The present research tested the hypothesis that everyday sadists show a distinct preference for violent video games and examined the relationship between everyday sadism and participant's mood after violent video game play. In Study 1, participants watched three trailers for video games that differed in their level of violent content. Whereas everyday sadists were attracted to a violent video game, there was no significant positive association between everyday sadism and attraction to the nonviolent video games. Study 2 showed that after playing a violent video game, there was a significant positive relationship between everyday sadism and participant's positive mood and a negative relationship between everyday sadism and participant's negative mood. In contrast, after playing a nonviolent video game, the relationship between everyday sadism and participant's negative mood was less pronounced. Overall, these studies show that everyday sadists specifically like to play violent video games and suggest that this tendency is adaptive in that they emotionally benefit from playing violent video games. For example, in one study : narcissism, Machiavellianism, psychopathy, and everyday sadism  provided 80% power to detect an effect of r = 0.18 . Given that the typical published effect size in social psychology is about r = 0.21 , we did not determine sample sizes by doing a priori power analyses but rather tried to run as many participants as possible during one semester. A sensitivity analysis showed that the current samples  each video game was assessed. One of the video games contained explicit violence. It was predicted that everyday sadism would be positively associated with liking, and interest in, this video game. We further examined whether these relationships remained significant when controlling for the influence of trait aggression and the Dark Triad. Such a destructive testing approach . There was no compensation for participation.After providing demographics, the Dark Triad was assessed. To this end, the Short Dark Triad by Jones and Paulhus  was emplAfterwards, participants watched three video game trailers. After each trailer, liking of the video game was assessed by three items . These items were assessed on a scale from 1 to 4. For each trailer, these three items were highly correlated and were thus collapsed into one liking scale . Participants had to watch each trailer for at least ten seconds before they could proceed. They were free to watch the entire trailer and the time participants watched each trailer was assessed. This measure was employed as a proxy for interest in the video game. The first trailer was for Splatoon, which is a third‐person nonviolent shooter game. Rather than shooting missiles, the player uses ink (or colors). The Entertainment Software Self‐Regulation (USK) organization approves the game for children aged six and above. The second trailer was for Sims 4, which is a life simulation game . The USK approves the game for children aged 12 and above. The third trailer was for Mortal Kombat X, which is a fighting game (violent video game). The USK does not approve the game for anyone under 18. All participants watched the three trailers in the same order.2.2F = 7.96, p < .001. Most importantly, everyday sadism was still significantly associated with liking the violent video game, β = .19, p = .020 . None of the other predictors received a significant regression weight .Descriptive statistics and intercorrelations of all measures are reported in Table Similarly, everyday sadism was positively associated with interest in the violent video game, whereas everyday sadism was not associated with interest in the nonviolent shooter game and was negatively associated with interest in the neutral video game. As before, the pattern of findings for trait aggression and the Dark Triad was similar, but less pronounced. However, everyday sadism did not significantly predict interest in the violent video game in the regression .z tests showed that the correlation between everyday sadism and liking of the violent video game was significantly different from the correlations between everyday sadism and liking the nonviolent shooter game, z = 4.45, p < .001, and between everyday sadism and liking the neutral video game, z = 5.75, p < .001, respectively. Moreover, the correlation between everyday sadism and interest in the violent video game was significantly different from the correlations between everyday sadism and interest in the nonviolent shooter game, z = 2.92, p < .001, and between everyday sadism and interest in the neutral video game, z = 4.48, p < .001, respectively.Fisher's 2.3Study 1 supports the hypothesis that everyday sadists show a distinct preference for playing video games where they can cause virtual harm. As expected, everyday sadism was positively associated with liking a violent video game and this relationship held when controlling for trait aggression and the Dark Triad. Most of these findings were corroborated with the behavioral measure of interest in the violent video game (with the exception that the influence of everyday sadism on interest in the violent video game did not hold when controlling for trait aggression and the Dark Triad).Everyday sadists were attracted to the violent video game, but they were not attracted to the nonviolent video games. The relationships between everyday sadism and liking of and interest in the two nonviolent video games were not significantly positive, and were less pronounced than the relationships between everyday sadism and liking, and interest in, the violent video game. There was even a negative relationship between everyday sadism and liking, and interest in, the neutral video game. Overall, Study 1 shows that everyday sadists are attracted to violent video games but that they are not attracted to video games that do not contain violence.3Study 2 examined the impact of video game play on mood change. Study 1 indicated that everyday sadists show a distinct preference for violent video games. It was reasoned that because playing preferred video games improves the player's mood . Participants either received course credit or 5 Euros (approximately 6 U.S. dollars).After providing demographics, participants completed the 18 items from the CAST (α = .82). We then assessed participant's amount of violent video game play. For each of their three favorite video games, participants indicated how often they play the game and rated how violent the content of the game is. The scale for both items was from 1 to 7. For each video game, frequency of game play was multiplied by violent content  to +5 (very good).Afterwards, we employed the PANAS . (This parameter is not meaningful for participants that got arrested because with the restart the number of kills was reset to zero.) In the nonviolent condition, participants took part in a triathlon and competed against computer opponents. To ensure that participants did not behave aggressively during video game play and in line with previous work showing that punished violent behavior within a video game reduces violent in‐game behavior  for ten minutes. In the violent condition, participants had to defend themselves against attacking police men. To minimize frustration and to limit the impact of previous game experience, the game was modified so that participants had unlimited access to weapons and ammunition and could not be killed. However, they could be arrested by the police. If they got arrested, the game had to be restarted. For those participants that were not arrested, the number of kills was recorded  to 5 (very much). To assess participant's previous GTA experience, they reported how often they played GTA before, using a scale from 0 (never) to 3 (more than 50 times). Because the study was part of a student project, additional measures were employed. For example, we examined whether everyday sadists were more likely to believe than others that violent video games do not cause aggression (they were). Because these measures are not relevant for the present purposes, we do not report them.After playing the video game, post‐game mood was assessed, using the same items . Participants then indicated how violent the video game was. This item was employed as a manipulation check. They also responded to four questions assessing liking of the video game . All video game ratings were assessed using a scale from 1 (3.2t(214) = 23.77, p < .001, d = 3.32. The content of the violent video game  was perceived as being more violent than the content of the nonviolent video game . Liking the two video games was relatively similar , t(214) = 1.69, p = .093, d = 0.22. Moreover, everyday sadism was positively associated with amount of violent video game play, r(216) = .33, p < .001.The manipulation check was successful, r(113) = .36, p < .001, and general mood, r(113) = .30, p = .001, respectively, were significantly positive, whereas the partial correlation between everyday sadism and negative mood was significantly negative, r(113) = −.27, p = .003. Unexpectedly, in the nonviolent video game condition, the partial correlation between everyday sadism and positive mood was also significant, r(97) = .20, p = .045. In contrast, the partial correlations between everyday sadism and general mood, r(97) = .18, p = .081, and negative mood, r(97) = .06, p = .535, respectively, were non‐significant.Our main hypothesis was that after playing the violent video game, everyday sadism should be related to participant's mood. Because the PANAS mood measures were not significantly correlated  = .47, p = .001. However, killing game characters may not only depend on the players’ motivation but also on their ability. Indeed, previous GTA experience was significantly associated with number of kills, r(49) = .45, p = .001. Hence, we tested whether the relationship between everyday sadism and number of kills would hold when controlling for previous GTA experience. In a multiple regression, everyday sadism and previous GTA experience were used as predictors of the number of kills. The overall regression was significant, F = 10.40, p < .001. Everyday sadism was still significantly associated with number of kills, β = .36, p = .009 .Next, we tested whether everyday sadism would be associated with number of kills during game play. The correlation was significantly positive, 3.3Study 2 showed that everyday sadists’ attraction to violent video games has some rational roots in that violent video game play has a positive impact on everyday sadists’ mood. As predicted, after playing a violent video game, there was a positive relationship between everyday sadism and participant's positive mood and a negative relationship between everyday sadism and participant's negative mood. However, after nonviolent video game play, there was also a positive relationship between everyday sadism and participant's positive mood. Moreover, the correlation coefficients between everyday sadism and participant's positive and general mood did not significantly differ between the two video game conditions. In contrast, after nonviolent video game play, the relationship between everyday sadism and participant's negative mood was non‐significant, and the correlation coefficient between everyday sadism and participant's