Intraprofessional collaboration (intraPC) between primary care (PC) doctors and medical specialists (MSs) is becoming increasingly important. Patient safety issues are often related to intraPC. In order to equip doctors well for their task of providing good quality and continuity of care, intraPC needs explicit attention, starting in postgraduate training. Worldwide, PC residents undertake a hospital placement during their postgraduate training, where they work in proximity with MS residents. This placement offers the opportunity to learn intraPC. It is yet unknown whether and how residents learn intraPC and what barriers to and opportunities for exist in learning intraPC during hospital placements.We performed an ethnographic non‐participatory observational study in three emergency departments and three geriatric departments of five hospitals in the Netherlands. This was followed by 42 in‐depth interviews with the observed residents and supervisors. The observations were used to feed the questions for the in‐depth interviews. We analysed the interviews iteratively following the data collection using template analysis.Hospital wards are rich in opportunities for learning intraPC. These opportunities, however, are seldom exploited for various reasons: intraPC receives limited attention when formulating placement goals, so purposeful learning of intraPC hardly takes place; residents lack awareness of the learning of intraPC; MS residents are not accustomed to searching for expertise from PC residents; PC residents adapt to the MS role and they contribute very little of their PC knowledge, and power dynamics in the hospital department negatively influence the learning of intraPC. Therefore, improvements in mindset, professional identity and power dynamics are crucial to facilitate and promote intraPC.Intraprofessional collaboration is not learned spontaneously during hospital placements. To benefit from the abundant opportunities to learn intraPC, adjustments to the set‐up of these placements are necessary. Learning intraPC is promoted when there is a collaborative culture, hierarchy is limited, and there is dedicated time for intraPC and support from the supervisor. While critically important, intraprofessional collaboration is not learnt spontaneously. Improvements in mindset, professional identity and power dynamics are crucial to its promotion. In many countries, PC residents, such as general practitioner residents and elderly care physician residents see Box , undertaBox 1This study aims to gain insight into the potential of hospital placements for learning intraPC, by answering the following questions: (a) When and how do PC residents and MS residents learn intraPC during hospital placements?, and (b) What are opportunities for and barriers to learning intraPC during these placements?2We carried out a constructivist ethnographic study. A constructivist approach acknowledges that researchers’ background assumptions, disciplinary perspectives and programmatic efforts along a line of study shape their research processes and conceptual emphases. Therefore, in our study, particular time and attention were paid to reflexivity throughout the research process on how our assumptions and perspectives have shaped our data collection and interpretation. The research group consisted of general practitioners, educational scientists, a psychologist, an internist, a geriatrician and a medical student. All group members were experienced in providing intraPE and/or conducting research into intraPE in different contexts. This multidisciplinary research group functioned as a form of triangulation as it brought together disciplines whose profession or training calls on highly different assumptions and knowledge areas.2.1We used a non‐participatory rapid ethnographic research approach.2.2Using purposeful sampling techniques, we sampled emergency departments and geriatric departments of both academic and regional hospitals in the Netherlands. After inclusion, we announced our visit with posters and emailed an information letter explaining the purpose of our study, including an invitation for the interview to all residents and supervisors. For the interviews, we applied purposive sampling, including snowballing. We sampled younger and older residents and supervisors and we talked about the results with participants. This allowed us to gather broad and deep information on learning intraPC during hospital placements. We excluded residents and supervisors who worked in the hospital department for less than 1 month.2.3Data collection through observations and in‐depth interviews was piloted by one researcher (NL). Two researchers (NL and MvW) then performed the observations and in‐depth interviews. Prior to our visit, we agreed with the supervisor, which moments would be observed. Work‐related activities and settings with potential intraPE moments were observed, for example, educational sessions, team meetings, mutual consultations and daily administrative work practice. Both researchers (NL and MvW) were familiar with the context of hospital placements.The interviews were semi‐structured; the interview script Appendix  was desi2.4Transcripts of the interviews were analysed using a template analysis method.The vast quantity of data, comprising 45 hours of observation and 42 interviews, made analysing data and finding patterns complex.The research group  discussed the data iteratively; all inconsistencies in applications of the codebook were discussed and resolved through consensus. Based on the discussions, NL adjusted the template. During the coding process NL discussed the results with CF, who is a researcher in the field of workplace learning. These discussions helped challenge NL’s interpretation of the data and introduce alternative interpretations. After analysing the interviews, the fieldnotes of the observations were re‐read to check for discrepancies between fieldnotes and interview data. The number of observations and interviews was determined by theoretical sufficiency.2.5This study was reviewed and approved by the NVMO  Ethical Review Board (NERB dossier number 983). Written informed consent of all participants was obtained before participation. In some cases, nurses, (para)medical professionals and medical students were visible during the observations and therefore they were asked for informed consent to be observed, after receiving an information letter on the day of our observations.3We conducted 45 hours of observations (10‐360 minutes per observation) and 42 interviews (18‐50 minutes per interview) with 14 PC residents, 14 MS residents and 14 supervisors at three emergency departments and three geriatrics departments of five hospitals from February to May 2018 Table .To me, it [intraPE] is super important, and it should receive more attention. (MS‐resident1_H3)A prevailing view amongst all PC and MS residents and supervisors was that intraPE is essential and needs explicit attention, including dedicated time.All participants indicated that hospital wards are rich in opportunities to learn intraPC. To actually benefit from these opportunities, interventions are needed. After categorisation of the results, we identified three main themes: (a) incidental and purposeful learning; (b) competing professional roles, and (c) work environment. In relation to these three themes, residents and supervisors mentioned clear recommendations for the introduction and implementation of intraPE during hospital placements.3.1Our data showed that learning intraPC on the hospital ward occurs by two routes: (a) incidental (implicit learning activities), and (b) purposeful (explicit learning activities).3.2Some attention is paid to intraPC, but it is not really high on the agenda. You do notice that they know PC‐residents are walking around and sometimes get questions for example ‘is that possible in the nursing home?’ or ‘how do you see that as GP?’ or ‘How would you feel if we discharge such a patient?’ This kind of interaction happens spontaneously. (PC‐resident1_H2)The majority of intraprofessional learning activities occurred implicitely during daily work activities. Though working, PC residents learned about the daily hospital routine: substantive medical skills; how to best refer a patient to the hospital, and how to formulate an adequate referral question. The PC residents usually learned these skills from the supervisors in the hospital department. Supervisors also operated as a role model in intraPC; MS residents often copy their behaviour. The MS residents learned about possibilities and limitations in the PC setting and referral patterns, mostly from PC residents. Residents mentioned that the learning of intraPC mainly occurred incidentally, without conscious reflection.3.3We observed that intraPE is purposeful and planned in some departments, especially in departments with a collaborative culture, dedicated time for intraPE and intraPE mindset of the supervisor see Box .Box 23.4I always try to share my PC‐vision […] It is a positive thing, that they really appreciate it that I have a vision as a general practitioner. And I also get to hear that they [supervisors] really like it that I contribute my PC‐opinion. That is of course stimulating. (PC‐resident1_H5).The PC residents indicated that some supervisors consciously stimulate interaction between PC residents and MS residents and encourage PC residents to show their PC expertise.What I mention about just [the use of] theories about collaboration, we haven't done it before, but lately occasionally. Yes as a doctor, you know very little about that, we just do it. Sometimes it goes well and sometimes it doesn't work. Things like that are, I should delve into it [theory about collaboration]. (Supervisor2_H2).Supervisors mentioned that they find it difficult to coach and assess the learning of intraPC for residents from different backgrounds. Their expertise is based on specialist medical knowledge and skills, and they feel competent in teaching in this area, but offering intraPE poses specific demands beyond their primary expertise. In order to provide intraPE, supervisors feel a need to study new knowledge and skills regarding collaboration. (Supervisor2_H2)3.5I can ask her [PC‐resident] ‘how are things organised in your PC‐setting?’ We do discuss such things, and she tells me a lot about that […] But really learning to collaborate, no. We are each on our own island, and you occasionally ask something about ‘how are things going on your island. ‘ (MS‐resident1_H2)Both PC residents and MS residents indicated that learning intraPC is essential, but they are not always aware of opportunities to learn this. Residents are accustomed to operating and learning separated from each other and indicate that intraPE is generally not in their mindset.Of course I learn a lot from his or her [MS‐resident] knowledge and skills […] Conversely I have the idea that they learn less from us. And that they do not really want it either. Then it is a bit of one‐way learning. (PCߚresident1_H5)The MS residents often teach PC residents about their medical specialty and how the hospital is organised, but they hardly ever ask for PC expertise, with the result that PC residents think that MS residents do not want to learn from them. This means that learning is predominantly unidirectional.PC‐residents have different [training] goals and portfolios than MS‐residents. And they [PC‐residents] steer very much on their own learning objectives. So then you might have to make a standard learning goal for them. That you say that the collaboration between PC‐residents and MS‐residents is one of the learning goals for all PC‐residents who come here. (Supervisor2_H6)Residents and supervisors reported that intraPC receives at best limited attention in the training programmes as a competency to be learned during hospital placements. Therefore, the learning predominantly depends on residents’ individual mindsets on learning intraPC to formulate learning goals within this domain. Some supervisors would like PC residents to be obliged to formulate a learning goal for intraPC, but supervisors indicated that they never insisted that MS residents should formulate such a learning goal.