There is good evidence that when people’s needs and expectations regarding behaviour change are met, they are satisfied with that change, and maintain those changes. Despite this, there is a dearth of research on needs and expectations of walkers when initially attending walking groups and whether and how these needs and expectations have been satisfied after a period of attendance. Equally, there is an absence of research on how people who lead these groups understand walkers’ needs and walk leaders’ actions to address them. The present study was aimed at addressing both of these gaps in the research.Two preliminary thematic analyses were conducted on face-to-face interviews with (a) eight walkers when they joined walking groups, five of whom were interviewed three months later, and (b) eight walk leaders. A multi-perspective analysis building upon these preliminary analyses identified similarities and differences within the themes that emerged from the interviews with walkers and walk leaders.Walkers indicated that their main needs and expectations when joining walking groups were achieving long-term social and health benefits. At the follow up interviews, walkers indicated that satisfaction with meeting similar others within the groups was the main reason for continued attendance. Their main source of dissatisfaction was not feeling integrated in the existing walking groups. Walk leaders often acknowledged the same reasons for walkers joining and maintaining attendance at walking. However, they tended to attribute dissatisfaction and drop out to uncontrollable environmental factors and/or walkers’ personalities. Walk leaders reported a lack of efficacy to effectively address walkers’ needs.Interventions to increase retention of walkers should train walk leaders with the skills to help them modify the underlying psychological factors affecting walkers’ maintenance at walking groups. This should result in greater retention of walkers in walking groups, thereby allowing walkers to receive the long-term social and health benefits of participation in these groups. Major health benefits are gained when people initiate and maintain physical activity on a regular basis ,2. HowevWalking in groups has been found to increase moderate physical activity among adults, for at least six months after initiation . WalkingA central idea in many theories is that a person’s decision to initiate a health behaviour change process is based on their assessment that the benefits afforded by the new behaviour compare favourably to their current situation ,8. FurthSeveral previous studies have elicited the views of walkers attending existing community groups –13. ThesEfficacy studies evaluating walking group interventions from different countries report similar findings. For example, studies conducted in Australia  and the Despite this evidence from previous research, it is all based on cross-sectional snapshots of reported needs, which limits our ability to understand whether and how the factors presented contribute to attendance at walking groups for longer periods of time. To our knowledge, no research has aimed to identify the factors that motivate walkers to join walking groups, whether these factors are the same after a period of time and whether and how these factors support maintained attendance. To address this gap in evidence there is a need for studies of longitudinal designs.The majority of walk leaders are lay people who lead walks voluntarily and have no professional role related to physical activity promotion . Walk leaders receive basic training, which focuses on ensuring that walks are safe, well run and friendly for walkers . They also deal with any issues that arise while walking in groups. Thus, it can be argued that part of their role is to implement practises to meet walkers’ needs and expectations.There is meta-analytical evidence that walking interventions implementing practises that were tailored to participants’ needs were more efficacious at promoting walking . Given tA survey that explored walk leaders’ experiences with their roles showed that walk leaders reported challenges concerning their abilities to provide support to walkers, organisation duties, and role responsibilities . AlthougThe aims of the present study were: (a) to elicit the needs and expectations from people when joining walking groups; (b) to identify the extent to which these needs and expectations were met three months later, and if not, walkers’ views on why not; (c) to identify how changes in initial needs and expectations might have happened; and (d) to elicit reports of actions taken by walk leaders to address walkers’ needs.This research was approved by Coventry University Ethics Committee before any participant was contacted. All participants were sent an information sheet describing: the aims of the research, participants’ right not to participate in this research or withdraw their data after participation, the interview process (e.g. the use of audio recording), possible benefits, risks and disadvantages of taking part in the research, how data will be used, confidentiality, and an introduction to the research team. All participants gave written consent confirming that they had read the information sheet and agreed to take part in this research.Semi-structured interviews were used to gather participants’ views . A longiAll participants above 18 years old were eligible to participate, apart from people with learning disabilities, other medical problems that may have impaired their cognitive abilities, and those with limited facility with the English language.Participants were recruited and interviewed between July 2009 and March 2010. Walk leaders’ contact details were provided to the research team by a walking scheme run by an urban local authority (Coventry City Council) in the Midlands of England, which was part of the Walking for Health (WfH) scheme. Further, walk leaders who agreed to participate in this research provided the research team with the contact details of walkers who had just joined their walking group. Both walk-leaders and walkers were sent an information sheet by post and asked to participate in a face-to-face interview.Nine walkers, when they first joined eight different existing walking groups were approached and asked whether they agreed to be interviewed. One person declined, providing work commitments as a reason. Eight walkers agreed to participate in this interview with a mean age of 60.5 years (sd = 3.9). All eight participating walkers were contacted again after a period of three months and asked whether they agree to be interviewed again. Five walkers agreed to participate in the second interviews. All five walkers reported that they had dropped out from the walking groups temporarily. Two of them reported Christmas holidays as a reason and three of them family commitments as reasons for dropping out. The other three walkers declined to participate in the second interview and indicated that they had permanently ceased attending walking groups: one provided the death of a spouse as a reason, the other hospitalisation and the third a lack of time. For information about participating walkers see Fourteen walk leaders from fourteen different walking groups were invited to participate. Three of them declined participation: One had dropped out from the walking scheme, one was excluded because he satisfied an exclusion criterion , two participants were not interested in participating and two had not yet established walks when approached. Eight of them agreed to be interviewed (five women and three men) with a mean age of 61 years (sd = 14.4). Most of the walk leaders had long-term experience in leading walking groups and only one had recently been trained. Information about participating walk leaders is provided in In line with the recommendations of Chamberlain and colleagues , recruitThe first author conducted and transcribed all interviews, and also conducted the analysis with input from the last author. The authors met to discuss coding and any discrepancies during the process of analysis. The first author had experience with qualitative research, the second author with qualitative methods and group based research and the last author with qualitative methods and physical activity research.Participants chose the place and time of the interviews. During the interviews only the interviewer and the interviewee were present. Each interview lasted approximately 45 to 90 minutes. All the interviews were audio taped using a digital audio recorder. Following transcription, all information that could lead to participant identification was removed or modified (e.g. using pseudonyms) to retain anonymity.An interview guide was developed by the first author based on relevant literature relating to both theory and research based evidence. The second and third authors commented on the interview guide. At the first set of interviews, walkers were asked to talk about their needs and expectations for joining walking groups. Three months later they were asked about whether their initial needs had been met or not and to elaborate on their reasons for this. Walk leaders were asked to talk about what they believed walkers’ needs were when they join walking groups, and walk leaders’ actions to address these needs.The period when interviews with walkers took place overlapped with the period when interviews with walk leaders took place. However, the set of interviews with walk leaders finished after the end of the follow up interviews with walkers. This was done so that issues that arose from walkers’ interviews could be followed up at some interviews with walk leaders. The researcher also aimed to follow up themes that arose from walkers’ interviews with walk leaders from all walking groups, i.e. not only with the leader from the group that a specific walker attended.Two preliminary thematic analyses were conducted, one with the cross sectional interviews with walk leaders and one with the longitudinal interviews with walkers. Thematic analysis is a method of systematically detecting, categorizing, interpreting and reporting patterns or themes within raw data . For theThe themes identified from separate analyses with walkers and walk leaders were then integrated in a longitudinal multi-perspective thematic analysis. The aim of this analysis was to identify walkers’ and walk-leaders’ perception about walkers’ needs when joining walking groups and whether these were satisfied three months later . Themes Broader themes were then explored in terms of whether they described separate phenomena at a specific time point, such as walkers’ needs, or the experiences or changes across time, such as processes to meet these needs. A more detailed description of the process of analysis is described in details provided in Supplementary Information.Three themes across walkers and walk leaders were identified by the longitudinal multi-perspective thematic analysis: social connectedness and healthy lifestyle, perception of control and actions to address walkers’ needs.Both walkers and walk leaders acknowledged that walkers joined walking groups to feel socially connected and to gain a healthy lifestyle. When they joined, walkers tended to compare their present unsatisfactory social or health situation with the one they desired to achieve.“Chatting to people making friends and hopefully getting a social circle which I don’t have at the moment” .“But the walk was a next step up from everything else I was doing, actually I thought I’ve got to start improving my fitness. So I wanted to lose weight as well” .“Well I think they are expecting to get fit and healthy, but apart from that the bonus is that they make new friends, which is good” .Walkers particularly tended to emphasize more the health benefits they expected to gain from joining walking groups, rather than the social aspects.“It was the fitness part cause I am looking in improving my health but the social side was an added bonus, it was, I am glad, I am glad I joined it” .“That again is good exercise and I can move, so why not, and would be fun, so if I can get fit and have fun at the same time why not, so I shall see how that goes” .When walkers first attended walking groups, they engaged in social comparisons, triggered by meeting other people in the walking group. A suitable model for social comparison was a person who was perceived to be similar to walkers, in terms of similar needs to themselves and similar expectations for the means to meet these needs .