negative mood did differ between the two video game conditions. It thus appears that violent more than nonviolent video game play is associated with decreases in everyday sadists’ negative mood, whereas positive affective states are less distinctly affected.Study 2 also provides a conceptual replication of Study 1 by showing that everyday sadists seem to be attracted to violent video games. As in previous research (Greitemeyer, 4The present studies had two main goals. First, previous research has shown that everyday sadists are attracted to violent video games (Greitemeyer, In fact, everyday sadism was not significantly associated with liking of and interest in the nonviolent shooter game and it was even negatively associated with liking of and interest in the neutral video game. Future research may examine whether everyday sadists are particularly disinclined to play certain kinds of video games. Given that everyday sadists enjoy causing harm, they may be less willing than others to benefit others. Many video games include the opportunity to behave prosocially, with the typical finding that these video games increase helping behavior (Gentile et al., When we refer to participants as being everyday sadists, it is important to keep in mind that the overall mean level of everyday sadism in both studies was considerably below the midpoint of the scale. That is, in our samples, participants who showed a preference for violent video games did not have high scores on everyday sadism; they just had higher scores than participants who were less favorable toward violent video games.not due to them being more aggressive and scoring high on narcissism, Machiavellianism, and psychopathy. This finding provides further evidence that everyday sadism is conceptually and empirically distinct from other forms of the dark side of human personality (see also Buckels et al., It is noteworthy that the relationship between everyday sadism and the preference for violent video games remained significant when controlling for the impact of trait aggression and the Dark Triad, suggesting that everyday sadists’ preference for violent video games is Our second goal was to address whether everyday sadists’ preference for violent video games would be adaptive in that playing those games is associated with increases in positive affect and decreases in negative affect. Previous research (Bowman & Tamborini, To conclude, the present studies provide evidence that everyday sadists are attracted to video games that offer an opportunity to satisfy their need for cruelty, whereas they are not attracted to video games where no virtual harm can be committed. These preferences seem to be adaptive in that everyday sadists emotionally benefit from playing violent video games. Playing violent games has been shown to increase aggression and thus has negative interpersonal consequences (Anderson et al.,


A national Do Not Attempt Cardiopulmonary Resuscitation policy was rolled out for the National Health Service in Wales in 2015. A national steering group led on producing information videos and a website for patients, carers and healthcare professionals, forming part of a quality improvement program. Videos were planned, scripted and produced with healthcare professionals and patient/carer representatives, and were completed with both English and Welsh language versions. The TalkCPR videos encourage and promote open discussion about Cardiopulmonary Resuscitation (CPR) and DNACPR in palliative care situations.We worked with patient/carer groups to evaluate whether video resources to convey the salient facts involved in CPR and DNACPR decisions for people with palliative and life-limiting illness were acceptable or not. We conducted a mixed-method design service review in five phases to evaluate whether this technological resource could help. After creating video and website materials, they were evaluated by doctors, nurses and a patient/carer group. We also sent out one lightweight TalkCPR video media pad to each practice in Wales. These rechargeable electronic video media pads had communication videos pre-loaded for easy viewing, especially in areas with poor roaming data coverage.Videos were demonstrably acceptable to both patient and carer groups, and improved healthcare professional confidence and understanding. Videos went live on the TalkCPR website, in all Welsh Health Boards and on Youtube, and are now used in routine practice throughout Wales.This is the first time that DNACPR information videos are aimed directly at palliative care patients and carers, to explore this sensitive subject with them, and to encourage them to approach their doctor or nurse about it. The website, app and video media pads were developed by patients, the Digital Legacy Association, Welsh NHS IT services, Welsh Government, the Bevan Commission and the Dying Matters Charity in Wales ‘Byw Nawr’. The GMC, the Royal College of General Practitioners and NICE have listed TalkCPR as a learning resource. There has also been a collaboration with Falmouth University Art College, who helped produce graphic designs to facilitate and encourage discussions about CPR and end of life care.The online version of this article (10.1186/s12904-018-0370-9) contains supplementary material, which is available to authorized users. How can a palliative patient express his or her views on future emergency care options, if they have little or no information about what procedures like CPR (Cardiopulmonary Resuscitation) involve, beyond what they have seen on television programmes and in fictional media? One of the authors of this research study, MT, outlined these challenges in a Guardian newspaper article, in particular how this area of medicine is often misrepresented on television ..The resuscitation council has stressed the importance of discussing DNACPR (Do Not Attempt Cardiopulmonary Resuscitation) decisions with patients and proxy, and two recent high profile court cases have changed practice . Not disFace-to-face discussions with a healthcare professional are important, but additional media resources can and should be used to help understand DNACPR towards the end of life and in palliative care settings . Such reCardiopulmonary Resuscitation or CPR is an emergency procedure, with the aim of restarting heart and breathing when these have ceased to function . This isWitnessing and undergoing CPR can be traumatic and involves chest compressions, intravenous lines being placed, insertion of airways, electric shocks to the bare chest. Rib fractures, internal bleeding, severe pain, collapsed lungs and brain damage can be short and longer term problems, if the patient survives.Talking about CPR is important, and in fact it is best to discuss it when a person is still reasonably well, and can express an opinion on whether the intervention is something they would consider appropriate for themselves or not, should it become necessary in future. There is a need to explain this procedure better within society, and also to create reproducible ways of giving clinicians opportunities to gain confidence in talking about this challenging topic more.We conducted a mixed-method design service review in five phases to evaluate whether this technological resource could help. These five implementation phases are described below.not as a formulaic process, but that they respect this as one of the most important conversations the patient and carers may have in their disease journey. They also felt that conveying a message that other measures, like antibiotics, chemotherapy, blood products, emergency fluids would not be left unconsidered, merely because of the presence of a DNACPR form. It was felt important by the group to convey this and that it would provide reassurance to patients. A Driver Diagram to further refine these aims and interventions was created  consisted at the time of enquiry of 24 individuals with either a background as a cancer patient, or as a current or past carer of someone with cancer. The hospital trust also has a patient and carer information and support co-ordinator, who was instrumental in setting up this meeting. Patients and carers unanimously agreed that these videos would be important and of high value and were very supportive of the TalkCPR project. Patient/carer representatives in our hospital felt it was important that healthcare professionals approach this topic ted Fig. .Fig. 1DrIn response to the aims collated and set out in Phase 1, the TalkCPR Implementation group, consisting of clinicians, patients, nursing staff, resus officers and a psychologist, evaluated the patient/carer suggestions and noted key aspirations for the TalkCPR project. The main author, Dr. Mark Taubert suggested the campaign name “Talk CPR” and the Twitter hashtag #TalkCPR and this name was unanimously agreed upon. An additional meeting with the chair of the patient group and a film production team was held, and also included a psychologist, a resuscitation officer, an oncologist, a palliative care nurse and the chair of the national Do Not Attempt Cardiopulmonary Resuscitation steering group.TalkCPR aims to improve healthcare professional communication and resources; and to improve patient/carer understanding about Do Not Attempt Cardiopulmonary Resuscitation (DNACPR) in palliative illnessThe key aim that was agreed on, as part of this baseline review, was outlined as follows:Increase doctor/nurse confidence in having DNACPR discussions, for example how to approach these conversations with patients and what to focus on.Encourage patients/carers affected by palliative, life-limiting illness in Wales to approach their clinical team about the issue of CPR/DNACPR.Two key drivers emerged during this baseline exercise. Patients and healthcare practitioners felt it was important to:DNACPR advice videos including ‘Top Tips on DNACPR communication’ to be made available in all Welsh health boards and trusts via Intranet, email and communications teamsPromote videos and TalkCPR website as a resource area for healthcare professionals who want more information on details of DNACPR in WalesMake videos available to healthcare professionals via online and offline means, in order to share them with patients/carers and to supplement face-to-face discussions about this important topicDevelop a strategic campaign to make videos widely accessible to general public, including on Youtube, Twitter, Facebook, charity websites and on television. Make videos smartphone compatible, but also develop resources that do not require internet access.All meetings were minuted and consensus was sought on what should emerge as content for the videos. The question: “What should the goal of this video information project be?” was asked at the start of the meetings, and all participants were asked to give their views. Aspirations were written down and then altered by participants until everyone was content that they represented what they considered most informative. From this, four aspirational interventions were agreed upon by the patients and healthcare professionals on the TAlkCPR group:Before creating the videos, the team also set out a process map on how DNACPR videos should be accessed and when Fig. . Help toAt appropriate time, patient/carer may wish to discuss appropriateness of potential future