We queried CAIRN, PubMed, CINAHL, MEDLINE, Scopus, and Google Scholar databases for this purpose. The search process followed the recommendations of Arksey and O'Malley. Items reported in this review follow the PRISMA-ScR guidelines. Sixty articles involving children from a total of twenty (20) countries, mainly Ghana and Nigeria, were included in the final analysis. The data used were predominantly cross-sectional and were mainly drawn from demographic and health surveys. The analysis revealed that chronic malnutrition in children is associated with sociocultural, economic, and healthcare factors related to the characteristics of children, mothers, households, and communities. The association with children's vulnerability to disease, maternal education, purchasing power, and autonomy need to be further investigated in West and Central Africa. Further analysis using longitudinal data is also needed to better understand the factors associated with chronic malnutrition in West and Central Africa.Chronic malnutrition is a major public health concern that is the focus of a large body of scientific research. However, there is no synthesis of knowledge about the factors associated with this disease in West and Central Africa, where its prevalence is particularly high. We conducted a systematic search for scientific articles published between January 1 Chronic malnutrition in children is a growth disorder resulting from a long-term deficiency in nutrient intake. The condition brings numerous adverse consequences to health and economic status which are long-lasting and sometimes irreversible . StudiesIn sub-Saharan Africa, chronic malnutrition is widespread, affecting one-third of children under the age of five . EvidencGiven the importance of the subject, several empirical studies have analyzed factors contributing to childhood chronic malnutrition. There are two review articles concerning the whole of sub-Saharan Africa ,12, whicTo date, there is no article synthesizing knowledge on the factors associated with chronic malnutrition in West and Central Africa, which is the most affected sub-region . The speStudy design: this scoping review was conducted according to the framework defined by Arksey and O'Malley [O'Malley  which isO'Malley . The revO'Malley .Protocol registration: there has been no protocol and registration of this scoping review.Research question: the scoping review was conducted around the following question: “according to the scientific literature, what are the factors associated with chronic malnutrition in children in West and Central Africa?”.Search strategy and data source: the search question was analyzed to identify keywords representing population, intervention, comparators, outcomes, timing, and study design (PICOTS), which were then used to build search strategies. These search strategies were then applied to the CAIRN, PubMed, CINAHL, MEDLINE, Scopus, and Google Scholar databases. The keyword searches used included free text and controlled vocabulary. Orthographic variants and synonyms of the keywords were also taken into consideration in the construction of the search equations. These equations enabled a systematic selection of all scientific articles that contained these words in their title, abstract, or keywords. The search equation used was:  and (child* or infant* or under-five or preschool).Eligibility criteria: eligibility criteria, both inclusion and exclusion, were defined to guide the selection of articles.Inclusion criteria: all articles included in this scoping review met the following criteria: i) the study concerned one or more countries in West Africa  or Central Africa ; ii) the study was published between January 1, 2000, and October 15, 2021; iii) the study addressed chronic malnutrition assessed by a height/age ratio; iv) the measurement of height respected the World Health Organization (WHO) standard of measuring height in the supine position for children under two and in the standing position for those aged two and over according to WHO and UNICEF [d UNICEF ; v) the d UNICEF  or the nd UNICEF ; vi) theExclusion criteria: although meeting the inclusion criteria described above, some articles were excluded for the following reasons: i) the articles were restricted to specific populations such as premature infants, children who were hospitalized or attending a given health service, or children suffering from a particular disease or condition ; ii) the articles were methodological guides or handbooks; iii) the data used were collected before January 1, 2000.Selection process: the selection of articles was carried out in several steps  articles was done by the whole team of authors during a working meeting. The remaining articles were distributed among the authors for extraction.Collating, summarizing and reporting the findings: the data extracted from the included articles were designed following the grid. The relevant information obtained includes references of the article, country, age of children, types of study and methods of analysis, and significant risk factors. Subsequently, these data were organized by country and by geographical area.Applying our search strategy identified a total of 6,915 articles: 2,122 in SCOPUS, 4,131 in PubMed/MEDLINE, 411 in CINAHL, 222 in Google Scholar, and 29 in CAIRN. There were 1,752 duplicates that were removed. Of the 5,163 remaining articles, 4,978 (96.4%) were excluded after reading their titles and abstracts. Also, 40 articles were initially classified as “undecided”, they were then re-evaluated, and 26 were excluded and 14 were retained. The 159 articles retained at this stage were read in their entirety and analyzed according to see if they met eligibility criteria. In the end, 68 articles were included. The entire selection process is presented in Characteristics of the selected studies: four studies [ studies -23 were  studies -87 in EnMethods for identifying factors associated with chronic child malnutrition: the characteristics of the selected studies, including the methods for analyzing the factors associated with chronic child malnutrition, are presented in Factors associated with chronic malnutrition in children: the analysis of the 68 selected articles showed that child, maternal, household, and community characteristics are statistically associated with chronic malnutrition in children in West and Central Africa , birth rank (n=3), birth weight (n=6), birth height (n=3), birth interval (n=2), disease frequency (n=13), vaccination status (n=7), breastfeeding (n=3), meal diversification (n=9), meal frequency (n=2), and vitamin A and iron supplementation (n=1) were also found. However, some studies that investigated these factors found that the sex or age of the child was not associated with chronic malnutrition in children. This was the case of two studies conducted in Nigeria [ Nigeria  and Burk Nigeria  which fo Nigeria ,38,60.Maternal characteristics associated with childhood chronic malnutrition: significant effects of maternal characteristics, including education, age, mobility, leadership, experience with domestic violence, decision-making autonomy, ethnicity, marital status, and health status were reported in the reviewed studies. The effect of maternal education was reported in 19 studies and showed a reduction in the risk of chronic malnutrition with an increased maternal level of education. These effects of education are particularly apparent when mothers have an education level from secondary school and up [l and up ,49,77,84The effects of a mother's purchasing power, as assessed by employment status or income level, were demonstrated in six studies in Angola, Burkina Faso, Cameroon, Ghana (n=2), and Nigeria ,57,67,80The effects of maternal autonomy, including leadership, participation in decision making, experience, or attitude toward violence were highlighted in five studies. They found that children of mothers with decision-making autonomy are less likely to suffer from chronic malnutrition ,72,79,84Some studies found that maternal and child characteristics are not statistically associated with chronic malnutrition in children. For example, four studies conducted in Ghana, Senegal, and Nigeria concluded that there was no effect of maternal education on chronic child malnutrition ,45,70,72Household characteristics associated with childhood chronic malnutrition: the analysis of the articles revealed that certain household characteristics are associated with child chronic malnutrition. These characteristics include socioeconomic status, family composition, and access to water and sanitation.The effect of socioeconomic status was found in ten studies and was assessed by the level of economic well-being ,66,80,86Eight studies conducted in Angola , BurkinaCommunity characteristics associated with childhood chronic malnutrition: community characteristics that were statistically associated with child chronic malnutrition are described in three studies. In Senegal, authors have established that an increase in the percentage of women in the district having decision-making power is statistically associated with reduced childhood chronic malnutrition [utrition . In Nigeutrition . Finallyutrition ,69.Our discussion first focuses on the methodological aspects of the selected studies. Secondly, it assesses the factors related to childhood chronic malnutrition identified in the selected articles.Methods used: nearly half of the data collected and analyzed in the studies were from the Demographic and Health/Multiple Indicator Cluster Surveys (DHS/MICS) that provide representative results at the national and regional levels. These surveys use a standardized data collection methodology that ensures the comparability of findings across the time horizon and geographic area.However, there are weaknesses related to the type of data collected. Almost all (63/68) of the study designs are cross-sectional. Thus, the data collected is not well adapted to examining the effects of ephemeral events such as episodes of disease in children on the onset of chronic malnutrition. Indeed, cross-sectional data do not ensure that the occurrence of the event (e.g. episode of diarrhoea) has preceded the resulting outcome . Therefore, it is difficult to prove that the associations made are not by chance. This is particularly problematic in a condition such as chronic malnutrition, where predisposing damaging factors are slowly and permanently established . Given tChildren's eating practices change dramatically in the first five years of life: from zero to six months, they are almost exclusively breastfed; from 6-23 months, they consume breast milk and light foods; and from 24 months, they share the family meal. Consequently, the factors associated with optimal feeding, a close determinant of chronic malnutrition, may differ according to their age. There is evidence to suggest that there are age-specific factors of malnutrition, as demonstrated by authors . NeverthFinally, it is important to note that 90% of the studies didn´t use a multilevel model although the theoretical hierarchical structure of malnutrition factors ,89 argueAssociated factors identified: the review of the articles demonstrated that children's characteristics and maternal characteristics are those most frequently associated with chronic malnutrition. This predominance of child and mother-related factors underlines the importance of these proximate determinants in predicting child chronic malnutrition. Our discussion will address the vulnerability of children to disease, maternal education, the purchasing power of parents, and the decision-making autonomy of mothers, which are the main determinants of chronic malnutrition in children highlighted in West and Central Africa.Children's vulnerability to disease: childhood illness is a major contributing factor to chronic malnutrition. Eleven studies describe a statistical association between the occurrence of diarrhoea, fever or anemia and the onset of chronic malnutrition [utrition ,81, 86. utrition . Most otutrition . For exautrition . Althougutrition . In contThe association between chronic malnutrition and children´s characteristics such as age, sex, twinship, birth size, and vaccination status is related (in part) that these factors  make children vulnerable to disease. Authors have demonstrated that the greater susceptibility of twins to malnutrition is due to a higher frequency of congenital malformations, low birth weight, and cerebral palsy, rendering them more susceptible to disease . SimilarThe effects of age on chronic malnutrition also express a vulnerability to childhood diseases. Indeed, the risk of chronic malnutrition is increased between 6 and 23 months of age when the child's immune system fails due to the decrease in maternal antibodies  and due In sum, a greater vulnerability to disease in boys, twins, children born with short stature, and unvaccinated children predisposes them to a greater risk of chronic malnutrition. Given that some of these vulnerability factors such as sex, age, twinship, and birth size are non-modifiable factors, research should be directed toward mitigating the effect of those factors that are most commonly associated with chronic malnutrition in children.Maternal education: maternal education is commonly associated with chronic malnutrition in children. Its effect has been highlighted in 19 studies, showing a reduction in the risk of chronic malnutrition when the mothers' education level increases. At school, lessons about nursing, biology, food hygiene, enable mothers and caregivers to acquire useful knowledge to care for and feed babies. In addition, educated people have access to more diversified sources of information , which can increase their knowledge. Thus, capitalizing on their schooling and better access to information, educated mothers have higher knowledge of nutrition [utrition . ConsequThe effect of education can also be mediated through a close-to-home diffusion within communities. Uneducated mothers can learn about good hygiene, care, and feeding practices from their educated counterparts, enabling them to overcome their skill deficits. This neighbourhood effect is often so strong that it leads to the elimination of differences in children's nutritional status caused by the individual educational level of their mothers. This