Video is a versatile and popular medium for digital health interventions. As mobile device and app technology advances, it is likely that video-based interventions will become increasingly common. Although clinic waiting rooms are complex and busy environments, they offer the opportunity to facilitate engagement with video-based digital interventions as patients wait to see their providers. However, to increase efficiency in public health, leverage the scalability and low cost of implementing digital interventions, and keep up with rapidly advancing technology and user needs, more design and development guidance is needed for video-based tailored interventions.We provide a tutorial for digital intervention researchers and developers to efficiently design and develop video-based tailored digital health interventions. We describe the challenges and solutions encountered with Positive Health Check (PHC), a hybrid app used to deliver a brief, interactive, individually tailored video-based HIV behavioral counseling intervention. PHC uses video clips and multimedia digital assets to deliver intervention content, including interactive tailored messages and graphics, a repurposed animated video, and patient and provider handouts generated in real time by PHC.We chronicle multiple challenges and solutions for the following: (1) using video as a medium to enhance user engagement, (2) navigating the complexity of linking a database of video clips with other digital assets, and (3) identifying the main steps involved in building an app that will seamlessly deliver to users individually tailored messages, graphics, and handouts.We leveraged video to enhance user engagement by featuring “video doctors,” full-screen video, storyboards, and streamlined scripts. We developed an approach to link the database of video clips with other digital assets through script coding and flow diagrams of algorithms to deliver a tailored user experience. We identified the steps to app development by using keyframes to design the integration of video and digital assets, using agile development methods to gather iterative feedback from multidisciplinary teams, and creating an intelligent data-driven back-end solution to tailor message delivery to individual users.Video-based digital health interventions will continue to play an important role in the future of HIV prevention and treatment, as well as other clinical health practices. However, facilitating the adoption of an HIV video intervention in HIV clinical settings is a work in progress. Our experience in designing and developing PHC presented unique challenges due to the extensive use of a large database of videos tailored individually to each user. Although PHC focuses on promoting the health and well-being of persons with HIV, the challenges and solutions presented in this tutorial are transferable to the design and development of video-based digital health interventions focused on other areas of health. In this article, we present a tutorial on the challenges and solutions for designing and developing Positive Health Check (PHC), a brief and interactive digital video counseling intervention that can be used on computers or mobile devices  for persons with HIV who are engaged in clinical care. To improve health outcomes, the US public health community is leveraging the nation’s ubiquitous use of digital media . Health The well-documented surge in the development of digital interventions for HIV prevention and care ,9 aims tConsequently, guidance is needed to help researchers and developers design, develop, and disseminate video-based digital interventions for HIV and other fields ,22. ThisPHC’s primary aim is to enhance HIV viral suppression by providing individually tailored video messages on relevant topics, which are delivered by virtual doctors (played by actors) and mimic a clinical encounter. PHC’s secondary aims are to engage people with HIV in clinical care more regularly and to reduce HIV transmission risk. The theoretical foundations of PHC include the Information-Behavior-Motivation model , motivatPHC is also designed to strengthen patient-provider communication and empower patients to voice their concerns and ask questions in medical appointments. This supports current trends in patient education, where the patient and provider collaborate to make treatment decisions . The vidMultiple design considerations aimed to maximize PHC’s digital platform. First, we chose video for its visually engaging qualities and the ability to create a personal experience by tailoring the delivery of video clips to each individual user. The PHC video modules share information through visual, verbal, and written cues (on graphic overlays) and closed captioning. Second, the digital platform facilitates easy updates to PHC video clips and content in the supplemental resources. Third, video clips can be repurposed. For example, the animated condom video  featuredPHC was designed to be used in the clinic setting, where staff provide earbuds and assign a tablet with a privacy screen to the patient. To use the intervention, patients log on to a device, answer four demographic tailoring questions, and then answer risk behavior assessment questions at the start of each intervention module. Based on this information, PHC generates tailored messages. A clip of the intervention that depicts an excerpt from the adherence module is available online .PHC is interactive and allows the patient to choose their “video doctor” by race and gender; if desired, they can switch doctors at every use. In each session, the patient can select intervention content and drill down into the topics they wish to learn about. For example, under the broad topic of adherence, the patient can choose to learn about side effects of medication or taking ART and drinking alcohol. PHC delivers tailored messages to each patient, drawing from a database of 700 unique video clips. Additionally, the patient engages with interactive graphics to select from a menu of behavior change tips. To reach people with disabilities, the tool features closed captioning and keyboard navigation capabilities instead of a mouse or touchscreen. Extra Info, offered at the end of the tool, features peer-delivered educational videos and other resources.Toward the end of the virtual clinic visit, PHC instantly generates two individually tailored handouts, a patient handout -5 and a This section of the tutorial describes guidance on key solutions and challenges we faced in developing PHC around three topical areas related to user engagement, integration of intervention assets, and building the app to deliver complex tailored content .We recognized that several key components were needed to create a video tool that would bring about user engagement and lead patients to become more active in their own health. In this context, user engagement refers to “the micro-level of moment-to-moment engagement with the intervention” designed to promote behavior change to improve health outcomes .User engagement is a critical process to improve intervention effectiveness and behavior change . Our chaWe approached this challenge to limit distractions by enhancing the “realness” of the virtual doctor visit. Although we considered using real-life doctors for the PHC, we ultimately decided to use actors because they are trained to deliver scripts verbatim from teleprompters in front of stage lights and crew. This ensured consistency of the message and delivery across the four virtual doctors embedded in the intervention. Actors appeared in clothing, hair, and makeup that would not reflect a specific decade and soon appear outdated. To mimic what a conversation might be like in a real doctor visit, instead of presenting all of the risk behavior assessment tailoring questions at the beginning of the tool, we embedded these questions throughout the experience. We used a “corner to corner” 16 × 9 video screen that most closely approximates human natural visual perception, thus using the entire screen of the devices that would deliver the intervention. We used a video game structure to model high levels of engagement and immersion, treating each PHC screen depicted in the intervention as a “window” into a virtual experience. Finally, we overlaid graphics on top of the video so the user could still see the video image. This allows users to process the onscreen information but remain engaged and be rooted in the virtual doctor visit.We also limited visual distractions by ensuring that the users’ focus stayed on the actor/doctor and their health messages, rather than on something in the background of the set. We accomplished this by filming in full high definition (1920 × 1080) on a Sony FS700 because the camera’s sensor size was large enough to provide a shallower depth of field, which would allow the background to be slightly blurred. Although the original scripts were developed collaboratively with end users and HIV providers , the creUser inputs resulted in the delivery of tailored video clips and layered multimedia digital assets. Achieving these tailored responses required filming 168 separate video scenes for each of the four actors playing virtual doctors, which eventually would be programmed to play in multiple combinations. The challenge was to correctly plan for filming by visualizing the flow of scenes for each patient as if they were all one seamless interaction. To break down the complexity of PHC, we used storyboards to help visualize the look, feel, and flow of the tool. We created storyboards for every major PHC scenario. The storyboards were created by shooting still images of the location, then overlaying representations of the actor and graphics that would appear on screen. Those images were then paired with the relevant scripting -7. Video is the basic medium of PHC, along with the other digital assets described above: interactive and tailored graphics, the repurposed Safe in the City animated video, closed captions, and user-generated handout templates. To make the intervention highly tailored and responsive to user input, we needed to connect scripted messages to a database of videos and digital assets so that when prompted by user input, the tool would correctly display these digital assets in congruity (or in sync) with the video.PHC features 168 video clips for each of the four virtual doctors, and 28 clips common across doctors. It quickly became clear that all clips needed to be coded so we could efficiently coordinate the project. The challenge was to track the filming of all scenarios and ensure the correct use of all video clips according to programming algorithms. We needed a tool to coordinate the filming of all the clips and to ensure that when a user made selections while engaging with the intervention, PHC seamlessly delivered the appropriate video clip. The shooting script links video clips with tailored messages and user inputs—such as when the user selects topics for behavior change tips—and provides direction to the video director on how to compose each shot. The shooting script also described any graphics that would appear on the screen. Coding the videos based on their respective PHC