[Question: Are learning intraPC and sharing PC expertise themes during release days?] Very few actually and that surprised me. I thought that there would surely be attention (for intraPC), also for the collaboration with the elderly care physician. But that [intraPC] is actually not discussed at all. (PC‐resident2_H6)The PC residents expected attention would be given to intraPC during release days, where PC residents learn amongst their peers at the PC specialty training institute once a week, but they indicated that intraPC receives only limited attention.3.6The PC‐resident steps into our MS‐role and that is also what is [implicitly] expected. It is a fact that they act just as MS‐residents; they have to drop their PC‐role to say the least. (MS‐resident1_H6)During handovers, when we discuss the patient's discharge we don't know if a GP can do anything with our suggestions. Then, we should explicitly invite PC‐residents to say how their view is; they don't do that on their own. (Supervisor2_H3)The observations and interviews showed that PC residents often adjust to the role of MS resident, providing specialist medical care during their hospital placement as if they are in training for that MS specialty. The majority of PC residents hardly ever share their PC knowledge and skills, except when invited.And he [MS‐supervisor] told me ‘you have to ask hard questions to the other disciplines like how far will we go in our decisions?’ And then I thought, of course, that is actually something I normally do in primary care. Well, I won't say that I really forgot it, but I think that I was too much in the MS‐role. (PC‐resident2_H6)The PC residents, who continued to adapt to the role of MS resident and barely expressed their professional PC identity, sometimes even were not aware of their PC knowledge and skills.I also give some kind of information back to the specialists which they can use. I see myself more as a general practitioner within the ER I know something about emergency cases, and I also know a lot about general practice. With that, I can also put them [MSs/MS‐residents] in the right direction. (PC‐resident1_H4)The PC residents, who easily switched between the MS role and their professional PC identity, were more explicit and proactive in demonstrating their PC expertise.3.7A prevailing view amongst participants was that learning intraPC between residents is only possible when a safe work‐learning climate and significant practicalities are secured.3.8The medical specialists are sitting at the head of the U‐form table, and the MS‐residents are sitting on the sides of the table. The PC‐resident is sitting on the second row, together with undergraduate students. The PC‐resident is the only doctor who have to take place second rank between the students. (Fieldnote_R2_H4)We observed that the placement of residents within the room during team meetings can reflect (in)equality. Within some departments, everybody was seated equally in the room. In other departments, PC residents were not sitting around the table amongst other MS residents, but sitting or standing in the second rank. The discussion took place at the table and the second rank (PC residents) were acting as spectators. The placement of residents was affecting the chances for intraPE.Sometimes medical specialists talk about primary care doctors in a negative way, like it's an inferior specialism. And sometimes I hear such comments during meetings between shifts, that is of course demotivating. (PC‐resident3_H1)Another essential aspect to creating a safe work‐learning climate is ‘knowing each other,’ for example, by having a drink together outside the ward. Residents and supervisors who know each other informally report that they get in touch with one another more easily, understand why people react the way they do and are more likely to invest in each other. Participants mentioned that hierarchy, such as that between supervisor and resident, is useful as it clarifies roles and responsibilities within the hospital. Nevertheless, they indicated that too much power dynamics in the hospital ward can lead to a lack of respect and inequality, which has a hindering effect on building a relationship in order to get to know each other's expertise. Supervisors and residents mentioned that the way MS speak about PC doctors can be responsible for creating a (un)safe work‐learning climate for intraPE.Participants mentioned that supervisors are in the position to steer power dynamics, and within some departments supervisors showed an active policy against unconstructive power dynamics.3.9We are in a set‐up in which we sit in a circle (behind computers) and where you easily pick up things from each other. And then an interesting  discussion, a case‐based discussion arises spontaneously. (Supervisor2_H5)Intraprofessional education can hardly take place when PC and MS residents are working in different shifts or having different offices. Supervisors and residents indicated that the opportunity to meet each other is necessary for intraPE to take place. This is possible by sharing physical space together.3.10It [intraPE] must be integrated in the placement/work structure. When it is something optional or incidentally, then it will not work out. (Supervisor2_H3)For example case‐based discussions, where we discuss the kind of cases that we all recognize. And that we also hear their [PC‐residents’] side of the story and also hear from them what they encounter when collaborating with us, and vice versa. I think that is very important. (MS‐resident1_H6)For us [MS‐residents], a placement in a nursing home would also be a very good idea. That is not at all in our training program […] It can sometimes be quite difficult if you have no idea at all about how it works in a nursing home […] I think it is very good that we make that more transparent and learn from each other. (MS‐resident1_H3)I think that we will only get a real collaborative relationship if they [MS‐residents] also come along when I am in the primary care practice. (PC‐resident1_H6)In relation to the above themes, residents and supervisors mentioned clear recommendations to identify the different workplace activities for learning of intraPC and to explicitly integrate workplace opportunities: (a) the specific organisation of work context by creating actual possibilities for learning intraPC; (b) explicit and purposeful learning of intraPC during workplace activities, by both PC residents and MS residents; (c) supervisors taking responsibility for intraPE by facilitating a constructive work‐learning climate and further professional development in intraPE; (d) intraPC as a placement goal for both PC residents and MS residents; (e) empowerment of PC residents to share their PC expertise; (f) empowerment of MS residents to ask for PC expertise, and (g) offering placements for MS residents in the PC setting.4All participants found intraPC essential for good health care and consider hospital wards to be rich in opportunities for learning intraPC. However, we also report that these opportunities are seldom exploited for various reasons. First, intraPC learning goals are often not apparent and both residents and supervisors lack awareness of the intraPC learning opportunities. When learning intraPC occurs, it is predominantly implicit. Second, PC residents often adapt to the role of MS resident and hardly ever share their PC expertise. The MS residents often neglect to search for PC expertise. Third, too much hierarchy led to inequity, which had a hindering effect on building relationships and formed a not‐safe‐enough work‐learning climate in which residents did not feel free to speak up. Therefore, improvements in mindset, professional identity and power dynamics are crucial to facilitate and promote intraPC.4.1When learning of intraPC occurs between PC residents and MS residents, this is mostly random through informal mechanisms: the learning occurs implicitly, spontaneously and with little conscious reflection, which is in line with the description by Watkins and Marsick of informal and incidental learning.Frequently, mindset is associated with the growth mindset theory from Dweck.The above indicates that intraPE is the responsibility of all parties involved: PC residents; MS residents; supervisors; teachers, and programme directors.4.2In the Netherlands, the purpose of hospital placements for PC residents is to gain expertise in emergency care and diseases that are not very prevalent in a PC setting and to learn intraPC with medical specialists. We found that PC residents often adapt to the role of MS resident. This is useful for learning medical skills and fitting into the hospital team. However, the majority of PC residents hardly ever share their PC expertise. This is counterproductive for learning intraPC. At first glance, the PC resident appears to have little influence on the dynamics of an expert team within the hospital ward. However, our results show that also temporary team members can bring a fresh eye to common practices. We found that PC residents who expressed their professional PC identity and easily alternated between the MS role and PC role, created intraPC discussions and bidirectional learning. Previous literature shows that pre‐existing teams are more receptive to the influence of newcomers when the newcomers are more assertive.4.3terPE confirm our findings.The participants mentioned that hierarchy is useful for clarifying roles and responsibilities within the hospital, but too much hierarchy can create inequity. Power is enhanced through the hierarchies in which residents interact.4.4A strength of this study is the use of four types of triangulation: (a) method; (b) data source; (c) investigator, and (d) research group triangulation.We consider the variability in the nature of the observations as a strength. The short observations consisted of five meetings lasting less than 15 minutes. These were meetings to start the day in an interprofessional way. Although short, these meetings provided us with very rich observations with respect to (opportunities for) intraPE. Because our observers were familiar with the context of hospital placements they could easily recognise relevant activities. Another strength is the cooperative attitude of residents and supervisors in participating in this study; we had to cancel some hospitals, which had applied to participate, after conceptual depth was reached. Because of this cooperative attitude, we could get a rich conception of the potential of hospital placements for learning intraPC.We acknowledge several limitations. Our presence during observations may have had an impact on the participant reactivity, which is defined by Paradis and Sutkin as: ‘a form of participant effect that comes from participants’ active engagement with the research and its aims, leading to behavioral adaptation that aligns with perceived social norms.‘4.5When organising the learning of intraPC through placements for residents from different medical backgrounds, we think the following should be kept in mind. First, informal learning can be planned or unplanned, but it involves at least some conscious reflection.5All residents and supervisors indicated that learning intraPC is essential and requires more explicit attention. IntraPC is not learned spontaneously during hospital placements. Even in a promising setting where PC residents and MS residents work together in the same department, intraPC receives at best limited attention as a competency to be learned. The MS residents are not accustomed to asking for PC expertise and PC residents often adapt to the role of MS resident and they hardly ever contribute their PC knowledge. Hierarchy and a lack of psychological safety in the hospital department negatively influence the learning of intraPC. We conclude that in order to benefit from the opportunities to learn intraPC during hospital placements, attention to mindset, professional identity and power dynamics is needed. Learning intraPC is promoted when there is a collaborative culture (with not too much hierarchy), dedicated time and goal setting for intraPC and support from the MS supervisor on the ward and PC teachers during release days.All authors  met all of the following conditions: substantial contributions to the conception and design of the work; the acquisition, analysis and interpretation of data for the work; drafting the work and revising it critically for important intellectual content; final approval of the submitted paper and the version to be published; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy and integrity of any part of the work are appropriately investigated and resolved.There are no competing interests.We asked the NVMO  Ethical Review Board for ethical review of our research proposal on 15 December 2017. The NVMO Ethical Review Board approved the study on 15 February 2018, dossier number 983.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.