DNACPR decision- CPR’s chances of success, likely outcomes, what CPR involves.And/or: At appropriate time, doctor/nurse/healthcare practitioner may wish to bring up topic of DNACPR in a palliative care situation- including chances of success, likely outcomes, what CPR involves.Framing what would trigger using this technology resource in the patient/carer journey, the following was agreed on by the project team:A healthcare practitioner (HCP) could discuss with a patient/carer that CPR would or would not have likelihood of success in a given situation, and explore the individual’s view on this topic. If appropriate, a suggestion could be made to write a DNACPR form and keep with patient/carer at all times, as per NHS Wales DNACPR policy. In addition, the healthcare professional should offer the patient/carer some thinking time before sharing their views, and offer TalkCPR website/videos as a resource to improve understanding and consent. This could be accessed via a webpage on patient’s/carer’s own computer device or on a video media pad left at house or by the hospital bed.) was created http://talkcpr.cymru. Lightweight video media pads were produced, and had videos pre-loaded onto them, in order for healthcare professionals who wanted to bring videos to patients/carers in areas with less Wi-Fi or data access to still be able to do so. Videos were also produced for the hard of hearing  created English language and Welsh language videos to inform anyone viewing these films what Cardiopulmonary Resuscitation (CPR) in life limiting and palliative illness involved. Scripting and production took 4 months in total, with a further post-production period of 2 months. Videos were hosted on the TalkCPR website A survey given to 25 nurses measuring pre- and post TalkCPR video viewing impact metrics (n = 15)A survey given to 15 junior doctors measuring pre- and post TalkCPR video-viewing impact metrics (n = 14)A summary of a hospital patient/carer focus group session (n = 100)A survey to evaluate TalkCPR video media pads with 100 healthcare professionals , then watched the TalkCPR videos and then filled in the questionnaire again (post intervention survey) on a separate sheet of paper, to measure if there had been any change in views and confidence after watching videos. They scored from 0 to 10 .We conducted a focus group session with the Velindre NHS Trust patient/carer liaison group. TalkCPR videos were presented to the Patient/Carer Liaison Group (PLG) at Velindre NHS Trust in December 2015. Fourteen members of the patient/carer group were present. Notes were taken during the meeting. Comments and suggestions were captured. Participants were asked a number of pre-prepared questions, for instance on the acceptability of the videos and whether they evaluated the videos as appropriate for viewers. Eight outcomes were eventually agreed on after discussion.https://www.youtube.com/watch?v=5Zw9mD3K0pU.TalkCPR video media pads survey: A survey was completed by 100 healthcare professionals in NHS Wales  to look at usability of light weight video media pads, onto which the TalkCPR videos were pre-loaded and ready to view at the click of a button. A short film of the video catalogue being used can be viewed here: The role of the personHow often they typically discuss DNACPR (0–3 / 3–6 / 6–10 / 10+ x per year)Whether they thought the video booklet would facilitate DNACPR discussions (Yes/No)How useful they thought patients/carers would find the video booklet (Yes/No)A questionnaire was designed in order to evaluate the video media pad product amongst healthcare professionals. Input was received from a clinical psychologist with expertise in questionnaire design. From the questionnaire it was aimed to attain the following information:Any nurse, junior doctor, consultant or General Practitioner working in Wales was entitled to take part and evaluate this video book product. Healthcare professionals were initially contacted via email, asking them to take part and were then met face-to-face, at which point they watched the videos on the video book, before finally filing in the survey.n = 25)Nurses (n = 25)Junior doctors (n = 25)Hospital consultants (n = 25)General Practitioners/General Practitioner trainees , and increased to 8.28 out of 10 after watching the TalkCPR videos. A summary is provided in Fig. n = 15) rotating through Velindre NHS Trust in Cardiff during 2015 and 2016 at the start of DNACPR teaching sessions, in induction week (Foundation Year 2- Core Training Year 2 level). They showed considerable increases in readiness to recommend videos to patients and proxy and they demonstrated increased confidence levels to show such a video, compared to pre-video survey. Figure The same survey was given to junior doctors n = 1 rotatingOutcome 1: PLG felt that videos were acceptable and not distasteful.Outcome 2: PLG took key educational messages from videos and interpreted them as was intendedOutcome 3: PLG felt initial dissemination was appropriate and that videos should get wide-spread coverageOutcome 4: PLG agreed that wider remit (i.e. not merely in cancer settings) of video accessibility was highly appropriateOutcome 5: Black & white video was seen as highly informative and with potential to alter patients/carers preconceptionsOutcome 6: Some PLG members felt that background music in videos was unnecessary and potentially distracting, others did not agree that this was a problem.Outcome 7: Mixed views on first (introductory) video showing a family in a waiting room, PLG felt that more detailed information on CPR/DNACPR could have been provided in this video- However acknowledged that this info was covered in other, longer video and the initial short video was aimed at generating interest in topic, suitable for social media channels as a ‘hook’.Outcome 8: Agreed that additional supporting information could be provided on TalkCPR website itself, for those who wanted more information after viewing first video.Listed below are the outcomes of the PLG focus group meeting. These were agreed topics during the December meeting and resulted from round table discussion. In summaryA later suggestion from a patient liaison group member was to add subtitles to videos, and this was also fed back from a nurse who had tried to view videos from an NHS Health board computer that had no loudspeakers. Videos were re-edited and subtitles were added in response to this feedback.n = 100; n = 25 per professional group; total of 4 professions). Table Frequency of DNACPR interactions per professional group in preceding year was recorded (n = 25 per profession) was recorded and is shown in Fig. Figures n = 25) in answer to the survey question: ‘Do you think that patients would find the video media pad helpful after an initial face-to-face consult?’ is shown in Fig. The number of ‘Yes’ respondents per profession , with one of them commenting that “DNACPR conversations are the bread and butter of palliative care” and therefore they would be unlikely to use such additional resources.Twelve consultants shared their comments. One wrote that in their job as a consultant breast surgeon they would use the booklet to improve their skills. A Rheumatologist wrote that if the occasion arose, he/she would be likely to use the video booklet, however they added “One GP wrote that “for some patients such as technological material would prove confusing and may enhance their feeling of alienation and confusion” however other than this the comments were only positive.often I arrange to revisit patients a few days after the initial discussion; it would be useful to leave something for the patient to watch between my visits to help them make an informed decision. I think this resource would be well placed in primary care”.Three GP’s wrote that they would be a very useful tool to keep in their bag for house calls, with one adding “it would help to reinforce the topics discussed during the consultation as a few patients continue to have unanswered questions and fears despite my best efforts at reassurance and explanation. I feel the video booklet would be helpful in addressing such issues”.Another wrote “http://talkcpr.wales website  and issues such as sound and loudspeaker access on computers in different healthcare settings. There were reports of variability in access to internet and Wi-Fi in different parts of Wales, with urban settings more likely able to gain access and bring the videos to the patient bedside or home. The creation of small lightweight video media pads that play all TalkCPR videos at the press of a button, without need for wi-fi or data access, enabled access for more remote areas. Video media pads have been sent out to all GP practices and clusters in Wales with a covering letter  and increasing patients’ education about the need for such discussions. These same interventions may also render discussions more meaningful.In order to have discussions about CPR/DNACPR that are The technology used, i.e. videos, website and video media pads received very positive comments. The high proportion of junior doctors and consultants who reported discussing DNACPR more than ten times in the last 12 months . It is hoped that this will provide an impetus for open and honest anticipatory face to face conversations on future ceilings of treatment in people with serious palliative illness.The campaign has been widely publicised on social media, for instance via the hashtag #TalkCPR on Twitter. After less than 6 months of being ‘live’ the site had reached nearly 100,000 hits. Comments on social media have been extremely positive and encouraging and no one has stated that the videos ae inappropriate or insensitive. By addressing the public directly with the TalkCPR videos and resource website, and also using videos as a learning resource for healthcare professionals, we hope to have created one of the steps towards an open, transparent process to capture people’s views on this sensitive topic. The project has won the NHS Wales Award 2016 in the Improving Quality Together using IQT Methodology category. It won a runner-up prize at the BMJ Awards in 2017. A collaboration with Falmouth University School of Graphic Design has meant that art students as part of the Moth art project have helped with innovative visual and video aids to encourage DNACPR discussions. Based on the survey we conducted on 100 Healthcare practitioners on the Talk CPR video media pads, we sent one pad out with a covering letter see Fig.  to each Project name: TalkCPR.http://talkcpr.walesProject home page: Programming language: PHP, HTML, centOS.Requirements: Open access via web. Smartphone and tablet compatible.License: NHS Wales. No third party/ongoing licenses or license costs.Restrictions: None.Additional file 1:Image 1. TalkCPR website- English language version. (PNG 479 kb)Additional file 2:Image 2. TalkCPR- Siarad am CPR – Welsh language version. (PNG 465 kb)Additional file 3:Image 3. Sign language video. (PNG 611 kb)