intervention modules and creating a video catalog (codebook) from the shooting script standardized a naming convention for the 700 video clips. Consequently, the shooting script became the ultimate tool to link PHC videos to all intervention scenarios and user inputs.PHC was designed as a highly tailored intervention with hundreds of different possible user pathways depending on user input. The challenge was to create a tool to plan for and monitor filming so that the team could see the logic and interconnection between all of the scenes and understand how the composition of each frame would inform the composition of subsequent video frames. The solution to this challenge was to create the PHC flow diagram to show all the different tailoring paths a patient could take through the intervention. The flow diagram was used by the creative team to monitor the filming of the intervention and by the intervention programmer to guide the development of the algorithms underlying the intervention structure. This ensured that each scene would flow smoothly and be perceived as a natural progression in a conversation between a doctor and a patient. The PHC architecture achieves the following: (1) an intelligent, “back-end” design that refers to the underlying software framework (the app and the database) to provide tailored content based on user input, (2) a user-friendly interface that performs efficiently on multiple devices, such as computers, laptops, and tablets, and (3) a streaming video delivery solution that delivers high-quality video content.The build-out started with the abovementioned coded shooting scripts and the tailoring flow diagram because these tools had applications for both filming and programming and were crucial linkage tools between the design and development teams at this stage. Building PHC involved finalizing technical functional requirements, prototyping, coding and programming, and performing internal quality testing (“sprints”). We also developed and tested each line of closed captioning that was paired with all script lines to ensure the captioning met Section 508 compliance requirements. Below, we describe the challenges and solutions encountered when building PHC, including programming the intervention tool, building a tool that performs efficiently on multiple devices, finding a streaming video solution, and planning for dissemination.Each PHC module is constructed as a sequence of scenes appearing one after another, posing questions and imparting health messages. Each scene incorporates videos; input elements (such as check boxes) to collect user responses; display overlays to show topics and questions for patients to consider; and audio prompts to encourage patients to reengage during moments of inactivity, complete all questions, or avoid an early exit from the intervention. All the visual, HTML, and audio elements were choreographed in a scene to appear and animate on cue to provide the user with a visually rich and engaging experience. The challenge was how to create a tool to plan for, visualize, and document the placement of all these elements so that the videographer would frame each shot to give programmers the needed space for each element on the screen. In film and video production, keyframes are pictures that define the timing and the starting and end points of transitions. Specifically, PHC keyframes in congruity with the scripts were the solution for defining the positioning and movement of the actors and displaying overlay images, check boxes, navigation buttons, and closed captioning. Keyframes proved to be a reliable tool for the graphic designers to communicate the intended look and user interface details to the developer who programmed the intervention. Multiple keyframes were created for each scene to show how visual elements would appear in the video at a specific point in time along with the narrative delivered by a doctor or nurse. The design of PHC had 200 functional requirements to guide the programming and delivery of 700 video clips to users. These functional requirements addressed the needs for specific intervention scenes, including the visual elements, data collected from or displayed to the patient, script to be conveyed through video, and all the probable user actions with corresponding responses from the system. Functional requirements also included rules to determine the next scene based on the user’s response to the current scene and/or all of the previous scenes. The challenge was to correctly develop, implement, and troubleshoot the functional requirements that would guide PHC programming and specify the interaction between user inputs and PHC outputs. To address the challenge to verify and test the 200 functional requirements, we conducted numerous rapid and iterative feedback sessions or “sprints” in the application of “agile” methodology via collaborative, cross-functional, multidisciplinary teams ,38. As nThe individually tailored content distinguishes PHC from other digital interventions such as those delivered through text messaging. As noted previously, user engagement is critical to intervention effectiveness and behavior change. The challenge was to figure out how to design PHC to deliver and display tailored messages in real time along with all other tailored content from a large database of videos and other digital assets.(such as adherence or retention in care) and the related scenes with video messages and graphical overlays are depicted in various configuration tables that reside in the SQL Server database.As noted earlier, the PHC back-end design refers to the underlying software and app, as well as a database with data sets that define the relationship between the scenes and videos, audio prompts, questions, and tips used to tailor PHC content to each user. This back-end design supports the delivery of individually tailored health messages based on user input. To achieve this end, our solution was to build an intelligent intervention engine in the back end that connects all app assets defined in data sets for a given scene and is fully data driven . It was built on the Microsoft ASP.NET MVC platform and an SQL Server database. The individual modules PHC was required to perform efficiently on mobile devices in spite of the challenge presented by the fact that, unlike desktop or laptop computers, mobile devices have limited memory and processing capabilities. These limiting features accommodate the small size of mobile devices and prevent overheating in the absence of fans and ventilation. However, the drawback is that these devices tend to crash when loading webpages with multiple media and code files. This is especially true when multiple full-page refreshes are needed during typical use of a website. In these mobile devices, memory and processing power are limited but valuable resources that need to be used efficiently to make intervention pages load and perform optimally.To address this challenge, PHC was developed as a single-page app that loads a webpage only once, at the beginning of the session; after that, the webpage content is manipulated through JavaScript programming. Additionally, the loading of required content is accomplished through asynchronous data transfers. The single-page app allows PHC to preload the video and JavaScript files, which are used across all or most of the intervention scenes during the initial page load. This approach also avoids full-page refreshes when transitioning from one scene to the next; instead, the scene-specific data, display overlays, and scripts are loaded asynchronously from the back end of the app based on the user inputs and intervention scenario into a screen area defined in the single-page app. The preloading of common videos and scripts into the tool significantly reduces load time for individual scenes. Additionally, the asynchronous loading of content into a single-page app decreases potential crashes by avoiding multiple-page refreshes involving videos, images, and JavaScript files.Another key requirement for PHC is to provide an engaging intervention user experience by transitioning from one scene to the next without the need for the user to manually start and stop videos. All videos need to play automatically. PHC also has audio prompts to keep the user focused by encouraging them to answer questions and alerting them to any periods of inactivity. These audio prompts are programmed to automatically play in sync with all the video questions and messages, which worked flawlessly in computer web browsers. However, a problem occurred with the autoplaying of videos on Apple and Android tablets. These mobile operating system vendors restrict video autoplay within their web browsers, such as Safari or Chrome. These restrictions prevent unexpected user data charges, which can be particularly expensive for cellular internet connections. Consequently, the challenge was to figure out how to play PHC videos on mobile devices. To address this issue, we developed hybrid apps for Apple iPads and Android tablets. Hybrid apps are web apps wrapped inside native apps that are deployed in mobile app stores, such as Google Play or Apple Store. This hybrid approach allowed for the autoplay of PHC videos on these devices. In addition, the PHC hybrid app will allow for continual enhancements to PHC and modification of the user experience. Most importantly, by using this solution, we can deploy changes to PHC through the web without needing to update and deploy mobile apps each time there is an update to PHC. This challenge involved identifying a server that would efficiently deliver video content to PHC users. Cost, flexibility, data security, and streaming speed were important considerations for the final solution. YouTube provides a low-cost streaming option but offers little flexibility when it comes to controlling and displaying content. Videos hosted by YouTube, for example, cannot be played directly through the PHC app’s HTML5 player, but instead must use Google’s YouTube player. Additionally, the YouTube player always includes Google’s advertisements and usage tracking code. As an alternate streaming solution, we considered Akamai’s cloud delivery platform, which uses caching to rapidly stream content from servers globally. However, Akamai pricing was prohibitive.After researching the above potential solutions to this challenge, we decided to implement a streaming video solution by hosting video content on a CDC internal web server. This decision was confirmed after evaluating video performance on delivering video content rapidly and securely using load testing with many concurrent users. The videos were fine-tuned to be of good quality yet lower file size to support delivery over standard internet connections. Although this approach is adequate for use within clinical settings, it may not scale well as the number of concurrent users increases. We recommend the use of cloud-based content delivery networks to stream videos for wider dissemination.We designed and developed PHC to create conditions for greater population impact, and decisions were made with the goal of disseminating PHC to HIV clinics, if it was found to be effective. We aimed to build an intervention that could be feasibly implemented in busy HIV clinic environments and would be relevant to clinic providers and patients with HIV engaged in clinical care. The challenge was to design an intervention tool that will be available and accessible on multiple commonly used devices.We achieved a solution for possible future compatibility by developing PHC using tools and technologies supporting the World Wide Web Consortium’s open web standards. Apps using the open web platform are scalable, cross-platform compliant, and supported by multiple browsers without sacrificing user experience . StandarVideo-based digital health interventions will continue to play an important role in the future of HIV care and other clinical health practices. This is, in part, due to the ever-increasing popularity of video as a way to consume information across all sectors of society and multiple disciplines, including medicine ,41. NotaOur experience in designing and developing PHC presented unique challenges because of the extensive use of a large database of videos individually tailored to each user, and also revealed other challenges that are well known to investigators who seek to develop digital interventions . We beli