The human microbiome research is with the notable exception of fecal transplantation still mostly in a descriptive phase. Part of the difficulty for translating research into medical interventions is due to the large compositional complexity of the microbiome resulting in datasets that need sophisticated statistical methods for their analysis and do not lend to industrial applications. Another part of the difficulty might be due to logical flaws in terminology particularly concerning ‘dysbiosis’ that avoids circular conclusions and is based on sound ecological and evolutionary reasoning. Many case–control studies are underpowered necessitating more meta‐analyses that sort out consistent from spurious dysbiosis–disease associations. We also need for the microbiome a transition from statistical associations to causal relationships with diseases that fulfil a set of modified Koch's postulates for commensals. Disturbingly, the most sophisticated statistical analyses explain only a small percentage of the variance in the microbiome. Microbe–microbe interactions irrelevant to the host and stochastic processes might play a greater role than anticipated. To satisfy the concept of Karl Popper about conjectures and refutations in the scientific process, we should also conduct more experiments that try to refute the role of the commensal gut microbiota for human health and disease. Microbiome research has with the exception of fecal transplantation not yet reached the level of industrial applications with respect to gut microbiome engineering. Part of the difficulty might be the\complexity of the system, but some problems might reflect imprecise definitions like that for dysbiosis. Refutations of concepts should be part of the scientific process. However, despite clear data on its efficacy . Furthermore, the basic concept of this approach predates microbiome research  Research Network Consortium,  de Vos, , and defThe problems confronting microbiome‐derived products for industrial applications are manifold. It might surprise that one hurdle is a question of basic scientific definition, an issue that should not be dismissed as of purely semantic importance. Nearly 2000 years ago, a founding father of medicine stressed the importance of clear scientific terms when writing ‘First, however, we must distinguish and explain clearly the various terms which we are going to use and to what things we apply them; and this will prove not merely an explanation of terms but at the same time a demonstration of the effects of nature’ . The proad hoc definition or specification. This neglect illustrates not only a lack of scientific rigour, but might in fact hamper progress in the microbiome field from a descriptive into a translational science. Over recent years, criticism of the undefined use of the ‘dysbiosis’ term has been repetitively articulated. In a short comment, ‘Dysbiosis is not an answer’, Olesen and Alm  ratio among obese and non‐obese individuals. A pioneer study was done with small subject numbers (12 obese and 5 lean subjects), showing that the B/F ratio increased with time on a calorie‐restricted diet  differed between the Chinese and Danish T2D cohorts, pointing to population‐specific effects  of this most widely used antidiabetic medication. Case–control studies must therefore be stratified for medication in order to provide reliable microbiota–disease associations. Otherwise, microbiota changes could simply be a consequence of disease treatment.Medication for everyday life conditions had the greatest impact on microbiota composition in the Belgian FGFP and the Dutch LifeLines DEEP studies  patients could be differentiated . Proponents of this concept state that all animals and plants establish symbiotic relationships with microorganisms, which are transmitted between generations and affect the fitness of the holobiont within its environment, thus leading to a type of superorganism and a new unit of selection in evolution  in Bangladesh, we realized that the faecal microbiome of the patients displayed a marked dysbiosis with increased streptococcal abundance compared with local healthy control children  and genome sequences from stool isolates showed no virulence factors (Sarker et al., et al., et al., et al., E. coli increase documented with both abundance increase and absolute titre increase compared with control children. While E. coli could be a pathogen for these forms of diarrhoea (Sarker et al., et al., Instead of ending with this rather theoretical outlook, I will shortly mention our own experiences in the dysbiosis–health and disease field. When we tried to treat children suffering from Acute diarrhoea is an interesting test case for microbiome research since the disease is of short duration and self‐limiting. It represents a natural perturbation of the physiological gut microbiota equilibrium and its disturbance by the pathological process and the re‐establishment of a new equilibrium holds promises to understand the mechanisms of microbe–microbe interaction in the gut.et al., Medical interventions, like antibiotic treatment or gut cleansing in preparation for colonoscopy represent interesting research opportunities for microbiome analyses since all enrolled subjects are informative and biological samples can be easily obtained before, during and after interventions (Fukuyama A critical test for a causative effect of dysbiosis on disease can only be provided by prospective studies where microbiota composition is regularly established for all participants before diseases are occurring. Such approaches are labour‐intensive and costly, even for diseases occurring with high frequency, like diarrhoea in children from developing countries. If the suspected disease‐causing dysbiosis is then observed in subjects before they develop the manifest disease, but not in age‐ and milieu‐matched children, who do not develop the specified disease, a causative role can be reasonably anticipated. A definitive proof will still need evidence for a mechanism of action by the dysbiosis. We are currently conducting a birth cohort study in Bangladesh with nearly 300 children who are clinically followed over a 2‐year observation period combined with regular microbiota sampling. Longitudinal studies and studies that fulfil the ‘commensal Koch's postulates’ are needed to put the gut microbiota dysbiosis connections with human health and disease on a firm scientific basis. Such claims might sound like scientific rigourism since it implicates many years of further research. However, we should be aware of what is needed for a proof in the dysbiosis/microbiome field, so as not to be tempted into premature conclusions and thus creating unrealistic hopes for translation of microbiome research into amelioration of human health.C. difficile infection is a sign of hope (Tariq et al., et al., Clinical intervention trials are finally the critical test for the practical value of microbiome research. The success of faecal transplantation in None declared.


A versatile and straightforward protocol is disclosed for controlled synthesis of complex lanthanide‐bridged heteroleptic porphyrin–phthalocyanine triple‐decker assemblies. Two porphyrins, linked by a flexible spacer chain of intermediate length, sequentially capture lanthanide ions and a phthalocyanine to efficiently form the triple‐decker complex. The bridge directs assembly, but also controls the mobility of the central macrocycle and further imparts a fully eclipsed arrangement of all three rings. A versatile and straightforward protocol is disclosed for the controlled synthesis of complex lanthanide‐bridged heteroleptic porphyrin–phthalocyanine triple‐decker assemblies (see scheme). The bridge directs assembly, but also controls the mobility of the central macrocycle and further imparts a fully eclipsed arrangement of all three rings. When metalation was complete, 2 equivalents of phthalocyanine were added and reflux continued overnight.As part of a wider project to assemble high‐order, machine‐like constructs from functional porphyrin and phthalocyanine building blocks, we performed model studies on the construction of multidecker systems on a porphyrin dyad platform. The experiment and key discovery are shown in Scheme 6 alongside the bridged triple‐decker 7. There was no evidence for formation of the corresponding bis‐double‐decker. Repeating the reaction using excess phthalocyanine led to 6, whereas using one equivalent of phthalocyanine led exclusively to 7 as a single product. The reaction is reproducible and the triple decker (TD) is robust. Full characterisation revealed several interesting features. X‐ray crystallographyThe resulting reaction mixture contained mostly just two products  and separation and analysis revealed formation of bis‐triple decker 1H NMR spectroscopy reveals a single pair of signals for the central phthalocyanine macrocycle, implying rapid rotation on the NMR timescale.In solution, absorption spectroscopy shows the TD arrangement with characteristic long wavelength absorptions extending to the near IR.8 and 9 . However, further detailed characterisation revealed that the two complexes are significantly different. TD 10, formed from the peripherally substituted phthalocyanine 8 gave an absorption spectrum that was essentially identical to parent TD 7. Its 1H NMR spectrum also shows characteristic features of the parent system, but it is clear that the long sidechains are no longer able to pass the bridging chain and therefore they prevent free rotation of the central phthalocyanine macrocycle. In this situation the aromatic protons of the central macrocycle are no longer equivalent and are observed as 4 singlets  previously observed in TDs for the inner phenyl protons of the porphyrins are absent. The absorption spectrum is also different and bears much closer similarity to the spectrum expected for a mixture of metalloporphyrin + phthalocyanine‐porphyrin double‐decker. The preliminary conclusion in this case is that the added steric demand of the central phthalocyanine, in which the sidechains are displaced above/below the macrocycle plane,The assembly formed from non‐peripherally substituted phthalocyanine 11 , neodymium (13), samarium (14) and europium (15) TDs proceeded smoothly in a single unoptimised operational step by directly heating a mixture of dyad 5, lanthanide salt and PcH2 in octanol  and the pure products gave absorption spectra characteristic of TDs, and as expected the inclusion of NdIII and EuIII led to dramatic expansion of the chemical shift range observed in the NMR spectra. The salts of smaller ions Tb (92 pm), Dy (91 pm) and Yb (87 pm) failed to produce easily isolable TDs suggesting a cut‐off point for stability in the constrained assemblies. More detailed examination of the reactions employing Dysprosium ions verified this preliminary conclusion. The reaction between porphyrin dyad 5, phthalocyanine and dysprosium (acac)3 in octanol was monitored carefully at intervals. The formation of two new (high mass) products was observed, but prolonged heating failed to give complete conversion, with unreacted porphyrin dyad 5 always present. MALDI‐MS analysis indicated that the newly formed products were the dysprosium linked TD 16 (m/z=2235) and the product from double‐decker formation between one porphyrin of the dyad with phthalocyanine . This second double‐decker product could be isolated in sufficient purity to allow preliminary characterisation, but the symmetrical TD complex 16 always contained traces of the double‐decker 17, and it was demonstrated that the TD 16 decomposed to 17 on storage and on silica. The difference in stability between one addition (double‐decker) and