could explain why, in communities where the majority of women are educated, maternal education is not a discriminating factor of children's chronic malnutrition ,72. SincPurchasing power of parents: purchasing power refers to the ability of parents to financially acquire the goods and services necessary for the appropriate growth of children. Some characteristics, such as the mother's employment and income, and the household's economic well-being reflect this effect of parental purchasing power. Women with higher incomes have greater access to food and health services, which is conducive to growth in their children [children ,57,67,80children ,64,66,86children .Despite this, some studies have found no effect of purchasing power estimated by the household income level on childhood chronic malnutrition ,66,70,86To be complete, the assessment of parental purchasing power on children's nutritional status must take into account the community income level, as access to goods and services is dependent on it. In fact, food and healthcare are relatively more expensive in upper-income communities found in large urban centers ,98. ThusMaternal autonomy: this concept refers to “a woman's ability to influence decisions about family, finances, and spending, work, social outings, health care, travel, family planning, and child care...” [care...” . The effcare...” ,72,79,84care...” . SimilarIn summary, the autonomy of mothers to make decisions concerning the care and feeding of their children is an important determining factor, especially in the contexts of child care and feeding which are almost exclusively their responsibility. Studies on chronic malnutrition should take into account the mothers´ autonomy whose effects have not yet been sufficiently analyzed in West and Central Africa.This systematic search and review of the scientific literature have identified child, maternal, household, and community characteristics that are associated with chronic malnutrition in children living in West and Central Africa. Despite the relative abundance of scientific evidence on the subject, the effects of some major factors such as children's vulnerability to disease, maternal education, and maternal autonomy are still poorly defined. Future studies should focus on a deeper investigation of the effects of these factors. In addition, the use of subgroups in analyzing age, and longitudinal data, would provide complete view of childhood chronic malnutrition factors in West and Central Africa.West and Central Africa, which is the most affected sub-region;Factors contributing to childhood chronic malnutrition are studied in several empirical studies.It is the first knowledge synthesis of factors associated with childhood stunting in West and Central Africa;It shows that there would be a contextual effect of mother's education and purchasing power that must be taken into account in future studies;Child´s vulnerability to disease is a factor of childhood chronic malnutrition.


Addiction to video games is a theme regularly mentioned and associated with the risks that concern adolescents. This study aimed to determine the prevalence of video game addiction (VGA) among adolescents and identify its associated factors.We conducted a cross-sectional study within public high schools, in Sousse, Tunisia in 2019. The target population was high school students. A structured self-administered questionnaire was used to collect data about sociodemographic characteristics, lifestyle behaviors, and mental health disorders, and we used the 21-item Game Addiction Scale to objectify video game addiction. Statistical analysis was carried out using the program SPSS v.20.A total of 1342 participants were recruited for the study,36.8% of whom were boys. The average age was 17.5 ±1.44 years. The analysis of mental health disorders showed that 67% were anxious, 66.8% were alexithymic, 65.4% were depressed and 39% had low self-esteem. The analysis of lifestyle behaviors showed that a lack of physical activity was reported by 57.6% of participants. Problematic use of the Internet (>2 hours per day) was reported among 72.4% of the students. The prevalence of video game addiction was 13%. Boys were more prone to be addicted than girls . ≥17 years old students had a higher rate of VGA than those aged<17 years old . VGA was higher among students who follow the non-scientific study section . Students who had moderate depression had the highest percentages of VGA . Students who had problematic use of the internet were more addicted to video games (80.3%) than those not having problematic use (19.7%), p = 0.009.This study allowed us to identify the students who are vulnerable to VGA. Also, a huge responsibility is accorded to school staff and parents to tackle this health problem by sensitizing their children and setting up some protective family rules.• Several factors were significantly associated with video game addiction, especially problematic internet use.• Further research is needed to understand the underlying mechanisms of video game addiction and to explore effective preventive or interventional strategies.


With the development of the Internet, more and more patients search for disease-related information on video platforms during the treatment process, and physicians also look for learning materials through these video platforms. Bilibili is one of the most popular video platforms in China. This study evaluated information on various interesting topics, and related surgical procedures searched through Bilibili.The Bilibili platform was independently queried for 12 common vascular diseases or related surgical procedures between October and November 2021 by two independent authors using the Baidu search engine. Information about the video and uploader was collected, and descriptive analyses of the overall and first-page results were performed.A total of 3,998 search results were retrieved by searching 12 vascular-related topics, of which 2,225 actual videos (55.7%) were finally confirmed to be related to medicine. Videos for the public accounted for 84.8% of these 2,225 videos. In addition, 50.5% of the video results were uploaded by vascular surgeons, 12.4% by other specialties, 17.7% by organizations, and 19.4% by other individuals. The total number of videos searched for varicose vein and peripheral vascular diseases was the largest, and the total number of leg amputation videos was the smallest. The largest number of videos for medical professionals was about pulmonary embolism, and the smallest was about leg amputation. On the first pages, 168 results (70.0%) were actually medically relevant, and only 7.7% of the videos were uploaded by vascular surgeons.On the Bilibili platform, videos about vascular diseases are extensive but not comprehensive. The videos uploaded by vascular surgeons are rare, and the results searched are not precise. The online presence of vascular surgeons needs to be improved, which may partially solve the problem of low-quality videos due to the lack of strict management and censorship. Traditionally, medical education requires a combination of clinical practice with textbooks, on-campus courses, laboratories, and lectures, and surgical education requires the accumulation of hands-on operative experience. Nowadays, Internet not only provides new methods for medical education but also provides new tools for patients and medical information providers. However, this is both an opportunity and a challenge, as the content of various video-sharing platforms has not undergone peer review and is not subject to strict regulations and censorship . TherefoEven with these drawbacks, there is no doubt that the video-sharing platform has become one of the important ways for people to obtain health information, and many new videos about diseases are uploaded every day . It is uRecently, a study explored the current state of vascular surgery on Google and YouTube . Bilibilhttp://www.bilibili.com)  , which iWe first excluded the videos that were not related to medical science and then analyzed the remaining videos. These videos about medical science are defined as actual videos. The number of results retrieved directly is defined as the total number, and the number of actual videos is defined as the actual number. Then, we evaluated each actual video to determine whether the information was popular science or professional knowledge. Video information containing details of disease diagnosis, procedures, and management is considered professional knowledge. The search results containing recommendations from specialties and hospitals, personal stories of patients, disease introduction, disease risk factors, and simple introduction of treatment methods were considered popular science. Furthermore, each individual result was reviewed to record the uploader. Uploaders are divided into four categories: vascular surgeons, other specialties, organizations, and other individuals. The uploaders of the organization category included people from websites, applications, and WeChat public accounts. Finally, we calculated the proportion of videos containing professional knowledge and the proportion of uploaders in each category in the actual videos. The proportion of each topic category was based on the actual video number.Given the use of publicly available data, institutional review board review and informed consent requirements were waived. Descriptive statistics were used to evaluate the results of the research. All analyses were performed using Microsoft Excel.A total of 3,998 search results were retrieved by searching 12 vascular-related topics, of which 2,225 videos were finally confirmed to be related to medicine. Of these 2,225 actual videos, 84.8% were directed to the public. In addition, 50.5% of the video results were uploaded by vascular surgeons, 12.4% by other specialties, 17.7% by organizations, and 19.4% by other individuals. For 4  of the 12 search topics, vascular surgeons were mentioned less frequently than the other medical/surgical specialties.The total number of videos searched for varicose veins and PVD was the largest, and the total number of results searched for leg amputation was the smallest. The number of actual videos searched for varicose veins and PVD was the largest, and the actual number of results searched for EVAR and CAS was the smallest. Nevertheless, the highest proportion of actual videos appeared when searching AD (78.3%), and the lowest proportion appeared when searching EVAR (13.1%) . The mosMost videos for medical professionals were about PE, and the least videos were about leg amputation. However, the highest proportion of videos for medical professionals was about AAA (64.0%), and the lowest was about varicose veins (4.8%) . ResultsThe least results uploaded by the vascular surgeons were about AD, AAA, CAS, claudication, and leg amputation; the lowest percentage was about AD, PE, AAA, claudication, and leg amputation; and the largest number and proportion of videos uploaded by vascular surgeons were about PVD .Of the 240 results, it was finally determined that 168 results (70.0%) were actually medically relevant, of which 46.4% (78/168) of the videos were directed to the medical professionals . The vidMost videos for medical professionals were about DVT, and the least videos were about leg amputation. The highest ratio was about DVT (68.4%), and the lowest ratio was about leg amputation (10.0%). In the results on the first pages, the videos uploaded by the vascular surgeons did not present topics of AD, PE, PVD, PAD, claudication, and varicose veins. The largest number of results and the largest proportion (44.4%) uploaded by the vascular surgeons were related to EVAR .In general, the number of results obtained by a direct search for each keyword was different , and the actual videos after screening did not account for a high proportion (55.7%) of the total. Only 15.2% of the videos were directed to the medical professionals, but the proportion in the first pages was three times that of the whole. The lack of videos uploaded by vascular surgeons results in insufficient technical quality, which may convey major biases to those who use these videos for training or even for laypeople to understand these diseases.The Internet has become an important way for patients to obtain disease and treatment-related information because it can be accessed conveniently and always free of charge. When evaluating vascular surgery information on the Internet in 2012, it was found that most of the information was of low quality .One study showed that average Internet users rarely clicked on results beyond the first page . Our resIn addition, the ranking of videos was also a problem, and researchers have raised the deficiency of a video ranking method of video platforms . The popOne study shows that shorter interactive courses are beneficial . InevitaResults vary with the nature of the retrieved part of speech. When searching for the name of a disease, the results were varied, including the introduction of the disease, etiology, risk factors, clinical manifestations, treatment, prognosis, and prevention. When retrieving disease treatment methods  and surgical video demonstrations, there is also popular science content such as surgical indications, conservative treatment indications, and medical expense reimbursement. However, this large number of materials can be overwhelming for the students. A study indicated that after searching for femoral artery access technology on YouTube, the clear and accurate results were less than 10% . Our resWe believe that our results will give people an understanding of the status of videos about vascular surgery on the Bilibili platform. This study also had some limitations: first, we used 12 common search terms related to vascular surgery diseases, and a more comprehensive search is necessary for the future; second, we only analyzed the audience and uploaders of the video, and a more detailed analysis of the video content is needed.Our research suggests that although there are plentiful videos about vascular surgery on the Bilibili platform, there are few videos for professionals. In addition, there are very few vascular surgeons among video uploaders, especially in the results on the first pages, so the online presence of vascular surgeons needs to be improved.