N-myc downstream-regulated gene 1 (NDRG1) has been shown to play a key role in tumor metastasis. Recent studies demonstrate that NDRG1 can suppress tumor growth and is related to tumor proliferation; however, the mechanisms underlying these effects remain obscure.Immunohistochemistry (IHC) was used to detect NDRG1 and p21 protein expression in colorectal cancer tissue, and clinical significance of NDRG1 was also analyzed. CCK-8 assay, colony formation assay, flow cytometry, and xenograft model were used to assess the effect of NDRG1 on tumor proliferation in vivo and in vitro. The mechanisms underlying the effect of NDRG1 were investigated using western blotting, immunofluorescence, immunoprecipitation, and ubiquitylation assay.NDRG1 was down-regulated in CRC tissues and correlated with tumor size and patient survival. NDRG1 inhibited tumor proliferation through increasing p21 expression via suppressing p21 ubiquitylation. NDRG1 and p21 had a positive correlation both in vivo and in vitro. Mechanistically, E3 ligase NEDD4 could directly interact with and target p21 for degradation. Moreover, NDRG1 could emulatively antagonize NEDD4-mediated ubiquitylation of p21, increasing p21 expression and inhibit tumor proliferation.Our study could fulfill potential mechanisms of the NDRG1 during tumorigenesis and metastasis, which may serve as a tumor suppressor and potential target for new therapies in human colorectal cancer. According to the latest globally statistics released in 2018, colorectal cancer (CRC) is estimated to be the third most commonly diagnosed malignant tumor  and the second leading cause of cancer-related deaths  . In addiN-myc downstream regulated gene-1 (NDRG1), a member of the NDRG gene family, is pervasively expressed in epithelial tissues and is a cytoplasmic and nuclear protein –6. NDRG1Dysregulated proliferation is a notable feature of tumorigenesis. Unlike the normal cells, whose proliferation is well balanced by growth and antigrowth signals, the tumor cells have their own growth signals, and their proliferation is precisely regulated by cell cycle regulators . p21, th.Therefore, based on the functions of NDRG1 and p21 in diseases, there may be interactions between NDRG1 and p21. In this study, we demonstrated that NDRG1 could inhibit tumor proliferation through increasing p21 protein expression in vivo and in vitroA total of 89 CRC cases were recruited from Ruijin Hospital  in accordance with the guidelines set by the Ethical Committee of Ruijin Hospital. All cases got the written, informed consent before the study. Staging of CRC was performed according to the UICC guideline (8th Edition). The cohort of 89 tumor tissues and paired normal colonic tissues were fixed with formaldehyde and embedded with paraffin. Tissue array was constructed for further immunohistological assay. The staining score of each tissue was calculated based on the widely used German semi-quantitative scoring system by three independent pathologists as described previously , 31, 32,2 at 37 °C. The plasmid vector containing NEDD4, Flag-p21 and HA-Ubiquitin were constructed by Genechem Inc. . Lentivirus vectors containing Flag-p21 were constructed by Genechem Inc. . For a transient transfection, cells were seeded in a 6-well culture plate 24 h before transfection, then cells were transfected with corresponding si-RNA or vector, using Lipofectamine 3000  following the manufacturer’s protocol. Sequences of siRNA used were as follows: p21: sense-1 5′-GAUGGAACUUCGACUUUGUTT-3′, antisense-1 5′-ACAAAGUCGAAGUUCCAUCTT-3′; sense-2 5′-CCUCUGGCAUUAGAAUUAUTT-3′, antisense-2 5′-AUAAUUCUAAUGCCAGAGGTT-3′; sense-3 5′-CAGGCGGUUAUGAAAUUCATT-3′, antisense-3 5′-UGAAUUUCAUAACCGCCUGTT-3′. NEDD4: sense-1 5′-GGAUGUUCCAACUCAUCUUTT-3′, antisense-1 5′-AAGAUGAGUUGGAACAUCCTT-3′; sense-2 5′-CCAAGAAGUCACAAAUCAATT-3′, antisense-2 5′-UUGAUUUGUGACUUCUUGGTT-3′; sense-3 5′-GCACAUCUCGGGUGCCUAUTT-3′, antisense-3 5′-AUAGGCACCCGAGAUGUGCTT-3′.The p53-wt HCT116, the p53-mutant SW1116 colon cancer cell lines and HEK 293 T cells were obtained from the American Type Culture Collection . The NDRG1 overexpression and knockdown clones were established as described previously . All celFor colony formation, after dissociated into single cell, 500 tumor cells (HCT116) or 1000 tumor cells (SW1116) were seeded in six-well plate, which were cultured in an incubator until visible cloning appeared. Then these six-wells plates were washed, fixed with 4% formaldehyde, stained with crystal violet dye, calculated and analyzed.For CCK-8 assay, six repeats of 1500 cells were seeded in 96-well plate and cultured in each group. The growth rates of the cells were determined at 24, 48, 72, 96 and 120 h using Cell Counting Kit-8 . The absorbance was measured at 450 nm using a model 3550 microplate reader .Cells were washed with PBS and then lysed with RIPA buffer. Insoluble materials were removed by centrifugation at 12,000 rpm for 15 min at 4 °C. Equal amounts of protein were separated using 10% SDS-PAGE, transferred to PVDF, and probed with the appropriate antibodies as indicated. Immunoreactive bands were visualized using an ECL kit . The extraction of proteins from nuclear and cytoplasmic fractions were performed using Subcellular Protein Fractionation Kit from Thermo Scientific . Primary antibodies including: NDRG1  from Abcam; p21  from Cell Signaling Technology; p21  from Abcam; NEDD4  from Cell Signaling Technology; NEDD4  from Proteintech; Flag  and Histone H3  from Cell Signaling Technology. The secondary antibodies implemented include horseradish peroxidase-conjugated anti-goat , anti-rabbit , and anti-mouse  antibodies from Sigma-Aldrich.RNA extraction and qPCR analysis were performed as described previously , 33. TheCells were washed twice with ice-cold PBS and lysed by RIPA buffer containing protease inhibitors . Protein (300 mg) was incubated with specific antibody overnight at 4 °C. This mixture was added to 30 ul of beads  from Santa Cruz Biotechnology  and incubated for 4 h at 4 °C. Then washed by ice-cold PBS, resuspended in loading buffer, and incubated over 90 °C for 10 min. The supernatant was separated on a 10% Bis-Tris gel. Then the mixture was detected by western blots.http://ubibrowser.ncpsb.org/). The co-expression analysis of NDRG1 and p21 in GSE33114 and GSE37892 profiles was performed via the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). The co-expression analysis of NDRG1 and p21 in the cancer genomic atlas (TCGA) was performed in The Gene Expression Profiling Interactive Analysis .UbiBrowser Database was used to screen possible E3 ligases related to NDRG1 or p21 .As described previously , cells w6 HCT116 cells  or SW1116 cells  were injected subcutaneously into nude mice . Tumor size was measured every 7 days. Tumor volume (V) was determined by measuring the length and width of the tumor and using the formula V = (width*width*length) / 2. Twenty-eight days after injection, all the mice were sacrificed and then tumor grafts were excised, fixed with formalin and embedded with paraffin for further immunohistochemistry research.Mice were cultivated under standard conditions following institutional guidelines. A total of 5 × 10t test. Differences with a P value < 0.05 were considered as statistically significant.IBM SPSS Statistical software (version 19.0) was utilized for statistical analysis. Differences were compared using a two-tailed Student p < 0.0001). Further analysis demonstrated that NDRG1 was positively stained (+) in 59.6% (53/89) of normal tissues, whereas only 29.2% (26/89) of carcinoma cases were positive to NDRG1 stain (p <  0.0001). To investigate the clinical significance of NDRG1 in CRC, the correlation between NDRG1 expression and clinicopathological variables was analyzed. The data showed a negative correlation between NDRG1 expression and local invasion, as well as lymphatic metastasis (Table p <  0.001), indicating that NDRG1 might be related to tumor proliferation. As for Overall Survival (OS) and Disease-Free Survival (DFS), patients in the NDRG1-negative group (−) had a significantly poorer prognosis than those in the NDRG1-positive group (+) . Results showed that NDRG1 expression was significantly lower in CRC tissues compared to their corresponding non-tumorous tissues . The results showed an obvious positive relationship between NDRG1 and CDKN1A in GSE33114 , GSE37892 , and TCGA   after NDRG1 overexpression, while NDRG1-knockdown obviously decreased the expression  of p21 in HCT116 and SW1116 cell lines. Besides, we checked the intensity of p21 by immunofluorescence staining. In agreements with the immunoblot results, immunofluorescence showed that the staining intensity of p21 was obviously increased and decreased in response to NDRG1 overexpression and knockdown, respectively . At various times after CHX treatment, aliquots of cells were removed, protein was extracted, and p21 protein in these extracts was quantified by immunoblotting analysis. As shown in Fig. Taken together, these results suggested that NDRG1 could increase p21 protein expression through suppressing p21 ubiquitylation and degradation.It is still not clear how NDRG1 regulates p21 ubiquitylation. E3 ligase is responsible for the ubiquitylation-induced protein degradation. As described above, p21 can be targeted by many E3 ligase. Therefore, we screened possible E3 ligases related to NDRG1 and/or p21 using UbiBrowser Database. Results showed that there were 29 and 211 possible proteins, acting as E3 ligases to target NDRG1 and p21, respectively Fig. c. FurtheTo further confirm the relationship between p21 and NEDD4, we overexpressed both Flag-p21 and NEDD4 in HEK 293 T cell line (293 T Flag-p21/NEDD4). When Flag-p21 was immunoprecipitated using anti-flag antibody, NEDD4 could be detected in this protein complex and vice versa. Besides, NEDD4 overexpression could decrease p21 protein levels , while the xenografts grew significantly bigger in the SH-NDRG1 group   . All these results above indicated that p21 overexpression could attenuate the tumor growth induced by NDRG1 silencing.To further determine whether p21 was responsible for the changes in tumor growth regulated by NDRG1, we constructed stably transfected SW1116 cell lines with p21 overexpression after NDRG1 was knocked down (SH-NDRG1/p21). The sh-Control, SH-NDRG1, and SH-NDRG1/p21 cells were subcutaneously injected into the flank of nude mice. Similar to HCT116 cells, when NDRG1 was silenced in SW1116 cells, the tumor weight and volume dramatically increased compared to the relative control group  and  Subcellular Fractionation Assay: expression of NDRG1 and p21 protein were detected in both nucleus and cytoplasm after NDRG1 overexpressed/silenced. Histone H3 and GAPDH were used as loading control for nucleus and cytoplasm, respectively. (B) qPCR analysis of NDRG1 and p21 mRNA levels after NDRG1 overexpressed/silenced. Error bar represents the mean ± SD of 3 independent experiments. ****p < 0.0001. n.s not significant.Additional file 3: Figure S2. NEDD4 could promote the ubiquitylation of p21 and induce p21 degradation. (A) Plasmids of Flag-p21 and NEDD4 were co-expressed in 293 T cells (293 T Flag-p21/NEDD4). Co-immunoprecipitation were performed to confirm their interaction. (B) NEDD4 overexpression could decrease p21 protein level. (C) We overexpressed HA-Ubiquitin, Flag-p21 or NEDD4 in 293 T cells. Flag antibody was used to immunoprecipitate Flag-p21. p21 ubiquitylation was then detected by immunoblotting by ubiquitin antibody.Additional file 4: Figure S3. The percentage of Ki-67-positive cells were quantified in Vector Control, NDRG1, sh-Control and SH-NDRG1 groups of Fig.