Faecalibacterium prausnitzii and Roseburia hominis were significantly in lower abundance in the drinking case group and were proved the protective effect against inflammatory liver damage in the former study. Ruminococcus gnavus exhibited the most positive association to alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and contributed to liver inflammation.Alcoholic liver damage has become a widespread health problem as alcohol consumption increases and is usually identified by elevated liver transaminase. We conducted this study to investigate the role of the gut microbiome in the individual susceptibility to alcoholic liver injury. We divided the participants into four groups based on alcohol consumption and liver transaminase elevation, which were drinking case group, drinking control group, non-drinking case group, and non-drinking control group. The drinking case group meant participants who were alcohol consumers with elevated liver transaminase. We found that alpha and beta diversities of the drinking case group differed from the other three groups. Species  Alcohol abuse is an important public risk factor for a variety of health problems and is related to more than 200 diseases, among which the liver is a major target organ –3. AlaniThe earlier investigations uncovered several mechanisms of alcohol-induced liver injury. Ethanol-induced oxidative stress  and the Individual susceptibility plays an important role in hepatocyte necrosis . Only abConsidering that normal ranges of ALT for men and women were different, and the number of women who drink alcohol is relatively low , we onlyIn this cross-sectional study, participants were recruited at the First Affiliated Hospital of Zhengzhou University, Eastern District of the Hospital between January 2018 and April 2019. Totally 531 individuals who had undergone routine physical examinations and had stool specimens collected in the Physician Health Center were included in our study. These were the exclusion criteria: (1) females, (2) participants under 18 years old, (3) cancer or systemic diseases patients, (4) hepatic cyst patients, (5) participants with histories of diarrhea in the previous 3 months, (6) participants who took any antibiotics 3 months prior to the collection of stools, (7) participants who have HBsAg positive illness, and (8) participants who drank alcohol only once a month in the drinking group .To clarify how alcohol regulates gut microbiota to cause liver damage, we designed four groups based on two variables: alcohol consumption and liver aminotransferase, namely ALT and AST. The four groups are as follows: (1) non-drinking control group: non-drinkers with normal ALT and AST; (2) non-drinking case group: non-drinkers with elevated ALT or AST; (3) drinking control group: alcohol drinkers with normal ALT and AST; and (4) drinking case group: alcohol drinkers with elevated ALT or AST. The four groups were matched with age and body mass index (BMI).via questionnaires. Drinking behaviors were evaluated according to drinking or not, drinking years, drinking frequency, drinking types, and alcohol consumption. If drinking frequency in the questionnaire showed once or twice a month, we calculated it by 1.5 a month. The past week was used as the time period for drinking types and alcohol consumption because individuals could recall their alcohol usage more precisely. Drinking types included liquor, wine, beer, and wine and liquor. Alcohol consumption was assessed based on the alcohol content of various drinking types and consumption of alcoholic beverages. To assess ethanol consumption, we selected the most frequent alcohol by volume for beer, liquor, and wine to calculate ethanol consumption, which are 5, 45, and 12%, respectively. For example, the calculation for beer is: ethanol consumption = beer consumption (ml) × 45% × 0.8 (g/ml).We collected participants' lifestyle information and personal history including drinking behaviors and smoking status Written informed consent was obtained from the participants. Participants were required to fast the night before the physical examination to draw blood tests and collect stool samples. Questionnaires, blood tests, and stool samples were collected on the same day. Blood samples were detected by Roche Cobas 8000 automatic biochemical analyzer . Both ALT and AST had normal ranges of 0–40 U/L. Stool samples were collected on the day of the physical examination. Indexes controlled attenuation parameter (CAP) and liver stiffness measure (LSM) were assessed by FibroTouch of HISKY Medical Technologies . The normal ranges for CAP and LSM are <240 dB/m and <7.3 kPa, respectively. A total of 20 participants performed the FibroTouch examination. The numbers of participants who underwent the examination in non-drinking control, non-drinking case, drinking control, and drinking case were 2, 1, 10, and 7, respectively. We obtained the liver function and FibroTouch information from the management system of the hospital. Stool samples from participants were all freshly collected at the hospital and stored at −80°C within 30 min after subpackaging.Following the manufacturer's instructions, DNA from the microbial population was extracted using the MagPure Stool DNA KF Kit B . DNA was quantified with a Qubit Fluorometer by using the Qubit dsDNA BR Assay Kit , and the quality was checked by running an aliquot on 1% agarose gel.About 1 μg genomic DNA was randomly fragmented by Covaris . The fragmented DNA was selected by magnetic beads to an average size of 200–400 bp. The selected fragments were through end repair, 3′ adenylated, adapters ligation, PCR amplifying, and the products were purified by the magnetic beads. The double-stranded PCR products were heat-denatured and circularized by the splint oligo sequence. The single-strand circle DNA (ssCir DNA) was formatted as the final library and qualified by QC. The qualified libraries were sequenced on BGISEQ 500 platform .Questionnaires and fecal samples were all collected on the same day. Women were excluded from our study due to gender variations in ALT and AST, and gut microbiota. The participants who had undergone physical examinations at the Physician Health Center of the First Affiliated Hospital of Zhengzhou University fasted the night before the examination. We only included drinkers who drank more than once a month to draw a clearer differentiation between the drinking groups and non-drinking groups.p-value < 0.05 was considered statistically significant. The difference of species and pathways of the two groups were calculated by the one-tailed Wilcoxon rank-sum test. Categorical variables, shown as counts and percentages [n (%)], were compared using Fisher's exact test, and a two-tailed p-value < 0.05 was considered statistically significant. Spearman's rank test was used for the correlation analysis.Statistical analyses were all performed on R version 3.6.1. Continuous variables in demographic characteristics shown as mean (SD) were compared using the Wilcoxon rank-sum test between two groups. Continuous variables in drinking behaviors shown as median  were compared using the Wilcoxon rank-sum test between two groups. A two-tailed p-value < 0.05.The Shannon index, the Simpson index, and the Gini index were used to estimate alpha diversity using the “vegan” package and “ineq” package of R. Beta diversity was assessed by the Pearson distance and Bray–Curtis distance, which were calculated by “vegan” package. We performed principal coordinate analysis (PCoA) to represent the statistically and visually microbial community profile differences using “ade4” package. Alpha diversity indexed and beta diversity distances were performed using the one-tailed Wilcoxon rank-sum test, with the statistically significant Venn diagrams conducted by “VennDiagram” packages were plotted to reveal the common and unique species or pathways in multiple samples and find differential species or pathways, displaying the similarity and overlap among the four groups.After a strict inclusion and exclusion process, a totally of 103 participants were enrolled in our study, which included 4 groups: non-drinking control group, non-drinking case group, drinking control group, and drinking case group, containing 18, 7, 55, and 23 participants, respectively . ParticiTotally 449 pathways and 694 species (including 215 viruses) were detected in the whole participants. Venn diagrams display the overlaps among the four groups . The fouSpecies alpha diversity indicated by Shannon index and Simpson index was decreased in drinking case group compared with non-drinking control group. Although there was no statistically significant difference, species Shannon index and Simpson index in the drinking case group tended to be a downward trend in microbiome diversity compared with the non-drinking case group and drinking control group . The GinWe performed beta diversity calculated by PCOA to display the overall diversity in microbiome composition among four groups. For species diversity, the Pearson distance and Bray–Curtis distance revealed substantial variations in the microbial community between the drinking case group and the other three groups . SamplesMetabolic pathway beta diversity was also assessed using the same analysis . The driBacteroidetes was reduced in the drinking case group vs. the drinking control group, whereas phylum Firmicutes was increased , one is associated with amine and polyamine degradation (GLCMANNANAUT-PWY), one is associated with pentose phosphate pathways (NONOXIPENT-PWY), one is associated with carrier biosynthesis (PWY_7371), one is associated with tetrapyrrole biosynthesis (PWY_5188), and one is associated with nucleoside and nucleotide degradation (PWY0-1296). For the four pathways decreased in the drinking case group, one is associated with galacturonate and glucuronate catabolism (GALACT-GLUCUROCAT-PWY), one is associated with β-D-glucuronide degradation (GLUCUROCAT-PWY), one is