two (TD) is likely to be a direct consequence of the role of the bridging chain, which directs an eclipsed arrangement in the (low stability) TD. The potential for controlled sequential assembly of heterometallic TDs was demonstrated through reaction of intermediate 17 with lanthanum (acac)3. The La‐Dy TD 18 was indeed formed as an inseparable mixture alongside the symmetrical La‐La TD 7 (the product of TD formation and further exchange of La for Dy). Prolonged reaction with an excess of lanthanum salt led to complete exchange and formation of the La‐La TD 7 exclusively.A particular attraction of the lanthanide‐bridged double and triple decker assemblies is the potential for generation of (single molecule) magnetic materials through selection of alternative ions from the lanthanide series. Ion size contracts across the lanthanide series so the combination of simple synthesis protocol alongside the strain and conformational constraints imposed by the link chain has the potential to offer control in the construction of bespoke TD systems. Initial experiments have been performed and indicate that the systems do indeed offer excellent potential for selectivity Scheme . RepreseIn conclusion, a versatile and straightforward protocol is disclosed for controlled synthesis of complex lanthanide‐bridged heteroleptic porphyrin–phthalocyanine–porphyrin triple‐decker assemblies. The protocol complements the few known approaches, importantly using a simple flexible link chain between the outer components of the structure to guide the assembly of complementary, size‐matched metal ions and central macrocycle. The system displays an intricate balance of steric, geometric and ion‐size effects that introduce further control. Assembly is spontaneous as the lanthanide series is spanned from lanthanum to europium and at this point the constraints of the bridge and porphyrin substituents prevent inclusion of smaller ions. The bridging chain can be passive or active in determining the final character of the complexes—it directs the ring–ring conformation and can also prevent free rotation of the (suitably designed) central unit of the molecular gyroscope. The combination opens a new, scalable approach to robust, highly functional single‐molecule materials.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Collision tumors are two distinct neoplasms seen together in same anatomic site. Management of such rare entity still lacks standardization with unknown prognosis. Here we are presenting one such rare case of invasive ductal carcinoma of breast and squamous cell carcinoma of anterior chest wall in a 31‐year‐old lady. Collision tumors are two distinct neoplasms seen together in same anatomic site. Management of such rare entity still lacks standardization with unknown prognosis. Here we are presenting one such rare case of invasive ductal carcinoma of breast and squamous cell carcinoma of anterior chest wall in a 31‐year‐old lady. The lesion was progressive in nature which developed into an ulcer associated with pain and serous discharge  chest showed well‐defined lobulated nonenhancing lesion (32 × 34 mm) situated in subcutaneous plane in anterior chest wall in right paramedian location with mild skin thickening and ulceration with bilateral axillary lymphadenopathy. Contrast‐enhanced magnetic resonance imaging (CE‐MRI) breast showed a well‐defined cystic lesion with thick enhancing wall with speculated margins in subcutaneous planes of midline and right paramedian area of anterior chest wall with a thin linear tract from the lesion reaching up to the skin.Wedge biopsy from the lesion was taken, and histopathological examination (HPE) report suggested squamous cell carcinoma.Patient then underwent wide local excision Figure B with flOn immunohistochemistry panel ER, PR, Her2neu were positive, p63 focally positive in tumor cells and Ki67 25%‐30% giving diagnosis of invasive ductal carcinoma with squamous differentiation. Decision for radiation therapy for chest wall SCC followed by definitive therapy for breast carcinoma was taken, and patient was sent to radiotherapy department after which patient lost to follow up.2Collision tumors are uncommon clinicopathological entities where two different malignant tumors having distinct histology coinciding in same anatomic site.Occurrence of such entities in breast is rare. Only 11 cases have been reported in literature Figure . UsuallyCutaneous SCC and primary SCC from breast is differentiated by various IHC markers one of which is p63 which is positive for cutaneous SCC.Management of these entities is still not standardized. In the present case, diagnosis could only be reached postoperatively on IHC. Multidisciplinary approach should always be present because sometimes we are not aware of the aggressive component. Other ancillaries such as histology and stage might help to prognosticate the entity.3On extensive search of literature, we can conclude that this case might be the third reported case. It should be always kept in mind that such rare case can occur in nature, and we should be able to manage and prognosticate the disease for the betterment of the patient by multidisciplinary approach.None declared.DM: collected data, structured the study and design, prepared the manuscript; BR: helped structuring the study and design and drafting the manuscript; NK: helped structuring the study and design and drafting the manuscript; DC: helped structuring the study and design and drafting the manuscript; AS: evaluated radiological data; PJ: evaluated pathological data.


Bacterial metabolosomes have been discovered for over 25 years. They play essential roles in bacterial metabolism and pathogenesis. In this crystal ball paper, I will discuss the recent advances in the fundamental understanding and synthetic engineering of bacterial metabolosomes. Bacterial metabolosomes have been discovered for over 25 years. They play essential roles in bacterial metabolism and pathogenesis. In this crystal ball paper, I will discuss the recent advances in the fundamental understanding and synthetic engineering of bacterial metabolosomes. Within cells, compartmentalization provides the structural basis for confining metabolic reactions in space and time. Compared with eukaryotic cells, prokaryotic cells have been thought to be simpler and less structurally defined for decades. It is now crystal clear that bacteria have complex, highly ordered subcellular structures and compartmentalized machines, such as outer membrane vesicles, intracellular thylakoids, chromatophores, membrane‐less organelles, as well as a range of regulatory systems that are involved in circadian clocks, motility, chemotaxis and secretion, etc. , the special metabolically functional machinery that is widespread among bacterial phyla  within the compartment. Depending on the types of encased Rubisco, carboxysomes can be divided into α‐carboxysomes and β‐carboxysomes. The shell facilitates accumulation of HCO3‐ that will be dehydrated by carbonic anhydrase to CO2 and prevents CO2 leakage into the cytosol. This allows the generation of a CO2‐rich microenvironment within the carboxysome to facilitate Rubisco carboxylation. Our recent studies have provided new insights into the assembly, structure, permeability, physiological regulation and bioengineering of carboxysomes  and catabolic BMCs (metabolosomes). Carboxysomes are the most intensively studied group of BMCs that function as the key COet al., et al., propanediol utilization) metabolosomes and Eut (ethanolamine utilization) metabolosomes in the pathogenic bacteria Salmonella enterica and Escherichia coli are the best‐studied metabolosomes. As a common feature, metabolosomes encase a core catabolic enzyme to generate acetaldehyde or propionaldehyde intermediates which are then converted to alcohol and acid derivatives  is a major metabolite produced by the gut microbiota through fermentation of fucose or rhamnose and can serve as a growth substrate for S. enterica under both aerobic and anaerobic conditions. Therefore, degradation of 1,2‐PD by Pdu metabolosomes in S. enterica provides a fitness advantage that promotes the growth of S. enterica in the inflamed gut, which is essential for enteric pathogenesis. In addition, degradation of phosphatidylethanolamine derived from ingested food and turnover of the intestinal epithelium and residential microbiota allows ethanolamine to be particularly prevalent in the gastrointestinal tract ‐dependent ethanolamine ammonia lyase (EutBC) catalyses the first step of ethanolamine utilization to produce ammonia (a nitrogen source) and acetaldehyde that is converted into acetyl coenzyme A (acetyl‐CoA) by aldehyde dehydrogenase (EutE) or into ethanol by alcohol dehydrogenase (EutG). Phosphotransacetylase (EutD) catalyses the conversion of acetyl‐CoA to acetyl‐phosphate that leaves the metabolosome and subsequently is converted to acetate. This pathway leads to the generation of ATP under fermentative conditions, while under respiratory conditions the resulting acetyl‐CoA promotes cell growth via the TCA (tricarboxylic acid) cycle. The degradation of ethanolamine also requires two Ado‐B12 recycling enzymes EutA and EutT. There are also two acetate kinases EutP and EutQ that is required for anaerobic respiration of ethanolamine with tetrathionate, and an ethanolamine permease EutH that mediates transport of protonated ethanolamine. Five shell proteins  are involved in the Eut metabolosome, along with a putative chaperone EutJ. A positive regulatory protein EutR regulates the transcription of the eut operon in the presence of both ethanolamine and Ado‐B12.Eut metabolosomes share similar structural principles with Pdu metabolosomes. Within the S. enterica, using QconCAT‐driven quantitative mass spectrometry  microenvironment created within the α‐carboxysome shell (Li et al., Advance knowledge of BMC assembly and function has allowed us to start the exploration of reprogramming BMC structures for biotechnological and biomedical applications in metabolic improvement, molecule delivery and therapeutics. It was shown that expressing the entire Pdu operon including 21 genes from the anaerobic gram‐negative bacterium H. ochraceum and demonstrated that shell proteins in the shell facets have the same sidedness (Sutter et al., in vitro (Faulkner et al., et al., Expressing individual shell proteins can also lead to formation of diverse higher‐order assemblies, e.g. two‐dimensional flat sheets, nanotubes and filament structures. We have applied high‐speed atomic force microscopy to visualize the dynamic assembly process of shell hexamers from Despite many exciting research advances, there are still open questions in the fundamental understanding and bioengineering of bacterial metabolosomes, for example, what the assembly pathways of distinct metabolosomes are, how metabolosomes are segregated and maintained in bacterial cells, how cargos are organized and interact with other cofactors within the shell, and how to control self‐assembly and shell encapsulation. Additionally, it is often that two or more types of metabolosomes coexist within one bacterium. How are their expression, assembly and function precisely coordinated in the same cell? On the other hand, the high similarity of shell protein structures and the shared building principles of BMCs offer the potential for rational design and engineering of chimeric BMC structures, with the capacity of fine‐tuning shell permeability and catalytic efficiency. Further efforts are also needed to gain an in‐depth understanding of the roles and controls of metabolosomes in pathogenesis and human health.The author declares no conflict of interest.