“I was expecting some people to be in the same boat as me. I thought they would be people that they are looking for something to do with the social aspect same as me” .Walkers who participated in the follow up interviews three months later, were people who had dropped out from walking groups temporarily (i.e. had missed some sessions). Some of them expressed an intention to continue attendance and some others expressed an intention to drop out from that walking group and/or join another group or activity.The walkers who expressed their intention to maintain attendance at walking groups indicated that satisfaction of their initial needs was their main motivation to do so. These walkers tended to focus their description on the social feedback they were getting from other people in the group and the physical feedback associated with health or fitness benefits of walking. For example, they tended to describe the other members of the groups as being pleasant and friendly, and to describe the short-term goal achievements  or subjective feelings after walking.“I suppose if the people weren’t nice I wouldn’t go so I suppose the people are the main reason that you go because otherwise you join another group or you wouldn’t go” .“You do feel healthier, it’s like you go to the gym and you got to work out of it, think for walking as a gym […] and you can feel the difference when you have done the walk, it’s three miles, takes an hour, you can feel different, you start feeling different, you start to feel better on yourself, your well-being” .“It’s very nice and encouraging to see how their health improves. They can do things that they couldn’t do before. People come down and say I can only do half a lap, I can’t do anymore. After a few weeks they are doing a lap, and then it goes to two laps, and people are amazed that they can do it […] it’s just a bunch of friends” .“You know, they enjoy the same things. You meet some nice people in same the position as you are” .The walkers who expressed an intention to drop out from that walking group and/or join another group or activity mentioned their dissatisfaction with meeting their initial needs. Where walkers’ needs for social connectedness were not achieved, some walkers focused their descriptions more on the expected health benefits. However, these walkers still acknowledged that their social needs remained an important motivation for continuous participation at walking groups.“I mean originally, we thought the social thing would be good […] but it’s not that we need more social life, we’ve got more than enough, so we really do go just for the walking” .Some walkers who intended to drop out tended to attribute dissatisfaction with social aspects of walking groups to other people having an established routine, due to frequent attendance over the longer term.“I wouldn’t say that people are as friendly as I hoped they would be maybe it’s because I don’t, the others they will be going for a long time […] but no it doesn’t bother me” .Walk leaders also acknowledged that dissatisfaction of walkers’ initial needs were reasons for walkers to cease participation from walking groups. It might be the case that walk leaders tended to report their accumulated knowledge from their experience with leading walking groups or could not distinguish that the processes walkers go through might differ from initiation to maintenance at walking groups. It could also be that walk leaders did not like to elaborate more on the reasons walkers stop attending walking groups, as this would not have been in favour to their co-ordinating scheme. It should also be noted that walk leaders were approached first by the co-ordinating scheme and then were referred to the researchers. A selection bias might have been introduced, so that only walk leaders with positive experiences with the scheme were selected for this study.Also, it might be the case that at initiation walkers emphasised their need to gain health benefits more than social benefits. It might be the case that walkers did not like to talk more about what made it difficult to them to continue participation in walking groups. Instead, when asked to evaluate their experiences with walking groups, they might have selectively reported the possible health benefits they would get. This could be due to socially desirable reasons: not to sound unpleasant, not to be unfair to walk leaders or to present themselves as someone who is more interested in getting healthier rather than having unmet social needs.Both walkers and walk leaders acknowledged that a perception of control over satisfying their initial needs via walking groups, was important for initiation of attendance at walking groups and that the other members of the group could be a source of feelings of efficacy for walkers. Verbal persuasion or vicarious information from people who had themselves participated in walking groups seemed to be an effective method for motivating walkers to join walking groups.“It was only because a friend at the church said “do you fancy doing that?” you know, we go to park on Saturday morning, we do a walk round there, if you fancy doing some walking, I thought that seems like a good idea, so that’s how that started really” .Walkers who participated in the follow up interviews acknowledged that perception of control seemed to be important for maintained attendance at walking groups. It seemed that walkers assessed both abilities for achieving social connectedness and fitness within walking groups, by comparing themselves with similar walkers in the group. When the outcome of this assessment was positive, then walkers tended to report that they were satisfied with their participation in walking groups.“Well it gives you confidence. Well you know if they can do it why can’t I?” .When the outcome of this assessment was negative, then walkers tended to report that they were not satisfied with their participation in walking groups and they intended to cease participation from that group.“If it would still be like the cold shoulder might think of blow this thing what if I do it on my own, you know with my wife, go around together and that would be it, but I don’t think it will come to that, I have a feeling that it will not come to that” .Some walk leaders acknowledged that other members of the groups are a key source of satisfaction and efficacy for walkers, however walk leaders seem not to fully report/acknowledge walkers’ perspectives about the importance of meeting other walkers with similar needs and expectations to themselves.“It makes you feel you are a better person. It gives you confidence that you’re able to do these things. Because everybody is going into the unknown, but if somebody has been there before they can help” .Some walk leaders also reported some other factors as important for walkers’ maintenance of attendance at walking groups, notably walkers’ willingness to participate in walking groups or environmental factors (e.g. weather).“But over the year, when the weather changes, people drop out, some come back, some don’t” .Walk leaders’ estimates of their ability to facilitate walkers’ needs seemed to depend upon their perceptions of the factors that influence walkers’ maintenance at walking groups. Those walk leaders who perceived that walkers’ participation at walking groups was not greatly influenced by their actions, reported low efficacy to address walkers’ needs.“I mean he [the walk leader] can do a bit with his personality. Some people they’ve got things to do, so they drop out but there is no solution to that one. I’ve never thought about it, but I can’t see anybody solving it. Really it is just a matter whether the newcomers can then think it is worthwhile, you see how could you convince them that it is worthwhile? You know you have to strike the balance and it is impossible to achieve some times. Oh dear it is difficult” .There were only a few walk leaders who reported several actions that they take to support walkers’ integration into groups and increases in walking when walkers join and/or maintain attendance at walking groups. Some of these actions were: assessing and tailoring the walk to walkers’ needs, introducing walkers to existing walkers, explaining the benefits of walking and the type of walking the group does, providing walkers with pedometers, providing walkers with feedback on behaviour, providing rewards, making telephone calls for supporting continued attendance, and encouraging walkers to increase their walking.“The encouragement to get people to speed up so they are getting the maximum benefit that they can from the walk […] to looked pleased to see them […] to introduce them to the group […] have to be respectful of how they wanted the walk to be and take the time to build the relationship with them and trust with them” .“My duties are, make sure they’ve done the health questionnaire, make sure I have first name knowledge and I know the history, where they are coming from and what their fears are […] we need to know why they are there […] they were using the pedometer to say how far they walk “that’s good for you” and then we were offering them something good at the end of it […] and I ring everybody so Saturday walk and so, come and join […] one person can talk about what benefits you have from this one […] explain what benefits you can get” .However, walk leaders attributed successful and continuous attendance at walking groups to uncontrollable factors (e.g. luck) or to walkers’ characteristics (e.g. pleasant people) and not to their or walkers’ actions.“I don’t know it’s something that we are lucky, all the long term walkers we’ve got are a nice group of people, so they are open and they are accepting to others you know” .On the other hand, the walkers who expressed their intention to continue attendance at walking groups expressed their satisfaction with walk leaders’ actions when they joined and maintained participation at walking groups. These actions were related to walk leaders taking into consideration walkers’ needs and expectations and tailoring their recommendations to these needs.“I think it’s nice they are taking interest in the people that are on their walks. Well they try to meet the expectations of other people […] it’s makes you feel like they know everybody’s needs […] they make it easy for you to ask things […] when I got the phone call from her we are meeting somewhere else […] it was nice to know that she recognized me as a new starter” .“It looks like he arranges some meals and it’s like you meet at that place, sit down and have a natural meal and it’s the social side of it” “He is very encouraging, he is very nice man, of course because he is enthusiastic you feel that you don’t let him down” .“She is very good anyway and she is friendly and at the minute she is trying to sort out different walk […] she does things like that you know gives you the choice, you know she is not “right we go so and so and that’s it”, no we discuss where we are going so that everybody is happy sort of thing” .However, the walkers who expressed their intentions to drop out from walking groups did not seem to acknowledge actions within walking groups, aimed at supporting their social integration or healthy lifestyle.