associated with D-galacturonate degradation (GALACTUROCAT-PWY), and one is associated with mixed acid fermentation (FERMENTATION-PWY).The distribution of the statistically differential species and metabolic pathways among the four groups was demonstrated in the Boxplot diagrams . CompareR. hominis showed a negative correlation with ALT and significantly decreased in the drinking case group. Among all the species in the heatmap, Ruminococcus gnavus showed the most positive correlation with ALT and AST.To identify the correlations between clinical variables and microbiota, associations between the gut microbiome and continuous clinical variables were calculated by the Spearman correlation coefficient in all participants. Species heatmap revealed the correlation between species and clinical variables . R. homiFor pathways, we noticed that NONOXIPENT-PWY, GLCMANNANAUT-PWY, PWY-5188, and PWY-7197 were all positively correlated with ALT or AST, and they were increased in the drinking case group .We firstly performed WGS in the comprehensive groups to explore the association between the gut microbiome and individual susceptibility in alcohol-related liver transaminase elevation. In our study, although the two drinking groups showed no differences in drinking behaviors, one group had raised transaminases and the other group had normal transaminases—indicating individual variability reflecting the liver injury caused by alcohol consumption. In our study, we observed fecal microbiome community in the drinking case group differed from the other groups and described the significantly different species and metabolic pathways.Liver function indexes ALT and AST are the easiest markers to assess hepatocellular damage and usually are the screen tests for liver disease . CompareMicrobiome alpha diversity of the drinking case group based on the Shannon index and the Simpson index was lower compared to HC participants. Alcohol use disorder patients were found to have a decreased alpha microbiome diversity in the former study . The redFirmicutes and decreased phylum Bacteroidetes in the drinking case group compared to the drinking control group. Phyla Firmicutes and Bacteroidetes are dominant and account for the majority of human gut microbiota 1-β, IL6, and tumor necrosis factor (TNF)-α -α . TNF-α i (TNF)-α , 39. Rum disease , 41. In ic cells . Therefoct TNF-α , 43, whiWe discovered 12 metabolic pathways that were statistically different among the drinking case group and the other three groups. Among the eight pathways, which increased in the drinking case group, 3 pathways were associated with nucleoside and nucleotide biosynthesis and 1 pathway was associated with nucleoside and nucleotide degradation. The activation of the processes might associate with the dysbiosis of gut microbiome and the possible bacterial overgrowth in patients with alcoholic liver injury , 45. TheThese are the advantages that only the participants without underlying diseases were included, and we designed four groups based on the alcohol consumption and liver aminotransferase. However, due to the strict exclusion criteria, the number of subjects was limited, and we only included male participants. Some participants forgot their drinking behavior resulting in missed information. In our study, the most important was that we discovered three altered species, which were related to alcoholic liver injury. The finding should be further verified through large-scale studies and animal experiments.F. prausnitzii and R. hominis exhibited a protective effect on the liver, and Ruminococcus gnavus showed a liver-damage effect. Further verification is needed in future studies. Since the three meaningful species were discovered in this study, we believe that after further verification, the probiotic administration or dietary patterns can be used to regulate intestinal flora, and thus protect the liver of alcohol drinkers.Alcoholic liver damage is usually noted early in the clinic with elevated aminotransferase levels. Our findings suggested that the gut microbiome contributes to the susceptibility of individuals to develop liver injury after alcohol consumption. Species https://db.cngb.org/search/project/CNP0002336/.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number (s) can be found below: The studies involving human participants were reviewed and approved by Ethics Review Committee of Scientific Research Projects in The First Affiliated Hospital of Zhengzhou University. The ethics approval number is 2018-KY-56. The patients/participants provided their written informed consent to participate in this study.AL, SD, and JL contributed to the conception and design of the study. MJ performed the analysis and wrote the manuscript. MJ, SY, and AL performed the data analysis. MJ and JL analyzed the participants' data. QS, YLiu, and YLi helped collect the data and edit the manuscript. All authors read and approved the final manuscript.This study was equally funded and supported by the Henan Province Key Science and Technology Program 172102310049, the Henan Province Medical Science and Technique Project 2018020001 Grant, and the Henan Province Postdoctoral Research Grant 001801005.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Although the postpartum period is suggested to provide an ideal opportunity for interventions to prevent hazardous drinking, evidence on the associations of education and income with hazardous drinking during this period is limited, including in Japan.We analyzed data from 11,031 women who participated in the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study in Japan. Hazardous drinking was defined as ethanol intake of ≥20 g/day 1 year after delivery. We conducted multiple logistic regression analyses to examine whether educational attainment or equivalent household income was associated with hazardous drinking, adjusting for age, parity, drinking status during pregnancy, work status, postpartum depression, breastfeeding, and income/education. We also conducted stratified analyses by income and education groups.The prevalence of hazardous drinking 1 year after delivery was 3.6%. Lower education was associated with hazardous drinking; the odds ratio  of high school education or lower compared with university education or higher was 2.17 (1.59–2.98). Lower income was also associated with hazardous drinking, but this association disappeared after further adjustments for education; the odds ratios  of the lowest compared with highest level of income were 1.42 (1.04–1.94) and 1.12 (0.81–1.54), respectively. A significant interaction was detected; lower education and lower income were associated with increased risks of hazardous drinking only in a lower income group and lower education group, respectively.Postpartum women with lower education and lower income had higher risks of hazardous drinking in Japan.The online version contains supplementary material available at 10.1186/s12199-021-00991-9. Harmful use of alcohol is one of the leading risk factors for disease burden and causes substantial health loss . ApproxiHazardous drinking is more common among women who are more highly educated and of higher income than among women of lower status in most Organisation for Economic Co-operation and Development (OECD) countries . The patConsidering the above circumstances, we aimed to examine the associations of education and income with hazardous drinking among postpartum women in Japan. We focused on hazardous drinking 1 year after delivery because there is evidence that parenting a child <1 year of age is associated with reduced alcohol use and that this protective effect diminishes after 1 year postpartum .We used data obtained from the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study (TMM BirThree Cohort Study), which has been described elsewhere , 16. PreBased on the questionnaire 1 year after delivery, educational attainment was categorized as high school or lower , college , and university or higher (university or graduate school). Using the questionnaire during pregnancy, the participants were asked to select their total annual household income from seven categories: ≤1.99, 2.00–3.99, 4.00–5.99, 6.00–7.99, 8.00–9.99, 10.00–11.99, and ≥12.00 million Japanese yen (JPY). The equivalent household income was calculated as the household income divided by the square root of the number of family members . The ressake (rice wine), shochu, beer, whisky, and/or wine. A score to each category of alcohol consumption frequency was assigned as follows: 7 for every day, 5.5 for 5–6 days/week, 3.5 for 3–4 days/week, 1.5 for 1–2 days/week, 0.5 for a few times/month, and 0 for almost never. The ethanol equivalent intake in grams was calculated as follows: 180 ml sake = 23 g, 180 ml shochu = 36 g, 633 ml beer = 23 g, 30 ml whisky = 10 g, and 100 ml wine = 12 g [Using the questionnaire 1 year after delivery, the participants were asked to select one of the following response options for their drinking status: current drinker, past drinker, never-drinker, and constitutionally never-drinker. Current drinkers were asked to report the types of alcoholic beverages consumed as well as the frequency and amount of each consumption per day in the past year: e = 12 g . Finallye = 12 g . FollowiAs covariates, we chose age, parity, drinking status during pregnancy, work status, postpartum depression, and breastfeeding. Age at delivery and parity were ascertained from the participants’ medical records. Age at delivery was categorized as ≤29, 30–34, and ≥35 years. Parity was dichotomized into nulliparous and multiparous. Using the questionnaire during pregnancy, the participants were asked to select one of the following responses concerning their drinking status: current drinker, past drinker, never-drinker, and constitutionally never-drinker. Responses were dichotomized into drinking (current drinker) and non-drinking . Information on the participants’ work status, postpartum depression, and breastfeeding was obtained from the questionnaire 1 year after delivery. The work status was dichotomized as working  and not working . The participants provided responses to the Japanese version of the Edinburgh Postpartum Depression Scale, which comprises 10 items assessing any symptoms of depression in the past 7 days . Each itThe characteristics of non-drinkers and drinkers 1 year after delivery were compared using the chi-square test. We conducted multiple logistic regression analyses to examine the associations of education and income with hazardous drinking 1 year after delivery. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for education and income, adjusted for age (model 1), as well as for parity, drinking status during pregnancy, work status, postpartum depression, and breastfeeding (model 2a for education and model 2b for income). Income and education were then entered simultaneously in the same model (model 3). We also conducted sensitivity analyses restricted to only drinkers 1 year after delivery.p < 0.01). Based on this result, we also conducted stratified analyses of the association between education and hazardous drinking by income groups (≥3.00 vs. 0–2.99 million JPY) and the association between income and hazardous drinking by education groups (college or higher vs. high school or lower).We entered the interaction term between education and income into the model and found that the interaction was statistically significant . For all analyses, a two-tailed Table Table p < 0.001).Table p = 0.0085).Table The present study was performed to examine the associations of education and income with hazardous drinking among postpartum women in Japan. The prevalence of hazardous drinking 1 year after delivery was 3.6%. Lower education was associated with an increased risk of hazardous drinking. Lower income was also associated with an increased risk of hazardous drinking, but this association disappeared after further adjustments for education. A significant interaction was detected between education and income; lower education and lower income were associated with increased risks of hazardous drinking only in a lower income group and lower education group, respectively.The prevalence of hazardous drinking among women 1 year after delivery was 3.6%. We could not directly compare the prevalence of hazardous drinking 1 year after delivery with corresponding data in Japan because there is little information on hazardous drinking among postpartum women. The National Health and Nutrition Survey in Japan reported that women who were hazardous drinkers comprised about 7% in their twenties  and about 11% in their thirties  . The 201Women with lower education were more likely to be hazardous drinkers. This result is inconsistent with the findings in Western countries, in which an association has been found between higher education and hazardous drinking among women in the general population  and amonThe association between lower income and hazardous drinking disappeared after adjusting for education. This result is inconsistent with the findings in Western countries, in which an association has been found between higher income and hazardous drinking among women in the general population  and amonThe present findings have implications for the design of effective interventions aimed at preventing hazardous drinking among postpartum women. Although most women reduce or abstain from alcohol use during pregnancy , 3.6% haLimitations of the present study should be noted. First, it was conducted in 1 of the 47 prefectures in Japan, which limits the generalizability of the findings. However, the prevalence of hazardous drinking among women in Miyagi Prefecture 8.0% in 2016)  was simi016  was The present study showed that the prevalence of hazardous drinking 1 year after delivery was 3.6%. Lower education was associated with an increased risk of hazardous drinking. Although the association between lower income and hazardous drinking disappeared after adjusting for education, this association remained only in a lower education group. Our findings can provide clues to designing effective interventions for preventing hazardous drinking among postpartum women, which would improve maternal and child health.Additional file 1: Table S1. Characteristics differences between 11,031 participants who were analyzed and 11,462 participants who were excluded from the analysis Table S2. Associations of education and income with hazardous drinking 1 year after delivery among drinkers


We report an experiment that considers the impact of emotional state on honesty. Using the die-rolling task created by Fischbacher and Föllmi-Heusi to detect the level of dishonesty in a sample of individuals, we study the effects of induced happiness on the incidence of self-interested lying. The experiment uses 360-degree videos to induce emotional state. We find that people behave more honestly in a state of happiness than they do in a neutral state. Since emotional states are malleable, interventions to do so may be feasible and cost-effective. The relationship between emotional state and honesty is the focus of the study reported here.Individuals differ from each other in their propensity to behave dishonestly. Indeed, the same person may behave with more integrity on 1 day than another. What causes some people to behave more dishonestly than others? Factors such as background, personality, decision history, managerial philosophy, and reinforcement have all been shown to correlate with ethical behavior in business settings  where an experimenter was present. The subject reported the roll to the experimenter with the room door closed and was paid accordingly. They were then asked to immediately leave the building. Each subject was sent out of room B to roll the die only after the previous participant had completed reporting her roll in the other room and had left the area. These procedures ensured that no other participants were within view or earshot when a report was made, and that subjects could not discuss their reports with each other before submitting them. In addition to the number rolled on the die, subjects were asked about their major (program of study) after they reported their roll. The experimenter also recorded their gender.Subjects were shown one of two 360-degree videos in virtual reality, depending on the treatment. The experimental design had a between-subject structure, in that each subject was only shown one video and performed the die-rolling task only once. No subject participated in the experiment more than once. Of the male participants, 25 were in the Neutral treatment and 23 were in the Happiness condition. Twenty-eight females were in the Neutral, and 30 took part in the Happiness, treatment respectively.https://www.youtube.com/watch?v=SmhuzTzUKQY.the video for the control treatment was a simple video of a tulip field on a sunny day. The video is taken from the perspective of an individual sitting in the field. There was no music, but there were soft noises, such as birds chirping and distant chatter. The video lasted for ~5 min and subjects were shown the video once before proceeding with the rest of the experiment. The video can be found at https://www.youtube.com/watch?v=MKWWhf8RAV8The video inducing a state of happiness was a video shown from the point of view of surfers in a tropical beach setting. Viewers would get a first-person viewpoint of surfing on waves, paddling out to sea, and swimming in the ocean. Accompanying the visual component of the video, there was upbeat, positive music playing, further promoting a pleasant experience. The video was approximately two and a half minutes long and subjects were shown the video twice; the video was immediately played again once it finished playing for the first time. This video was played twice to maintain consistency among video lengths across treatments. This video can be viewed at We conducted a manipulation check during several earlier sessions, with different individuals than those who participated in the experiment, to verify that the videos increased the level of the targeted emotion while not increasing any others. In the manipulation check, we asked individuals to report the levels of five emotions: Happiness, Fear, Sadness, Anger, and Disgust, that they were currently experiencing. We asked these participants to indicate, on a scale of 1–7, the strength with which they felt each emotion. They did so both before and after viewing one the videos. The data are given in the t-test rejects the hypothesis that the two means are equal . The Neutral video did not yield a level of self-reported happiness significantly different from that recorded prior to the viewing of a video . Those who viewed the Happiness video reported a greater degree of happiness afterward than those who had viewed the Neutral video , though the effect is only borderline significant. The average level of each of the other four emotions after viewing a video is not different between the two treatments.The table shows that the Neutral video did not increase the strength of any of the emotions (other than an insignificant increase in happiness). The Happiness video raised average reported happiness while not increasing any of the other emotions.Before conducting the experiment, we formulated the following hypothesis regarding our treatment differences. Since there are no prior results, to our knowledge, to guide our a priori beliefs, we have no basis to hypothesize a sign for a treatment effect. Thus, our hypothesis is a two-sided claim that there would be no treatment effect.Hypothesis: People behave equally honestly in the Happiness and Neutral treatments. The average reports, as well as the percentage of individuals reporting a roll of 6, are not different between the two treatments.t-test of the hypothesis that there is no difference in average report between treatments, our sample size yields a power of 73% to detect a medium sized treatment effect of 0.5 standard deviations at a significance level of 0.05. In terms of the proportion of individuals claiming a roll of 6, our sample size yields a power of 60% of detecting a difference between treatments at a significance level of 0.05, if the true means are 0.17 and 0.35 in the two treatments. Although the experiment was not designed specifically to do so, in our analysis of the data, reported in section Results, we also consider whether there are differences in the level of honesty between women and men, and between economics/business majors and those pursuing other programs of study.For a two-sided The distribution of reported dice rolls in each treatment can be seen in t = 7.07, p < 0.001). There was also a greater than random incidence of the reporting of 5 or 6. Thirty-eight of the 53 subjects reported rolling either a 5 or a 6. If people had been honest, we would expect 17.67 out of 53, one-third of, subjects to report either a 5 or a 6. We reject the hypothesis that the proportion reporting 5 or 6 is equal to 1/3, using a binomial test . On the other hand, if all participants were selfish and willing to be as dishonest as needed to maximize their monetary payment, all players would report a six. This is also clearly not observed, with only a minority of participants reporting a 6. Gender differences are small and insignificant, with 40% of men and 42.8% of women reporting a 6, and the average reports being 4.89 and 4.76 for women and men, respectively.In the Neutral treatment, the average report was 4.83 (std. dev = 1.369), significantly greater than the average under honest reporting of 3.5 . z = 4.17, p < 0.01). However, only 12 subjects in the Happiness treatment reported rolling a 6 (22.6%). In the Happiness treatment, 20% of women and 26.6% of men claimed a six, and the average report was 4.5 and 4.69 for women and men, respectively. The difference in the average report between the two treatments is therefore 0.39 for women and 0.06 for men. There is a 18.9% point difference between treatments in the incidence of claiming a roll of 6, with almost twice as many claims of 6 in the Neutral treatment. Because women make slightly lower average reports than men under the Happiness treatment, while making slightly higher reports than men under Neutral, there is no overall gender effect.In the Happiness treatment, the average reported roll was 4.58 (std. dev = 1.20), 0.25 lower