The purpose of this study was to explore the latent conditions of cooperation and conflict in intra‐hospital patient transfers .N = 13), nurses (N = 10) and support staff (N = 6) from a single, large academic tertiary hospital in the Northeastern United States.Secondary qualitative analysis of 28 interviews conducted with 29 hospital staff, including physicians  patient flow policies created imbalances of power; (b) relationships were helpful to facilitate safe transfers; and (c) method of admission order communication was a source of disagreement. Hospital quality improvement efforts could benefit from a teaming approach to minimize unintentional power imbalances and optimize communicative relationships between units. Thousands of transfers between hospital emergency departments, inpatient floors and intensive care units occur in USA hospitals each day  from the sending and receiving units consisted of 13 physicians , 10 nurses  and six support staff . The parent study analysis team was surprised by the frequency of cooperation and conflict codes—not only did the codes occur often, but these two seemingly contradictory codes occurred frequently together. Twenty‐seven of the original 28 interview transcripts included both the cooperation and conflict codes. The team felt that the prominence of these codes warranted further analysis to understand how cooperation and conflict were experienced during care transfers.The quality improvement project—herein referred to as the parent study—intended to: (a) better understand the process of transferring patients from the emergency department and medical intensive care unit to the general internal medicine floors of an urban teaching hospital; (b) identify challenges and opportunities for improvement in the transition process; and (c) develop interventions to reduce adverse patient events as well as increase staff satisfaction with the transition process. Details of the parent study including the sampling process, data collection  and data analysis, are well‐described in the parent publication  immersed themselves in the interview data coded with conflict and cooperation Patton, . The twoAnalytic rigour was supported through coding procedures, including maintaining an audit trail through memos kept in Atlas.ti to document the analytic process. The trustworthiness of data analysis was established by reviewing themes in a series of feedback sessions between the secondary analysis team and the parent study team  at the senior author's institution reviewed and exempted the parent study as quality improvement. This secondary analysis of the parent study was permitted as the institution's IRB has published guidelines permitting the publication of works related to quality improvement initiatives. Participants were asked for verbal consent to conduct and record the interview before it began. The consent process included a discussion about what types of information may be collected and analysed, including interpersonal communication related to the in‐hospital transfer process.3The research team identified three themes in the data: (a) patient flow policies created imbalances of power; (b) relationships were helpful to facilitate safe transfers; and (c) method of admission order communication was a source of disagreement Figure . Illustr3.1Senders, receivers and support staff identified several hospital policies that complicated the transition process by creating imbalances of power and conflict among groups. These patient flow policies epitomize the struggle of working within the mandates of a complex system that staff perceived as sometimes impeding high‐quality and safe care transfers.At the time of the parent study, there were two specific hospital policies in place designed to improve patient flow and reduce crowding in the emergency department: the unit transfer privilege and the four‐hour mark. Both policies focused on the transfer process between the emergency department and general medicine service. Both policies were aimed at decreasing length of stay in the emergency department. The four‐hour mark is a controversial performance indicator that stipulates that all patients presenting to emergency departments should be seen and treated or admitted to a hospital bed within 4 hr of arriving  change in admission order communication—an electronic medical record‐based checklist had replaced the verbal component of transfers between emergency and general medicine nurses. This change was a frequent source of debate among participants. When valuable information that influenced a patient's placement and, thus, the receiving unit's ability to care for patient was left out of an admission order, conflict ensued.Participants espoused two schools of thought on the necessity of verbal transfer communication in addition to the electronic medical record‐based checklist. Staff who had been at the hospital for a long time remembered the pre‐electronic medical record days. The electronic medical record‐based checklist was intended to be a streamlined communication mechanism, replacing the need for face‐to‐face or phone communication between providers. However, some clinicians valued the extra verbal exchange with a receiving provider and saw these conversations as valuable to their safe preparation for a new patient.Other staff felt that the checklist replaced the need for face‐to‐face transfer communication between providers. A sending nurse, on the other hand, considered verbal exchange of information as unnecessary and a waste of time Table . This nuIn addition to mode of communication being a source of disagreement, the content of admission orders hindered transfers. Nursing scope of practice policies—policies that dictate what nurses can and cannot do—varied across units. Sending providers’ lack of knowledge about variation in policies contributed to transition inefficiencies and were marked by more conflict than cooperation. Sending providers were sometimes unaware of limitations in receiving units’ scope of practice and therefore did not always include pertinent patient care in the admission orders. Receivers described receiving patients that they were unable to treat, such as patients requiring additional telemetry monitoring for specific cardiac medications. Often, this resulted in a re‐booking of the patient to a different unit. Occasionally, scope of practice policy was interpreted as lack of confidence in caring for specific patients. While a sending physician interpreted units not accepting patients as within the control of the receiving nurses, a receiving nurse described having to serve as an arbiter for the frequent conflicts that arose when sending teams were unaware of these unit‐specific policies and did not understand limitations in scopes of practice.4This study sought to understand and characterize the multidisciplinary staff experience of cooperation and conflict during intra‐hospital transfers. The directed content analysis generated three themes: (a) patient flow policies created imbalances of power; (b) relationships were helpful to facilitate safe transfers; and (c) method of admission order communication was a source of disagreement.Sending and receiving staff were asked to adhere to transition policies that facilitated transfers for some providers, but left others feeling disempowered. Despite having opportunities to voice concerns in formal venues including huddles and safety reporting, conflict was simmering at the staff level rather than moving up to administration. The mechanisms that allowed staff to file complaints with the administrators were indeed functioning. Relationships with staff on other units helped them navigate the myriad challenges of intra‐hospital transfers, but not having these relationships complicated the transfers for some clinicians. Staff also acknowledged the importance of sharing information about patients but reported that incomplete communication frequently occurred. Specifically, lack of patient information in admission orders and lack of senders’ awareness of receiving unit nurse scope of practice policies complicated appropriate patient disposition. These findings have important implications for the development of interventions to improve intra‐hospital care transfers.This study—along with the parent study—builds on the existing literature that had focused on transfers and communication between only‐physicians  to enhance the rigour of our findings (Miles, Huberman, & Saldaña, 5Our findings have implications for improving the day‐to‐day experience of staff navigating transfers of patients between units. In a report by the Joint Commission Center for Transforming Healthcare (Joint Commission, Hospital staff involved in intra‐hospital transfers must navigate a complex healthcare system to get patients to the appropriate site of care. Nurses, physicians and support staff described episodes of cooperation and conflict—some complicated by hospital policy—pervading the relationships and communication that occurred between units. The emergence of these domains across different types of staff underscores the importance of integrating the whole healthcare team in organization‐wide problems and solutions.The authors declare no conflicts of interest to disclose.