“Depends on who you are walking with and who is sitting next to you during coffee time after walking […] you are only talking to whoever pops in with you, at your speed […] not really, I wouldn’t say welcome really, you know, a greeting, a smile, a hello and shaking hands with the man, that sort of things, but if you don’t get any of those, you think grrr terrible, isn’t it?” .In these cases, a possible explanation could be that walk leaders reported their roles as walk leaders and not their actions to address walkers’ needs, since walkers seemed generally not to acknowledge that their leaders’ actions were effective at integrating new walkers to the walking group.The decision to join a walking group was facilitated by walkers’ needs to be both socially connected and healthy. It appeared that walkers engaged in comparison processes with similar others in the group in order to make sense of their experiences and appraisals of their ability to emotionally and proactively cope with challenges during the first weeks of attendance at walking groups. When these processes of social comparison provided walkers with feelings of efficacy to cope with challenges while making changes, then walkers’ initial needs were satisfied. This feeling of satisfaction was associated with meeting the other people in the group. Further, to maintain this feeling of satisfaction, walkers tended to maintain participation at the walking group in which these other people were present.Walk leaders also acknowledged that social connectedness and healthy lifestyle were the main reason for walkers to join walking groups and that satisfaction with meeting other members of the group was the most important reason for walkers’ maintenance at walking groups. However, walk leaders appeared not to view these processes as important sources of efficacy for walkers to continue attendance. Walk leaders seemed to also report some other factors important for walkers’ maintenance at walking groups. These were relevant to walkers’ willingness, environmental factors, or their actions. Walk leaders’ perceptions of the factors that influence walkers’ maintenance at walking groups seemed to have an effect on their perception of their ability to facilitate walkers’ needs. Those walk leaders who perceived that walkers’ participation was less influenced by their actions reported low efficacy to address walkers’ needs.The sample employed for this study consisted of walkers and walk leaders recruited from a single urban location in England. Moreover, the sample recruited for this study mainly consisted of older women. Thus these findings should be treated with caution when generalising to other non-urban areas and demographic groups. Despite this, the majority of people who participate in WfH group walks in England are older women , so it iIrrespective of this, the primary aim of this qualitative study was not to recruit a representative sample and generalize findings to the whole population, but to generate the most relevant information to answer the research question and apply recommendations tailored to the included population.A strength of this study was that the majority of potential participants agreed to participate in the interviews, with only a minority of walkers not willing to be followed up (three out of eight). The three people who declined participation in the follow up interview had ceased attending walking groups and were unwilling to be interviewed. Although their views would be valuable for this study, the problem of dropouts being unwilling to participate in research is well known . It shouTo our knowledge this is the first study that has explored both the needs and expectations of people when they joined walking groups and whether these needs and expectations had been met three months later. This study not only identified needs and expectation at two time points, but also the psychological processes that facilitated or challenged the satisfaction of these needs during this period of time. However, it should be mentioned that saturation was not achieved for follow up interviews with walkers.An important strength of this study is its multi-perspective approach, which provided the opportunity to generate a better understanding of walkers’ and walk leaders’ needs, and to explore how walk leaders understand walkers’ needs and what actions they take to address these needs. This is an important approach especially when aiming at evaluating service effectiveness and generating suggestions for improvement.Previous research has identified motivation at initiation ,14,26 anPrevious research did not however explore the stability of motivations and challenges at initiation and at three months maintenance of attendance at walking groups. Moreover this previous research did not explore the psychological process underlying walkers’ motivations at initiation and three months maintenance of attendance. The present research identified that satisfaction of walkers’ initial needs was an important factor for maintenance at walking groups and that walkers’ reporting of initial needs changed over time. For example, even though walkers reported that they joined walking groups to gain a healthy lifestyle, at the follow up interview they reported that social connectedness seemed to be more salient motivation for their participation at a particular walking group. The longitudinal design of the present study facilitated the identification of underlying psychological process over time, which could not be done by previous research employing cross-sectional designs.Previous studies have not used a multi-perspective approach to investigate walkers’ reasons for participating in walking groups. Multi-perspective interviews can show different facets of walkers’ needs and ways to address them based on the role that each participant has. The present research identified that walkers perceived other members of the group as important source of both satisfaction and efficacy. It also showed that walkers engaged in social comparisons with other walkers in the group to assess their progress towards social integration and healthy lifestyle. However, even though walk leaders acknowledged the importance of other walkers, they did not seem to acknowledge that this was the process that walkers went through when they joined walking groups and that this process was important for their maintenance.Moreover, the multi-perspective approach provided different perspectives on the services, which could contribute to formulating tailored recommendations for improving these services. This research identified that although some walk leaders reported several actions to address walkers’ needs, walkers who expressed their intention to drop out from walking groups did not report that these were effective at promoting maintenance at walking groups. For example, walkers were dissatisfied when their needs to socialise with similar others were not met, and walk leaders agreed with this point of view. Despite this, walk leaders attributed difficulties walkers faced in joining new groups as being beyond their control and their actions, instead of walk leaders tending to report low efficacy to address these difficulties. The lack of reported efficacy and/or effective strategies to address walkers’ needs also suggested the need for further actions that might be taken to improve the current situation, such as training. These strategies can be drawn from those walk leaders’ actions who effectively addressed walkers’ needs and relevant literature.The findings of this study provide some useful insights on the role of walkers’ needs and expectations to support maintenance of attendance during the first three months after joining a walking group. These findings have been supported by a longitudinal quantitative study, building on the findings of the present study, which found that satisfaction with social connectedness and health benefits predicted maintenance of attendance at walking groups .Walking groups can promote moderate physical activity in the long term, i.e. more than six months  and are Interventions are likely to be more effective if they target the underlying psychological processes that produce walkers’ desired outcomes . This stProviding training to walk leaders with techniques to promote walking within walking groups might be a useful way of helping walkers to achieve social connectedness. However, in doing so, consideration should be given to walk leaders’ perceptions of how changes in walkers occur and the actions needed to address walkers’ needs. For example, the present study showed that some walk leaders were apparent not aware of the processes underlying walkers’ social connectedness and satisfaction, and how these influence uptake and maintenance of attendance at walking groups, or how walk leaders’ actions facilitate or not these processes.In conclusion, to date no research has been done to identify whether and if the techniques mentioned above are effective at increasing physical activity, group connectedness or any other outcomes such as quality of life in efficacy or effectiveness studies within lay-led, community based walking schemes. Future research should identify what type of training is effective for promoting those behaviours of walk leaders that are likely to result in walker satisfaction, what strategies are effective at promoting specific outcomes for walkers, and aim to identify potential contextual influences. Future research might also usefully investigate whether training interventions for walk leaders are effective at increasing satisfaction and retention of walkers, and are cost effective.S1 File(PDF)Click here for additional data file.


Fermentation of dietary oligofructose-enriched inulin fibers by the microflora leads to acidification of the intestinal lumen and by this enhances mineral uptake. This study aimed, therefore, to improve mineral absorption by application of dietary inulin to counteract PPIH.Proton-pump inhibitor-induced hypomagnesemia (PPIH) is the most recognized side effect of proton-pump inhibitors (PPIs). Additionally, PPIH is associated with hypocalcemia and hypokalemia. It is hypothesized that PPIs reduce epithelial proton secretion and thereby increase the pH in the colon, which may explain the reduced absorption of and Mg2+ and Ca2+ homeostasis was assessed by means of serum, urine and fecal electrolyte measurements. Moreover, the mRNA levels of magnesiotropic and calciotropic genes were examined in the large intestine and kidney by real-time PCR.Here, C57BL/J6 mice were supplemented with omeprazole and/or inulin. Subsequently, Mg2+ and Ca2+ levels. However, concomitant addition of dietary inulin fibers normalized serum Ca2+ but not serum Mg2+ concentrations. Inulin abolished enhanced expression of Trpv6 and S100g in the colon by omeprazole. Additionally, intestinal and renal mRNA levels of the Trpm6 gene were reduced after inulin intake.Treatment with omeprazole significantly reduced serum Mg2+ absorption upon PPI treatment. In contrast, inulin did not increase intestinal absorption of Mg2+ sufficiently to recover serum Mg2+. The clinical potential of dietary inulin treatment should be the subject of future studies.This study suggests that dietary inulin counteracts reduced intestinal Ca PPIs detected , 11. An detected , 12, 13. PPI use .in vitro data suggest that omeprazole inhibits passive paracellular Mg2+ fluxes, predominantly present in the small intestine [in vivo studies indicate that omeprazole directly interferes with important transcellular Mg2+ transport mechanisms of the colon [2+ channel, transient receptor potential melastatin member 6 (TRPM6), facilitates the absorption of Mg2+ [Trpm6 as well as Atp12a, which encodes the non-gastric proton potassium adenosine triphosphatase  [2+ transport is dependent on the protonation of the channel itself, PPI-induced pH increases will reduce TRPM6-mediated Mg2+ influx [The exact mechanism by which PPIs cause mineral deficits is currently under debate. Recent ntestine , 16. Morhe colon . In the  of Mg2+ –20. Omepr cHK-α) . It has r cHK-α) . Given t+ influx –23.2+ absorption and, therefore, provides a promising approach to prevent PPIH. Interestingly, the dietary application of the fructan fiber inulin has been proposed to reduce intestinal pH [2+ absorption by reducing the luminal pH has already been described decades ago, but has been largely overlooked since then [2+ and Ca2+ absorption in the colon of mice and humans [Trpm6 mRNA expression [To date, defined intervention strategies preventing PPIH have not been established in clinical practice. Local luminal acidification of the colon may rescue intestinal Mgtinal pH . Ingestetinal pH . The stince then , 27. Howd humans , 29. Morpression .2+ and Ca2+ absorption in order to counteract omeprazole-induced defects in mineral uptake. Additionally, the mRNA expression pattern of the Mg2+ and Ca2+ transporting proteins expressed in the cecum, colon and kidney was determined by qRT-PCR.Here, an intervention study was performed using dietary oligfructose-enriched inulin fibers in control- and omeprazole-treated wildtype C57BL/6J male mice. The aim was to enhance intestinal Mgad-libitum access to standard pellet chow  and drinking water. The control diet consisted of standard pellet chow, the experimental inulin diet additionally contained 10% (w/w) inulin fiber product . Omeprazole  was dispersed in a solution (vehicle) containing 0.5% (w/v) methylcellulose and 0.2% (w/v) NaHCO3 (adjusted with NaOH to pH 9.0). During the 14 days of the experimental phase, the mice received a daily dose of 20 mg omeprazole per kilogram bodyweight, administered via oral gavage once a day, or vehicle. The health of the animals was regularly checked. For urine and feces collection, animals were housed individually in metabolic cages for 24 h. At the experimental endpoint, animals were anesthetized with isoflurane (5% v/v), blood sampling was performed by orbital sinus bleeding and subsequently the mice were sacrificed by cervical dislocation. Kidneys, cecum and colon segments were extracted and cleaned, fecal contents of cecum and colon were preserved and all samples were snap frozen in liquid nitrogen.This study was carried out in strict compliance with the legal Dutch animal welfare act. All experimental procedures were approved by the animal