing, designed for glass curtain wall, aluminum curtain wall, glass daylighting roof and metal structural engineering structural assembly silicone sealant. Show the effective physical properties and bonding performance Key Features 1. 100% silicone 2. No sag 3. Strong bonding strength 4. Water & weatherproof 5. Primerless adhesion to most building materials 6. 25% movement capability Basic Application...
Product Detail
Product Tags
Our company since its inception, always regards product quality as enterprise life, continuously improve production technology, improve product quality and continuously strengthen enterprise total quality management, in strict accordance with the national standard ISO 9001:2000 for Hot sale good quality SV-999 Structural Glazing Silicone Sealant for Southampton Factories, Our final goal is "To try the best, To be the Best". Please feel free to contact with us if you have any requirements.
SV – 999 silicone structural sealant is a one-component, neutral curing, designed for glass curtain wall, aluminum curtain wall, glass daylighting roof and metal structural engineering structural assembly silicone sealant. Show the effective physical properties and bonding performance
Key Features
1. 100% silicone
2. No sag
3. Strong bonding strength
4. Water & weatherproof
5. Primerless adhesion to most building materials
6. 25% movement capability
Basic Application
1.Glass curtain wall, aluminum curtain wall structure adhesive seal
2.Glass daylighting roof, metal structure engineering
3.Insulating glass bonding
Technical data sheet
|Test standard
|Test project
|Before curing——25℃,50%R.H.
|specific gravity
|Flow, sagging or vertical flow
|Operating time
|surface drying time(25℃,50%R.H.)
|Sealant curing speed and operating time will have different with different temperatures and temperature, high temperature and high humidity can make sealant curing speed faster, rather low temperature and low humidity are slower.21 days after curing——25℃,50%R.H.
|Durometer Hardness
|Shore A
|The ultimate tensile strength
|Tensile strength(23℃)
|Tensile strength(90℃)
|Tensile strength(-30℃)
|Tensile strength(flooding)
|Tensile strength(flooding – ultraviolet)
300ml in cartridge * 24 per box, 500ml in sausage *20 per box
Shelf life
12 months
If you want the TDS or MSDS or other details, please contact with our sales person.
GAF – Mastering The Roof (chapter 09)
This video demonstrates the proper installation technique for GAF starter strips; a superior alternative to starter shingles • Starter strips are important for wind resistance • Use WeatherBlocker or ProStart starter strips.
One of the most common sources of failure in a roof system is the improper installation of starter shingles. This starter course is very important, especially for wind resistance.
GAF offers Pro-Start and WeatherBlocker starter strips that are manufactured with a perforated line. This feature is a real time saver by eliminating the need to manually cut and cleanup shingles tabs.
Along with convenience and savings; the Pro-Start and WeatherBlocker products utilizing advanced Dura Grip adhesive to achieve maximum protection against wind blow-offs. Now that we have covered the importance and benefits of GAF precut starter strips lets take a look at they install.
For shingles exposure less than six inches, the use of either Pro-start or Weatherblocker starter strips can be used. For such applications; begin by separating the strip at the perforated line.
When applying shingles with exposures greater than 6 inches, such as the Camelot collection, only full un-split WeatherBlocker starter strip can be used.
Place the appropriate sized starter strip with the sealant facing up at the eave edge.
For proper water drainage, Both Pro-Start and Weatherblocker starter strip require a one quarter to three quarter inches overhang at all eave and rake edges.
Every Starter strip requires a minimum of four nails driven in a linear fashion one and a half to three inches above the shingle's bottom edge. Begin with the first nail at a distance of one and a half to three inches from the beginning side edge. Space the remaining three nails ten to twelve inches from the previous nail, and the fourth nail one to two inches from the ending edge.
When installing Metric dimensioned shingles, you must trim five inches off the first WeatherBlocker starter strip or three inches off the first ProStart starter strip. This critical step ensures that the starter strip butt joints and overlapping shingles butt joints are off-set a minimum of four inches. Depending on the length of the shingle being installed, A 10 to12 inch section of starter strip may periodically need to be cut and installed to meet the required 4 inch offset. Fasten the adjustment starter strip with two nails one to two inches from each side end.