We sought to assess the degree, nature and impact of poor audio quality during ward rounds for individuals attending remotely using Microsoft Teams. We aimed to evaluate attendee experiences of audio quality against our expectation that due to the existing ward microphone system using a cardioid polar pickup pattern that attendees would have difficulty hearing all members of the multidisciplinary team, as well as the patient, gathered in the conference room. We also hypothesised that after switching to an omnidirectional sound recording system, we would observe an improvement in attendee satisfaction with audio quality during ward rounds.Individuals who had remotely attended ward rounds at Lesney Ward, Oxleas NHS Trust between 01/11/21 and 01/01/22, mainly patient family members and community care co-ordinators, completed a digital feedback questionnaire regarding audio quality. There was no exclusion criteria. Data from Likert scale questions were analysed with descriptive statistical tests (mode and distribution of responses). As minimal demographic data were obtained, inferential statistical tests were not used. Qualitative data were analysed using thematic sorting based on prevalence of themes in the data.Feedback was provided by three family members, one ward team member and six members of the community mental health team prior to the intervention. Pre-intervention feedback indicated high levels of dissatisfaction with 6/10 respondents reporting they were “dissatisfied” and 1/10 “very dissatisfied”. Only 3/10 of attendees reported being able to hear and understand all individuals physically present in the room. In addition, respondents agreed that the audio quality was poor , and that the sound quality impacted upon their experience of the ward round .The three most common main issues reported by respondents were: people speaking too far from the microphone (7/10), voices sounding muffled (6/10), and too much background noise (4/10). Using their own words, respondents described how the ward round sound quality made them feel. Common themes identified through thematic sorting included: distress, difficulty in understanding information / management plan, ward round prolongation and inability to comprehend the patient or staff.In conclusion, we found that when using an in-built laptop microphone with unidirectional pick-up remote ward round attendee satisfaction was poor, though this improved with the introduction of an omnidirectional system. Key areas for improvement include assessment of optimal positioning for adequate audio pick-up, and the introduction of automatic transcription for individuals with hearing impairments.


Persuasion can be defined as an active attempt by a person to change the behavior and attitudes of others. The purposive attempt to influence one's behavior can originate from different areas, and people who are able to do so are often referred to as influencers. Social media platforms such as Instagram or YouTube have become crucial platforms for influencers who generate their income by recommending products and services to their followers, including cosmetics, multimedia articles or clothing. Studies indicate that influencers actively try to persuade the viewer to adopt specific desirable behavior by strategically altering their displayed behavior on social media. Such strategies have mainly been explored in the context of beauty products, where lack of expertise and misinformation might have few negative consequences. Less is known about strategies used in a health-sensitive context, such as nutritional supplements. This research addresses this gap and aims to understand persuasive techniques used by health professionals on YouTube to promote the use of protein supplements. This study is based on an interpretive paradigm using interpretive grounded theory to analyze 60 YouTube videos. We developed a three-step model of persuasion for YouTube videos consisting of the steps: reaching the message, staying on the message, and performing the action that the persuader desires. Our analysis resulted in five core themes that contributed to the persuasiveness of the analyzed YouTube videos. These themes included: Quality, curiosity, engagement, concretization, and genuineness. We conclude the paper with reflections on our model's theoretical and practical implications. Many theories try to explain why humans behave the way they do and how their behavior can be steered toward an “ideal” or desired one. The widely cited Social Learning Theory is a comprehensive model that integrates behavioral and cognitive learning theories. The theory proposes that people can learn new behaviors through observation and imitation of others instead of learning by direct experience    and marketing who actively create new persuasive technology. For example, a detailed understanding of techniques for online persuasion could guide social media content creation for positive food consumption. Furthermore, researchers and practitioners could use the model to design persuasive interfaces, apps or even chatbots.The manuscript is structured as follows: We first outline current findings regarding SMIs and persuasive strategies. We then describe the methodology and steps undertaken during the iterative data collection and analysis process. In a consequential step, we describe our three-step persuasion model. We conclude the paper with a discussion of the model and point out potential limitations.In this section, we describe related work conducted in the context of SMIs. We would like to emphasize at this stage that we followed a grounded theory approach that focuses on the literature review once the data collection and analysis are completed to avoid bias. Therefore, the outlined frameworks and theoretical findings were not taken into consideration as part of the study design and analysis process but are presented to provide an overview of current research efforts.YouTube can evoke a sense of community among followers  and its creators, such as age, gender, and professional occupation. This kind of additional information helps form a thorough understanding of the social context in which the video is embedded. Furthermore, the comment section also provided contextual insight into the video's audience.Grounded theory uses different kinds of sampling techniques, often starting with purposive sampling  Open coding stage, including initial data; (2) selective coding stage, including focused data; and (3) theoretical coding.The way codes are developed, and a theory is formulated can differ between the different schools of grounded theory . The researcher starts the process by familiarizing themselves with the data and coding the data excerpts by adding short labels to the data . The first author reviewed the developed codes to search for concepts and categories by increasing the level of abstraction. We noticed in the process that using three levels of abstractions  was more effective and sufficient in the context of this study. We, therefore, did not develop concepts during the analysis process.The abstraction process concluded with 25 codes and eight categories. As the last step of the open coding, the researcher should ideally select a single core theme. However, despite repeated abstraction, we did not find one core theme that adequately explained most of the data. Therefore, we ended the initial analysis by selecting three themes.During the second stage of the coding process, the goal was to add more data to achieve theoretical saturation and refine the categories and themes. We, therefore, added 10 more videos to our analysis. We selected these videos based on the criteria described above. After these 10 videos, we noticed codes starting to repeat. Furthermore, the number of categories increased from eight to twelve, codes increased to thirty, and a new theme emerged. We analyzed 10 additional videos, after which we were confident of having reached theoretical saturation of the data, completing our selective coding stage.Finally, in the theoretical coding stage, we focused on examining the relationships between the four core themes and the 12 categories. Through constant comparative analysis, we established multiple links between the categories. We also reviewed the literature as part of this process to refine the conceptual elements of our model. The result of this stage was a three-step model of persuasion, which we present in the next section.Our model proposes that on YouTube, persuasion is a process that can be divided into three main steps: (1) “Reaching the message”; (2) “Staying on the message”; and (3) “Performing the action that the persuader desires,” see .reaching the message. This step is crucial because there is an abundance of videos on YouTube related to fitness and nutrition. For the viewer to see the persuasive message embedded in a given video, they should select it first amongst millions of other videos. Through our literature review conducted during the theoretical sampling stage, we noted that thumbnails, titles, and categories are influential criteria in the decision making process  Aesthetics; and (3) Catchiness. These three categories were grouped under the two themes “Quality” and “Curiosity: Entertainment” ” and "Dietitian Reacts to Ashley Graham What I Eat In A Day ”.To conclude, the first step of our model focuses on encouraging viewers to click on the video and “reach the message.” This step consists of establishing the perception of quality by linking it to professional expertise and aesthetic features. This step also aims to persuade viewers by evoking elements of curiosity and entertainment.stay on the message. We identified “Engagement” as the main theme of this step. We define engagement to take place when the viewer of a video experiences any emotion while watching it. In this research, we focus on positive emotions such as enjoyment. We identified the four categories of (1) Humor; (2) Aesthetics; (3) Curiosity; and (4) Personal Relationship  How to use it; (3) Why to use it; and (4) Where to access it.Once a viewer has decided to watch a video and paid close attention to its content, they need to do the desired action, such as buying the presented protein supplement. Therefore, the third step of our model focuses on performing the action that the persuader desires. Our analysis suggests that persuasive features concentrate on describing the desired actions precisely and in a concrete manner. The main theme of this step, labeled “Concretization” comprised of four categories: (1) Concretizing an action entails giving compelling arguments on “Why” the viewer should do the activity and what its results could look like. This display can be seen as a source of