ethics board of the Radboud University Nijmegen (permit-no: RU-DEC 2014–032) and all efforts were made to minimize suffering of the animals. Wild-type C57BL/6J mice  were purchased from Charles River, the Netherlands. The animals were randomly allocated into four experimental groups of n = 10 mice. Before the experiment was started, the animals underwent acclimatization for one week under temperature- and light-controlled conditions with 2+, Ca2+, K+ and Na+ concentrations were determined at the university hospital central clinical lab on an automated system according to the manufacturer's protocol . Feces were homogenized and digested in nitric acid (concentrated with 65% (w/w) Sigma-Aldrich, USA) by a 2 h pre-incubation at 50°C, followed by an overnight incubation at room temperature. Urinary and fecal Mg2+ concentrations were determined with a colorimetric xylidyl-II blue kit  on a Nanodrop 2000c spectrophotometer  at 600 nm wavelength. Urinary and fecal Ca2+ concentrations were spectrometrically determined with a colorimetric chromogenic/buffer dual-component kit  on a Biorad plate reader  at 570 nm wavelength. The obtained values for Mg2+ and Ca2+ were cross-verified using a serum standard solution .Serum MgThe SCFA profile of cecal and colonic contents was determined on a Chrompack Model CP 9001 gas chromatograph  equipped with a 2 m x 2 mm column, packed with 10% SP 1200/1% H2PO4 on 80/100 Chromosorb W AW . Samples were centrifuged at 15,000 x g for 10 min. Subsequently fecal water (supernatant) was extracted and 1:1 (v/v) diluted with a solution  of 30 mmol/L of 2-ethylbutyric acid in 100% formic acid, resulting in a 9% (v/v) formic acid suspension used for injection into the gas chromatograph.Total RNA was extracted from tissues using TRIzol reagent  according to the manufacturer’s protocol. Obtained RNA was subjected to DNase treatment . Subsequently, the purified RNA was reverse transcribed with murine leukemia virus reverse transcriptase .Gapdh) as an endogenous control. Relative mRNA expression was analyzed according to the Livak method (2-ΔΔcT) and annotated as times-fold change of expression compared to control [The mRNA expression was quantified by SYBR Green  real-time PCR on a CFX96 real-time detection system . Real-time PCR primers  were designed with Primer 3 software . Primer sequences are provided in  control .P < 0.05. The analysis of the datasets was performed using GraphPad Prism (PC version 6).Values are expressed as means ± SEM. The differences between single groups of control, inulin-only, omeprazole-only and omeprazole + inulin treated mice were tested by using one-way ANOVA with a Tuckey correction. Differences between groups were regarded to be statistically significant when P < 0.05).In this study, we aimed to evaluate the application of dietary oligofructose-enriched inulin fibers as a means to prevent PPIH. Therefore, wildtype C57BL6/J mice were supplemented with vehicle or omeprazole for 14 days. In addition, the vehicle group and the omeprazole group were subdivided into two groups fed with a normal diet or with a diet containing 10% w/w oligofructose-enriched inulin fructan fibers, making a total of four experimental groups . After 12+ homeostasis, serum Mg2+ concentration, 24 h urinary Mg2+ and fecal Mg2+ excretion were determined. Omeprazole-treated and inulin-omeprazole-treated mice had significantly lower serum Mg2+ levels compared to control mice  and 1.39 ± 0.02 mmol/L, respectively, P < 0.05 2+ excretion was significantly increased in the inulin-omeprazole group compared to omeprazole-treated group Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Refractory coeliac disease (RCD) is a rare complication of coeliac disease (CD) and involves malabsorption and villous atrophy despite adherence to a strict gluten-free diet (GFD) for at least 12 months in the absence of another cause. RCD is classified based on the T-cells in the intra-epithelial lymphocyte (IEL) morphology into type 1 with normal IEL and type 2 with aberrant IEL  by PCR (polymerase chain reaction) for T cell receptors (TCR) at the β/γ loci. RCD type 1 is managed with strict nutritional and pharmacological management. RCD type 2 can be complicated by ulcerative jejunitis or enteropathy associated lymphoma (EATL), the latter having a five-year mortality of 50%. Management options for RCD type 2 and response to treatment differs across centres and there have been debates over the best treatment option. Treatment options that have been used include azathioprine and steroids, methotrexate, cyclosporine, campath , and cladribine or fluadribine with or without autologous stem cell transplantation. We present a tertiary centre’s experience in the treatment of RCD type 2 where treatment with prednisolone and azathioprine was used, and our results show good response with histological recovery in 56.6% of treated individuals. Enteropathy related to coeliac disease (CD) occurs in genetically predisposed individuals upon exposure to toxic gluten resulting in various gastrointestinal and extra-gastrointestinal manifestations . The symet al. observed a histologic response in some of the few cases with RCD type 2 following treatment with methotrexate or anti-tumor necrosis factor α [RCD is divided into primary (absent response to gluten-free diet (GFD)) or secondary (previously responded to GFD but has now relapsed) . Anotherfactor α . Treatmefactor α ,8.We report a single centre retrospective study of all cases of RCD type 2 using the coeliac database in a single centre between 2000 and 2015. We have concluded that Prednisolone combined with azathioprine can be used successfully to treat RCD type 2. Our experience shows it is a safe and successful approach to improve prognosis.n = 30), were treated with azathioprine and prednisolone (n = 27). The other patients did not tolerate azathioprine and/or prednisolone or had side effects and were given alternative treatment with thioguanine (n = 1), methotrexate (n = 1) or mycophenolate mofetil (n = 1). The initial dose of prednisolone we used was 20 mg daily which is reduced to 15 mg/day, and if necessary to 10 mg/day, if the patients experience side effects. The standard dose of azathioprine used was 2–2.5 mg/Kg per day, but we checked the thiopurine methyltransferase (TPMT) levels to adjust the dose if necessary depending on the patient’s methylation activity. Duodenal biopsies were immunostained and PCR of the TCR was performed. The molecular signature of the clones in each repeat biopsy was compared. We looked at the patient clinical outcome after follow up as (1) improvement or (2) remains RCD type 2 on ongoing treatment. We define improvement as conversion from RCD type 2 to RCD type 1 or responsive coeliac disease as indicated by improved symptoms of coeliac disease and malasborption in addition to evidence of downgrading of RCD type 2, including: improved histological Marsh criteria to less than 2, improved CD8 positivity on immunohistochemistery or change of TCR from monoclonal to polyclonal.We reviewed the cases of RCD with negative coeliac serology retrospectively over a period of 15 years from 2000 to 2015. The information was collected from patient case notes and the hospital electronic patient records. Thirty-seven patients were diagnosed with RCD type 2 . The age range was 30–87 (mean age 58). We excluded 7 patients from the study: one was a recent diagnosis and was yet to commence treatment, 2 were diagnosed with RCD type 2 and referred to our centre, but we diagnosed established EATL, one had major comorbidities and opted not to start treatment, and 3 relocated abroad. The human leucocyte antigen (HLA) calls II gene, or HLA-DQ2, which is known to have a strong association with coeliac disease, was found in 86% of the cases. The patients with RCD type 2 (Eighteen out of 30 patients (60%) completed treatment  and demoOur data show that treatment of RCD type 2 with steroids and azathioprine show good response with a histological recovery in 16 out of 30 patients (53%). The TCR clonality improved converting to polyclonal in 17/30 (56.6%). None of the patients in our cohort developed ulcerative jejunitis or EATL, providing them with a better 5-year survival.p-value 0.017).All patients continued to be on regular follow up after completing their treatment. We observed that all patients who completed treatment were asymptomatic and the percentage of CD8 positivity improved. We also observed persistent clones in the repeat duodenal biopsies of most of the refractory cases not responsive to treatment. Of the 12 patients who have not recovered, nine had persistent identical clones on repeat biopsies. Compared to the group who have completed treatment and improved to either RCD type 1 or responsive coeliac disease (18 patients), seven had identical clones at some point during surveillance  has a normal intra-epithelial lymphocyte phenotype whereas type 2 RCD (RCD type 2) has an abnormal clonal population. This classification is important not only to guide treatment but is of prognostic value. RCD type 2 carries a poor prognosis with a five-year mortality in RCD type 2 of about 55% [Complications of RCD include ulcerative jejunitis and enteropathy associated T-cell lymphoma (EATL), the latter being the major cause of mortality in these patients. RCD type 2 has a female preponderance with a female to male ration of 3:1, similar to responsive CD, but the ratio is reversed in EATL ,12,13. HDiagnosis of RCD may be challenging and is in many cases a diagnosis of exclusion. Clinical assessment, pathological, histological, laboratory and radiological findings all aid in the diagnosis and enable to differentiate between the two subtypes.When making the diagnosis, the patient needs to be on a strict GFD with a negative anti-enterocyte antibody result. Dietary assessment of compliance to GFD is key and instruction and education by a specialized dietitian is advised.Upper gastrointestinal endoscopy to obtain small bowel biopsies for tissue analysis. The samples are used for Marsh scoring.Assessment of IEL phenotyping and polymerase chain reaction (PCR) for TCR (T-cell receptor) monoclonality at the β and/or γ loci.In our centre, we follow a strategy when making a diagnosis of RCD type 2: 4.All cases of RCD type 2 should have a capsule endoscopy to exclude EATL (enteropathy associated T cell lymphoma). It is our practice if there is any suggestion of possible EATL to undertake a small bowel magnetic resonance imaging (MRI) in the first instance to exclude an obstructing lesion, which would be a contra-indication to capsule endoscopy. The capsule endoscopy should be repeated after a year to exclude the development of EATL in view of the high risk. It has been proposed that RCD type 2 should be renamed pre-EATL . Double 5.Cross sectional imaging including small bowel MRI, computed tomography (CT) scan and positron emission tomography (PET) scan are recommended when suspecting EATL. This can identify abnormal areas within the bowel, abnormal lymph nodes and other organ involvement.The presence of abnormal  IEL in the small bowel supports a diagnosis of RCD type 2. Transient TCR clonality can be detected in patients at diagnosis and with poor compliance ,16. The Patients with RCD presenting with abdominal pain, weight loss or evidence of malnutrition should undergo urgent investigation. Cross-sectional imaging by CT +/− PET or MRI for the presence of lymphadenopathy or intestinal tumours should be carried out and capsule or balloon enteroscopy should be performed to diagnose any cases of EATL .Several approaches to management of RCD type 2 have been trialed in different centres . Treatmeet al. reported 32 patients treated with cladribine, of whom 18 had a good response [et al. reported 61% histological improvement with this treatment in a group of 13 patients with RCD type 2 [There have been other reports of different treatment options, some with limited cases and variable results. Methotrexate has been used as a single agent  or in coresponse . Cyclospresponse ,27,28. WD type 2 . There hD type 2 ,31,32. An = 1), thioguanine (n = 1) or mycophenolate mofetil (n = 1); the latter responded to treatment and converted to RCD type 1. Our data show improved outcomes with this management, which could be that we may be seeing affected subjects earlier through the UK tertiary referral system such that they may be less ill. In some centres, RCD type 2 patients are seen to have a serum albumin that we do not see. This suggests the patients that are referred to us are earlier in the disease process, and this may explain the improved outlook with less risk of developing EATL. In the azathioprine/steroid group, we block the aberrant immune response to gluten proteins that occurs in coeliac disease resulting in a better outcome than using other drugs with a different mechanism of action. The percentage of CD8 negative cells and/or the presence of persistent identical clones may have a role in persistent features of RCD type 2. Further studies are required to confirm this. The immunophenotyping is less sensitive than PCR TCR clonality testing. However, immunostains for CD3/CD8 are easier to be carried out in a routine pathology laboratory and almost always available in non-tertiary centres. This method can be used as a screening tool. Although the majority of IELs in RCD type 2 show an abnormal CD3+/CD8− immunophenotype, a normal CD3+/CD8+ phenotype has been seen in up to 52% of RCD type 2 cases [et al. found that only one out of eight RCD type 2 patients showed a loss of CD8 expression and concluded that other more sensitive ancillary tests such as flow cytometry may be necessary in the diagnosis of some RCD2 patients [Prednisolone combined with azathioprine can be used successfully to treat RCD type 2. Our experience shows it is a safe and successful approach to improve prognosis. We successfully treated 18 out of 30 patients with RCD type 2 with this regimen, converting either to RCD type 1 or responsive coeliac disease. Where azathioprine and/or steroids are not tolerated or patients experience side effects, alternatives may be used. In our cohort, we used methotrexate ( 2 cases . Howeverpatients . In flow