than in the Neutral treatment. This is also significantly different from the average under honesty of 3.5 . The Neutral treatment generates a higher average report than the Happiness condition.We conducted a number of formal statistical tests to compare the average report, the incidence of extreme lying, and the distribution of reports, between the two treatments. A p-value of 0.038. The probability of claiming 6 is significantly lower in the Happiness than in the Neutral treatment.To compare the amount of extreme lying between treatments, we conduct a binomial test of the hypothesis that the proportion of 6s is equal in the two treatments. The test yields a Finally, we conducted a chi squared test to determine whether there were significant differences in the distribution of reported rolls between treatments. This test results in a statistic of 18.795. At five degrees of freedom, this is significant at 1%. Thus, the distribution of reports differs between the two conditions.Happiness was coded as a 1 for the Happiness treatment and 0 otherwise. To create the variable “Major,” business and economics majors were coded as a 1 and all other majors were coded as a 0. For the “Gender” variable, all males were coded as a 1 while all females were coded as a 0.To evaluate the hypothesis, while controlling for influences that might affect the comparison between treatments, we conducted regressions with two different dependent variables. The first is a dummy variable, which takes on a value of 1 if the participant rolls a 6 and 0 otherwise. These regressions consider the determinants of extreme lying. The second is the actual reported roll, a measure of the general tendency to lie. The dummy variable p < 0.1, and that is robust to the specification. It confirms, albeit at a marginal significance level, that controlling for gender and major, there is more honest behavior in the Happiness than in the Neutral treatment. Splitting the sample between women and men, however, reveals that the treatment effect is specific to women. The variable Happiness is significant in equation (4) though not in (5). In specifications (1)–(3) in which both women and men are included, the coefficient for Happiness is marginally significant, indicating that there is a treatment effect for women (the base category for gender). However, the sum of the coefficients for Happiness and Gender*Happiness is not significant, indicating that there is no treatment effect for men.In The regressions also show that there is no overall effect of gender on honesty. There is also no effect of program of study for the sample as a whole. However, there is an effect of major if only women are considered. Women who are studying business or economics submit lower reported rolls than those pursuing other majors.We observe some evidence that people are more honest in a state of happiness than in a state of neutrality. In the laboratory, emotions can have an effect on the extent of ethical behavior. We do not know, for now, how general this relationship is. However, if the effect transfers to a workplace environment, it would indicate that a creating a more positive workplace environment would lead to more honest behavior on the part of employees. Research on how to create a positive workplace culture is well-developed. Some of these strategies include caring for colleagues on a personal level, providing support and compassion when others are struggling, avoiding blame, forgiving mistakes, and emphasizing the meaning of the work being done (Seppala and Cameron, We observed no significant difference in honesty by gender. The conclusion that there was not a significant effect of gender on honesty provides yet another rationale for the equal treatment of the genders in the workplace. There is no reason to believe, based on what we have observed in this study, that an employee or a student of one gender would be more or less ethical than an individual of another gender.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board of the University of Arizona. The patients/participants provided their written informed consent to participate in this study.Both authors were involved in designing and conducting the experiment, analyzing the data, and writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Castor fiber) project in England and identify how governance of coexistence with reintroduced species may differ from the governance of coexistence with species that are already present in the landscape. Using a qualitative thematic analysis of an online survey, we identify a series of lessons in six key areas: (1) project governance, (2) stakeholder engagement, (3) research and monitoring programme, (4) strategy to manage arising conflicts, (5) public engagement, and (6) broad perspectives on reintroduction trials. We advocate for reflective evaluation as an essential component of reintroduction projects to enable knowledge-sharing from experiences, leading to improved practices in the future. Reflecting on our analysis, we identify and define ‘Renewed Coexistence’—a new term that draws on pre-existing coexistence knowledge but identifies the unique elements that relate to governing coexistence with reintroduced species.Species reintroductions are growing in popularity, and example motivations include supporting species populations or the restoration of ecosystem function. Interactions between humans and the reintroduced species are likely to occur post-reintroduction. Coexistence between humans and wildlife is adaptive and dynamic, in part requiring management of conflicts between humans and wildlife, or of conflicts between humans over wildlife management. We seek to learn from the experiences of steering group members in a Eurasian beaver (The online version contains supplementary material available at 10.1007/s10344-021-01555-6. Coexistence between humans and wildlife ‘entails the behaviour of living together’ Frank . It is dUsually, coexistence refers to coexistence between humans and wildlife that is already present in the landscape. In wildlife reintroductions, however, there is a ‘new’ coexistence for the humans in the locality with a species with which they are unlikely to have prior experience. Wildlife reintroduction is the process of returning a species to an area where it was previously present but is now extinct  reintroduction are favoured amongst the public in the western USA, some groups hold more negative views, e.g. farming and ranching groups whose economic interests may be affected by predation  to England were rejected, partly as UK Government felt the efforts to engage with stakeholders and reduce concerns were insufficient  is a large mammal which lives in terrestrial aquatic environments and was historically present in Britain  was a reintroduction trial in England between 2015 and 2020 in the catchment of the River Otter, Devon. The catchment is mostly rural with 50% of land use comprised of improved grassland, and 27% arable and horticulture. Only 5% is urban or suburban; human settlements are generally small and there are only three towns  was granted a 5-year licence to monitor the population   meeting on 13th February 2020 (prior to COVID-19 restrictions), at which SG members supported the proposal. Members indicated a preference for a questionnaire-based study to facilitate participation around work commitments. Proposed topic coverage was determined pragmatically and outlined to the SG in the meeting as a set of ‘key questions’ in the proposal presentation . The proWe designed the survey in Qualtrics software. Questions were based around the key questions presented to the SG and their response. We piloted the survey internally to ensure balanced question framing and coverage. We anticipated participation would take up to 30 min. Questions  were desOriginally, this was proposed for steering group (SG) members only as key informants. This group comprised of a wide range of stakeholders with high levels of representation and knowledge of their organisational interests who had not had opportunity to participate in the earlier social studies (outlined in Study Context). At the 13th February 2020 meeting, SG members requested the invitation be extended to the Beaver Management Strategy Framework Working Group (BMSF) and Science and Evidence Forum (SandE), thus including groups responsible for research, monitoring, and developing key document outputs : fourteen SG, ten BMSF, and nine SandE members  and piloted with two colleagues who had no ROBT involvement;the lead author was excluded from participation;a ‘Findings Report’ of key points  was sharanonymised participant responses are available in full in the the final text was subjected to peer review.Researcher positionality was an important consideration; the lead author  had been an SandE member and conducted previous research within the ROBT . They contributed by checking study context details and reviewing structure and presentation of material. The funders had no study oversight.Prior to taking part, respondents were given details of the study and informed that participation would be voluntary and anonymous  project governance, (2) stakeholder engagement, (3) research and monitoring programme, (4) strategy to manage arising conflicts, (5) public engagement, and (6) broad perspectives on reintroduction trials. Each is outlined in this section, using the participants’ words and relevant literature.We refer to participants using their participant numbers, i.e. P1 = participant 1, P2 = participant 2.First, participants identified a need for clearly defined objectives. Clear objectives facilitate successful planning and assessment . If this is not possible, the transparency of leadership will increase in importance; there may always be factors which affect trust in wildlife managers but where stakeholders perceive greater transparency, trust levels are likely to increase  were highlighted as a challenge in governing the project, requiring ‘personnel, time and money’ (P16). Any similar reintroduction should consider efficient resource use, with P18 stating the Trial could be ‘streamlined’ (see theme 6). Costs of reintroduction can be high  suggested scientific peer review may be one avenue through which a demonstration of objectivity and rigour could be achieved. When asked about risks/challenges of trial participation, they said ‘Maintaining scientific integrity and impartiality. Whilst our role as University researchers is to undertake independent research, our position as the main project partner has at times led some to question this. Have sought to address via peer review of results etc.’