Real-time video streaming over vehicular ad-hoc networks (VANETs) has been considered as a critical challenge for road safety applications. The purpose of this paper is to reduce the computation complexity of high efficiency video coding (HEVC) encoder for VANETs. Based on a novel spatiotemporal neighborhood set, firstly the coding tree unit depth decision algorithm is presented by controlling the depth search range. Secondly, a Bayesian classifier is used for the prediction unit decision for inter-prediction, and prior probability value is calculated by Gibbs Random Field model. Simulation results show that the overall algorithm can significantly reduce encoding time with a reasonably low loss in encoding efficiency. Compared to HEVC reference software HM16.0, the encoding time is reduced by up to 63.96%, while the Bjontegaard delta bit-rate is increased by only 0.76–0.80% on average. Moreover, the proposed HEVC encoder is low-complexity and hardware-friendly for video codecs that reside on mobile vehicles for VANETs. Vehicular ad-hoc networks (VANETs) can provide multimedia communication between vehicles with the aim of providing efficient and safe transportation . VehicleThe demanding challenges of VANETs are bandwidth limitations and opportunities, connectivity, mobility, and high loss rates . BecauseConcurrently, the main video codecs are High Efficiency Video Coding  that are developed by the Joint Collaborative Team on Video Coding (JCT-VC) Group, and AV1 that is developed by Alliance for Open Media (AOMedia) . NeverthWe propose a low-complexity and hardware-friendly H.265/HEVC encoder. The proposed encoder allows the encoding complexity to be reduced significantly so that low delay requirements for video transmission in power-limited VANETs nodes are satisfied.A novel spatiotemporal neighboring set is used to predict the depth range of the current coding tree unit. The prior probability of coding unit splitting or non-splitting is calculated with the spatiotemporal neighboring set. Moreover, the Bayesian rule and Gibbs Random Field (GRF) are used to reduce the encoding complexity for H.265/HEVC encoder with the combination of the coding tree unit depth decision and prediction unit modes decision.The proposed algorithm can balance the encoding complexity and encoding efficiency successfully. The encoding time can be reduced by 50% with negligible encoding efficiency loss, and the proposed encoder is suitable for real-time video applications.To address this issue, spatial and temporal information is widely used to reduce the computation redundancy of the H.265/HEVC encoder. However, the spatiotemporal correlation between the coding unit and the neighboring coding unit is not better used. To the author’s best knowledge, complexity reduction in real-time coding with the available computational power at the VANET nodes has not been well studied, especially from the viewpoint of hardware implementation. The key contributions of this work are summarized as follows:The rest of paper is organized as follows. Related works are reviewed in Real-time Video transmission over VANETs can improve emergency responses’ effectiveness for road safety applications. The high rates and low delay are the most challenging aspects of video transmission over VANETs . RecentlTo reduce the encoding complexity of the H.265/HEVC encoder, some fast algorithm of coding unit (CU) size decision and prediction unit (PU) mode decision is presented. In previous works, the main spatiotemporal parameters that are used for fast CU size decisions include the neighboring CU depth, rate-distortion (RD) cost, motion vector (MV), coded block flag (cbf), and sample-adaptive-offset (SAO) information. Moreover, some other statistical learning based CU selection methods are proposed include Bayesian classifier, support vector machine (SVM), decision tree (DT), AdaBoost classifier and artificial neural network (ANN).Jiang et al. presented a fast encoding complexity method based on the probabilistic graphical model ,20. ThesTai et al. introduced three novel methods including early CU split, early CU termination and search range adjustment to reduce the computation complexity for H.265/HEVC . This prAll in all, this paper focus on the development of video encoder that supports real-time video streaming over VANETs. We design a low-complexity and hardware-friendly encoder to allow video transmission to adapt to the VANETs environment. In addition, compared with the current literature, the proposed encoder can achieve better performance trade-off.H.265/HEVC standard was released in 2013 by JCT-VC, which can reduce bit-rates by about 50% over H.264. In addition, H.265/HEVC adopts hybrid video compression technology, and the typical structure of H.265/HEVC encoder is shown in The video frame is divided into a lot of coding tree units (CTUs) in H.265/HEVC standard. A CTU includes a coding tree block (CTB) of the luma samples, two CTBs of the chroma samples, and associated syntax elements. The CTU size can be adjusted from The prediction unit (PU) can be transmitted in the bitstream, which identifies the prediction mode of CU. A PU consists of a prediction block (PB) of the luma, two PB of the chroma, and associated syntax elements. A CU can be recursively divided into transform units (TUs) according to the quadtree structure, and CU is the root of the quadtree. The TU is a basic representative block having residual or transform coefficients. In TU, one syntax element named coded block flag (cbf) indicates whether at least one non-zero transform coefficient is transmitted for the whole CU. When there is a non-zero coefficient, cbf is equal to 0. When there is no non-zero coefficients, cbf is equal to 1. Moreover, cbf is an important factor for the CU size decision .The advantage of block partitioning structure is that the arbitrary size of CTU enables the codec to be readily optimized for various contents, applications, and devices. However, the recursive structure of coding block causes lots of redundant computing. In order to support the real-time video transmission over VANETs, the redundant computing of the H.265/HEVC encoder should be decreased significantly.The object motion is regular in video sequences and there is some continuity in the depth between adjacent CUs. If the depth range of the current CU can be inferred from the encoded neighboring CU, then some hierarchical partitioning is directly skipped or terminated. Therefore, the computational complexity has been reduced, significantly.In order to utilize the spatiotemporal correlation, the four neighborhood set G is defined asG is shown in Set G are used to predict the depth range of current CTU, and the predicted depth of current CTU is calculated ask is the index of neighboring CTU in set G, G, and G. In the H.265/HEVC standard, the range of CTU is depth 0, 1, 2, and 3. Hence, the calculated depth of the current CTU when the predicted depth of current CTU (2)when the predicted depth of current CTU (3)when the predicted depth of current CTU Due to the predicted depth of the current CTU, each CTU can be divided into three types: In this case, the minimum depth of current CTU i = 0, 1. In this work, x represents the RD-cost of the PU. According to the Bayes’ rule, the posterior probability The CU splitting or non-splitting is formulated as a binary classification problem According to Bayesian decision theory, the prior probability Otherwise, CU splitting φAt the encoding time for inter prediction, first of all, look up the statistical parameters in LUT. Then, the RD cost of the inter (2)RD cost of the inter (3)RD cost of the inter (4)Other PU modes are checked according to the H.265/HEVC reference model.Through the above analysis, the proposed PU decision based on Bayes’ rule includes the CU termination decision  is used to represent the average bit-rate , and theIn this work, the scenarios have been chosen carefully. This work focuses on the development of a video codec that supports real-time video transmission over VANETs for road safety applications. The common test conditions (CTC) are provided to conduct experiments . The tesVideo objective quality evaluation can be expressed by rate–distortion (R–D) curve. The R–D curve is fitted through four data points, and PSNR/bit-rate are assumed to be obtained for QP = 22, 27, 32, 37. In addition, when an error on the predicted depth of the current CTU occurs, the bit-rate will increase. In this paper, the video objective quality is evaluated by using bit-rate and PSNR. Then the lower the accuracies of the predicted depth of the current CTU algorithm, the more the bit-rate increases. The performance comparison of the proposed method is shown in In order to develop the low-complexity and hardware-friendly H.265/HEVC encoder for VANETs, based on a novel spatiotemporal neighborhood set, the Bayesian rule and Gibbs Random Field are used to reduce the encoding complexity for the H.265/HEVC inter-prediction in this work. The proposed algorithm consists of CTU depth decision and PU mode decision methods. Experimental results demonstrate that the proposed approach can reduce the average encoding complexity of H.265/HEVC encoder by about 50% for VANETs, while the increasing of BDBR is less than or equal to 0.8% on average.


The study on the impact of video lecturing on clicker-assisted English flipped class was necessary because it has seldom been explored.This study studied the impact of video lecturing on student satisfaction and English proficiency, plus correlations between student satisfaction levels and English proficiency.Randomly recruited Chinese participants  from a university in China received both pre and post College English Test Band 4 and satisfaction measurements, together with a semi-structured interview.p < .001, Partialη2 = .216); there were significant differences between video- and non-video-assisted cohorts for post interaction , post efficacy , and post regulation ; there were strong, positive relationships between post English proficiency and post student interaction , self-efficacy  and self-regulation  levels in both cohorts at the .05 level. However, no strong, positive correlations were found in both cohorts at the .05 level between pre English proficiency and pre student interaction , self-efficacy  and self-regulation  levels. Future research into video-assisted English flipped class may need interdisciplinary cooperation.We concluded that the video-assisted class could cause significantly higher English proficiency than the non-video-assisted class (F = 23.17,  The flipped or flipped pedagogical approach is a method which flips the teaching and learning process–2. ThrouClicker system, also referred to as classroom response system, is a popular information technology mainly composed of remote controllers, hand-held emitters, receivers, computers, multimedia projectors, screens and software. Using this system, students can anonymously poll and join learning activities involving peer discussion and problem solving, by which learning efficiency can be improved and learAssisted with video lecturing designed and provided by teachers, the concepts hard to be perceived through texts, and graphics or PowerPoint slides may be easily understood. Before Considering the contradictory results, it is therefore important to determine the effectiveness of teaching tools, e.g. videos, slides, blackboard, multimedia projector, and online communicative technologies in the cVideo-assisted learning was endowed with powerful features to constitute an effective pedagogical approach to improve academic achievements and student satisfaction. With raVideos provided students with convenience in English listening and speaking practice, together with easiness of relearning contents. Videos-Video lecturing also enabled students to understand previous lectures before they attended the physical or virtual class, facilitating their understanding and improving academic activities. Video lFurthermore, in the context of online learning, video lecturing through images, sound, and other multiple inputs might enlarge capacity of working memory than the traditional blackboard and chalks-aided teaching. They miTeachers and students were more interested in classes assisted with videos than in those without aid of videos. The vidBesides watching video lecturing, students could also discuss and finish the academic tasks assigned by teachers in the flipped classroom that involved blended learning. This isThe appropriate selection of pedagogy plays an important role in the asynchronous learning assisted with video lecturing. The video assisted pedagogy was beneficial to students’ academic activities. StudentAs a popular teaching style, the flipped class integrated the asynchronous video lecturing into assignment, and cohort-based learning activities. As a poFurthermore, video lecturing-based pedagogical approach enjoyed students, reduced their anxiety and lessened their workload. Video-aIn the flipped pedagogical approach, student participation in learning activities