This study investigated responses to near-wins  in a decision-making task, measuring (a) luck ratings, (b) adjustment of bet amount, and (c) facial muscle reactivity at zygomaticus and corrugator sites. Compared to full-misses, near-wins decreased self-perceived luck and near-losses increased self-perceived luck, consistent with the effects of upward versus downward counterfactual thinking, respectively. Wins and losses both increased zygomaticus reactivity, and losses selectively enhanced corrugator reactivity. Near-wins heightened zygomaticus activity, but did not affect corrugator activity, thus showing a similar response pattern to actual wins. There were no significant facial EMG effects of near-losses. We infer that near-wins engender some appetitive processing, despite their objective nonwin status. Gambling is a widespread form of entertainment where a monetary wager is placed upon the uncertain prospect of a larger monetary win. Its allure can provide insight into the psychological mechanisms of human decision making. Previous research has shown that near-wins—nonwin outcomes that are proximal to a jackpot—foster persistent play  and heart rate acceleration, in comparison to full-miss outcomes  from the student population at the University of Cambridge for a study of gambling behavior. We determined this sample size based on previous facial EMG studies  in the range of previous studies in recreational gamblers ; (3) the belief in good luck scale . No time constraints were imposed on bet selection or luck ratings. During a variable intertrial interval (8–12 s), only a fixation cross was displayed, to allow for recovery of physiological signals.The specific trial timings were as follows  and loss segments (−10×), thus offering a neutral expected value. The other two wheel types offered only a win or loss segment , generating a positive and negative expected value on those wheels. These were included in order that participants should vary their bet on a trial-by-trial basis (see the results in the online supporting information). The outcomes were fair, such that each segment was selected five times, with wins and losses on one in eight trials (12.5%), and near-wins, near-losses, and full-misses each occurring on two in eight trials (25%). Near-wins were zero outcomes in the segment either side of the win. Similarly, near-losses were zero outcomes in the segment either side of the loss. Both near events were compared against a baseline of “full-misses,” where the highlighted null segment was not adjacent to either the win or loss segment. An equal number of near-wins and near-losses were delivered on either side of the win and loss, respectively.Following the gambling task, participants viewed screenshots of near-wins that stopped before and after the winning segment, and near-losses that stopped before and after the losing segment. For each screenshot, they were asked to list their first thought about how the outcome could have been different.Facial EMG data were collected via a BIOPAC  MP36R, recording at 1,000 samples per second. The BIOPAC was connected to a stimulus delivery computer and a second administrator computer running Acqknowledge v4.1. Events occurring on the stimulus delivery computer (including the outcomes on the task) were synchronized to the facial EMG recording using digital channels. Facial EMG recordings were collected through 4-mm shielded chloride electrodes attached to the skin over the left eye  and left cheek  via 4-mm adhesive disks, according to the standard procedures established previously . The data were filtered through a 30 Hz high-pass filter to remove low frequency noise and artifacts recorded during the task. The filtered data were then rectified, converting negative values into positive values. Mean values were extracted for a baseline period in the final 2 s of the spin, and for 4 s following the wheel stopping (the outcome). Percentage change from baselines was calculated, in order to compare activity at the two muscle locations.nlme  modeling via restricted maximum likelihood for all repeated measures analyses to reduce information loss when evaluating large, unbalanced data sets after signal standardization  = 241.76, p < .001, M = 0.70, SD = 0.42, whereas near-losses elicited downward counterfactuals, M = −0.82, SD = 0.32. Neither the main effect of near-miss position nor the interaction term were statistically reliable, both F < 1.We coded the counterfactual statements given by the participants as +1 for an upward counterfactual , 0 for no counterfactual (“I don't mind”), −1 for a downward counterfactual  = 146.12, p < .001, with participants feeling luckier following wins compared to null outcomes, b = 1.93, t(88) = 7.61, p < .001, and following null outcomes compared to losses, b = −2.31, t(88) = −9.12, p < .001.The first model assessed the impact of the different objective outcomes (three levels: win vs. loss vs. null) on the luck ratings (see Table 2(2) = 30.13, p < .001. Near-losses significantly increased luck ratings relative to full-misses, b = 0.29, t(88) = 2.91, p < .01. Conversely, luck ratings were lower following near-wins compared to full-misses, b = −0.31, t(88) = −3.06, p < .01.The second model compared the three types of null outcomes .The near-loss effect was further moderated by the trait level of beliefs in luck, on the BIGL scale. The BIGL score was positively correlated with the increase in luck ratings following a near-loss compared to a full-miss, 2(1) < 1.The third model decomposed the four types of near-misses by near-miss type (near-wins vs. near-losses) and near-miss position (before vs. after). The main effect of near-miss type was already established in the second model, and, similar to the manipulation check for counterfactual thinking, neither the main effect of near-miss position nor the interaction term reached significance, both χ2(2) = 11.23, p < .01, with participants reducing their bet following wins, compared to both null outcomes, b = −4.67, t = −2.49, p = .01, and losses, b = −6.04, t = −3.23, p < .01.In the first model looking at the objective outcomes (see Table 2(2) = 1.28, p > .1. Given this result, the third model decomposing the null events by position was not run on betting behavior.In the second model (see Table 2(2) = 4.45, p = .1, which was driven by losses  significantly increasing zygomaticus reactivity compared to null outcomes , b = 10.55%, t(88) = 2.11, p < .05. An a priori test comparing zygomaticus activity following wins  and null outcomes confirmed a significant response to wins, χ2(1) = 5.82, p = .01. There was no difference between wins versus losses, b = −4.79%, t(44) = −0.81, p > .1.The first model assessing the objective outcomes  = 5.70, p = .05, with near-wins  eliciting higher zygomaticus activity than both near-losses , b = 2.54%, t(88) = 2.05, p < .05, and full-misses , b = 2.60%, t(88) = 2.10, p < .05.The second model tested for differences between three types of null outcomes  = 0.21, p > .1, nor the interaction term, χ2(1) = 0.007, p > .1, was significant.The third model confirmed the main effect of near-miss type in model 2, but neither the main effect of near-miss position, χ2(2) = 4.50, p = .10, driven by losses  significantly increasing corrugator reactivity relative to wins , b = 9.14%, t(88) = 2.02, p < .05, consistent with our a priori prediction.In the first model looking at the objective outcomes , χ2(2) = 1.67, p > .1, and therefore the third model further decomposing near events by position was not performed.In the second model  = 1.03, p > .1, and losses elicited stronger responses compared to near-wins, b = 8.84%, t(88) = 2.05, p < .05. For the corrugator, there was no difference between wins and near-wins, b = 3.08%, t(88) = 0.83, p > .1, and losses elicited greater responses compared to both objective wins, b = 9.14%, t(88) = 2.19, p < .05, and at trend, near-wins, b = 6.06%, t(88) = 1.63, p = .1.Thus, both objective wins and losses increased zygomaticus activity, whereas corrugator activity was selectively sensitive to losses. Near-wins increased zygomaticus but not corrugator activity, thereby showing a similar pattern to the actual wins. As a direct test of the similar profile, we ran a supplementary model to compare near-wins, objective wins, and objective losses. For the zygomaticus, near-wins and wins did not differ from each other, By using a wheel of fortune task, the present study investigated the effects of near-wins and near-losses on self-perceived luck, betting behavior, and facial muscle reactivity. Subjectively, participants reported greater feelings of luckiness following near-losses, relative to full-misses, and this effect was further correlated with the trait beliefs in luck using Darke and Freedman's BIGL scale. Near-wins exerted the opposite effect on luck ratings, decreasing self-reported luck. While this effect did not scale with BIGL score, it is notable that the BIGL selectively assays positive aspects of luck. Betting behavior was primarily sensitive to the objective outcomes on the task, with participants reducing the amount of the bet following wins. Near-wins and near-losses had no significant influence on this adjustment in betting behavior in the next round of the game.Our facial EMG data provide an important proof-of-principle demonstration for the differential sensitivities of zygomaticus and corrugator. Zygomaticus activity increased after objective wins and losses, whereas corrugator activity selectively increased following objective losses. Near-win outcomes significantly increased zygomaticus activity, relative to both full-misses and near-losses, but did not affect corrugator response, thus showing a similar response pattern as actual wins. This interpretation was confirmed in a model that directly compared objective wins and losses against near-wins.Previous facial EMG research indicates that zygomaticus activity is a sensitive marker of appetitive processing  in a random task (Ayton & Fischer, Despite the marked adjustment of betting following wins, we did not observe any reliable change in betting following near-wins or near-losses. As such, our results do not replicate Wohl and Enzle , who repIn the present study, we did not find any position effects of near events either side of the win/loss segment, either on subjective luck ratings or on facial muscle reactivity. Using a slot machine simulation, Clark et al.  previousTo our knowledge, this is the first study using facial EMG to investigate near-miss effects in a laboratory setting. Zygomaticus was sensitive to objective wins and losses, whereas corrugator was only sensitive to objective losses. Near-wins were perceived as unlucky, but heightened zygomaticus activity, showing a similar facial muscle response pattern as actual wins. This is consistent with previous literature showing the bivalent emotional nature of near-wins. This supports the utility of facial EMG as a marker of emotional reactivity in gambling and decision-making research. Near-wins and near-losses elicited downward and upward counterfactuals, respectively, and drove luck perceptions in opposing directions, which emphasizes the role of counterfactual thinking in the near-miss effect.