For maximum wind resistance, starter strips should be installed along all rake edges. Begin by laying the strip with the sealant side up, closest to the rake edge. Position the starter strip so that it overlaps the eave starter strip by at least three inches. As with the eave edges, the Starter strip must overhang the rake edge one quarter to three quarters of an inch. Begin the line of nail fasteners one to two inches from the side edge of the starter strip, and one and a one half to three inches from the rake edge. Complete the rake edge starter course by nailing along a vertical line every ten to twelve inches up the rake until reaching the ridge.
The finished product of our Adams Polishes video shoot! Huge shout out to Blake for filming and editing everything and to Hunter for being gorgeous and washing my car haha everyone will be tagged below so check them out and their channels!
Products – @AdamsPolishes instagram
Youtube channel – https://www.youtube.com/user/Adamspolishes
Filmed by – @StevenBlakeM Instagram
Youtube channel – https://www.youtube.com/channel/UCzTTU70hQ8VbMQdzNg4CdZg
Model – @ihunter_Rae instagram
Youtube Channel – https://www.youtube.com/channel/UCfbEerwCw3vkl0IutVnKtWg
Song – https://soundcloud.com/illeniumofficial/odesza-always-this-late-illenium-remix-1


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The adoption homestudy is a detailed written report and screening of the home and life of prospective adoptive parents prior to allowing an adoption to take place. In the U.S, and in all international adoptions, a home study is required by law. This can take three to six months to finish. The adoption home study requires the prospective adoptive family to gather different documents, answer several questions, and explore their reasons for adopting. Through a series of visits and interviews the social worker can get a complete picture of who prospective adoptive parents are and what life is like in their family. Once this criteria is met an official legal document is filed with the state and good for one year.
Specific home study requirements and processes vary greatly from agency to agency, State to State, and (in the case of international adoption) by the child's country of origin. The adoption home study can include the following pieces of information depending on their location.
Autobiography/Family Background: Some states or agencies have a social worker working very closely with the family to finish this part of the home study, while others have a detailed list of questions that the agency wants answered.
Neighborhood/Community/Schools: prospective adoptive parents need to know what their school system is like. Which schools their children will attend and what resources does their community have to offer.
Physical Health: You may need a physical or health exam and/or a Tuberculosis(TB) test or chest x-ray. The social worker will also be interested in hearing how you have any health issues under control.
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References: Adoptive parents will need the names, addresses, and phone numbers of three or four references. Some agencies will contact by phone, others will require that your references write a letter of recommendation.
Interviews: Interviews are a time for the social worker to go over the prospective adoptive parents paperwork, learn more about them, clarify anything in their autobiography and see that their home is a safe and healthy place for a child.
If You Already Have Children: Some agencies will have the children write a statement about their feelings toward adoption. If they are too young, they may be asked to draw a picture. Workers will want to know about prospective adoptive parents children's interests, hobbies, and grades. Their children will be involved in interviews, and may meet with the social worker individually.


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Okay, so sue me, I know it is kind of cheating but hey it's my birthday and they're British, they won't mind.
Exploding in 2013, both of these fine actors have taken the states by storm starring in huge blockbuster films but also remaining close to their theater roots on the London stage. Here a just a few of my favourites.
The Fifth Estate
Benedict Cumberbatch brings the character of Julian Assange to life. As a man in search of the truth and only the truth, he believes that it is his mission to bring it to the masses, no matter what consequences it may bring. Adopting a rather alarming hair style for this role, Benedict embodies the man to a T. : everything from mannerisms to language pattern, so truly scary that at times you forget you are watching an actor play this man and not actually watching the man himself. It's a story that embodies the saying " The road to hell is paved with good intentions". When is enough truly enough? Is it really our right to know everything or is it for our own good that we are kept in the dark?
The Deep Blue Sea
Tom Hiddleston portrays the dashing Freddie Page, a fighter pilot in the Royal Air Force. He embarks on a twisted love affair with Hester Collyer played by the enchanting Rachel Weisz. Even though she is married to a British judge, their passion for each other is too much to deny. In spite of Freddie's horrible choices and theway he deals with Hester as she loses herself to grief for her wrong doings and indiscretions, he makes you understand why Hester would abandon all reason and logic to be with the man she so deeply loves.
War Horse
Bringing the two together, as Captain Nichols (Hiddleston) and Maj. Jamie Stewart ( Cumberbatch), this epic Steven Spielberg drama was released Christmas Day 2011 about the life of a horse named Joey during the war. Joey is bought by a family that really can't afford the purebred but after their young son Albert begins to train the horse an inseparable bond is formed. Needing money and owing debts in order to survive, Joey is sold to the British cavalry where he is put into the care of Captain Nichols. I've only seen this film once because that's all I can bear. I have a hard time with animals stories but I am glad I saw this one. It's a beautifully told story of a young man and his horse, their bond, and the soldiers that Joey carries through the trenches of war.