was encouraged and student satisfaction was also enhanced. The vidHowever, video lecturing did not prove the best medium of instruction for introductory political science course. In the non-video-assisted course, the teacher and the course were more highly evaluated and students scored higher on exams than the video-assisted course, and theDespite that use of videos has been widely studied in the flipped class, the results were inconsistent and the effect of videos on clicker-assisted flipped English class has seldom been explored. Considering the research status quo, research questions are thus raised as: (1) Could the video-clicker-assisted flipped English class cause significantly better academic achievements than the non-video-clicker-assisted flipped English class? (2) Could the video-clicker-assisted flipped English class cause significantly higher student interaction, self-efficacy and self-regulation levels than the non-video-clicker-assisted flipped English one? (3) Are the post interaction, self-efficacy and self-regulation levels positively correlated with the post English proficiency in both pedagogical approaches? In order to answer this question, the pre interaction, self-efficacy and self-regulation levels (pretests) should be equivalent in both cohorts, and so should the pre-CET 4 results (pretests).Most literature supported the effectiveness of videos in flipped classes in terms of academic achievements and satisfaction. It has also been largely demonstrated that there is a positive relationship between academic achievements and satisfaction. Based on previous findings, three null hypotheses are raised as follows:H1: The video-clicker-assisted flipped English class could not lead to significantly higher English proficiency than the non-video-clicker-assisted flipped English class.H2: The video-clicker-assisted flipped English class could not cause significantly higher student interaction, self-efficacy and self-regulation levels than the non-video-clicker-assisted flipped English class.H3: The post interaction, self-efficacy and self-regulation levels are not positively correlated with the post English proficiency in both pedagogical approaches. We assume that the pre interaction, self-efficacy and self-regulation levels are positively correlated with the pre-English proficiency in both pedagogical approaches.The research lacks consent because the data were analyzed anonymously. The research has been approved by the authors’ institutional review board-School of Foreign Languages of Hohai University, which waived the need for written informed consent from the participants. All experiments conform to the relevant regulatory standards.http://dx.doi.org/10.17504/protocols.io.7sjhncnThe DOI link: This research attempts to integrate quantitative into qualitative research methods using several scales to measure English proficiency and satisfaction levels, together with a semi-structured interview to collect qualitative data. English was the linguistic medium of administration of questionnaires and interviews.Randomly selected 87 Chinese undergraduates  from a public university in China were willing to join the research, whose formal English education endured for approximately eight to nine years. They were normally literate and were in a normal psychological state based on their self-reports. They ranged from 19 to 22 in age . They were assigned to two cohorts on a random basis, who received English teaching in the video-clicker-assisted flipped English class (N = 41) and the non-video-clicker-assisted flipped English class (N = 46) respectively. Both cohorts, under the instruction of the same teacher, had similar English proficiency identified through College English Test Band Four (CET 4). Both cohorts were assumed to be equivalent to each other in terms of pre-interaction, self-efficacy and self-regulation levels because they were randomly divided into two groups from the same population who shared the same educational and social backgrounds. This assumed similar English proficiency and interaction, self-efficacy and self-regulation levels established a basis for comparison.Pre English proficiency and pre interaction, self-efficacy and self-regulation levels of the randomly selected participants were then determined via CET 4  and the interaction, self-efficacy and self-regulation scales after they were randomly assigned to either intervention or control cohorts.The teacher was awarded a Ph.D. degree in English language. Before the study commenced, he also received specialized training regarding both video-assisted and non-video-assisted pedagogical approaches.Participants in the non-video-assisted cohort were asked to read textbooks before class without access to videos, while those in the video-assisted cohort had access to videos plus textbooks when preparing for class. The video-assisted cohort was required to complete all of the tasks that the non-video-assisted cohort completed and vice versa. Both cohorts of participants were instructed via clickers. The only difference was whether students could learn English assisted with videos. In the video-clicker-assisted flipped English class, students learned English aided with videos, while in the non-video-clicker-assisted flipped English class, they learned English without aid of videos.Four scales, including a CET 4 to determine English proficiency and three scales to identify interaction feasibility, self-efficacy and self-regulation levels, were used as research instruments in both the video-clicker-assisted flipped English class and the non-video-clicker-assisted flipped English class.CET 4 in June, 2012, designed under the guideline of Ministry of Education of China, was used to identify English proficiency. CET4 was internally reliable and externally valid. Its identification of English proficiency has been generally accepted since it came into being decades ago. Test itAs shown in I strongly disagree, I disagree, neutral, I agree, I strongly agree, were used to identify interaction feasibility, self-efficacy and self-regulation respectively  Clickers-assisted English class provides a discussion platform for interaction; (2) Clickers-assisted English class presents an easy access to frequently asked questions; (3) Clickers-assisted English class provides a place to discuss questions.Cronbach’s alpha is a method to examine reliability, which was proposed by Lee Cronbach in 1951. It overcomes the shortcomings of partial halving method and is the most commonly used reliability analysis method in social science research. In general exploratory studies, the Cranbach’s alpha coefficient is above 0.6, and the benchmark study is above 0.8.The internal consistency was demonstrated by Cronbach’s alphas see . Three sA semi-structured interview, designed by professors in English language teaching, was designed in order to collect qualitative data. Researchers informed participants that all the information in the interview would remain confidential and would merely be used in the research. All the interviewees would be properly rewarded for their cooperation after the interview.The interview is made of three sections involving demographic information, interview questions and acknowledgment. Section 1 focuses on interviewees’ demographic information including names, ages, genders, educational backgrounds, working experiences, literacy and psychology. Section 2 is mainly concerned with interview questions. For the interaction feasibility, we selected 2 to 3 questions for interviewees to answer. For the scale of self-efficacy, 3 to 4 questions were selected. For the scale of self-regulation, we selected 2 to 3 questions See . We alsoLearning tasks of participants were to improve their English proficiency through an intensive English reading course for two semesters. Both cohorts learned English assisted with Clicker system.The teaching progress assisted with Clicker system is visualized in As shown in The teaching progress was decided based on the answers of students. If most of students (>80%) answered correctly, the teacher would move on after correcting the wrong answers. If nearly half students answered correctly, the teacher would stop to explain the question in details and continue the teaching progress after they understood them. If most of the students answered wrongly, the teacher would repeat the teaching progress after most of them learned the knowledge, understood the questions and provided correct answers.The intervention cohort was instructed through the video-clicker-assisted flipped English class, while the control cohort was taught via the non-video-clicker-assisted flipped English class instruction.The video was used to both present the image of the teacher and show educational materials. The intervention cohort was provided with teaching videos through the communicative platform. They were required to download videos from the online space. The activities were automatically recorded by the platform. In both the physical and virtual classes, the teacher designed quizzes or tests in the content of the videos in order to encourage students to view and understand videos. Task and key knowledge were included in the videos. Students could not successfully perceive the knowledge and complete the task without watching videos, which ensured that the intervention cohort truly made use of the videos.After two academic semesters, both cohorts were tested in terms of English proficiency and interaction, self-efficacy and self-regulation levels. The pre and post test results were entered into a computer for further analysis and discussion. The only difference between the intervention cohort and the control cohort was that the former was instructed assisted with videos, while the latter was not instructed with the aid of videos. Consequently, differences in student interaction, self-efficacy and self-regulation levels and English proficiency were due to whether video lecturing was used. The effect of videos on clicker-assisted flipped English class would thus be revealed.The videos, enduring 10 to 50 minutes, were all designed by the teacher, who aimed to inspire and encourage students to improve their listening, speaking, reading, writing and translating skills. Cultural awareness and linguistics-related knowledge would also be integrated into language teaching via the videos.This section includes the results of the assumption and hypotheses, together with those from the semi-structured interview.In order to test the hypotheses using an appropriate statistical test, the pre interaction, self-efficacy and self-regulation levels and pre English proficiency were assumed equivalent in both cohorts.F = .739, p = .392), self-efficacy  and self-regulation levels  and pre English proficiency  at the .05 significance level for video or non-video assisted conditions. Taken together, pre interaction, self-efficacy and self-regulation levels and pre English proficiency are not significantly different in video or non-video assisted conditions. Our results suggest that the pre interaction, self-efficacy and self-regulation levels and pre English proficiency are equivalent in both cohorts.A one-way between-subjects ANOVA was conducted to compare the effect of video or non-video assisted conditions on pre interaction, self-efficacy and self-regulation levels and pre English proficiency. There was insignificant effect of pre interaction  between video and non-video lecturing-assisted cohorts. Post hoc tests showed there was a significant difference between video lecture and non-video lecture assisted cohorts (p<0.001). Comparing the estimated marginal means showed that the video lecturing-assisted cohort (M = 5.058) obtained significantly higher English proficiency than the non-video lecturing-assisted cohort (M = 5.009). Therefore, we rejected the null hypotheses “the video-clicker-assisted inverted English class could not lead to significantly higher English proficiency than the non-video-clicker-assisted inverted English class”.As shown in To test Hypothesis 2, we included video or non-video lecturing condition as a fixed factor, and post satisfaction levels  as dependent variables see .Pillai’s Trace = .169, F = 5.41, df = 3, p = .002, indicating a significant difference in the satisfaction levels of student use of video lecturing between both cohorts. The univariate F tests showed there were significant differences between video lecturing-assisted and non-video lecturing-assisted cohorts for post interaction , post efficacy , and post regulation  with respect to satisfaction levels. Therefore, we rejected the null hypotheses “the video-clicker-assisted inverted English class could not cause significantly higher student satisfaction than the non-video-clicker-assisted inverted English class”.As shown in In order to test the relationship between English proficiency and student interaction, self-efficacy and self-regulation levels, data collected from the corresponding scales were entered into WarpPLS 6.0 for analysis since WarpPLS is especially reliable for small