After publication of this work , it was From page 16 “By approximate numerical integration, we derived…” to page 17 “Specifically for Fig. 2b, each data point represented the…”, the correct text is as follows:By approximate numerical integration, we derived:z) in z-stack .Where nρ/2, nρ/2], ΣSince we observed that in the range of [−And we could further simplify this formula as follows:Where 2+]i was approximately positively proportional to From the formula, we knew that i in the branch. The mean value of these 50–100 branches was calculated to represent the level of [Mg2+]i in the AOI. For each coverslip, several AOIs were measured and the mean value of these AOIs was calculated to represent the average level of [Mg2+]i in the coverslip. For each data point, several coverslips were used. For each statistical data point, the mean ± SEM of all coverslips was presented. Specifically for Fig. 2b, each data point represented the [Mg2+]i (MgGrn fluo. a.u.) value in an individual branch.In the experiments, given a randomly selected AOI of dendritic area (the image was obtained as described above), 50–100 branches were randomly selected from each AOI and the mean intensity of fluorescence  A segment of branch was modeled as a cylinder characterized by diameter (d) and length (L). (B) Z-stack of MgGrn fluorescent images was taken in the range of –h to h at z-axis. d). (E) After correction, the value Additional file 1: Figure S1. Modeling the measurement of [MgIn addition to the incorrect equations, it was noted on page 20, reference 39 [Nat Neurosci. 2007;10(2):186–95. doi: 10.1038/nn1837.Futai K, Kim MJ, Hashikawa T, Scheiffele P, Sheng M, Hayashi Y. Retrograde modulation of presynaptic release probability through signaling mediated by PSD-95-neuroligin.


Globozoospermia is a rare disorder characterized by the presence of sperm with round head, lacking acrosome. Coiling tail around the nucleus has been reported since early human studies, but no specific significance has conferred it. By contrast, studies on animal models suggest that coiling tail around the nucleus could represent a crucial step of defective spermatogenesis, resulting in round-headed sperm. No observations, so far, support the transfer of this hypothesis to human globozoospermia. The purpose of this work was to compare ultrastructural morphology of human and mouse model globozoospermic sperm. Sperm have been investigated by using scanning and transmission electron microscopy. The images that we obtained show significant similarities to those described in GOPC knockout mice, an animal model of globozoospermia. By using this model as reference, we were able to identify the probable steps of the tail coiling process in human globozoospermia. Although we have no evidence that there is the same pathophysiology in man and knocked-out mouse, the similarities between these ultrastructural observations in human and those in the experimental model are very suggestive. This is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia. Globozoospermia represents a rare and severe type of teratozoospermia, first described by Schirren et al. in 1971 . UnfortuThe aim of this study is to compare ultrastructural morphology of human and mouse model globozoospermic sperm.6/mL. One aliquot of the cells suspension was cultured on poly-L-lysine coated coverslips (18 mm diameter)  and incubated at 37°C in a 5% CO2 atmosphere, for 1 h. Cells were fixed with 2.5% glutaraldehyde in PBS, pH 7.4, at room temperature for 20 min, rinsed in PBS, and postfixed in 1% osmium tetroxide for 30 min. Fixation was followed by rinsing in PBS and then by dehydration through graded ethanol. Samples were transferred to a critical point dryer  in 100% ethanol and dried through CO2. Coverslips were mounted on aluminum sample stubs and gold-coated by sputtering . SEM micrographs were acquired by a Leica Stereoscan 430i scanning electron microscope . For preparing samples for optical and transmission electron microscopy, sperm (5 × 106 cells) in PBS were centrifuged at 500 ×g for 5 min and subsequently the pellet was fixed for 30 min at room temperature in a solution of 2.5% glutaraldehyde  in 0.1 M sodium cacodylate buffer (pH 7.4) containing 0.03 M calcium chloride. Samples were washed twice with sodium cacodylate buffer (pH 7.4) and then postfixed with 1% osmium tetroxide for 1 h at 4°C. Postfixed cells were dehydrated with a graded ethanol series ending with 100% ethanol and then embedded in Dow epoxy resin  and DER732 . Thin sections for optical microscopy and ultrathin sections for TEM were cut with an Ultrathome III . Thin sections were stained with toluidine blue at 70°C and observed with a common optical microscope. Ultrathin sections were treated with double staining with lead citrate and uranyl acetate and visualized with a transmission electron microscope .Sperm were obtained from an infertile man with globozoospermia. The initial semen analysis was performed following the standard procedure . Briefly6/mL, rapid progressive motility 1%, slow progressive motility 12%, and normal morphology 0%. All spermatozoa had round heads without acrosomal cap, showing complete globozoospermia. Sperm antibody testing with the mixed agglutination reaction (MAR) method was negative for all antibodies. Peroxidase test was negative for leukocytospermia. In order to investigate sperm ultrastructure, SEM and TEM were used, in addition to optical microscopy on thin sections  as in Figures Sperm having normally straight tails, without tail sections in the perinuclear cytoplasm Figures .Sperm showing the probable start of the tail coiling with the nucleus rolled down the midpiece, with the membrane covering the tail progressively expanded and joined to the membrane covering the perinuclear cytoplasm. In this step, a tail segment lay by the nucleus side Figures .Sperm with conical neck filled with mitochondria .Sperm with tail totally coiled around the nucleus as Figures Sperm with tail coiled around the nucleus more than once Figures .Sperm with several tail encirclements of the nucleus Figures .We compared several images of our specimens with the images obtained in a mouse model by Suzuki-Toyota et al. , 6. ThesFew studies have focused on sperm tail coiling in human globozoospermia. Pedersen and Rebbe  first reOur hypothesis has been tested by reviewing the animal models of globozoospermia. Several animal models have been proposed to investigate pathogenesis of globozoospermia .Male mice deleting gene encoding GOPC show globozoospermia with a coiled tail around the nucleus . A straiHrb-deficient mice are infertile and their sperm lack acrosome and present globozoospermia . During Zpbp1-null sperm exhibit typical morphological defects of globozoospermia such as round-headed appearance and the absence of an acrosome . Zpbp1-nSperm acrosome associated 1 (Spaca-1) has recently been found to be a new member of the globozoospermia-related genes . Sperm fDPY19l2 is a transmembrane protein expressed specifically in spermatids and localized only in the inner nuclear membrane –22. Dpy1In other animal models of globozoospermia tail coiling is not present. However, it should be noticed that in these models the sperm are lacking the fundamental criteria of globozoospermia, such as round head and absence of acrosome.In male mice homozygous for the blind-sterile (bs) mutation, acrosomes do not develop but the nucleus is elongated .Gba2 knockout mice  and Pick−/− mice have also been proposed as a valuable model for studying human globozoospermia [α show flagellum wrapping around nucleus. However, in a subsequent report, the authors observed that the shape of sperm head was not round but more or less oval, or hooked [Csnk2a2ospermia . Proteinr hooked . Lackingr hooked .On the basis of this review, it can be concluded that only those animal models that show both typical characteristics of globozoospermia, round nucleus and absence of acrosome, present coiled tail around the nucleus . Therefo DPY19L2 deletion [ DPY19L2 homozygously deleted men and those of spermatids from knockout mice was remarkably similar [Genetic analysis of our patient was not performed. Therefore, our hypothesis is based only on morphological evaluation. However, it is also supported by observation in patients with aWe found that a minority of sperm (7%) showed a normally straight tail. It can be hypothesized that, in these sperm, tail coiling process has not yet started, similar to what was described in the animal model . It shouThe precise mechanism of the coiling of the flagellum around the nucleus is not known yet. Sperm with coiled tail around the head were found in 91% of infertile patients, although at very low percentages (3%) . The sigIn conclusion, to the best of our knowledge, this is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia. Therefore, the significance of sperm tail coiling in human globozoospermia should be reconsidered. Our observations further support the validity of animal model for investigating the pathogenesis of this rare particular disorder and should encourage further research in this direction.