motivation for doing the desired action. In YouTube videos, clips of men and women with exercised muscles lifting heavyweights in a gym indicate the anticipated results of consuming protein, including increased muscle strength and a “pleasant” physical appearance. Mentioning academic articles as part of the video can be seen as a persuasive strategy that convinces the viewer to buy protein supplements because they help establish the product and its associated benefits to be trustworthy and backed by scientific studies. The theme of concretization also includes telling the audience precisely “What” to purchase. For example, a video might convince a viewer to buy a supplement because of the category “Why” based on the persuasive features used by the video creator. However, this particular viewer could still get confused and overwhelmed by the vast choice of protein supplements, thus giving up on buying them.The category of “Where to access” focuses on persuading the viewer to buy a specific brand of supplements and reducing the risk of feeling overwhelmed during the purchase decision process. Discount offers and links to e-commerce websites for protein supplements can encourage and simplify the process of executing the purchase.The fourth theme also describes “How to use” the product. As part of the video, the viewer should receive information that gives a clear idea of how they could practically and realistically incorporate this product into their everyday life. This included providing quick recipes and concrete meal plans that involved supplements.scientific information and reliability. Furthermore, the category of personal relationships included in step 2 of our model contributes to elements of genuineness.Our model consists of one additional theme, labeled “Genuineness,” that does not fit exclusively into a single step but instead influences all three steps . We define genuineness to be based on a natural, unstaged and trustworthy perception of the video. The persuasive message of the video should not come across as pushy, artificial or as a blatant advertisement. The theme of genuineness is influenced by the categories of The persuaders (video creators) should appear to believe in what they are telling the audience. By appearing more relatable, shooting the video in a natural environment such as a kitchen and sharing personal stories, the video creators establish an image of being just an “average human being” that the viewer can relate to and less like a salesperson. By emphasizing one's flaws through strategies such as self-deprecating jokes, the video creator becomes more relatable to the viewer. Supplying the viewers with logical reasons (such as scientific information) for acting can contribute to genuineness. These factors help counteract any ulterior motives the video creator may have  and make the message more trustworthy. Genuineness can help the audience to perceive the video as trustworthy.When thinking of YouTube design, a term that comes to mind is “attention economy,” coined by psychologist and economist Herbert Simon . The conIn this study, we used interpretive grounded theory to analyze the persuasive strategies employed by SMI as part of vlogs on YouTube to persuade viewers. We focused on protein supplements as a research focus due to their nature of combining food and health. Arguably, this context requires different persuasive strategies compared to SMI, where a level of expertise of the SMI is of little concern . In this context, it also should be noted that using social media to disseminate health-related messages is a topic where care should be exercised. For example, the online distribution of statements that are not backed up by scientific findings. In a worst-case scenario, this can affect an individual's health  to the overarching theme of “Genuineness.” The essence of PRT is that human beings enjoy their freedom and autonomy Brehm, . When inOne main difference we noticed when reviewing current research efforts  based influencers has attracted increased attention in research and commercial areas. This kind of disruptive technology requires a better understanding and customer engagement when used as a marketing strategy. Application areas in the context of social media marketing vary and range from identifying optimal promotion of products to providing customized support . Instagram alone has 35 verified virtual SMIs, including SMIs with a human-like appearance, such as “Lu of Magazine Luiza” (5.9 million followers), the brand representative for the Brazilian retail conglomerate Magazine Luiza. However, there are also several SMI with little resemblance to humans, including cartoon-style characters such as “Nobody sausage” (2.7 million followers)  and time of the video post (business hour vs. non-business hour) can influence a video's popularity , companies who advertise through YouTube and end consumers about the persuasive strategies displayed in the vlogs could provide further insights. Currently, this introspective view is missing from our model.We presented our model to subject experts. Based on five open-ended interviews, we offer three suggestions for future work. First, the experts mentioned that examining factors that decrease persuasion on social media platforms might be interesting. Exploring such aspects could be combined into our model, which so far solely focuses on the factors that increase persuasiveness. Second, future research should also examine the relative importance of the categories included in the three steps, as well as the relative importance of each step in the overall effectiveness of a persuasive message. Finally, future studies could investigate if the proposed model translates to contexts outside of social media. For example, in political speeches or offline advertisements.Social media advertisement often relies on influencers to promote services and products. This area has become a million-dollar industry using different persuasive strategies to make viewers follow their purchase advice. While research has focused on the area of beauty and fashion SMI, little is known about the techniques used to advertise products with associated health benefits. We proposed a three-step persuasion model for YouTube videos through an interpretive grounded theory methodology. While there are considerable amounts of persuasion theories that are in agreement with parts of the model, there is no single theory that combines all the factors we discovered into one model. Our model describes persuasion in this context as based on a multi-factorial process to make the viewer click on a video, watch it and act on the advice provided. Future empirical studies are needed to validate the theoretical components of our model.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by University of Twente. The patients/participants provided their written informed consent to participate in this study.JT conducted the data collection, analysis, and wrote the first draft of the manuscript. RV and ML reviewed and revised the initial draft. Initial codes and themes were discussed among the three authors and refined. All authors have approved the final version of the manuscript.This research was funded by the Pride and Prejudice Project by the 4TU federation under Grant No. 4TU-UIT-346.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.


Since 2000, adolescent alcohol use has declined substantially in many high-income countries, particularly in Northern Europe. This study examined whether birth cohorts in Norway who experienced different levels of alcohol consumption in mid-adolescence differed in drinking behaviour when they reached young adulthood. We analysed data from annual population surveys in Norway (2012–2021). The analytic sample comprised data from respondents aged 20–29 years (N = 5266), and we applied four birth cohorts . We applied age categories with two- and five-year intervals and tested whether drinking frequency, heavy episodic drinking (HED) and usual number of drinks per drinking occasion during the past 12 months differed by birth cohort in age-specific strata. Possible cohort differences within age groups were tested using Pearson’s Chi square. There were no statistically significant differences between cohorts with respect to drinking frequency or HED frequency. However, the youngest cohort had fewer drinks per occasion when in their early 20s compared to older cohorts. This study showed that birth cohorts who differed substantially in levels of alcohol consumption in mid-adolescence, only to a little extent differed in drinking behaviour in young adulthood. Since 2000, adolescent alcohol use has declined substantially in many high-income countries, particularly in Northern Europe . The decAdolescent alcohol consumption is correlated with consumption in young adulthood, and thus heavier consumption in adolescence is found to predict heavy consumption and alcohol problems in young adulthood ,7,8. StuIn this study, we will add to the sparse literature on this topic by analysing data from Norway. The minimum legal age for purchasing alcohol is 18 years for beer, wine and other alcoholic beverages below 20% and 20 years for stronger alcoholic beverages. From the European School Project on Alcohol and Drugs (ESPAD), surveys among 15–16-year-old students in Norway have shown that the prevalence of past year drinking declined in a fairly linear fashion from 78% in 1999 to 45% in 2015, and the corresponding figures for the prevalence of past month drinking were 55% and 23%, respectively [“Twenty years later: Explanations and consequences of the decline in adolescents’ drinking in the Nordic countries” which was established in 2020. We employed data from the annual general population surveys on alcohol and drug use in Norway from 2012 through 2021 (average response rate 58.4%). The survey samples comprise adults aged 16–79 years from the general population in Norway. Sampling procedures, measurements and data collection methods are identical over time, and Statistics Norway conducts the surveys on behalf of the Norwegian Institute of Public Health. The surveys were conducted according to the guidelines of the Declaration of Helsinki, the Personal Data Act and the Statistics Act. Each survey year, respondents in the age group 16–25 years were oversampled. For the present study, we applied only data for respondents aged 20 through 29 years. The number of respondents by birth cohort and survey year is displayed in This study is part of a Nordic research collaboration project entitled Age in years at survey date was recorded from the population registry, from which the survey samples were drawn. Birth cohort was calculated from age (continuous measure) and