Sherlock Holmes (BBC)
I have never seen Sherlock Holmes brought to life in this way. You can't help but look at Benedict Cumberbatch and see the iconic character. I know that I have said a similar thing about Robert Downey, Jr. as Holmes but they play the same character so differently. Having just wrapped up season 3...we now more than likely have to wait another 2 years for season 4, if it happens at all. We have seen Sherlock face Moriarty, Adler, and most recently Charles Augustus Magnusson. The show's writing style requires Cumberbatch to be constantly on his toes with quick dialogue and a clever witiness woven throughout. He likes to think he is perfect but as his character has evolved he discovers his vulnerabilities. If you have not seen this show and if you are thinking that it's just another Sherlock Holmes adaptation, I dare you to watch one episode and not be hooked for life!
Thor: The Dark Work/ The Avengers/ Thor
Loki, how can you not adore him? Oh wait, he's the bad guy! But I will let you in on a little secret, it does not matter. The way Hiddleston brings Loki to life, you can't help but love. Yes, this is a villain that you are supposed to be against and even hate, but this is no ordinary villain; this is one with quick one liners, sarcastic remarks, and sexy little smirks. It is great testament to the ability of the actor that when his character is not on screen you find yourself wondering what he's doing, where he is, and what his next move will be. That's what Hiddleston brings to the screen. A must see - all three!!
Wrapping up my British obsession, if you have a chance you can also catch Tom playing Henry the IV in The Hollow Crown, and don't forget Benedict as the evil Khan in Star Trek: Into Darkness.


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Troubleshooting Connection Issues in Neo4j Browser and Cypher Shell
August 7, 2020 2 mins read
This page describes common issues users may encounter in connecting Neo4j Browser or cypher-shell to a Neo4j database, and how to address them. Connection Timeout Symptom: connection attempts lag for a long time, and then fail with connection timed out… Read more →
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When using $NEO4J_HOME/bin/cypher-shell at the command line and authentication is enabled via the setting $NEO4J_HOME/conf/neo4j.conf: # Whether requests to Neo4j are authenticated. # To disable authentication, uncomment this line dbms.security.auth_enabled=true a username and password can be provided on the command… Read more →
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If yor prepare a file with a Cypher statement that includes either a profile or explain clause and then want to pipe that file to bin/cypher-shell, to produce the profile/explain output you must include –format verbose on the command line.… Read more →
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Enabling TLSv1.2 with IBM JDK9
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Neo4j 3.4.0 only supports TLSv1.2 by default. IBM JDK9 uses the TLSv1 protocol by default. When attempting to run cypher-shell, users will be unable to connect to Neo4j. To enable TLSv1.2 in the IBM JDK, set the following JAVA OPT:… Read more →
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I got a fundraising e-mail from Amnesty International the other day. It was from a celebrity, as so many non-profit fundraising e-mails are. But this one really made me stop and look. It was from actor Patrick Stewart and it was about supporting Amnesty International's campaign concerning violence against women. He wrote: I know too much about violence against women – as a child ...Read More »
Monthly Archives: December 2010
Stacey Armato Responds To The TSA Post On Her Breast Milk Detention
Stacey Armato, the mom who was detained by the TSA when she requested that her pumped breast milk be given an "alternate" screening, agreed to an interview with me to answer some questions that might be lingering since the TSA posted its statement on her case. Q. In any earlier interview you said you hadn't received an apology from the TSA but ...Read More »
The TSA Responds To Breast Milk Mom, Stacey Armato
[UPDATE: After the original publication of this blog post, the following went up on The TSA Blog: "Updated on 12/9/2010 at 8:25 P.M. to add that proper procedures were followed." Oh, really? ]Over at The TSA Blog, they have posted TSA Response to "TSA Breast Milk Screening" Video. It is quite short but has already acquired a large number of comments, the vast majority ...Read More »
Breast Milk Mom Prepares to Sue the TSA Over Detention
Stacey Armato, the mother who was detained by TSA agents in Phoenix last February when she asked for "alternate" screening for her pumped breast milk, has filed a TSA Tort Claim Package, the last step before she can file a lawsuit against the government employees who she claims detained her in retaliation for filing a complaint against them. Under the Federal Tort Claims ...Read More »


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Vietnam Law & Legal Forum Magazine
New Official Gazette issue
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Friday, August 7, 2020
Domestic enterprises need protection from takeovers: experts
Updated: 09:43' - 19/05/2020
Vietnamese businesses reeling under financial stress due to the COVID-19 pandemic have become vulnerable to foreign takeovers and need the Government's support, according to experts.