Pseudomonas aeruginosa.Microbial biofilm colonies will in many cases form a smart material capable of responding to external threats dependent on their size and internal state. The microbial community accordingly switches between passive, protective, or attack modes of action. In order to decide which strategy to employ, it is essential for the biofilm community to be able to sense its own size. The sensor designed to perform this task is termed a quorum sensor, since it only permits collective behaviour once a sufficiently large assembly of microbes have been established. The generic quorum sensor construct involves two genes, one coding for the production of a diffusible signal molecule and one coding for a regulator protein dedicated to sensing the signal molecules. A positive feedback in the signal molecule production sets a well-defined condition for switching into the collective mode. The activation of the regulator involves a slow dimerization, which allows low-pass filtering of the activation of the collective mode. Here, we review and combine the model components that form the basic quorum sensor in a number of Gram-negative bacteria, e.g.,  Over thVibrio fischeri, which populates the light organs of squids. QS ensures that visible light is only synthesized when a sufficient number of bacteria have amassed to meet the production requirement  are linked by the diffusion equation:Consider a spherical micro-colony of bacteria in a biofilm matrix of radius i.e., D is the diffusion constant and Δ is the Laplacian operator. The second term is the production of signal molecules per volume written as the product of the intracellular production, sκ, and the volume fraction occupied by cells, vρ. The signal molecules are assumed to diffuse freely through the cell membrane, and the diffusion constant in the biofilm matrix is similar to that in water.The first term is the diffusion term, 3.P. aeruginosa produces a regulator that appears to be dimeric in its active form , the production of signal molecules proceeds at a low baseline level, sb. At a higher concentration, the activated regulator is able to bind to the promoter site for the signal molecule with dissociation constant sK. The signal molecule production may therefore be expressed as:sb, when ar is lower than sk, when the concentration ar is above sK. In particular, note that the expected ignition point for feedback is at sK to produce a significant effect. In sk = 100sb.At a low concentration of activated regulator, 5.2. The signal molecule concentration is eliminated by inverting In the quasistatic limit of the reaction-diffusion equation, a simple dimensional analysis allows us to connect ar = [R2S2], as a function of the proper size, Σ = vρ2, of the system. The resulting plot is shown in mr = [R2S2]max. As expected, the ignition point is at This approximate form allows us to plot the activated regulator concentration, We refer to  for a de6.2S2, which targets the promoter site with the effect of increasing the signal molecule production. As indicated in 2 and R2S. Reports indicate that the monomer form in the LuxRI homologues is susceptible to proteolytic degradation, while the dimer forms are resilient , r2 = [R2], r3 = [R2S] and r4 = [R2S2]. The regulator formation and binding to the ligand is described by the kinetic equations:The signal sensor system in b1tR normalized to intracellular volume. On-rate constants are superscripted “+”, while off-rates are superscripted “−”. The dilution from cell growth is included in the proteolytic degradation, λi, of each form of the regulator. The combinatorial factor two has been explicitly included, with this normalization K3 = K4 for non-cooperative ligand binding.The regulator is continuously produced at rate r2s = K3r3, and r3s = 2K4r4.In comparison with the overall timescale set by the proteolytic degradation and the growth rate, the association and dissociation given in these equations can be regarded as fast. This results in the approximate quasistatic approximations The slow timescale can be solved for by regarding the total regulator economy, which is derived by summing i.e., λ2 = λ3 = λ4 = dλ. For this illustrative calculation, let us assume cooperative binding of signal molecules to the dimerized regulator. We then have:To simplify, let us assume that all dimer forms are equally resilient to proteolytic degradation, r1λ1 ≫ 2r2dλ, we will get a slow response to a sudden increase in S concentration. This is effectively a low-pass filter. In line with the result for a first order system [By changing the concentration of signal molecules, we can change the distribution between regulator forms rapidly, while changes in the total concentration of regulator will proceed slowly. This means that if, in the absence of S, we have r system , the lowr1λ1 ≪ 2r2dλ, we will get a fast response to an increase in S concentration. In the latter case, the introduction of signal molecules affects only the distribution between dimer forms, while the overall concentration of the regulator remains unchanged. In this limit, the introduction of signal molecules will not change λ, and we therefore get an all-pass filter and:On the other hand, if we have  K=K3K4 .et al. [et al. [P. aeruginosa [The low-pass and all-pass filter designs described here are analogous to the result derived for monomer binding by Garde et al.  and Clau [et al. . The all [et al. . Howeverruginosa ,21. The ruginosa . The det7.We have presented a view of a biofilm as a smart material and demonstrated how a size-aware sensor system, capable of producing an on/off switch at a critical system size, can be modeled. The generic quorum sensor consists of only two genetic components, one component coding for the production of a diffusible signal molecule and one component coding for a regulator protein capable of binding the signal molecules. A positive feedback leads to a well-defined ignition point:sk ≫ sb. If this is the case and if growth conditions allow efficient binding of the regulator to the promoter site for signal molecule production, mr ≳ sK, the switch will ignite when the activated regulator concentration reaches the critical value.The positive feedback loop is effective when the induction factor for the feedback is much larger than the background production, The role of the quorum sensor system plays its primary role during micro-colony formation, while most systematic studies of constitutive promoters are conducted on planktonically growing cells –27. TherPseudomonas aeruginosa [Pseudomonas aeruginosa, Pseudomonas fluorescens, Klebsiella pneumoniae, and Stenotrophomonas maltophilia range from (37 ±12) μm with flow to (51 ± 19) μm without flow [Staphylococcus aureus (30−40 μm thick) [Staphylococcus epidermidis (240−590 μm diameter) [Typical biofilm thicknesses range from 13 to 60 μm mean (34 ± 10) μm in ruginosa . Thickneout flow  with cor flow to 1 ± 19 μmsb ∼ 1000−10, 000nM/h [K ∼ 10−1000 nM [D ∼ 1 mm2/h. This results in the very rough estimatein vivo biofilm micro colonies.An estimate of the geometrical size needed to ignite the quorate state may be obtained using the above expressions. At the ignition point,  000nM/h , K ∼ 10−−1000 nM ,32, and P. aeruginosa, where several quorum sensors are thought to be organized in a hierarchy, where the rhl system acts under the control of the las system [As previously mentioned, the geometrical size and density at which the ignition occurs will depend on the internal state of the cells, e.g., growth rate and temperature. The size estimate given above may therefore vary, even within the same microbial strain. The complexity of the response of the biofilm to external conditions is further increased in, e.g., s system ,34.8.We have reviewed the key properties of a single-loop quorum sensor with positive feedback and the main properties of the ligand binding. The quorum sensor changes the biofilm from a passive material to an active material capable of responding to external threats in accordance with the quorate state.A proper measure for the size of the quorum sensing biofilm combines the geometry, cell density, and boundary conditions. When the maximal regulator concentration is sufficiently large to bind to the promoter for signal molecule production and the induction of the signal molecule production is at least an order of magnitude, a functioning quorum sensor with an all-or-none switch results.