survey year, and we applied four birth cohorts . Thus, the oldest cohort, born in the period 1983 to 1987, corresponds roughly to the cohort participating at age 15–16 years in the ESPAD surveys in 1999 and 2003 when alcohol use was most prevalent. The youngest cohort, born in the period 1997 to 2001, corresponds roughly to the cohort participating in the 2015 ESPAD survey, when alcohol use was far less prevalent among 15–16-year olds [Respondents reported whether they had consumed any alcohol in the past 12 months, and if so, how frequently they had consumed alcohol. Among past year drinkers, responses to one of eight frequency options, from ‘once’ to ‘every day’ were used to construct a semicontinuous measure of past year drinking frequency. We categorized drinking frequency into three dichotomous measures of drinking behaviour: any drinking during the past 12 months, monthly drinking  and weekly drinking . Moreover, a frequency measure of heavy episodic drinking (HED)  was also categorized into three dichotomous measures: (i) during the past 12 months, (ii) monthly and (iii) weekly. Finally, usual number of drinks per occasion (past 12 months) was measured, with response options 1–2, 3–4, 5–6, 7–9 and 10 or more. Notably, the risk of overall health harm increases with increasing volume of consumption  and withWe included only respondents with valid responses on the measures of drinking frequency (missing observations = 2), HED frequency (missing observations = 89) and usual number of drinks per occasion (missing observations = 28). Hence, the analytic sample comprised a total of 5266 respondents.p < 0.05.In the initial step, we visualized drinking behaviour by age and cohorts in diagrams. For these analyses, we applied age categories with two-year intervals. We excluded observations from cell counts < 100. Next, we tested whether drinking behaviour differed by birth cohort in age-specific strata. For these analyses, we grouped age into two categories: 20–24 years and 25–29 years to obtain larger cell counts and reduce random variation. We tested possible cohort differences within age groups, using Pearson’s Chi square. Statistical significance level was set to Although young people were over-sampled in the series of Norwegian surveys, we employed weighted data in the main analyses to achieve the most comparable samples possible.By visual inspection of diagrams, there was little difference between birth cohorts in drinking behaviour by two-year age groups in the span 20–29 years. The only exception was weekly drinking at age 20–21 years, for which we found a significantly lower proportion in the youngest cohort .Correspondingly, we examined the proportion reporting past year monthly and weekly heavy episodic drinking (HED) by age and birth cohort. As can be seen from In a similar vein, we explored mean annual drinking frequency and usual number of drinks per occasion by age group and cohort . Usual nNext, we tested possible cohort differences in drinking behaviour within broader age groups . There wMoreover, a test of whether mean annual drinking frequency differed by cohort within the two age groups revealed no statistically significant difference . With reWhile a large number of studies have demonstrated a substantial decline in adolescent alcohol use in many high-income countries since 2000 ,24,25,26Our findings mirror those of the few previous studies on this topic ,15, demoIn other words, when following drinking trajectories of cohorts  over an age span, a very different pattern emerges compared to when we follow individual trajectories. At the population level, there seems to be a substantial convergence between cohorts in drinking behaviour with increasing age as judged from the extant literature. At the individual level, on the other hand, the trajectories—as described in previous studies—do not converge much, and the drinking level in mid-adolescence tends to predict drinking level in young adulthood ,7,8,9,10Our findings indicate that younger cohorts ‘catch up’ with older cohorts when reaching young adulthood. This may suggest that the factors which have driven the substantial decline in adolescent drinking are mainly of importance during adolescence. This assumption is in line with some findings from previous studies. Parental rules, practices and norms seem to be among these factors, as they contribute to explain—in statistical terms—the decline in drinking among adolescents ,20,27,29The public health implications of the decline in adolescent drinking have been discussed, both with respect to the immediate short-term implications as well as possible long-term effects . There iSeveral study limitations should be noted. First, we did not have comparable data series prior to 2012; hence we could not describe drinking behaviour of the older cohorts when they were in their early 20s. Correspondingly, it is not yet possible to track drinking behaviour of the youngest cohorts more than a few years into young adulthood. Thus, based on available data, we can only to a limited extent describe cohort trajectories over the age span from 20 to 30 years. The present analyses were also limited by statistical power to perform fine-grained analyses with respect to more narrow categories of birth cohorts and age groups. Finally, some general limitations with population surveys on alcohol consumption should be noted. Heavy drinkers are often under-represented in survey samples, and responses to questions about drinking behaviour are often inaccurate due to memory bias, problems in conceptualizing alcohol consumption and a tendency to downplay one’s own drinking  . Thus, oResearch on the implications of the declining trends in alcohol use among adolescents for drinking in young adulthood is sparse. This study showed that birth cohorts in Norway who differed substantially in levels of alcohol consumption in mid-adolescence, only to a little extent differed in drinking behaviour as young adults. This suggests that the decline in adolescent drinking since 2000 mainly implied a delay of drinking onset. However, compared to older cohorts, the youngest cohort reported drinking less frequently and lower amounts of alcohol per occasion when in their early 20s. Thus, in addition to the potential health gain related to the delayed onset of drinking in adolescence, the youngest cohort may possibly also experience a positive health impact of keeping their consumption at a slightly lower level into adulthood than older birth cohorts.


Neurodegenerative diseases such as Parkinson’s disease (PD) are known to be related to oxidative stress and neuroinflammation, and thus, modulating neuroinflammation offers a possible means of treating PD-associated pathologies. Morin  is a flavonol with anti-oxidative and anti-inflammatory effects found in wines, herbs, and fruits. The present study was undertaken to determine whether a morin-containing diet has protective effects in an MPTP-induced mouse model of PD. Mice were fed a control or morin diet for 34 days, and then MPTP  was administered daily for 5 days to induce a PD-like pathology. We found that dietary morin prevented MPTP-induced motor dysfunction and ameliorated dopaminergic neuronal damage in striatum (STR) and substantia nigra (SN) in our mouse model. Furthermore, MPTP-induced neuroinflammation was significantly reduced in mice fed morin. In vitro studies showed that morin effectively suppressed glial activations in primary microglia and astrocytes, and biochemical analysis and a docking simulation indicated that the anti-inflammatory effects of morin were mediated by blocking the extracellular signal-regulated kinase (ERK)-p65 pathway. These findings suggest that morin effectively inhibits glial activations and has potential use as a functional food ingredient with therapeutic potential for the treatment of PD and other neurodegenerative diseases associated with neuroinflammation. Parkinson’s disease (PD) is a common age-related neurodegenerative disorder characterized by progressive dopaminergic neuronal cell loss in the substantia nigra (SN), tremors, rigidity, and bradykinesia . In addiAvailable evidence also indicates that PD is related to the activations of microglia and astrocytes involved in immunity in the striatum (STR) and substantia nigra (SN) of PD patients , and it Moraceae family . Computational docking simulations were conducted between ERK2 protein and morin, LY3214996, or ATP using AutoDock Vina 1.2. in AMDock version 1.5.2 in ‘Simple Docking’ mode [PMID: 32938494]. The grid box was set to 70, 70, and 70 Å for x, y, and z, respectively, and centered on the following xyz-coordinates to explore the entire predicted binding site: Morin, 1.10, −9.10, and −11.10 Å; LY3214996, 0.70, −10.50, and 4.90 Å; ATP, 0.20, −6.20, and −13.70 Å. Results of the docking conformation and receptor–ligand interactions were visualized using Discovery Studio Visualizer v21.1.0.20298 .The reported three-dimensional (3D) protein structure of ERK2 was downloaded from AlphaFold Protein Structure Database  and t-test were used to determine the significance of differences between groups, and post hoc tests were performed to compare groups using Tukey’s multiple comparisons. The analysis was performed using Prism ver. 7.0. , and