.With co-creation in mind, the research focus will need to give a ‘holistic understanding’ (P13) of a reintroduction, beyond single interests of contributors. This is for the social and ecological consequences of a project to be sufficiently understood, as recommended by IUCN Guidelines. Additionally, the programme may need to be reactive to emerging issues and changing circumstances: ‘Balancing the need for a clear programme of research with the value of being able to be reactive so research can focus on areas that emerge as being of key importance or lacking in existing evidence’ (P16).However, there can be limitations. Three were evident in our findings, which are reported in Table In addition, P18 felt future projects should ‘learn from other projects/experiences to build on knowledge, don’t reinvent the wheel, take more things as red with confidence ‘(P18). This suggests that research could build upon prior knowledge, rather than cover topics addressed elsewhere. Indeed, P8 said in the ROBT it ‘Sometimes felt like lessons and experience/expertise from Scotland were not being fully taken into account or utilised and potentially, therefore, re-inventing the wheel when this was not needed’.Reintroduction projects should anticipate and seek to prevent or manage conflict issues, with proactive action likely to be received well . These partners sat on the BMSF, formed by the SG  when there is a desire for management to be accessible. Indeed, in a previous nationwide questionnaire, several respondents indicated stronger levels of legal protection may make management of negative beaver impacts more difficult, . This includes potential for education via the press, which P15 cited as a ROBT success: ‘generating press interest and articles that show beavers can play a role in creating more flood resilient landscapes and create habitats for a wide range of biodiversity’. This may be important for garnering community support, with engagement leading to education and influenced attitudes .Although participants were generally favourable of the principle of a reintroduction trial, further lessons could be learned. P11 believed ‘[reintroduction trials] need to be developed but this was a very important first start’. Nonetheless (as discussed), there will still be room for more learning at the end of a trial phase. For example, P16 stated ‘trials such as this are invaluable for providing context-specific evidence. The caveat is that it will not be possible to answer all the questions within the scope of such trials. The end of these kinds of trials does not indicate that no more research is required’.Some participants had issue with how the trial began. P12 noted the beavers were already present prior to commencement and felt it’s not really a model for re-introduction as [it is] a model for how to deal with escaped and feral animals. If looking for how to do a introduction properly from the start it would not be the recommended approach to let the animal loose and then deal with it’. This was cited by P5: ‘the dubious nature of how the beavers arrived is always a bone of contention amongst farmers’. This is a notion that was also reported in Scotland resulting from the appearance of a population of unlicensed beavers in Tayside; the lack of formal process in the reintroduction was observed to be the main driver of post-reintroduction conflict, leading to a lack of trust between stakeholders .Respondents suggested population sizes may limit the ability to collect necessary evidence to meet trial objectives. Thus, depending on objectives defined at the outset, some research may require time before a larger population can exist ’ (P16). Therefore, as discussed above, research may need to continue beyond the end of any trial, but trials may ‘provide a starting point for decision making and management and this should be made clear to all stakeholders involved’ (P16).Finally, some participants, although favourable towards trials, felt that they risk resource-intensive processes which inhibit the potential of reintroductions. P18 believed reintroduction trials should ‘not be overly cautious […] This type of trial set-up has a time and place but […] we are in danger this sets a precedence that conservation translocations require this level of investment every time, when other similar sectors have no such requirement or expectations thereof […] we need to be careful we aren’t holding up reintroductions as overly complicated and expensive and therefore subject to continued scrutiny’. P6 agreed: ‘We are facing a climate and ecological crisis—species reintroductions need to be done well—but the barriers organisations face are often prohibitive and costly’.We have identified a series of themes from the points of view of stakeholders involved in steering a reintroduction project. In this section, we will discuss how this relates to the previous study and examine what is different in reintroductions for conflict and coexistence issues. Prior to doing so, however, we would like to advocate for reflective evaluation to be an essential part of future reintroductions. All reintroduction projects provide opportunities to learn from the process undertaken. Such learning may inform and improve the steering and expectations of future reintroduction projects. As reintroductions are growing in popularity and uptake increases Corlett , many paIn this analysis, there are points identified which reinforce the findings of research from pre-existing conflict and coexistence literature but in the reintroduction context, particularly about stakeholder engagement. In 2016, Decker et al. outlined ten ‘governance principles for wildlife conservation in the twenty-first century’. These include the incorporation of multiple, diverse perspectives in governance, as well as governance that is transparent and accountable. This is reflected in the participants’ assessments of the ROBT, with thirteen participants citing stakeholder engagement as a key trial success, but with a perception among some that group leadership should have been independent to facilitate transparency. This may help build trust between parties; trust building is essential for the resolution of conflict issues, which can be built through demonstrable efforts to recognise and respond to issues Madden . Riley eAs stated above, the increasing uptake of reintroduction projects means many practitioners are likely to be ‘learning-as-they-go’. We believe that the relatability between our findings and previous study can provide practitioners and stakeholders with reassurance that preexisting knowledge from non-reintroduction-related experiences is applicable also within this emerging field. That said, by eliciting the views of the ROBT Steering Group stakeholders in a way that was meaningful for them, we have identified an important distinction between reintroduction and pre-existing research, regarding conflicts and coexistence.Coexistence with a reintroduced species is a specific form of coexistence as humans in the locality are likely to have no prior experience of the historically present species. Hence, coexistence challenges in reintroduction begin from a different start-point; projects seeking to facilitate coexistence in response to conflict issues start from the point at which the issue exists in the present, whereas in reintroductions potential coexistence challenges are in the future (post-reintroduction). In our participants’ responses, this sense of the future context is evident. Under theme 3, for example, participants identified outstanding research questions at the trial end, particularly regarding interactions between beavers and fish. Research questions which remain unanswered can lead to a sense of uncertainty which in turn can lead to worry or concern about future consequences, influencing decision-making Hudenko . In reinWhen a reintroduction does take place, theme 4 identified the importance of a planned management strategy to respond to conflicts if coexistence is to be achieved. Where management is developed to facilitate coexistence with present species they are reactive to challenges that exist and are able to engage with stakeholders who hold experience of those challenges  will provide the necessary emphasis for groups steering reintroduction projects to consider future coexistence challenges in this specific context, engaging with affected stakeholders early to address uncertainty, and encouraging an a priori attention towards the management of potential future conflicts to achieve coexistence with reintroduced species, if and where reintroductions occur Supplementary file2 (DOCX 26 KB)Supplementary file3 (DOCX 43 KB)Supplementary file4 (DOCX 264 KB)Below is the link to the electronic supplementary material.


P was successfully synthesized and characterized as Mg2+-selective fluorescent probe. The binding of P with Mg2+ caused an obvious fluorescence enhancement at 482 nm. The fluorescent, UV-vis spectra, 1H-NMR, and IR spectra confirmed the formation of P-Mg2+ complex, and the formation of a 1:1 stoichiometry complex was proved by Job’s plot and mass spectrometry. The recognition mechanism of P to Mg2+ was owing to the photoinduced electron transfer effect (PET). The fluorescent response was linear in the range of 0.9–4.0 µM with the detection limit of 0.3 µM Mg2+ in water–ethanol solution . In addition, the results of cell imaging of Mg2+ in Hl-7701 cells was satisfying.A simple benzoyl hydrazine derivative  It also has been involved in many cardiovascular and neurological processes in human body +, 328.26 [M + Na]+. IR (cm−1): 3402.75 , 1624.98 (C=O). 1H-NMR : 12.97 , 11.99 , 9.48 , 8.18 , 7.92 , 7.67 , 7.62 , 7.42 , 7.26 , 6.81 , 6.65 , 6.62 . 13C-NMR : 165.57, 158.77, 151.36, 146.79, 133.61, 133.37, 132.53, 129.90, 129.01, 128.71, 128.61, 124.42, 121.35, 119.87, 117.58, 115.60, 113.11, and 109.52. ( methods . 2-amino 109.52. .P with deionized water and dimethyl sulfoxide (DMSO), respectively. The testing solutions were freshly prepared using the stock solutions. For all fluorescent measurements, the slit widths of emission and excitation were both 5 nm; excitation wavelength was set as 415 nm.One-millimeter stock solutions were obtained by dissolving metal salts and 2+ (1 μM) (in PBS) were added to the cells and incubated for 30 min at 37 °C, and then washed with PBS three times followed by the addition of probe P (10 μM) and incubation for 30 min at 37 °C. Fluorescence imaging in Hl-7701 and 4t1 cells was recorded by a fluorescence microscope (Olympus FluoView Fv1000).Hl-7701 cells grown in 6-well coverslips were washed with phosphate-buffered saline (PBS). MgP to Mg2+, the pH titration experiment was performed firstly .In order to investigate a suitable pH value for the sensing of  firstly . The resP (10 µM) was studied in water–ethanol solution  with the addition of tested metal ions Na+, K+, Mg2+, Ca2+, Cd2+, Co2+, Zn2+, Pb2+, Ni2+, Hg2+, Cu2+, Cr3+, Fe3+, and Ag+ (10 µM)  a. The red anions . It was P (10 μM) increased regularly as the concentration of Mg2+ from low to high in water–ethanol solution , and the fluorescence intensity of P was proportionate to the concentration of Mg2+ in the range of 0.9–4.0 µM with a detection limit of 0.3 µM Mg2+  were carried out to further examine the coordination process of Mg2+ and P in water–ethanol solution . The addition of Mg2+ to the solution of P induced an apparent red-shift of absorbance in the UV-vis region and a new peak at 430 nm appeared . . b. The stP with Mg2+, Job’s plot experiment was carried out, and the study revealed that a P-Mg2+ complex was formed in 1:1 molar ratio  m/z: 328.0 assigned to [P + Mg2+ − H+]+, 346.1 assigned to [P + Mg2+ + OH−]− and 374.3 assigned to [P + Mg2+ + CH3CH2OH − H+]−  mechanism and caused the fluorescence enhancement of P.To investigate the binding mode, onducted . Upon ad complex . In the mplex  in the growth medium for 30 min, which led to a very weak fluorescence , a significant fluorescence change was recorded after the addition of P (10 μM) to the cells  and Hoechst 33,342 (1 μg·mL−1) for 30 min in the same conditions as those used in the cell imaging experiment. It could be concluded that P located primarily in the cytoplasm of these living Hl-7701 cells, as represented in The subcellular localization of MgP in living 4t1 cells was also studied by conducting co-localization experiment with mitochondria-specific dye Mito Tracker Red . We believe that this study will significantly promote the development of effective Mg2+-selective probes for both studies on the effects of Mg2+ in environmental or in biological systems.In summary, a simple structure probe derived from benzene hydrazine was characterized as a Mg