The Royal Dutch Dental Association (KNMT) developed a reporting code for dental professionals (RCD). Moreover, an e-learning module about DV has been developed. A web-survey was conducted to investigate how general dental practitioners (GDPs) deal with the RCD and what their experiences are with (signs of) DV and CA.On July 1In April 2014 1038 GDPs were invited by e-mail to participate in a web-survey consisting of 24 items, through the KNMT Data Stations Project. The data was analyzed using SPSS .Of all GDPs invited to participate 264 (25 %) responded. 82 % of these GDPs are aware of their obligation to use the reporting code. 54 % of the GDPs are in favor of this obligation. 76 % of the GDPs have taken notice of the KNMT’s RCD and 51 % of the GDPs have implemented the reporting code in one form or another in their practice. 24 % of the GDPs stated having suspected DV during the last twelve months in the case of 2.4 patients on average. 81 % took note of this in the patient’s record and 58 % also took action in different ways. 54 % wants to complete the e-learning module.Most GDPs are aware of the new legislation and have taken cognizance of the RCD. Even though the majority of GDPs are not opposed to using a reporting code, over half of them have not yet implemented the code in practice. An important factor in this regard seems to be that a substantial minority of the GDPs says they are not sufficiently informed about aspects of reporting a case and about the steps they have to take. Domestic violence (DV) can be defined as threatening behavior, violence or abuse between adults who are relatives, partners or ex-partners. It also includes violence or child abuse (CA) from adults to children or abuse from (adult) children to parents . It can DV is a serious public health problem, which particularly affects women. Overall, 30 to 35 % of women worldwide have experienced either physical and/or sexual abuse, most of which is intimate partner violence . In the In general, the majority of physical injuries from domestic and other forms of violence are inflicted to the head, face or neck , 9. BecaHow often primary care clinicians and in particular general dental practitioners (GDPs) suspect DV or CA cannot be concluded from available literature. Ramsay et al. (2012) state that 71 % of primary care clinicians in the United Kingdom have diagnosed one or more new cases of DV in the last 6 months . In 2010However, it appears that in many cases GDPs who suspect DV or CA do not report their suspicions , 17–20. st 2013. This act makes it compulsory for organizations and independent  professionals to have a reporting code that meets the statutory requirements [Meanwhile, in the United States reporting child abuse and neglect is mandatory for health professionals in all 50 states . In seveirements . MoreoveIn order to support oral health care professionals to fulfill their obligations according to the Act, the Royal Dutch Dental Association (KNMT) developed the reporting code for dental professionals (RCD). The RCD contains an information brochure which explains the different forms of DV and neglect, the new legislation, the judicial aspects, how to ask for advice and how to report abuse. The RCD also contains the five step action plan. In 2012 this RCD was sent to all dental practices in the Netherlands. It is up to the GDP to decide how to fulfill the requirements, how to tailor instructions and how to decide what kind of training the dental team needs. In cooperation with two other dental professional associations and with Augeo Academy, the KNMT developed an e-learning module about DV and the reporting code called ‘The Next Page’ . The modThe aim of this study is to investigate to which extent Dutch GDPs are aware of the RCD, if they have implemented the RCD and if so, how they implemented it. This study will also investigate how often dentists have had suspicions of DV or CA and how they dealt with their suspicions in the most recent case.By means of its Data Stations Project the KNMT periodically collects data on delivery of oral health care, on practice management and on GDPs’ opinions and views regarding current issues in dentistry in the Netherlands . For thiThe online questionnaire covered 24 items. GPDs were asked about their acquaintance with the RCD and whether or not they had actually perused this or a different reporting code, how they evaluated the content and the ‘look and feel’ of it and if they had implemented the RCD in their practice. Subsequently, their opinions on the effect of the RCD were gauged using four Likert type items. Furthermore, questions were asked about any suspicions of DV or CA in the last 12 months. If GDPs stated that they have had suspicions, some specific details were asked about their professional behavior concerning the most recent case in which suspicion of DV or CA was raised. GDPs who did not respond after 2 weeks and consecutively after 5 weeks, received a reminder e-mail.p < 0.05), but all associations proved to be very weak (Cramèr’s V < 0.10 and eta2 < 0.05).Ultimately, 264 (25 %) of the 1038 GDPs in the sample completed the questionnaire. With regard to some individual characteristics, the total group of respondents proved to be fairly representative for the population of GDPs in the Netherlands  in order to determine if one or more additive scales could be formed.A Cronbach’s alpha of 0.80 or greater was considered as a criterion for a reliable scale.Chi-square tests were used to test the correlation between nominal items and by analysis of variance differences between group means were tested.st, 2013, dentists and other care workers are compulsory to adhere to a reporting code in case of suspicion of DV or CA. It appears that 54 % is more or less positive about this obligation, while 35 % holds a neutral position. All others (11 %) are negative about the obligation to use a reporting code, most of whom (69 %) stated that they regard themselves not adequately equipped to judge on DV and CA.Most (82 %) respondents stated that they were aware of the fact that since July 1The majority (76 %) of GDPs indicated that they received information in some way about the brochure and/or the action plan of the RCD Table . Most GDp < 0.01). They also more often answered that they are sufficiently informed about the background of the reporting code , the aim of the reporting code , the various forms of DV and CA , the difference between asking advice and reporting a case  and the implications of reporting a case . Furthermore they more reported to be well informed about the five steps .Table All in all, 30 % of GDPs stated that the RCD had influenced their alertness and the way in which they take action Table . Almost n = 58) stated that they have had suspicions of DV or CA in the last 12 months in the case of 2.4 patients on average. These GDPs are more often female  and on average younger  than GDPs who have not had any suspicions of DV or CA. Moreover, these GDPs have implemented the RCD much more often .Twenty-four percent of the GDPs (Table p < 0.01) and have implemented it in their practice more often .GDPs who did not take action said they felt unsure about whether or not their suspicions were correct (88 %). GDPs who wrote down their suspicions of DV or CA in the patient’s record and/or took action are, in comparison to those who did not, more often aware of the RCD . Moreover, they have implemented the RCD more often  and they have had suspicions of DV or CA in the last 12 months . In more cases, they take a positive stance with regard to the legal obligation to use the reporting code . The RCD also has had more influence on them .Sixty-one percent of GDPs wants to complete the e-learning module ‘The Next Page’ and/or thinks it is important that their team complete the module Table . These GThis study shows that a large majority of GDPs was aware of the obligation to use a reporting code in 2014. Over 50 % of GDPs viewed this as positive. Those who were not positive usually felt unsure about the issue of DV and dealing with it. Most GDPs are also aware of the RCD. The majority of GDPs who have had suspicions of DV or CA took some form of action.The study gives some insight in why almost half of the GDPs have not implemented the reporting code into their practice. It appeared that these GDPs more often failed to take notice of the RCD than those who implemented the code. Moreover, there seems to be a connection between failure to implement and the information in the RCD. After all (proportionately) many GDPs who did not implement stated that the RCD insufficiently informed them about the background and aim of the reporting code, about the various forms of DV and CA, about aspects regarding asking advice and reporting a case and about the five steps they have to take.The information in the RCD is apparently not able to clarify the issue to all GDPs. Admittedly, Dutch legislation is complicated. GDPs, even though they are not qualified to diagnose DV or CA, are expected to judge suspicions based on their own observations and to follow the guideline which consists of five rather difficult steps. This starts with ‘identifying the signs’. According to this study, a large number of GDPs are unsure about the correctness of their suspicions. They are required to talk about their suspicions with experts. But the apparent unclarity regarding the task and the function of the Advice and Reporting Centers can form a barrier here and the same holds true for the judicial aspects of the confidentiality agreement. The next step is talking to the patient or their parents. This is not an easy task either when it comes to suspicions of DV or CA. Lastly, the GDP is supposed to decide whether or not to report his or her suspicions. What is best for the patient? How can I make sure not to damage the relationship with the patient and between the patient and their family? And are the signals strong enough to justify taking action? As becomes clear from the literature, GDPs and caretakers from other countries are also wrestling with these problems. For example Scottish GDPs, whose failure to take action is related to lack of certainty of the diagnosis  and or fThis does not change the fact that the reporting code is a legal obligation and that the inspectorate can ask individual GDPs to provide concrete details of how they implemented the code in their practice, what they have done to train the dental team in order to promote awareness and use of the reporting code and what their plans are for the coming year. It is therefore important for GDPs to be aware of their obligations. The KNMT should inform GDPs about these matters. Offering practical support with regard to using the RCD is also of pivotal importance when it comes to helping the GDP and the dental team in the diagnosis and documentation . After aFurthermore, it appears that educational interventions such as online tutorials and an e-learning module seem to have positive effects in particular when it comes to knowledge of DV and recognizing it –32. BaseMoreover, the Mandatory Reporting Code DV and CA makes it compulsory for health professionals like GDPs to promote the awareness and use of the code within their practice. This means that by offering an e-learning method the KNMT meets these educational needs. Most GDPs appear to appreciate this initiative.Finally, a critical evaluation of the results of this study is needed. The relatively high number of non-response raises questions. Regardless of a certain ‘research fatigue’ GDPs may experience (which plays a role in every survey), the subject of this research may also have been a reason not to respond. For example because of disinterest, but more importantly, insecurities regarding how to handle suspicions of DV and CA and fear of acting legally insufficient may play a role. Presumably, the percentage of GDPs in the Netherlands that have not implemented the reporting code in their practice is higher than the 49 % this study found. In relation to this, a certain amount of ‘socially acceptable answers’ should be taken into consideration. This is likely with regard to for example implementing the reporting code in their practice as well as taking action when GDPs have suspicions of DV or CA.All in all, it can be concluded that the large number of GDPs who do not (yet) comply with the new legislation do not fail to do so because of unwillingness, but merely because of inexperience and insecurities with regard to handling signs of DV and CA. This survey shows that a reporting code like the RCD can be a good support to GDPs and can encourage them to be alert on signs of DV and to report these. Moreover, the survey shows that the KNMT should pay close attention to the ways in which GDPs are coping with their legal obligations and to possible problems that may arise. Meanwhile, the legislation in the Netherlands has been adjusted in a number of ways. The KNMT is working on an updated version of the RCD, which will be published in 2015. In this version, partly in response to the conclusions of this study, clear information on the new legislation and steps that need to be taken is of pivotal importance.st 2015 the Dutch government transferred the responsibility for the approach of DV and CA to municipalities. They have to make their own agreements with certified agencies responsible for child protection measures in their region, which leads to regional differences. Fortunately, the Dutch government is aware of the importance of monitoring this issue. In 2015, the Ministry of Health Welfare and Sport (VWS) ran a quick scan in order to investigate the workings of the Mandatory Reporting Code DV and CA. This quick scan should provide insight into how professionals experience work with the Reporting Code.In the future the KNMT should investigate whether or not GDPs are better informed as a consequence of the updated RCD and whether or not they meet the legal requirements. In this context, the KNMT should also take stock of possible problems regarding dealing with (suspicions of) DV and CA. However, this is also a public health issue, and more attention should be paid to making this an issue of public interest. When society regards these issues as highly important, it will be easier for GDPs to implement this new legislation in their daily practice. Future research should then focus on the effect of (early) reporting DV and CA. A complicating factor is that on January 1