Living organisms have never been solitary individuals and symbiotic relationships are challenging our very conception of the individual. Symbiosis, initially defined as a living together of different organisms  represenChlamydomonas reinhardtii and diverse ascomycete fungi that provide nitrogen to the algae  which challenges the long-held paradigm of dominance of cyanobacteria interactions with microalgae over NCDs and brings the first hints on how heterotrophic proteobacteria thrive in surface waters and oxygenated areas  genomes of Dinoflagellates Symbodinium, essential symbionts of corals. Their in silico analyses demonstrate that a scalable k-mer approach largely agrees with the phylogenetic signal inferred from the LSU rDNA sequence. The combination of genomic and experimental data sometimes allows us to hypothesize about the metabolic bases of coexistence, as in the case of coexistence between Roseovarius and the green alga Ostreococcus tauri, where the bacterial genomes encodes the metabolic pathway to produce the vitamins needed by the microalgae . This study reports a stable coexistence maintaining the microalgae and the bacterium over several years, unlike the dynamic associations reported between Dinoroseobacter shibae and the microalgae Prorocebtrum minimum (Emiliania huxleyi (The articles included in this Research Topic provide a view of how symbiotic interactions can help bypass environmental stresses such as in  minimum , or Sulf huxleyi .in silico and experimental approaches, and many novel insights into species interactions, their ecology and evolution are expected in the near future.The field of symbiosis in aquatic habitats is expanding quickly with both All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Ka value between pH 6.0 and 7.0, suggesting that chaperone effects can be differently affected by physiological pH variations.Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but to what extent the respective mechanisms are overlapping is not fully understood. The BRICHOS domain constitutes a disease-associated chaperone family, with activities against amyloid neurotoxicity, fibril formation, and amorphous protein aggregation. Here, we show that the activities of BRICHOS against amyloid-induced neurotoxicity and fibril formation, respectively, are oppositely dependent on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families. The conserved Asp in its ionized state promotes structural flexibility and has a p Activities of the BRICHOS domain against amyloid fibril formation and amyloid-induced neurotoxicity are affected by pH. DNAJB6b and HSPB1.7,8 Amyloidogenic polypeptides can be expressed as proproteins which are subjected to proteolytic cleavage to release the amyloid forming fragments.9–11 Some amyloid-generating proproteins contain the domain BRICHOS, initially found in dementia-associated integral membrane protein 2B , cancer-relevant Chondromodulin-1, and ILD-related prosurfactant protein C (proSP-C).12,13 The BRICHOS domain is suggested to promote the correct folding and prevent amyloid formation of the amyloid-prone region of its proprotein during biosynthesis,14–16 and is itself released by proteolysis.17–19Proteins and peptides can self-assemble into highly ordered fibrillar structures as well as into smaller oligomers with less well-defined structures but apparently stronger ability to cause toxicity to cells. These phenomena are linked to amyloid diseases, which encompass about forty severe human diseases including interstitial lung disease (ILD), Alzheimer's disease (AD) and type 2 diabetes (T2D).19–21 and has thus emerged as a model compound in studies of amyloid fibril formation.22–25 For instance, recombinant human (rh) BRICHOS domains recognise and bind to amyloid fibrils of amyloid β-peptide  and islet amyloid polypeptide , and reduce their cellular toxicity,25–27 bind the smallest emerging toxic Aβ oligomers,24 bind to amyloid fibrils of Huntingtin (linked to Huntington's disease) and α-synuclein (linked to Parkinson disease),28 and can be used to efficiently reduce Aβ neurotoxicity in mouse hippocampal slice preparations and in vivo in animal models of AD.27,29–33 This broad anti-amyloid spectrum makes BRICHOS a potential candidate for amyloid disease prevention and even treatment.Recently, the BRICHOS domain has been shown to have chaperone activities against fibril formation and neurotoxicity of “alien” client peptides associated with human diseases but not part of BRICHOS containing proproteins,14,34,35 but the underlying pathogenic mechanisms are largely unknown. The rh BRICHOS domain from proSP-C is an efficient inhibitor of amyloid toxicity of Aβ42 in mouse hippocampal slice preparations and a Drosophila fruit fly model, reducing the generation of toxic Aβ42 oligomers, but it is not very competent to reduce the overall amyloid fibril formation rate.20,23,31,32 Rh Bri2 BRICHOS, on the other hand, assembles into differently sized species, of which monomers potently prevent Aβ42 neuronal toxicity, dimers strongly suppress Aβ fibril formation and large oligomers inhibit non-fibrillar protein aggregation.20,30,32,36 A Bri2 BRICHOS mutant (R221E), designed to stabilize the monomeric conformation prevents Aβ42 neurotoxicity rather than its overall fibrillization rate.30 A corresponding mutation in proSP-C BRICHOS (T187R) generates monomers that bind to the smallest emerging Aβ42 oligomers and is more efficient in vitro than the wildtype against amyloid fibril formation.24 Interestingly, another mutation at the same location in human proSP-C BRICHOS (T187N) leads to ILD with amyloid deposits.37 Thus, understanding the molecular mechanisms that regulate BRICHOS anti-amyloid chaperone activities are of interest from a basic science point of view, and for the development of treatments against amyloid diseases.Mutations in the BRICHOS domain or in the proproteins are associated with different protein misfolding and amyloid diseases,12,30 The physiological function of the Asp is unknown, yet two mutations of D105 in human proSP-C BRICHOS are linked to ILD.14,18,38 Here, we focused on this evolutionarily conserved Asp residue and investigated Asp to Asn mutations of two BRICHOS domains.In the BRICHOS superfamily, the secondary structure elements are highly conserved but the amino acid sequence conservation is rather low, and only one aspartic acid residue  and two cysteine residues that form a disulphide bridge were previously found to be strictly conserved, from analyses of a rather small number of sequences.i.e., ILD and familial dementias, respectively, possess different chaperone activity spectra, and have only ∼17% amino acid sequence identities.13,32,36The observation that mutations of the conserved Asp in the BRICHOS domain can result in amyloid disease prompted us to mutate it to Asn. The BRICHOS domains from proSP-C (D105) and Bri2 (D148) were selected , since tKa of the transition of about 6.1  migrated as a dimer on size-exclusion chromatography (SEC). In contrast, cleavage of differently sized NT*-Bri2 BRICHOS D148N species resulted in rh Bri2 BRICHOS D148N species with similar chromatographic profiles as the corresponding wildtype states  rh Bri2 BRICHOS D148N showed a distribution between dimers and monomers, approximately at a ratio of 1 : 1  and allowed to stabilize for 30 min prior to any recordings. As previously observed,23,32,45 100 nmol L−1 wildtype proSP-C BRICHOS prevented Aβ42-induced degradation of γ oscillations, which were remained at control levels . By contrast, surprisingly, rh proSP-C BRICHOS D105N showed a complete loss of the preventative efficacy at the same concentration . Apparently, mutating the conserved Asp to Asn in proSP-C BRICHOS dramatically impaired its capacity to prevent Aβ42-induced neurotoxicity.We recorded  BRICHOS . γ oscil47 fluorescence to monitor the kinetics of Aβ42 fibril formation in the absence and presence of different concentrations of rh proSP-C BRICHOS  BRICHOS  Fig. S6entration , while t decline . Interesand rmax , which iand rmax . These rγ oscillations30,36 and we therefore tested rh Bri2 BRICHOS D148N monomers. Rh Bri2 BRICHOS D148N monomers (50 nmol L−1) showed reduced potency to prevent Aβ42-induced neurotoxicity compared to wildtype monomers (50 nmol L−1), but did not completely lose its preventative efficacy . In the ThT assay, rh Bri2 BRICHOS D148N monomers were significantly more efficient in inhibiting Aβ42 fibril formation compared to wildtype monomers  and/or primary nucleation (kn) in addition to secondary nucleation (k2). However, the effects on secondary nucleation (k2) of rh wildtype proSP-C and the D105N mutant did not show striking differences (ESI49 and surface plasmon resonance (SPR). With seeding, the fibrillization traces typically follow a concave aggregation behavior  delays Aβ42 fibril formation without changing the total number of oligomers generated, while suppressing the secondary nucleation (k2) efficiently prevents the generation of oligomers, and blocking elongation (k+) significantly increases the number of oligomers formed.23 Of relevance for the results on Aβ42 neurotoxicity of wildtype vs. D105N proSP-C BRICHOS , which shows that the conserved Asp148 is buried and not involved in intersubunit interactions . These observations provide a likely explanation for the different effects the rh Bri2 BRICHOS D148N mutation on anti-amyloid activity versus ability to suppress amorphous protein aggregation.All the so far studied BRICHOS domains are able to suppress amyloid fibril formation, but the activity against amorphous protein aggregation is only found for oligomers of rh Bri2 and Bri3 BRICHOS.ractions . This isractions  Fig. S7c53–55 while the functions of BRICHOS domains in group II are unknown.The unexpected diverse effects of mutating the conserved Asp to Asn in proSP-C and Bri2 BRICHOS inspired us to analyse all so far deposited BRICHOS containing proproteins. Amino acid sequences of 3355 BRICHOS domain precursors were extracted from the SMART database, covering 537 different metazoan species spanning worms to humans . After sInterestingly, 1% of all analysed BRICHOS containing proproteins contain multiple BRICHOS domains along with their corresponding amyloid-prone regions, and these were found in GKN1, proSP-C, CNMD and group II families  Fig. S10Ka between 6 and 7 in both BRICHOS domains studied.In this work, we find that the capacity of the BRICHOS domain to inhibit amyloid-associated neurotoxicity and fibril formation, respectively, is oppositely affected by mutations of a phylogenetically conserved Asp, whereas the capacity to suppress non-fibrillar, amorphous protein aggregation is not affected by Asp to Asn replacement. Moreover, conformational changes occur as a result of Asp to Asn mutations in both proSP-C and Bri2 BRICHOS, and those changes can partly be mimicked by lowered pH and the conserved Asp titrates with an apparent p14,18,38 Based on molecular dynamic simulations, monomeric wildtype proSP-C BRICHOS and a D105N mutant behaved differently: only minor conformational changes were seen in the mutant, but several large-scale changes occurred in the wildtype protein at moderately elevated temperatures, which resulted in a more loosely folded structure.14 D105N mutation in rh proSP-C BRICHOS results in a more ordered conformation, as judged by CD spectroscopy, and apparently more efficient trimer formation, while D148N mutation of rh Bri2 BRICHOS results in transition of monomers to more compact dimers  are linked to ILD.t dimers . Similart dimers . This sut dimers . The diswith Asn .56 It has been shown that the small heat shock protein αB-crystallin uses different interfaces to bind to amyloid and amorphous substrates, respectively, but it is not known how different conformational states of αB-crystallin are regulated to affect activities against different types of substrates.57 The chaperoning capacities of BRICHOS domain against amyloid neurotoxicity and fibril formation can apparently be modulated by a conserved Asp in response to pH changes, suggesting the possibility that the microenvironment may affect BRICHOS function. For example, pH in the secretory pathway