Workers on production line of headsets for smartphones of South Korean-invested Glonics Vietnam Co Ltd.__Photo: VNA
At Prime Minister Nguyen Xuan Phuc's meeting with businesses this month, Minister of Planning and Investment (MPI) Nguyen Chi Dung mentioned the risk that domestic firms could be acquired at cheap prices after the pandemic.
Data from the ministry's Foreign Investment Agency showed the number of newly-registered foreign direct investment (FDI) projects in the first four months decreased 10 per cent against 2019's same period to 984, but deals in the forms of share purchase and capital contribution increased 33 per cent to 3,210.
More than 2,600 equity acquisitions took place without increasing the charter capital of companies worth USD 1.6 billion while only 580 transactions contributing USD 0.9 billion to domestic companies were recorded.
Japan, South Korean and China were the most active investors with deals from them increasing 40 per cent year-on-year, of which Japan ranked first with USD 743 million, followed by South Korea at USD 365 million.
Foreign investors are eyeing processing and manufacturing industries with 822 deals worth more than USD 1 billion, wholesale and retail, automobile and motorbike repair services with more than 1,000 deals worth USD 500 million.
Dung said difficulties caused by supply disruptions could not be overcome soon and would continue impacting many businesses and drive up M&A transactions, posing risks that businesses could be bought at low prices.
The Vietnam Chamber of Commerce and Industry (VCCI) has recently proposed the PM temporarily suspend merger and acquisition (M&A) deals during the pandemic to limit foreign takeovers of Vietnamese enterprises.
Do Nhat Hoang, head of the MPI's Foreign Investment Agency, said the ministry has reported to the Government on the situation and solutions countries are applying to support domestic enterprises. However, according to him, Vietnam should only limit the takeover of key businesses while still allowing investment activities in ordinary companies.
"We are currently working with ministries and agencies to prepare specific measures to limit takeover risks and advise the Government on this issue," Hoang was quoted on
Many governments have warned of the risk of businesses being acquired through M&A deals. Some have made moves to limit foreigners' efforts to buy their domestic companies amid the pandemic.
Last week, Japan introduced new rules restricting foreign investment, forcing foreign investors to report in advance when they plan to buy a more than 1 per cent stake in more than half of the listed companies in the country (the previous threshold was 10 percent).
In April, India tightened its policy on FDI that requires investment from neighboring countries to go a government approval process.
Similar to India, Australia and Germany have recently stipulated that during the COVID-19 pandemic, all foreign investment must be considered by the governments to prevent "opportunistic takeovers".
According to economist Nguyen Tri Hieu, this is the time when "shark" investors hunt for small enterprises at low prices. He has suggested the Government build policies and plans to attract foreign investment but should also tighten control over areas that may pose political and socio-economic risks.
The selection of industries calling for investment must also go along with setting the limit of foreign ownership at a reasonable rate which may be adjusted in accordance with supply and demand of the market, he said.-
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When we survey the whole cultural history of humankind we find we have to define religion in much broader terms to do justice to the great variety of forms it has taken.
My preferred definition of religion is that of Italian scholar, Carlo Della Casa: "Religion is a total mode of the interpreting and living of life."
In this new secular age, religion is manifesting itself in a much more humanistic and naturalistic way than in the past.
We are coming to value what it is that all humans have in common, irrespective of class, race, religion, gender, or age. We are developing a growing concern for human rights. We have come to see that what used to be regarded as the divine attributes are actually human value judgments, expressing what our forbears found to be of ultimate concern to them.
It is these values which came to the fore when the modern secular world began to emerge from the 18th century onwards, and they gave rise to a whole series of emancipations - the democratic emancipation from absolute monarchy, the emancipation of slaves, the emancipation of women from male domination, the emancipation of the humankind from racist prejudice and the emancipation of homosexuals from homophobia.