samples. A WarpPWhen assessing the model fit with the data, several criteria need to be recommended. In this study, average path coefficient (APC) is 0.466, p < 0.001; the average R-squared (ARS) is 0.323, P < 0.001; average adjusted R-squared (AARS) is 0.315, P < 0.001. The model fit is thus recommended since the P values for the APC, ARS and AARS are all equal to or lower than 0.05. FurtherBoth pre and post analyses were conducted simultaneously through the correlational analysis of WarpPLS model See .R2= .70; β = .84; p < .01), self-efficacy  and self-regulation  levels under both the video-clicker-assisted and non-video-clicker-assisted inverted English class models at the .05 level. However, no strong, positive correlations between pre English proficiency and pre student interaction , self-efficacy  and self-regulation  levels were found in both video- and non-video-clicker-assisted cohorts at the .05 level. As a result, we rejected the null hypothesis “the post interaction, self-efficacy and self-regulation levels are not positively correlated with the post English proficiency in both pedagogical approaches”, and the assumption “the pre interaction, self-efficacy and self-regulation levels are positively correlated with the pre English proficiency in both pedagogical approaches” was violated.It can be found from To enhance the reliability of the correlational analysis, a bivariate correlation program was conducted. The results are shown in r = .021, p = .845), self-efficacy  and self-regulation  levels, while there was a strong, positive correlation between the post English proficiency and the post student interaction , self-efficacy  and self-regulation  levels. The results are consistent with those obtained from the WarpPLS model and therefore enhance the reliabilities of rejecting the third null hypothesis and the assumption.Pearson correlation coefficients were computed to assess the relationship between student interaction, self-efficacy and self-regulation levels and English proficiency. As shown in The interview was recorded and the recording was then transcribed for further analysis. The results are generally consistent with the quantitative research findings based on CET 4 and satisfaction scales. Specifically, the results were divided into two sections: opinions of interviewees and the information revealed.We randomly selected 35 interviewees on a voluntary basis. Most of them felt that the video-clicker-assisted flipped English class could help them reach a higher English proficiency level than the non-video-clicker-assisted flipped English class. They reported that the video-clicker-assisted flipped English class was more satisfactory than the non-video-clicker-assisted flipped English class. It was believed that the post satisfaction might exert an important influence on the post English proficiency in either video or non-video assisted approach. They assumed that their pre satisfaction levels were similar and their satisfaction levels with the video and non-video assisted approaches were not significantly different before they joined the research.Many participants thought that videos could expedite the pedagogical process since teachers do not need to painstakingly write with chalks on the blackboard. Instead, they could deliver knowledge assisted with educational technologies such as online recording, typing, and presenting. Participants said, “Videos light us up; They are time-saving; They energize us.” They could also share resources among students asynchronously. Videos including large quantities of contents could undoubtedly increase learning efficiency. Videos could also act as a tutor guiding students through the learning process. Many participants said, “Videos are highly efficient; Videos share information quickly and widely; Videos carry a large amount of information.”It was reported by most interviewees that video lecturing provided them with flexibility, accessibility, and diversity when they achieved academic achievements by meeting various individual learning needs via pieces of knowledge and by accommodating individual needs via rich resources. Video lecturing was also available to students day and night, and students were able to watch video lectures from any internet-connected, video-play device such as smart phones and laptops. Interviewees thus believed that their learning styles were flexible with regard to their timetables, with which students with different learning styles were satisfied since they could flexibly choose the time and venue to watch or review lectures. Many interviewees thought that they could apply and appreciate the videos that enabled them to pause, rewind or replay at their will.Note taking and note reviewing were essential learning activities, which was demonstrated by a tremendous number of studies. InterviNevertheless, interviewees also showed their worry about the possibility of increasing teachers’ cognitive load in that teachers had to spend a huge amount of time and energy in recording and designing the videos. Furthermore, the benefits of video lecturing failed to be demonstrated as better results in English intensive reading courses. They complained that they preferred to read the static books rather than read books when walking. They also sometimes considered video lecturing a way to distract them from reading due to the noise along with the video play. When forced to read the mobile materials, they tended to suffer from eye strain, which discouraged them from continuously watching the videos.The qualitative accounts provided by the participants also revealed some important information. A phenomenon worth attention is participants’ report on their feelings about use of videos. For example, numerous students gave detailed accounts of experiencing pleasure and enjoyment about video viewing. They said, “I like watching videos; I enjoy watching videos; Learning through the video is interesting.” They also expressed their negative feelings about the lecturing without videos, e.g., “I hate reading the boring texts; I don’t like following the teacher; Listening to the teacher is sometimes boring.”Besides, participants detailed their positive feelings about English learning with the assistance of videos. Many of them expressed similar feelings. Examples are “watching videos can improve my English listening skills; I can improve my sense of English language through viewing videos; Videos can improve my English pronunciation; Watching videos can improve my English skills.” By contrast, they disliked the English learning style without aid of videos. They said, “Learning without videos disgusts me; No lively pictures can be seen without videos; No videos, no English.”This study arrived at the conclusion that the video-clicker-assisted flipped English class could lead to significantly higher levels of English proficiency and student interaction, self-efficacy and self-regulation than the non-video-clicker-assisted flipped English class; the post interaction, self-efficacy and self-regulation levels are positively correlated with the post English proficiency in the video or non video-assisted pedagogical approach; the pre interaction, self-efficacy and self-regulation levels are not significantly correlated with the pre English proficiency in both pedagogical approaches. The findings of this study are generally consistent with previous works, 42, 15.Assisted with videos, students could freely learn the difficult contents they feel necessary. They may speed up, rewind or pause to meet their own needs. Videos made of interesting stories, vivid sounds and colorful pictures could have attracted student attention, due to which students might concentrate on the learning contents. In this way, their English proficiency could doubtlessly be improved. Pieces of knowledge in the videos could also be helpful for students to construct organized and systematic schema. It is therefore reasonable to find that video-assisted academic achievements are significantly higher than those without videos.Interaction, self-efficacy and self-regulation constitute the elements that play key roles in student satisfaction. To deteThe positive correlation between post English proficiency and post interaction, self-efficacy and self-regulation levels indicates that the higher English proficiency is, the more satisfied students will feel and vice versa. In the clicker-assisted flipped English class, student interaction, self-efficacy and self-regulation levels are important factors influencing post English proficiency. To improve student interaction, self-efficacy and self-regulation levels may be a decisive strategy to raise the English proficiency level in the clicker-assisted flipped English class whether or not videos are in use. Interaction, self-efficacy and self-regulation play important roles in student satisfaction. English proficiency is closely related with self-efficacy, self-regulation, peer interaction, and student-content interaction. Thus interaction, self-efficacy, and self-regulation are strongly correlated with English proficiency.This part aims to summarize the research in terms of advantages, disadvantages and future research directions.Despite this study finds that video lecturing is positively correlated with student interaction, self-efficacy and self-regulation levels and academic achievements, no evidence of this finding has been found in previous empirical studies, 44. FinAlthough extensively explored, satisfaction is still an elusive variable that can hardly be fully measured. Measurement of satisfaction may need highly advanced and precise technologies. Questionnaires may never be perfect scales to identify satisfaction. The elements of satisfaction may not be limited to interaction, self-efficacy and self-regulation. Other elements may also be important factors related to satisfaction. Additionally, features of videos may vary from one to another. Features of video lecturing may be an inescapable factor in this study.Many video lectures are simply videos of the face-to-face lecture and therefore, many of the advantages may not be applicable. There may be no rationale for the video lecturing design if interesting pictures and other engaging techniques can’t be presented in the classroom. Video lectures can be rewound or sped up, but they may fail to offer the possibility of clarification that can be realized through face-to-face pedagogy.The double blind approach was not used since it was impossible that the researchers who interacted with the participants did not know which cohort was the intervention group and which was the control group.Students with various economic statuses may perform differently on the video lecturing-assisted teaching. Those who are better-off may have more convenient access to technologies and have better skills than those who are economically lower. The economic status may be considered an important factor in the research of educational technologies.The Type of videos is also a topic worth exploring. As mentioned, rich moving pictures, vivid voice, connected plots, and background music may help students perceive the main contents and command the linguistic knowledge. Future research may thus focus on the effect of videos carrying different information in the flipped pedagogy. Measurement of psychology may be in need of advanced technologies and interdisciplinary cooperation. Interdisciplinary research involving psychology, education, statistics, linguistics and mathematics may be necessary in the future research.S1 Dataset file(RAR)Click here for additional data file.