span from 7.2 to 5.7.58 The results here are based on in vitro and ex vivo experiments, and further work that addresses the different activities of BRICHOS are motivated.Molecular chaperones or chaperone-like domains have not been studied extensively as regards sensitivity to pH. One exception is clusterin, the activity of which is enhanced at mildly acidic pH, which appears to result from an increase in regions with solvent-exposed hydrophobicity, but independent of any major changes in secondary or tertiary structure.59 3355 BRICHOS sequences were downloaded while the sole bacterial BRICHOS precursor (Paeniclostridium sordellii ATCC 9714) was not included. Incomplete sequences were filtered out, resulting in total of 3190 sequences. Identical sequences were filtered by means of CD-HIT,60 with a threshold of 100% of sequence identity, which gave 3093 amino acid sequences. The hidden markov model profile (HMM) of BRICHOS (PF04089) was extracted from PFAM database.61 The CD-HIT filtered BRICHOS protein sequences were then scanned against the HMM profile using HMMER software v3.3.262 with an E-value cut-off less than 1.0 × 10−5. An in-house python script was written to filter out the significant BRICHOS proteins from the HMM result file and to extract the BRICHOS domain sequences (each BRICHOS amino acid sequence was extended six residues upstream from the BRICHOS domain starting position defined by PFAM). Further, BRICHOS domain sequences less than 68 aa were removed as one rodent BRICHOS domain is just 69 residues and still functional against amyloid fibril formation,63 which eventually generated 2019 BRICHOS sequences. The multiple sequence alignment of the 2019 BRICHOS domain sequences were created using MAFFT alignment server with the default settings,64 and the sequence logo was generated using Weblogo webserver.65 RAxML HPC (v8.2.10)66 was employed for constructing the phylogenetic tree using the PROTGAMMAAUTO model with 100 times bootstrap iterations. The tree shown in this study was visualized using the Interactive Tree of Life (iTOL) server67 and Geneious software. The taxonomy tree common for species that contain BRICHOS precursors were generated by NCBI Taxonomy (https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi) and visualized by Geneious software.From the SMART database,36,39) plasmid as PCR template Bri2 BRICHOS D148N was obtained with QuikChange II XL Site-Directed Mutagenesis Kit , and the DNA sequence was confirmed . Similarly, the amino acid Thr at position 206 was mutated to Trp using KAPA HiFi HotStart ReadyMix PCR Kit  together with the designed complementary primers (5′-CCTATCTGATTCATGAGCACATGGTTATTTGGGATCGCATTGAAAAC-3′ and 5′-GTTTTCAATGCGATCCCAAATAACCATGTGCTCATGAATCAGATAGG-3′) and verified by sequencing (Eurofins Genomics). As described,30,36 the Bri2 BRICHOS variants were expressed in SHuffle T7 E. coli cells. Briefly, the cells were incubated at 30 °C in LB medium with 15 μg mL−1 kanamycin until an OD600nm around 0.9. For overnight expression, the temperature was lowered to 20 °C, and 0.5 mmol L−1  isopropyl β-d-1-thiogalactopyranoside (IPTG) was added. The induced cells were harvested by centrifugation  and the cell pellets were resuspended in 20 mmol L−1 Tris pH 8.0. After 5 min sonication  on ice, the lysate was centrifuged  for 30 min and the protein of interest was isolated with a Ni-NTA column. To remove the His6-NT* part, the target proteins were cleaved with thrombin  at 4 °C overnight and loaded over a second Ni-NTA column. Different species of rh Bri2 BRICHOS variants were isolated and analysed by size-exclusion chromatography (SEC) with Superdex 200 PG, 200 GL or 75 PG columns  using an ÄKTA system  with buffer of 20 mmol L−1 NaPi (Sodium Phosphate) with 0.2 mmol L−1 EDTA at different pHs. For generating rh proSP-C BRICHOS D105N, the PCR primers 5′-CACTGGCCTCGTGGTGTATAACTACCAGCAGCTGCTGATCGC-3′ and 5′- GCGATCAGCAGCTGCTGGTAGTTATACACCACGAGGCCAGTG-3′ were synthesized. With the wildtype proSP-C BRICHOS (corresponding to the solubility tag thioredoxin followed by proSP-C residues 86–19714) plasmid as PCR template, proSP-C BRICHOS D105N was obtained with QuikChange II XL Site-Directed Mutagenesis Kit , and the DNA sequence was confirmed . The expression and purification were performed as described.14,68 Briefly, both wildtype proSP-C BRICHOS and the D105N mutant were expressed in Origami 2 (DE3) pLysS E. coli cells. The cells were grown at 37 °C in LB medium containing 100 μg mL−1 ampicillin until an OD600nm around 0.9. The temperature was lowered to 25 °C and 0.5 mmol L−1  IPTG was added for overnight expression. The cells were harvested by centrifugation at 7000 × g for 20 min, and the cell pellets were resuspended in 20 mmol L−1 Tris pH 8.0. The protein was purified using Ni-NTA column and ion exchange chromatography . Thrombin  was used to remove the thioredoxin tag. The purified rh proSP-C BRICHOS variants were analysed by Superdex 200 GL columns  using an ÄKTA system . The BRICHOS mutants in this study were expressed and purified in parallel with their wildtype counterparts.For generating rh Bri2 BRICHOS D148N, the amplification primers 5′- ATAGTGATCCTGCCAACATTGATAACTTTAACAAGAAACTTACA-3′ and 5′- TGTAAGTTTCTTGTTAAAGTTATGAACAATGTTGGCAGGATCACTAT-3′ were synthesized. With the wildtype NT*-Bri2 BRICHOS . At 100% D2O, 1 mL LB was used to inoculate 100 mL M9 in 100% D2O enriched with 15N H4Cl and 13C glucose, and was grown over night at 31 °C. After overnight incubation, the 100 mL was added to 900 mL M9 in D2O enriched with 15N and 13C and grown at 30 °C until OD600nm was around 0.8. The temperature was lowered to 20 °C and 0.5 mmol L−1  IPTG was added for overnight expression. The purification was performed as described above.For the NMR experiments, gene fragment encoding human proSP-C BRICHOS was transformed into SHuffle T7 1H–15N TROSY-HSQC experiments were obtained at 37 °C on Bruker 800 MHz spectrometer equipped with a TCI cryogenic probe. Spectra were processed with the software NMRPipe and visualized using Sparky NMR. The concentrations of 2H, 15N,13C-labeled proSP-C was 288 μmol L−1 in 20 mmol L−1 ammonium acetate pH 7.2 and 229 μmol L−1 in 20 mmol L−1 ammonium acetate pH 5.5, both in 90% H2O/10% D2O.2D −1. The bandwidth was set to 1 nm, data pitch 0.5 nm, and scanning speed 50 nm min−1. The spectra shown are averages of five consecutive scans.Circular dichroism (CD) spectra were recorded in 1 mm path length quartz cuvettes at 25 °C from 260 to 185 nm in a J-1500 Circular Dichroism Spectrophotometer  with a protein concentration of 4.2–10 μmol L−1 HEPES/KOH pH 7.5 to 600 nmol L−1  and then equilibrated at 45 °C with and without different concentrations of rh Bri2 BRICHOS D148N oligomer or proSP-C BRICHOS variants. The aggregation kinetics were measured by reading the apparent increase in absorbance at 360 nm under quiescent conditions using a microplate reader .Citrate synthase from porcine heart  was diluted in 40 mmol L−1, calculated for the monomeric subunit) in 20 mmol L−1 NaPi pH 8.0 or pH 6.0 were incubated at 25 °C with 2 μmol L−1 bis-ANS --5,5′-disulfonic acid dipotassium salt) for 10 min, and the fluorescence emission spectra were recorded from 420 to 600 nm after excitation at 395 nm with the Infinite M1000 plate reader . Rh Bri2 BRICHOS T206W monomers were diluted to 2 μmol L−1 by using 20 mmol L−1 NaPi containing 0.2 mmol L−1 EDTA with different pH in the final samples in the range of 6.3–8.0. For tryptophan fluorescence measurements, samples were prepared in duplicates with a volume of 150 μL. Samples were excited at 280 nm (5 μm bandwidth) and fluorescence emission from 300–400 nm  was recorded on black polystyrene flat-bottom 96-well plates (Costar) using a spectrofluorometer (Tecan Saphire 2). For the final fluorescence intensities, the results were corrected by subtracting the background fluorescence of the buffer.Different rh BRICHOS . For each image, the contrast transfer function (CTF) parameters were estimated on boxed out regions  using e2ctf.auto.py program. A reference-free 2D classification based on the selected 10 223 phase-flipped particles (low-pass filtered to 20 Å) was performed using e2refine2d.py. The 2D classes show an approximate 2-fold symmetry, which is consistent with both the results of rh wildtype Bri2 BRICHOS oligomer and the biochemical data. Generated 2D classes were used as the input for building the 3D initial model using e2initialmodel.py. 3D refinement was performed in several rounds using e2refine_easy.py applying D2 symmetry aiming at a final resolution of 15 Å. The first two rounds of 3D refinements were performed with pixel size of 3.52 Å after binning the data by a linear factor 2. In the last round of refinement, the data was resampled to 2.464 Å per pixel. The final map from the first round of refinements was used as model in the second, and the final map from the second round of refinements was used as model in the third. The resolution was determined based on a Fourier shell correlation (FSC) value of 0.143,71 following the gold standard FSC procedure implemented in EMAN2.72Rh Bri2 BRICHOS D148N oligomers after SEC isolation were immediately stored on ice followed by grid preparation. Aliquots (4 μL) were adsorbed onto glow-discharged continuous carbon-coated copper grids  for one min. The grids were subsequently blotted with filter paper, washed with two drops of Milli-Q water, and negatively stained with one drop of 2% (w/v) uranyl acetate for 45 s before final blotting and air-drying. The sample was imaged using a Jeol JEM2100F field emission gun transmission electron microscope  operating at 200 kV. Single micrographs for evaluating the quality of the sample were recorded on a Tietz 4k × 4k CCD camera, TVIPS  at magnification of ×85 200 (1.76 Å per pixel) and 1.0–2.8 μm defocus. A total of 16 micrographs were recorded for single particle analysis. All 16 micrographs were imported to EMAN2 (version 2.3) for further processing.−1 with 20 mmol L−1 sodium phosphate pH 8.0 containing 0.2 mmol L−1 EDTA as running buffer, and the other details were set according to the manufacturer's instructions. After immobilization with a final reponse level of 727 RU, the flow-cells were stabilized over night in running buffer (10 mmol L−1 HEPES pH 7.5 containing 150 mmol L−1 NaCl and 0.2 mmol L−1 EDTA) at a flow rate of 20 μL min−1. For binding analysis, 25 μmol L−1 rh wildtype proSP-C BRICHOS or the D105N mutant in 10 mmol L−1 HEPES pH 7.5 containing 150 mmol L−1 NaCl and 0.2 mmol L−1 EDTA were injected in over the chip surfaces at 25 °C at a flow rate of 20 μL min−1 for 3 min, respectively. 