Sadly, the churches have often been initially opposed to these emancipations. This showed they were no longer on the side of freedom and human justice, as they should have been, because they had become committed to outworn dogmas of the past.
This is why the churches have become increasingly marginalised from society. The new forms of religion, appropriate to the secular age, are more often to be found outside the churches than within them.
It is also the reason why para-religious movements are springing up outside the churches, such as the Ephesus groups in New Zealand and the Sea of Faith Network in several countries.
The erosion of church institutions does not mean the end of Christianity, for the latter needs to be seen not as something eternally fixed but as an ever-changing and developing process. The modern secular world is all part of that evolving process.
When Christianity emerged out of Judaism, Christians claimed it to be the legitimate continuation and fulfilment of the Jewish path of faith. Similarly, the modern, secular and humanistic world may be regarded as the legitimate continuation of the Judeo-Christian path of faith.
The churches must stop treating the secular world as an enemy to be fought and conquered and welcome it as the new form of the Christian tradition out of which it has come.
One of the first theologians to recognise this was the Anglican J. R. Illingworth, a man being read and valued when I was a student. As long ago as 1891 he warned Christians not to regard secular thought as the enemy of Christianity.
In an essay on the incarnation he said: "Secular civilisation has co-operated with Christianity to produce the modern world. It is nothing less than the providential correlative and counterpart of the incarnation."
Ludwig Feuerbach had implied the same back in the 1840s but it was to be more than a century before he began to be valued. The failure of the churches to recognise that the secular world has evolved out of their own central doctrine - the incarnation of the divine within the human world - and their claim that they alone are the true interpreters of Christianity, have meant that those outside the churches (now more than three-quarters of New Zealanders) are often failing to realise how much they owe to the Christian past.
The churches must now bear some of the blame that such people are cutting themselves off from their cultural and spiritual roots. As plants without roots wither and die, so cultures which forget their past do the same.
The most important task of the churches on entering the 21st century is to help the secular world to understand its Christian origin. The study of the past illuminates the present, but it does not dictate the future.
That is why the Bible remains an invaluable set of documents. We learn much from it but we are not bound by it.
During the course of the 20th century the idea of God as a supernatural, controlling, personal being was fading from general human consciousness. Oxford Professor of Theology John Macquarrie said: "There was a time in Western society when 'God' was an essential part of the everyday vocabulary ... but in the West and among educated people throughout the world, this kind of God-talk has virtually ceased ... People once knew, or thought they knew, what they meant when they spoke of God, and they spoke of him often. Now in the course of the day's business we may not mention him at all. The name of God seems to have been retired from everyday discourse."
We may illustrate this by noting how our common everyday speech has been changing. Once, on parting from others, we always said, "Goodbye," which is derived from "God be with you." Now we say, "Have a good day."
In the very last year of the 20th century, the radical theologian Don Cupitt made a study of the way our everyday language is reflecting the religious change that has taken place. He found, for example, that where once we would have spoken of God, we now refer to "life". We have created a whole new set of phrases including it, such as"Get a life."
Within the space of 100 years New Zealand has changed from being a predominantly churchgoing God-believing society to a predominantly non-churchgoing secular society. In 2000, the main-line churches showed little of the triumphant confidence they manifested in 1900 and they have now entered survival mode. Religion has become privatised and God has lost his public face. That much is plain for all to see.
But how are we to interpret all this? If we define religion in the supernatural terms encoded in Christian dogma, then New Zealand is fast becoming non-religious. But if we define religion in much broader terms (as I do), and if we acknowledge the secular world view to be the product and legitimate continuation of the ever-developing path of faith we call Judeo-Christian, we may come to an entirely different judgment.
New Zealanders generally, by their passion to combat apartheid and racism, to condemn war and violence, to promote tolerance of diversity ... manifest in these and other ways a genuine religious zeal. Insofar as New Zealand has been called the most secular country in the world, then we may well be in the vanguard of the new secular phase of religion, just as we were in the forefront in giving the franchise to women.
The most widespread religion in New Zealand today is what I have called elsewhere "religion without God", and which our leading citizens often refer to as "Christian values".* This is an edited versions of Lloyd Geering's Hocken lecture given in Dunedin this month.
* Lloyd Geering is a former principal of the theological hall at Knox College, Dunedin, and was the first professor of religious studies at Victoria University, Wellington.
When we survey the whole cultural history of humankind we find we have to define religion in much broader terms to do justice to the great variety of forms it has taken.
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