Mg2+ can regulate vascular functions, yet the mechanistic insight remains ill‐defined. Data show that extracellular Mg2+ enters endothelium mainly through the TRPM7 channel and MagT1 transporter. Mg2+ can act as an antagonist to reduce Ca2+ signaling in endothelium. Mg2+ also reduces the intracellular reactive oxygen species (ROS) level and inflammation. In addition, Mg2+‐signaling increases endothelial survival and growth, adhesion, and migration. Endothelial barrier integrity is significantly enhanced with Mg2+‐treatment through S1P1‐Rac1 pathways and barrier‐stabilizing mediators including cAMP, FGF1/2, and eNOS. Mg2+ also promotes cytoskeletal reorganization and junction proteins to tighten up the barrier. Moreover, Mg2+‐deficiency enhances endothelial barrier permeability in mice, and Mg2+‐treatment rescues histamine‐induced transient vessel hyper‐permeability in vivo. In summary, Mg2+‐deficiency can cause deleterious effects in endothelium integrity, and Mg2+‐treatment may be effective in the prevention or treatment of vascular dysfunction.Mg It participates in >500 metabolic reactions as a cofactor, including DNA and RNA formation, protein composite, cellular energy supply, reproduction, and mitochondrial membrane stabilization.2+‐deficiency has been linked to many disorders, such as Alzheimer's disease, migraine headaches, cerebrovascular abnormalities, diabetes, hypertension, and other cardiovascular diseases. Most Mg2+ exists in bone, muscle, and soft tissues, and serum Mg2+ concentration (0.7–1.05 mmol L−1) cannot represent the total body Mg2+ content with taking only 1% of that. Thus, the body can be in a state with severe Mg2+‐deficiency while the serum values could be within the normal range. According to this, medical influence of Mg2+‐deficiency could be largely underestimated. Moreover, Mg2+ levels in serum and other tissues are not routinely measured in clinical practices. Therefore, Mg2+ is often regarded as the “neglected” cation in human health.[qv: 1a]As the second most abundant intracellular divalent cation, magnesium , Mg2+ transporter 1 (MagT1), solute carrier family 41 member 1/2 (SLC41A1/2), cyclin M2/3/4 (CNNM2/3/4), and mitochondrial RNA splicing 2 (MRS2).[qv: 1a] Some of these transporters are ubiquitously expressed whereas most are highly tissue, cell, and organelle specific. Mechanism of endothelial handling of Mg2+ is largely unknown although it plays a critical role in endothelial function and barrier integrity.Whether Mg2+ may regulate the endothelial barrier function.The endothelial monolayer performs a pivotal role in regular tissue–fluid homeostasis, vascular tone, angiogenesis, and host defense.2+ in different pathophysiological conditions remains elusive, Mg2+ has been used as a treatment for asthma, stroke, preeclampsia, and myocardial infarction in several large‐scale clinical trials over the last decade.[qv: 1a] Such therapeutic effects of Mg2+ are probably because Mg ion is a natural antioxidant, antiinflammation agent, and Ca2+/Na+ antagonist.2+ can regulate vascular endothelial and smooth muscle cell functions related to hypertension and other cardiovascular diseases.2+ may regulate endothelial barrier functions with the underlying mechanism by examining its effect on cell survival/growth, Ca2+ signaling, intracellular oxidative stress, endothelial permeability, cytoskeletal and junction protein organization, and endothelial barrier‐integrity mediators both in cellular models and Mg2+‐deficiency mouse model.Although the exact role of Mg22.12+ influx can be controlled by various plasma membrane transporters including SLC41A1, MagT1, TRPM6, and TRPM7, and their expression might be tissue and cell specific. We demonstrated previously that TRPM7 and MagT1 expressed in brain vasculatures.[qv: 3b] Here, we first showed the existence of SLC41A1, MagT1, and TRPM7 in vascular endothelial cells  changes in cells. Data showed that the addition of Mg2+ into the Mg2+‐free bath induced a notable increase in [Mg2+]i, suggesting the entry of Mg2+ into cells. However, Mg2+‐entry was significantly inhibited after treatment with siRNA specifically against MagT1 and/or TRPM7, respectively, but not SLC41A1  as confirmed by increased fura‐2 fluorescence. Basal [Ca2+]i was unchanged in all groups with different treatment. Mg2+ blocked the ionomycin‐mediated [Ca2+]i responses significantly, indicating Mg2+ is an antagonist of Ca2+ signaling by binding to Ca2+ channels competitively  by DHE fluorescence after Mg2+ deprivation with or without H2O2 treatment. Mg2+‐deficiency induced significantly higher level of ROS over time regardless the presence of H2O2  was required to significantly abolish the oxidative stress induced by addition of H2O2. MagT1 and/or TRPM7 knockdown by siRNA attenuated such rescue effect of Mg2+ on intracellular oxidative stress , which are upregulated during vascular inflammation. In the Mg2+‐deficiency mouse model, the mRNA levels of ICAM‐1 and VCAM‐1 were significantly higher than that of normal controls in brain vasculature . Significantly lower TER and higher permeability were observed after Mg2+ Figure6A,B. T2.72+‐deficiency were significantly lower than that of Mg2+‐treatment  compared to Mg2+‐deficiency .[qv: 3a] Due to its importance to human body, Mg2+ plays an essential role in disease precaution, treatment, and entire health. Hypomagnesemia has been linked to many chronic disorders such as Alzheimer's disease, Parkinson's disease, stroke, hypertension, migraine headaches, cardiovascular abnormalities, and diabetes.[qv: 1b]Mg2+, thus intracellular concentrations of Mg2+ are strictly adjusted.[qv: 1a] Demonstrating the molecular properties of transporters involved in Mg2+ homeostasis has been the main target of research over the last two decades. A few Mg2+ transporters have been identified through genetic screenings on human diseases. The exact role of these transporters need further investigations and several of them have been characterized functionally. TRPM7, MagT1, SLC41A1, and CNNM3 are ubiquitous and expressed on plasma membrane. SLC41A2 is mainly on Golgi membrane while MRS2 is on mitochondrial membrane though they are also ubiquitously expressed. TRPM6 is primarily located in epithelial cells in kidney and intestine. CNNM1 is found to be mainly in brain tissue. CNNM2 and CNNM4 are primarily located on the basolateral plasma membrane of kidney and intestine, respectively. The specific Mg2+ transporters responsible for Mg2+ homeostasis in endothelium is still unknown. In this study, we found that TRPM7 and MagT1 are the major plasma membrane transporters facilitating extracellular Mg2+‐entry into cells and regulating subsequent Mg2+‐signaling.Many cellular processes like the synthesis of DNA and protein are highly dependent on the availability of intracellular Mg2+ is a known natural antioxidant, and reduces inflammation in various pathological conditions. Therefore, it has protective effect on different cells including neurons and vascular cells due to inflammation and oxidation. For example, MgSO4 treatment reversed lipopolysaccharide‐mediated inhibition of apolipoprotein E mRNA expression in both rat and human placentas.[qv: 5a] Oral Mg2+ supplementation induces favorable antiatherogenic changes in apolipoprotein E‐deficient mice.2+ could overcome the deleterious metabolic consequences related to apolipoprotein E4.2+‐deficiency induced a decrease in the percent composition of apolipoprotein E in rats.[qv: 13a] Consistent with these findings, our data also demonstrated that Mg2+ can suppress ROS production in endothelium with or without pro‐oxidant H2O2.Mg2+ channel blocker and competing with Na+ for binding sites on cells are one of the mechanisms that Mg2+ protects cells.[qv: 5b] Thus, it decreases intracellular Ca2+ and Na+, binds to K+ in a cooperative manner, increases prostaglandin E level, improves endothelial dysfunction, induces endothelial‐dependent vasodilation, and reduce blood pressure.[qv: 6c] K+ is vasoactive and increases blood flow when infused into the arterial supply of a vascular bed. The vasodilation results from hyperpolarization of the vascular smooth muscle cell subsequent to K+ stimulation by the ion of the electrogenic Na+–K+ pump and/or activating the inwardly rectifying K+ channels.2+ can regulate this rectification through blocking the K+ channel, functioning in at least four potassium channels.+ channels in an outward direction.2+ channels which become more prominent when going down from conduit arteries to resistance vessels embedded in parenchyma.2+, Na+, and K+, Mg2+ can control cellular pH, insulin sensitivity, left ventricular mass, as well as arterial compliance. It also holds back circulating Na+/K+‐ATPase suppressive activity that decreases vascular tone.[qv: 6c] Mg2+, as an analogous Ca2+ channel blocker, results in formation of vasodilator prostacyclin and NO, and alters the vascular reaction to vasoactive agonists. These biochemical processes administer vascular contraction and relaxation, survival and growth, differentiation, and inflammation. Variation in Mg2+ hemostasis and transport system may predispose patients to hypertension and other cardiovascular dysfunctions.Acting like a natural Ca2+ controls the endothelial barrier function is by regulating barrier‐stabilizing mediators. Results showed Mg2+ enhanced the protein expression of cAMP, FGF1/2, Akt, Ang, and eNOS. Upregulation of cAMP levels by Mg2+ diminishes vascular leakage via activating PKA, in line with a previous study.2+ to enhance endothelial barrier functions.Another mechanism by which Mg42+ regulates the endothelial barrier function through TRPM7, MagT1, and S1P1‐Rac1 pathways both in vitro and in vivo ‐2,5‐diphenyltetrazolium bromide (MTT) assay as described before. Cell proliferation was measured using a BrdU cell proliferation kit as described before.Intracellular ROS Measurement: Dihydroethidium (DHE) was used to determine reactive oxygen species production following a modified protocol as described before.Cell Adhesion and Migration: Cell adhesion and migration assays were carried out as described before.[qv: 6k] The average cell migration rate was calculated.cAMP and PKA Activity: Cellular cAMP activity was measured using a direct immunoassay kit following manufacture's instruction.Real Time PCR: After different treatments, cells were harvested and total RNA was extracted by using an RNeasy Mini kit  and subsequently quantified using a spectrophotometer  with OD260/OD280 ratios between 1.9 and 2.1. Real time PCR was used to estimate the mRNA levels of different gene expression in a CFX96 Touch RT‐PCR Detection System . Data was analyzed by Bio‐Rad CFX Manager 3.1 . The 2−ΔΔCt method was used to calculate gene fold changes. The level of specific mRNA was normalized to the endogenous control, β‐actin.Gene Silencing: Small interference RNA was used to knockdown specific gene expression. Briefly, cells were transfected with siRNA using Lipofectamine 2000  according to the manufacturer's protocol, and used 48 h post transfection. Small interfering RNA (siRNA) targeting human TRPM7, MagT1, and S1P1 were purchased from Santa Cruz Biotechnology. The corresponding scrambled siRNAs were used as controls.Transmonolayer Electrical Resistance (TER): The cells were cultured on inserts with collagen‐coated polycarbonate in transwell, and TER was measured using an Endohmeter  as described before.2+]I and [Ca2+]iMeasurement of I and [Ca2+]I, respectively, as previously described in the article.[qv: 6b] [Mg2+]I responses to increasing concentrations of extracellular Mg2+ (2 mmol L−1) were measured in cells incubated in Mg2+‐free, Ca2+‐containing modified Hanks' buffer. Cells were exposed to Mg2+‐free buffer for 15 to 20 min before addition of extracellular Mg2+. [Ca2+]I reaction to ionomycin (10−6 mol L−1) were performed in cells incubated for 15 to 20 min in Ca2+‐containing modified Hank's buffer with Mg2+ (1.3 mmol L−1).Permeability of the Endothelial Barrier: Fluorescein‐isothiocyanate (FITC)‐labeled dextran  was used to measure endothelial permeability in transwells as described before.Protein Expression and Phosphorylation: Protein expression and phosphorylation were determined by Western blot as described before.Animal Model and In Vivo Blood Vessel Permeability Measurement Using Miles Assay: All procedures were approved by institutional IACUC following NIH guidelines for the care and use of laboratory animals. C57 mice of 8–10 weeks old (Jackson Labs) were selected for equal age and weight (n = 6). Additionally, equal numbers of male and female mice were used, thus sex was not a biological variable in this specific study. Mg‐deficient mouse model was created by an Mg‐deficient diet for 15 days.−1 and 1,000 mg kg−1 for Mg‐deficient and control diets, respectively. The intravenous injection of Evans Blue in mouse animal model is a fundamental step of Miles assay.Mouse Brain Vessels Isolation and Expression of Cell Adhesion Molecules: Isolation of whole mouse cerebrovascular fragments was performed as described.Transient Vessel Permeability Increase and Mg Treatment In Vivo: To evaluate the rescue effect of Mg2+ on endothelial permeability in vivo, a transient leaky vessel model in mice was created by histamine treatment with or without Mg2+.n = 6). Mice were first pretreated with Mg2+ or saline control. MgSO4 injection was administrated by an IP dose of 350 mg kg−1 followed by 50 mg kg−1 every 20 min for 4 h; injections were given in a volume of 0.1 mL. A second dose of 350 mg kg−1 was given at the end of the 4 h period. Mice were then injected with Evans Blue solution. To induce vascular hyper‐permeability, 5 mg histamine (Sigma‐Aldrich) dissolved in 500 µL PBS was injected via tail vein. After 30 min, mice were sacrificed and subjected to Miles assay. The S1P1 specific inhibitor W146 on Mg‐mediated endothelial integrity was also assessed by pretreating mouse with W146 (20 mg kg−1) together with MgSO4.Statistical Analysis: All data were presented as mean ± standard deviation. Each cellular experiment was repeated independently minimal of three time (n = 3), and each in vivo study was performed using six animals (n = 6). One‐way or two‐way analysis of variance (ANOVA) followed by Tukey post hoc test was used to determine statistically significant differences. Unpaired Student t‐test was also used to compare difference between two groups as appropriate. P < 0.05 was considered statistically significant.The authors declare no conflict of interest.Supplementary.Click here for additional data file.