This paper explores trends in beverage preference in adolescents, identifies related regional differences, and examines cluster differences in key drinking measures. Data were obtained from the European School Survey Project on Alcohol and Other Drugs (ESPAD), covering 24 European countries between 1999 and 2019. Trends in the distribution of alcoholic beverages on the participants’ most recent drinking occasion were analysed by sex and country using fractional multinomial logit regression. Clusters of countries based on trends and predicted beverage proportions were compared regarding the prevalence of drinkers, mean alcohol volume and prevalence of heavy drinking. Four distinct clusters each among girls and boys emerged. Among girls, there was not one type of beverage that was preferred across clusters, but the proportion of cider/alcopops strongly increased over time in most clusters. Among boys, the proportion of beer decreased, but was dominant across time in all clusters. Only northern European countries formed a geographically defined region with the highest prevalence of heavy drinking and average alcohol volume in both genders. Adolescent beverage preferences are associated with mean alcohol volume and heavy drinking at a country-level. Future approaches to drinking cultures need to take subpopulations such as adolescents into account. Alcohol consumption is one of the most important risk factors for adverse health effects and mortality . AdolescThere is evidence for an association between the preference for different types of alcoholic beverages and several aspects of alcohol consumption among adolescents. Indicators of risky drinking such as high mean alcohol volume and heavy use are related to beverage choice. Consumers of beer and spirits typically drink more and show higher rates of heavy use as compared to wine drinkers. A preference for wine, in contrast, is known to be associated with less risky drinking ,9,11,12.Beverage choice is subject to temporal changes and differs between countries ,16,17,18Studies on beverage choice in different drinking cultures have primarily been conducted in the adult population, while research among adolescents is lacking. As beverage choice and related drinking behaviour vary substantially across age and sex ,31, largData on alcohol consumption were obtained from six waves  of the European School Survey Project on Alcohol and Other Drugs (ESPAD). In brief, cross-sectional school surveys were conducted every 4th year since 1995 for 15- to 16-year-old students. With the exception of Germany (Bavaria), in each participating country, sampling was carried out nationwide, using a stratified cluster sampling design. Data were collected in classrooms with either paper-and-pencil or online questionnaires. Participation was anonymous and voluntary. Different school types were included, with the class constituting the last unit of the multi-stage stratified sampling design . For 24 The consumption of five different types of alcoholic beverage, beer, cider, alcopops, wine and spirits, was assessed for the last drinking day. To this end, participants were asked for each beverage separately, ‘If you drank beer/cider/alcopops/wine/spirits the last day you drank alcohol, how much did you drink?’. Beer/cider/alcopops were categorised as ‘<50 cl’, ‘50–100 cl’, ‘101–200 cl’ and ‘>200 cl’. The response categories for wine were ‘<10 cl’, ‘10–30 cl’, ’37 cl’, ‘≥75 cl’ until 2003, and changed to ‘<20 cl’, ‘20–40 cl’, ‘41–74 cl’ and ‘≥75 cl’ in 2007; the response categories for spirits were ‘<5 cl’, ‘5–10 cl’, ‘11–25 cl’ and ‘≥30 cl’ until 2003, and ‘<8 cl’, ‘8–15 cl’, ‘16–24 cl’ and ‘>25 cl’ from 2007. Quantities of pure alcohol per beverage type were converted into centilitres of pure alcohol using midpoints of the range of beverage quantities multiplied by the following alcohol concentrations: 5 vol% for beer, cider, and alcopops, 12 vol% for wine and 38 vol% for spirits . For theDue to the well-known sex differences in alcohol consumption, all analyses were conducted separately for girls and boys. Trends in the distribution of beverage types were analysed using fractional multinomial logit regression ,35,36,37Based on the results from the fractional multinomial logit regression models for each country and sex, the trend coefficients for beer, wine and spirits, and the average predicted beverage proportions of total consumption  across all waves were used as variables in a hierarchical cluster analysis. The Ward’s method for linking and the Squared Euclidian Distance, an agglomerative approach, were applied on the basis of the variance of clusters. In each step, the sum of the squared distance of the individual cases from the respective cluster centroid was calculated for all pairs of clusters that were possibly being merged. The two clusters with the smallest increase in the total sum of squared distances were merged in the respective step ,44.Data from countries in the resulting clusters were pooled and the trend analyses, as described above, were applied within all clusters in girls and boys. Changes in beverage proportions across time were further examined by the difference between the highest and lowest rate between 2019 and 1999. The cut-off of 10 percentage points (pp) was used to consider the observed changes as diverging (positive difference ≥ 10 pp) or converging (negative difference ≤ 10 pp). Clusters were compared in terms of the prevalence of 12-month drinkers, mean alcohol volume and prevalence of heavy drinking on the last drinking day. Except for the prevalence of drinkers, the estimates were based on the subsample of the last 12-month drinkers. To account for different, country-specific sample sizes, these indicators were calculated for each cluster as the mean of the arithmetic country’s means across the whole observation period.The results of the fractional multinominal regression analyses, as well as the model-predicted beverage choice by beverage, country and year, on which the following analyses are based, are depicted in The cluster analysis, which was conducted using the trend coefficients for each beverage type and the predicted proportions of beverage choice, yielded four distinct clusters for both girls and boys . Among tAmong boys , the firCompared to the remaining clusters, the fourth cluster for both sexes, largely representing northern European countries, showed a higher overall mean alcohol volume and a higher prevalence of heavy drinking . ConversWe examined the beverage choices of 15–16-year-old adolescents on recent drinking occasions in 24 European countries and how they evolved over time. We investigated these changes separately for girls and boys and performed a cluster analysis to detect commonalities and differences between countries. Four clusters emerged for both sexes. Remarkably, in the majority of clusters, the preference for cider/alcopops increased over time, while the preference for more traditional beverages decreased. We found that risky drinking behaviours, in terms of high mean alcohol volume and high prevalence of heavy drinking on the most recent drinking day, were more common in northern European countries, Estonia, and Ukraine (in the latter two countries only among girls), the cluster where cider/alcopops were most  preferred.Previous research has found an association between a preference for spirits and heavy use in adolescents ,9 and adAmong girls in northern European countries (cluster 4), high and increasing proportions of cider/alcopops were associated with higher mean alcohol volume and a higher prevalence of heavy drinking on participants’ most recent drinking occasion. An increase in the proportion of cider and alcopops among the youngest cohort could also be found in a recent study on beverage preferences according to age, period and cohort in Sweden . In contBeverage choice among adolescents is a function of various aspects, including taste or predilection, as well as the availability of specific beverages. Accordingly, the distribution of the beverage types consumed on drinking occasions may reflect changes in availability. In Sweden, Norway and Finland, alcohol, with the exception of low-alcohol beverages, can only be purchased in government-run alcohol monopoly stores, where age limits are controlled more strictly than in grocery stores, in which beverages with a low alcohol content are sold . Laws onMost importantly, recent discussions on drinking cultures have already extended beyond the subject of traditionally preferred beverages ,29,58. WA generic view of drinking cultures tends to result in stereotypes that do not reflect the variation in actual consumption behaviour. The notion of there being ‘many drinking cultures’ within a society has recently come to the fore. Kilian and colleagues postulated the existence of several combinations of drinking cultures within a country rather than only one . DescripESPAD data on alcohol consumption derive from self-reported consumption on the last drinking day. Hence, the reported amount and type of alcohol consumed may not be representative of an individual’s drinking behaviour ,69 and mTemporal trends in beverage preferences among adolescents vary considerably between European countries and between girls and boys. Adolescent beverage choices and changes in beverage choice are associated with alcohol volume and heavy drinking, particularly with the high and increasing proportions of the use of cider/alcopops. This points to the necessity of considering beverage choice in alcohol policies. Measures such as the adaptation of taxes, and minimum pricing policies for beverages associated with risky drinking behaviour, might be effective here. In addition to clusters consisting of primarily northern European countries, the resulting clusters do not match the previous findings of geographically distinct regions, representing particular drinking cultures. The present results support the recent debate on drinking cultures, suggesting the importance of beverage choice and considering various drinking patterns within countries. Future approaches to the study of drinking cultures should broaden their scope by acknowledging adolescents as a distinct subpopulation.