Hop stunt viroid (HSVd) is the type species of the genus Hostuviroid (family Pospiviroidae). The other species of this genus is Dahlia latent viroid, which presents an identical central conserved region (CCR) but lacks other structural hallmarks present in Hop stunt viroid. HSVd replication occurs in the nucleus through an asymmetric rolling‐circle model as in the other members of the family Pospiviroidae, which also includes the genera Pospiviroid, Cocadviroid, Apscaviroid, and Coleoviroid.Hop stunt viroid consists of a single‐stranded, circular RNA of 295–303 nucleotides depending on isolates and sequence variants. The most stable secondary structure is a rod‐like or quasi‐rod‐like conformation with two characteristic domains: a CCR and a terminal conserved hairpin similar to that of cocadviroids. HSVd lacks a terminal conserved region.HSVd infects a very broad range of natural hosts and has been reported to be the causal agent of five different diseases . It is distributed worldwide.HSVd is transmitted mechanically and by seed. Hop stunt viroid, a polyphagous pathogenic RNA that has shed light on significant aspects of viroid–host interactions.This pathogen profile summarizes the current knowledge on  The disentangling of the molecular biology and physical properties of viroids has been mainly carried out using Potato spindle tuber viroid, the first viroid discovered and type species of the family Pospiviroidae  plants showing abnormal dwarfing of bines    , Rosaceae , Anacardiaceae (pistachio)   RNAs (HSVd‐Nb plants) were obtained  strand transcripts of the type member of each genus of the family Pospiviroidae. Northern blot hybridization revealed the correct processing to the circular monomer in all cases, and multimeric (−) RNAs of HSVd were also detected, indicating that the first RNA–RNA transcription of the rolling‐circle mechanism occurs in Arabidopsis. Furthermore, the second step of the rolling‐circle is possible because transgenic Arabidopsis expressing HSVd dimeric (−) transcripts accumulated the circular (+) monomers. These results suggest that the inability of HSVd to infect Arabidopsis may be related to systemic movement, although it cannot be excluded that low levels of replication might be hindering viroid spread.6How viroids induce disease has been a prevalent question in the field. Early studies suggested a sequence‐specificity of the symptoms, as point changes could greatly influence the degree of severity  activity  was constitutively silenced (rdr6i‐Nb) were unable to develop symptoms even though the HSVd mature forms accumulated at levels comparable to those observed in infected controls with symptoms. These results were reinforced by the observation that HSVd‐Nb plants showing severe symptoms at 28 °C were symptomless when maintained at low temperatures (14 °C) at which the RNA silencing pathways are inhibited  mechanism  was reported, which is responsible for the removal of acetyl groups from the N‐terminal part of the core histones  and gives a tag for epigenetic repression (Liu et al., In addition to these alterations in vegetative tissues, HSVd infection is also associated with drastic changes in gametophyte development (Castellano et al., 8The large number of studies involving HSVd have resulted in a considerable knowledge about the replication, systemic movement, and host range of this ubiquitous pathogen, as well as concerning its interaction with RNA silencing and epigenetic mechanisms of the host. HSVd might be the second most studied viroid after PSTVd. However, some aspects of HSVd pathogenesis are yet to be unravelled. For example, host transcripts effectively silenced by HSVd‐sRNAs have not been described yet despite the well‐proved relation of viroid pathogenesis with RNA silencing (Gómez et al., The authors declare no conflict of interest.


N = 171, and a relational processing task, N = 865) and both univariate and multivariate approaches to voxel‐wise correlations. We created subsamples of different sizes and calculated correlations between task‐related activity at each voxel and task performance. Across both data sets the magnitude of the brain‐behavior correlations decreased and similarity across spatial maps increased with larger sample sizes. The multivariate technique identified more extensive correlated areas and more similarity across spatial maps, suggesting that a multivariate approach would provide a consistent advantage over univariate approaches in the stability of brain‐behavior correlations. In addition, the multivariate analyses showed that a sample size of roughly 80 or more participants would be needed for stable estimates of correlation magnitude in these data sets. Importantly, a number of additional factors would likely influence the choice of sample size for assessing such correlations in any given experiment, including the cognitive task of interest and the amount of data collected per participant. Our results provide novel experimental evidence in two independent data sets that the sample size commonly used in fMRI studies of 20–30 participants is very unlikely to be sufficient for obtaining reproducible brain‐behavior correlations, regardless of analytic approach.Limited statistical power due to small sample sizes is a problem in fMRI research. Most of the work to date has examined the impact of sample size on task‐related activation, with less attention paid to the influence of sample size on brain‐behavior correlations, especially in actual experimental fMRI data. We addressed this issue using two large data sets (a working memory task,  Limited statistical power due to small sample sizes is a problem in fMRI research. Most of the work to date has examined the impact of sample size on task‐related activation, with less attention paid to the influence of sample size on brain‐behavior correlations, especially in actual experimental fMRI data. Our results provide novel experimental evidence in two independent data sets that the sample size commonly used in fMRI studies of 20–30 participants is very unlikely to be sufficient for obtaining reproducible brain‐behavior correlations, regardless of whether a univariate or multivariate approach is used. Although there is some disagreement on this topic Friston, , it is gSeveral recent papers have addressed the sample size question in both resting state and task‐based fMRI studies, with highly variable results depending on the analysis method and specific brain measure assessed Table . In term2 of 0.5 was achieved with an N of 40 participants , with maximum values in quite different locations in the brain. These HCP results were consistent with the simulations and supported the idea that small sample sizes can provide results that may not be stable across subsamples and may not reflect the actual underlying “true” correlations. This inflation of correlational effect sizes has been called the “winner's curse” and has been discussed as one type of mistaken inference that can result from low power due to small sample sizes  with adding additional participants if the original result is a false positive . High‐resolution anatomical images were also collected with a T1‐weighted MP‐RAGE sequence with 160 sagittal slices .Participants were scanned on a 3T Philips Achieva scanner equipped with a 32‐channel head coil. Blood oxygenation level dependent (BOLD) data were collected using a T2*‐weighted echo‐planar imaging sequence with 29 interleaved axial slices per volume providing full brain coverage and acquired parallel to the AC‐PC line,  along with in‐house Matlab scripts . Additionally, the ArtRepair toolbox in SPM (RRID:SCR_005990) was used to determine motion parameter estimates. Functional images were adjusted for slice acquisition time and motion correction (using six directions of motion‐estimates from ArtRepair included as nuisance regressors), and each participant's T1‐weighted anatomical image was used to co‐register the functional maps to standardized MNI space. The resulting normalized images were smoothed with an isotropic 8 mm FWHM Gaussian kernel. The final voxel size was 3 mm isotropic.2.2The relational task data from the HCP 1200 release were used as the second data set. We did not use the working memory task from the HCP data set because accuracy was near ceiling. We accessed 865 nonrelated participants, between the ages of 22 and 36 years for the analyses described here . The relational task was adapted from a task developed by Christoff and colleagues  and selecting equal numbers from each age‐group for each subsample of the data. For this data set the sample sizes were 12 (10 subsamples of 12 participants each), 24 (7 subsamples), 36 (4 subsamples), 48 (3 subsamples), 60 (2 subsamples), 72 (2 subsamples) and 84 (2 subsamples). In the HCP data set the sample sizes were 20, 40, 60, 80 (10 subsamples for each), 100 (8 subsamples), 120 (7 subsamples), 140 (6 subsamples), 160 (5 subsamples), 210 (4 subsamples), 280 (3 subsamples), and 420 (2 subsamples).2.4www.rotman-baycrest.on.ca/index.php?section=84). SPM uses the general linear model on voxel‐wise contrasts, with FWE corrections for multiple comparisons. PLS uses singular value decomposition to determine latent variables present in the data and determines the robust voxels contributing to the LVs in a single step, so there typically are no corrections for multiple comparisons .All analyses were carried out with SPM8 and PLS , and for PLS we report voxels with BSR ≥ 4.0 . These thresholds were chosen because they are qualitatively similar and somewhat conservative . Thus, these analyses were as similar as possible between SPM and PLS, allowing both univariate and multivariate approaches to the same question of how activation during the n‐back conditions  and the relational task (HCP), relative to their respective control tasks, correlates with performance accuracy.r = 0.46, p < .0001), and both age and FD were negatively correlated with accuracy on the n‐back tasks . Because we wanted to explore the effects of sample size independent of any effect of aging, we corrected for age and head motion in the brain‐behavior analyses for the Dallas data. This was done in the PLS analyses by regressing age and mean FD from both the accuracy measure and the brain image (per voxel) for each participant and using the residual values in the brain‐behavior PLS analyses. For the SPM analyses, age and mean FD were entered as covariates of no interest.The Dallas data set had a large age range in the participants, which could be problematic because previous studies have found that older adults move more in the scanner than younger adults , between age and FD was 0.12 (p < 0.001), and between accuracy and FD was −0.19 (p < .00001). Therefore, we used the same procedure to regress age and FD out of the brain and behavioral data for both PLS and SPM as described above for the Dallas data set.Although the HCP data set had a much more restricted age range, there nevertheless were weak, but significant correlations among age, FD and accuracy on the relational task. The correlation between age and accuracy was −0.09  across all voxels in the maps for each possible pair in a given sample size. The second metric was the Jaccard index, which is the number of voxels in the intersection (overlap) between two thresholded spatial maps divided by the union of all above‐threshold voxels across both maps. We only report the Jaccard indices for voxels with positive correlations with accuracy, as there were very few above‐threshold negative correlations with accuracy in either data set. To calculate these Jaccard indices an uncorrected threshold of three was used for both SPM t maps and PLS BSR maps (approximate voxel‐level p = .003); this threshold was chosen to provide a reasonable estimate of the above threshold voxels without being too liberal.We report the results of the brain‐behavior analyses in several ways. From the PLS analyses we obtained a Finally, to determine the voxels that contributed consistently to the brain‐behavior correlations as a function of sample size, we