10 mmol L−1 NaOH was used for further chip surface regeneration. For kinetic analysis, different concentrations of rh proSP-C BRICHOS D105N mutant in running buffer, i.e. 0, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μmol L−1, were individually injected in over the chip surfaces at 25 °C at a flow rate of 20 μL min−1. 30 mmol L−1 NaOH was used for further chip surface regeneration. In all experiments, the response from the blank surface was subtracted from the immobilized surface response, the baselines of the sensorgrams were adjusted to zero and buffer spikes were excluded.Aβ42 monomers were immobilized by amine coupling onto flowcell 4 on a CM5 sensor chip  using a BIAcore 3000 instrument (BIAcore AB). A reference surface was prepared on flowcell 3 using the same coupling protocol but without protein injected. The immobilization was performed at a flow rate of 20 μL minversus BRICHOS concentrations. The baseline of the sensorgrams were adjusted to zero and buffer spikes were excluded for global fits to reflect the binding affinity. Since the response signals of the two lowest protein concentrations  used in kinetic analysis were too weak, only sensorgrams obtained from rh proSP-C BRICHOS D105N mutant ranging from 6.25 μmol L−1 to 100 μmol L−1 were included in the global fits. The dissociation was globally fitted to a biexponential model as described by 24,73R1 is the response signal at the starting time for the dissociation phase t1, and x is between 0 and 1. kd1 and kd2 are the dissociation rate constants for the fast and slow dissociation phases, respectively. The global fitted value kd2 was used to calculate the apparent KD value.Steady state apparent affinities for rh proSP-C BRICHOS D105N mutant to immobilised Aβ42 monomers were estimated by plotting the maximum binding response 24R0 and Rf are the initial and final response signal of the association phase, respectively. kobs is the observed rate constant described by 24,73c is the protein concentration, ka is the association rate constant, and kd from this analysis is related to secondary binding artifacts corresponding to kd1. Linear regression was used to determine ka. The apparent KD value was calculated as ratio of the dissociation rate constant and association rate constant.The association phase was fitted to E. coli (B strain) cells and purified by ion exchange.36 The purified Aβ42 peptides were lyophilized and re-dissolved in 20 mmol L−1 Tris pH 8.0 with 7 mol L−1 Gdn-HCl, and the monomers were isolated in 20 mmol L−1 sodium phosphate pH 8.0 with 0.2 mmol L−1 EDTA by a Superdex 30 column 26/600 . The concentration of monomeric Aβ42 was calculated with using an extinction coefficient of 1424 M−1 cm−1 for (A280–A300). For Aβ42 fibrillization kinetics analysis, 20 μL solution containing 10 μmol L−1 ThT, 3 μmol L−1 Aβ42 monomer and different concentrations of rh BRICHOS at molar ratios 0, 10, 30, 50, 70 or 100% (relative to Aβ42 monomer molar concentration), were added to each well of half-area 384-well microplates with clear bottom , and incubated at 37 °C under quiescent conditions. The ThT fluorescence was recorded using a 440 nm excitation filter and a 480 nm emission filter using a microplate reader . For Aβ42 seeds preparation, 3 μmol L−1 Aβ42 monomers were incubated for about 20 h at 37 °C, and the fibrils were then sonicated in a water bath for 3 min. For seeding of Aβ42 fibrillization, 20 μL solution containing 10 μmol L−1 ThT, 3 μmol L−1 Aβ42, different concentrations of rh BRICHOS at 0, 10, 50, 70 and 100%, and 0.6 μmol L−1 seeds  were added at 4 °C to each well in triplicate of 384-well microplates with clear bottom  and immediately incubated at 37 °C under quiescent conditions. The elongation rate constant k+ in the presence of rh BRICHOS was calculated from the highly seeded experiments. The initial slope of the concave aggregation traces was determined by a linear fit of the first ∼20 min traces. For all the experiments, aggregation traces were normalized and averaged using four or three replicates, and data defining one dataset was recorded from the same plate at the same time, and all the ThT data was from the same batch of Aβ42 peptide.Recombinant Met-Aβ1–42 here referred to as Aβ42, was produced in BL21*(DE3) pLysS 30,36 where the half time τ1/2 and the maximal growth rate rmax were extracted:A the amplitude and F0 the base value.Fibrillization traces of Aβ42 with and without different concentrations of rh BRICHOS were fitted to a sigmoidal equation 23M(t) is the total fibril mass concentration, and the intermediate coefficients are functions of λ and κ, and nC and n2 are the reaction orders for primary and secondary nucleation, respectively:To dissect the molecular mechanism underlying BRICHOS counteracting Aβ42 aggregation, the fibrillization traces were globally fitted by kn, k+, and k2 are for primary nucleation, elongation, and secondary nucleation, respectively. The kinetic data were globally fitted to kn, k+ or k2) is the sole fitting parameter.30,36 To investigate the generation of nucleation units, according to the nucleation rate rn(t):23rn(t) over the reaction.The microscopic rate constants −1 Aβ42 monomer was incubated at 37 °C with 100% rh wildtype proSP-C BRICHOS and the D105N mutant overnight, and the fibrils were collected at 4 °C by centrifugation for 1 h at 22 000 × g. The fibrils were gently resuspended in 20 μL 1 × TBS, of which 2 μL were applied to carbon coated copper grids, and incubated for about 5 min. Excess solution was removed and the girds were blocked by incubation in 1% BSA in 1 × TBS for 30 min, followed by 3 × 10 min washing by 1 × TBS. The grids were then incubated with polyclonal antibody against human proSP-C (SFTPC)  overnight at 4 °C, and washed 3 × 10 min with 1 × TBS. Finally, the grids were incubated with anti-rabbit IgG-gold coupled to 20 nm gold particles  for 2 h at room temperature, and washed 5 × 10 min with 1 × TBS. Excess solution was removed, and 2 μL of 2.5% uranyl acetate was added to each grid (kept about 20 s). Excess solution was removed, and the grids were air-dried at room temperature, and analysed by transmission electron microscopy .Five μmol LAll the animal experiments were carried out in accordance with the ethical permit granted by Norra Stockholm's Djurförsöksetiska Nämnd (dnr N45/13). C57BL/6 mice of either sex  were used in the experiments. Before sacrificed, all the mice were anaesthetized deeply using isoflurane.−1 NaCl, 24 mmol L−1 NaHCO3, 25 mmol L−1 glucose, 1.25 mmol L−1 NaH2PO4, 1 mmol L−1 ascorbic acid, 3 mmol L−1 NaPyruvate, 2.5 mmol L−1 KCl, 4 mmol L−1 MgCl2, 0.5 mmol L−1 CaCl2 and 75 mmol L−1 sucrose. Horizontal sections (350 μm thick) of the ventral hippocampi from both hemispheres were sliced with a Leica VT1200S vibratome . The sections were immediately transferred to a submerged incubation chamber containing standard ACSF: 124 mmol L−1 NaCl, 30 mmol L−1 NaHCO3, 10 mmol L−1 glucose, 1.25 mmol L−1 NaH2PO4, 3.5 mmol L−1 KCl, 1.5 mmol L−1 MgCl2 and 1.5 mmol L−1 CaCl2. The chamber was held at 34 °C for at least 20 min after dissection and it was subsequently cooled to room temperature (∼22 °C) for a minimum of 40 min. Proteins (Aβ42 and rh BRICHOS) were first added to the incubation solution for 15 min, and then the slices were transferred to the interface-style recording chamber for extracellular recordings. During the incubation, slices were supplied continuously with carbogen gas  bubbled into the ACSF.The brain was dissected out and placed in modified ice-cold ACSF . The ACSF contained 80 mmol Lγ oscillations) were recorded at 32 °C in an interface-type chamber (perfusion rate 4.5 mL per minute) and elicited by applying kainic acid . The oscillations were stabilized for 20 min before any recordings. No Aβ42, rh Bri2 BRICHOS R221E species or combinations thereof were present in the recording chamber either during γ oscillations stabilization, or during electrophysiological recordings. The interface chamber recording solution contained 124 mmol L−1 NaCl, 30 mmol L−1 NaHCO3, 10 mmol L−1 glucose, 1.25 mmol L−1 NaH2PO4, 3.5 mmol L−1 KCl, 1.5 mmol L−1 MgCl2 and 1.5 mmol L−1 CaCl2.Recordings were performed with borosilicate glass microelectrodes in hippocampal area CA3, pulled to a resistance of 3–5 MΩ, filled with ACSF and placed in stratum pyramidale. Local field potentials . The signals were sampled at 10 kHz, conditioned using a Hum Bug 50 Hz noise eliminator , software low-pass filtered at 1 kHz, digitized and stored using a Digidata 1322A and Clampex 9.6 software .Power spectral density plots (from 60 s long LFP recordings) were calculated using Axograph X  in averaged Fourier-segments of 8 192 points. Oscillation power was calculated from the integration of the power spectral density from 20 to 80 Hz.Q = 1%) followed by D’Agostino & Pearson omnibus normality test. Based on the previous experience with the outliers and overall sample behaviour, sample size was determined based on previous studies performed in an interface-type chamber.23,30,36,46,74,75 Each experimental round was performed with parallel controls (Control KA and Aβ42) from the same animal and randomized preparations . For comparison purposes data from Control KA and Aβ42 was pooled from interleaved slices recorded in these conditions. The number of recorded slices per condition (at least from 3–5 mice) are shown in the corresponding figure legend and source data file. Kruskal–Wallis test followed by Dunn's multiple comparisons were carried out due to the non-parametric nature- or relatively small size of some data. Comparison of the preventative efficacies of rh Bri2-BRICHOS D148N monomers and rh proSP-C BRICHOS D105N was assessed with the Mann Whitney test. Significance levels are * p < 0.05, ** p < 0.01, and ***p < 0.001. The ThT assay data are presented as means ± standard deviation, and the aggregation traces are averaged from four or three replicates.The electrophysiological data is presented as means ± standard errors of the means. Prior statistical analysis all the data was subjected to outlier determination and removal with the ROUT method (The density map of the Bri2 BRICHOS D148N oligomer have been deposited in the Electron Microscopy Data Bank (EMDB) under the accession code EMD-13005. All data and materials related to this paper are available from G. C. (gefei.chen@ki.se).G. C., Y. A. T., X. Z., H. P., H. B., A. L., and N. K. performed experiments. S. H. and G. C. carried out bioinformatic analyses and visualization. G. C., Y. A. T., X. Z., H. B., H. P., A. A., N. K., A. R., H. H., P. K., A. F., and J. J. analysed the data. G. C. and J. J. conceived the study. G. C. and J. J. wrote the paper. All authors discussed the results and commented on the manuscript.The authors declare no competing financial interests.CB-003-D2CB00187J-s001CB-003-D2CB00187J-s002