calculated penetration maps for each sample size using the spatial maps obtained from the SPM and PLS accuracy analyses for the voxels with positive correlations with behavior. These penetration maps indicate the voxels where some number of thresholded spatial maps overlap within each sample size, from a minimum of 2 to a maximum equal to the total number of samples of a given size. A threshold of 3 was used for all images entering into the penetration maps, and no clustering was done on the thresholded images prior to computing the penetration maps. Prior to calculating the penetration maps, images from both the PLS and SPM analyses were masked using a gray matter mask that also removed voxels estimated as “NA” .33.1rho values for all samples sizes were quite high (≥ 0.8) even for the subsamples of only 12 participants. However, when comparing thresholded images resulting from the SPM analyses the overlap for those regions with increased activity during the working memory tasks, as indicated by the Jaccard index, was low for the smallest sample size, but increased to >0.6 at the largest sample size. PLS identified a similar group of frontoparietal regions with more activity during the n‐back conditions, but also showed more extensive activation in these regions, as well as activation in occipital cortex not found by SPM .SPM identified a set of frontoparietal regions with more activity for the cognitively demanding n‐back conditions relative to the 0‐back condition. As seen in Figure Both the task‐positive and the task‐negative activity for this data set show that univariate and multivariate approaches can sometimes result in somewhat different spatial maps. For example, SPM identified occipital regions with more activity during 0‐back, whereas PLS showed more activaton in occipital areas during 2,3,4‐back .As with the task activation patterns, the brain regions that correlated with accuracy on the n‐back conditions were similar for SPM and PLS  and the smallest size where all of the PLS accuracy LVs had p < .05 (72 participants). At the relatively small sample size of 24 there was overlap across PLS maps .Penetration maps for the accuracy analyses from two subsample sizes are shown in Figure s Figure  in left s Figure  were limIn addition to the overlap identified by the penetration maps, there also were unique regions identified by the spatial maps at the small sample size of 24 participants. Figure S3.2rho; the effect was less robust according to the Jaccard metric, which reached 0.6 only when 160 participants were included. The PLS task effect was similar . Maximum Jaccard values for the relational task with 420 participants exceeded 0.8 for both SPM and PLS. The spatial maps also were quite similar but slightly more extensive in the PLS result. Overall, the relational task effect in terms of rho value was similar in strength to the working memory task effect in the Dallas data set for subsamples of similar size when using PLS . These rho and Jaccard values were similar to those from similarly sized subsamples in the Dallas data set, although the Dallas values tended to be higher, and the PLS Jaccard values were slightly higher than those resulting from SPM  and accuracy on the relational task showed positive correlations in frontoparietal areas, a broad expanse of occipital cortex, and the caudate nucleus in the largest subsamples for both SPM and PLS Figure . A smalls Figure , peaking Figure S.p values associated with the LVs decreased with increasing sample size, such that with a sample size of 80 or more the LVs from all the subsamples had p‐values below the .05 level. Therefore, the number of participants required to obtain relatively stable correlation magnitudes and significant LVs in the PLS accuracy analyses was roughly 80 participants in both the Dallas and HCP data sets.In the PLS analyses of the HCP data Figure , the corp < .05, there was overlap of positively correlated voxels in the medial occipital, parietal, and frontal regions for both SPM and PLS, although this overlap was much more extensive for PLS. In the PLS analyses a precentral region was identified in 7 of 10 of the 20‐participant subsamples, medial occipital cortex showed overlapping correlations in 6 of 10 subsamples and bilateral parietal regions had 5 of 10 subsamples with overlapping voxels. The maximum amount of overlap in the SPM maps was lower, and was found in medial occipital cortex, which showed overlap in 4 of the 10 subsamples. Right parietal and frontal cortex showed overlap in 3 maps. At a larger sample size of 80, where all the accuracy PLS LVs had p < .05, the occipital area was considerably larger in extent, as were the frontal and parietal cortical regions of overlap in both the PLS and SPM maps. In the PLS maps the right parietal region showed the maximum amount of overlap (10 of 10), and the frontal and medial occipital regions showed overlap in 8–9 maps. In the SPM penetration maps 8 of 10 maps overlapped in the medial occipital region and right frontal cortex, with the maximum amount of overlap (9/10) in a small region of left cerebellum. As with the Dallas data set, the mean number of overlapping voxels identified in these spatial patterns increased with increasing sample size . Thus, as with the Dallas data set, larger sample sizes in the HCP data were associated with a greater extent of regions reliably found across subsamples and techniques, revealing more frontal, parietal, and occipital cortex involvement. The PLS and SPM maps were similar, but the PLS maps showed more overlapping voxels.The penetration maps for the HCP accuracy analyses Figure  showed tThe 10 individual maps from the 20‐participant subsamples  are shown in Figure S4As expected, all of our analyses showed an increase in the stability of the derived spatial maps as sample size increased, as well as a decrease in the size of the correlation between brain activity and task accuracy identified by PLS. These task and brain‐behavior effects appeared to be similarly robust in the Dallas and HCP data sets, with slightly greater similarity measures in the Dallas data set when using SPM to analyze task effects. Overall, our results are consistent with the literature on the disadvantages of small sample sizes and extend this work by showing in two independent data sets: (a) sample size influences brain‐behavior correlations in a similar way regardless of whether one uses a univariate or multivariate analytic approach, although the multivariate approach identified more consistent and extensive correlational patterns; and (b) the effects of small sample size on interpretation of brain‐behavior patterns include type I and type II errors, as well as inflated correlation effects.rho and the Jaccard index  was ~90% whereas the overlap of the two thresholded images was ~40%. This difference in similarity metrics calculated on task activations also was noted by Turner et al. . This finding is consistent with evidence that multivariate analyses can be more sensitive than univariate ones . The analyses reported here are limited to the effects of sample size in healthy adults, removing the effects of age, and although the results generalize across the two experiments used here it is not clear how well our findings would apply to studies using patient populations, or different tasks. For example, the working memory and relational tasks that we used here are typical examples of an externally driven task in which stimuli are presented visually and participants are required to make a judgment about a stimulus property. Other types of task rely on cognitive processes that are internally driven, such as autobiographical memory retrieval or social/emotional decisions, and it is unknown whether the results reported here would characterize internally driven kinds of tasks. An additional limitation of this work is that for our aim of examining the effect of sample size on brain‐behavior correlations we were limited in the task data that could be used from the HCP study. A number of other tasks with in‐scanner performance measures are included as part of the HCP release, including social cognition tasks, but are either prone to ceiling effects in performance and/or have a restricted range of performance values, making them ill‐suited to the study of individual differences in brain‐behavior relations. We also note that the results reported here with whole brain analyses might not be applicable to brain‐behavior correlations measured with pre‐defined ROIs, whether these are defined anatomically or functionally. Future work will be needed to determine adequate sample sizes across a broad range of cognitive tasks and different ways of extracting brain activity.Finally, Turner et al (Turner et al., In conclusion, we have presented evidence from two human fMRI data sets supporting the idea that small sample sizes can be particularly troublesome for brain‐behavior correlations. These results are consistent with previously reported simulation studies, but also provide novel experimental evidence from two independent data sets of the importance of sample size in obtaining stable results. An important aspect of our results is that multivariate approaches, such as the PLS approach used here, are not only more sensitive than a univariate approach to task‐related activations but also produce more extensive and consistent correlations between brain activity and behavior. This greater sensitivity might provide some benefit with smaller sample sizes, but should not be used as a substitute for giving full consideration to estimating a sufficient sample size when planning one's experiment. We hope that researchers will be able to use the results of this study to guide them in planning experiments and choosing a sample size appropriate to their scientific question, and to help evaluate the likelihood of true brain‐behavior effects reported in published work of varying sample sizes. Multicenter studies and large collaborative fMRI efforts, such as the HCP data set utilized here, are becoming more common and will mitigate the problem of small sample sizes for some types of research question. Nevertheless, it likely will still be the case for some time to come that many cognitive neuroscience experiments probing the neural correlates of specific cognitive processes will be carried out in single laboratories and researchers will need to ensure that issues of sample size and power are adequately addressed when brain‐behavior correlations are employed.The authors declare no conflicts of interest.AppendixS1: Supplementary InformationClick here for additional data file.


Acute brainstem strokes can present a diagnostic challenge due to its variable clinical presentation. MRI with diffusion‐weighted  imaging is highly sensitive for diagnosing ischemic lesions however even that can fail to identify early lesions in the brainstem. Combining coronal section to standard axial MRI‐DWI can facilitate early diagnosis in these cases. Acute brainstem strokes can present a diagnostic challenge due to its variable clinical presentation. MRI with diffusion‐weighted  imaging is highly sensitive for diagnosing ischemic lesions however even that can fail to identify early lesions in the brainstem. Combining coronal section to standard axial MRI‐DWI can facilitate early diagnosis in these cases. The clinical examination was only significant for paralysis of adduction in his left eye. CT of the head without contrast, axial MRI of the brain without contrast Figure , 3, 4.In a case series reported by Oppenheim et al, 19% of vertebrobasilar stroke had false‐negative initial DWI study.Herein, we present a case to emphasize upon importance of using a combination of standard axial and thin‐section coronal DWI to facilitate the diagnosis of brainstem infarction. An argument can be made to include thin‐section coronal DWI of brainstem as standard protocol for all stroke workup.None declared.PSB and KJK: wrote the manuscript. PBL: procured and labeled the images. VKS: involved in literature review and edited the manuscript.


The Golden Retriever Lifetime Study (GRLS) is one of the largest canine cohort studies undertaken in the United States to date. This study design allows for evaluation of multiple exposures and outcomes throughout the lifetime of each dog, but relies on participants to comply with study requirements over a long period of time. Failure to do so can lead to biased reporting of results.To examine factors associated with dog owner compliance for GRLS.Golden Retrievers (n = 3044) whose owners elected to participate in GRLS.Prospective, cohort study. A logistic regression model was constructed to examine associations between data collected at the time of initial enrollment in GRLS and the outcome of failure to fulfill all study obligations at the end of the first year after enrollment in GRLS.There were 192 (6.3%) owners who did not comply with study requirements 1 year after enrollment. Owners of dogs without a record of vaccination had nearly 4 times higher odds  of being noncompliant than owners of vaccinated dogs and owners of dogs that slept in the garage had nearly 6 times higher odds  of being noncompliant than owners of dogs that slept in their bedroom.Survey questions about a dog's sleeping location at night and vaccination status are important indicators of an owner's odds of compliance in a prospective study. Use of similar questions during enrollment in cohort studies might help to predict owner compliance that can aid in subject selection. AAHAAmerican Animal Hospital AssociationC‐BARQCanine Behavioral Assessment and Research QuestionnaireGRLSGolden Retriever Lifetime StudyORodds ratio95% CI95% confidence interval1Prospective cohort studies like the Framingham Heart Study and the Nurses' Health Study have proven to be valuable for collecting a large amount of epidemiological data about human populations over an extended period of time.The Golden Retriever Lifetime Study (GRLS), predicted to be a 15‐year cohort study of more than 3000 Golden Retrievers, is currently being conducted by Morris Animal Foundation.22.1All 3044 dogs enrolled in GRLS were included in this study. A logistic regression model was constructed with the primary endpoint being failure to fulfill research obligations at the end of the first year after enrollment in GRLS. Data collected from the owner at the time of enrollment in GRLS were used to examine associations with the outcome of non‐compliance.2.2Enrollment into GRLS was an owner‐driven process that has been described previously.Owners could begin the annual study processes 90 days before their enrollment anniversary date and had about 6 months after that date to fully complete the requirements. A time‐structured reminder protocol that utilized email, phone, and postcard reminders was employed both in advance of the enrollment anniversary and after the anniversary date if the survey had not been completed. Completion of all study requirements at the initial enrollment visit was a study inclusion criterion. For year 1 after enrollment, dogs were placed into 1 of 4 compliance status categories: open (owner still has opportunity to comply), owner noncompliant (owner did not fulfill any requirements or completed survey only), veterinarian noncompliant , or fully compliant (owner was fully compliant and veterinarian had submitted survey and samples) , but individual behavior‐related questions were combined to form composite scores for behavior variables . Composite scores were calculated using a method outlined in the Canine Behavioral Assessment and Research Questionnaire (C‐BARQ).Variables included in the modeling approach for owner noncompliance were the dog's sex and age at enrollment, geographical region of residence , dog insurance , clinic American Animal Hospital Association (AAHA) status , number of study dogs enrolled with the same veterinarian, number of study dogs with the same owner, where dog was acquired , activity level , primary activity , where dog spends most time , where dog sleeps at night , home type , travel frequency (assessing if owner traveled with dog for 2 weeks or more within previous 12 months), leash walk frequency (daily or less than daily), aerobic activity frequency (daily or less than daily), professional grooming frequency , home bathing and/or grooming frequency , heartworm prevention frequency , flea/tick prevention frequency , rabies 1 year vaccine given in past 12 months , rabies 3 year vaccine given in past 12 months , no vaccines given , and the composite scores for stranger‐directed aggression, owner‐directed aggression, stranger‐directed fear, nonsocial fear, dog‐directed fear and aggression, separation anxiety, attachment behavior, trainability, chasing behavior, excitability, and pain sensitivity.P ≤ .25) was required for inclusion in the multivariable model. The final multivariable logistic regression model was constructed using a critical α for retention ≤0.05. Excluded variables were reintroduced to the final model to evaluate confounding effects (identified by ≥20% change in parameter estimates). First‐order interaction terms for variables included in the final multivariable model were evaluated. The Pearson chi‐square goodness‐of‐fit test was used to evaluate model fit. Odds ratio (OR) and 95% confidence intervals (95% CI) were calculated using the final multivariable logistic regression model.A logistic regression model was constructed using a backwards, stepwise procedure with commercially available software in order to estimate the odds ratios using owner noncompliance as the outcome of interest . Univariable logistic regression models were used to screen individual exposure variables and a wide statistical association with the outcome of noncompliance , yearly , never , >monthly (reference)] and unvaccinated P = .031 . With the interaction term, home grooming P = .005  and unvaccinated P = .0041 . Although not included in the final model, the number of dogs enrolled in GRLS with the same veterinarian approached statistical significance . The Pearson chi‐square statistic indicated that the model fit the data sufficiently well .Variables remaining in the final multivariable model were age at enrollment, where dog spends most time, where dog sleeps at night, home grooming frequency, no vaccines given, and excitability Table . No confIn the adjusted model, owners of dogs who sleep anywhere other than the owner's bedroom at night had higher odds of being noncompliant at their second study visit than were owners who allowed their dogs to sleep in their bedroom. Owners of dogs who sleep in the garage had nearly 6 times higher odds of being noncompliant  compared to owners of dogs who sleep in the bedroom. Additionally, owners of dogs that are never bathed and/or groomed at home had almost 2 times higher odds of being noncompliant  than owners of dogs who are groomed at home at least once a month. Owners of dogs without record of vaccination had nearly 4 times higher odds of being noncompliant with study protocols  than owners of vaccinated dogs. Owners had slightly higher odds of being noncompliant  with each additional month added to their dog's age at the time of enrollment in GRLS. Owners of dogs who spend most of their time both inside and outside had 1.5 times higher odds of being noncompliant  than owners of dogs who spend most time indoors. Finally, owners of dogs who were more excitable (composite score > 1) had nearly 2 times higher odds of being noncompliant  than owners of dogs who were less excitable (composite score 0‐1).4Out of an extensive baseline survey, 6 variables were statistically significant predictors of owner noncompliance by the end of year 1 after enrollment of GRLS. These predictor variables are simple questions that might help guide future efforts for recruiting dog owners for other cohort studies by predicting owner compliance with study requirements. The pool of owners of potential participants that is predicted to be more likely to be noncompliant based off of screening questions such as “Where does your dog sleep at night?” could either be excluded or oversampled in order to maximize data collection. Oversampling owners predicted to be noncompliant would potentially avoid bias that would be introduced by simply excluding potentially noncompliant owners altogether.Another strategy that could increase owner compliance is the use of incentives, such as rewards or gifts, being given at the completion of the annual survey and veterinary visit or prepaid cash incentives sent with the reminder in advance of the due date for the annual survey. However, the use of such incentives in longitudinal studies has been shown to be variably effective and can disproportionately attract different types of respondents.The survey question with the largest OR for owner compliance was where an owner allows their dog to sleep at night. The answer to this question might be a surrogate for questions about how the owner perceives the relationship between themselves and their dog. An association has already been established between the strength of an owner's bond with their pet and an increased likelihood to follow veterinarian recommendations and to seek preventive care for their pet.Vaccination status was another statistically significant indicator of owner compliance, which might indicate that the level of preventive veterinary care a dog receives, is associated with owner study compliance. Conversely, a dog's level of excitability might have an impact on an owner's likelihood of taking their dog to the veterinarian as owners of dogs who are more excitable might not be comfortable or willing to take their dog to the veterinarian as compared to owners of dogs who are less excitable. Additionally, although all dogs were relatively young at enrollment, perhaps owners of the youngest dogs were better able to establish GRLS compliance as an integral part of their care regimen when their dog was still a puppy. The greater the number of dogs in the study cared for by the same veterinarian approached statistical significance. It is possible that veterinarians with more dogs involved in GRLS positively influenced owners to be more compliant through year 1, but it is also possible that highly motivated owners (who might be more likely to be compliant through year 1) are more likely to go to a high‐quality and engaged veterinarian who cares for more dogs in GRLS. The practical significance of this factor could become clearer as the study progresses.An important limitation to this study is the potential for information bias introduced by having the owner answer lifestyle and behavior questions about their own dog. Not only can dog owners view their dog's behavior with bias due to their close relationship with the dog, but dog owners might also be impacted by social desirability bias, tending to answer survey questions in a way that they believe might be viewed more favorably by others.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Authors declare no IACUC or other approval was needed.Authors declare human ethics approval was not needed for this study.