Delivering access to sufficient food, energy and water resources to ensure human wellbeing is a major concern for governments worldwide. However, it is crucial to account for the ‘nexus’ of interactions between these natural resources and the consequent implications for human wellbeing. The private sector has a critical role in driving positive change towards more sustainable nexus management and could reap considerable benefits from collaboration with researchers to devise solutions to some of the foremost sustainability challenges of today. Yet opportunities are missed because the private sector is rarely involved in the formulation of deliverable research priorities. We convened senior research scientists and influential business leaders to collaboratively identify the top forty questions that, if answered, would best help companies understand and manage their food-energy-water-environment nexus dependencies and impacts. Codification of the top order nexus themes highlighted research priorities around development of pragmatic yet credible tools that allow businesses to incorporate nexus interactions into their decision-making; demonstration of the business case for more sustainable nexus management; identification of the most effective levers for behaviour change; and understanding incentives or circumstances that allow individuals and businesses to take a leadership stance. Greater investment in the complex but productive relations between the private sector and research community will create deeper and more meaningful collaboration and cooperation. Delivering access to sufficient food, energy and water resources to ensure human wellbeing, both now and in the future, is a major concern for governments worldwide  and that they should be formulated as a question that might result in a research process that could generate an answer over the next 5 years. Questions should be either useful for, or relevant to, business. Not all research questions were anticipated to directly result in changes to business practice. Some, when answered, might provide evidence to help regulatory bodies or consumers encourage or drive change in business behaviour. Contributed questions also, therefore, included those that help us to understand aspects of regulation, policy or consumption choices that affect business practice and sustainability.Various methods were used to prompt participants to produce researchable questions, including the sharing of example questions to all participants, speaker-based events, peer-to-peer discussions, and workshops in which facilitators worked with participants from problem statements through to specific research questions. To avoid bias formed from sampling a group with a narrow range of geographic interests, we ensured a broad range of international experience amongst contributors and workshop attendees. In total 722 questions were submitted by at least 238 individuals from at least 152 institutions or companies (some were submitted anonymously). Sixteen questions were excluded prior to the first round of voting for being incomplete or clearly unrelated to the topic. The nature of nexus thinking is that any one element cannot be researched or acted upon in isolation. Forty-eight per cent of questions did not explicitly mention food, energy, water, or the environment but, of those that did, the majority focused on just one , followed by two (eleven per cent), three (five per cent) or all four elements (five per cent).Once question submission was closed, three of the authors  independently grouped the questions into research areas, before coming together to agree on twelve broad categories, which each received approximately sixty questions: (1) Consumption, consumer behaviour and demand-side issues; (2) Measuring, reporting and transparency; (3) Technological solutions, instruments and innovation; (4) Policy, regulation and governance; (5) Awareness, education and communications; (6) Ecosystem services, valuation and externalities; (7) Resource efficiency, waste and the circular economy; (8) Collaboration, stakeholder engagement and supply chain influence; (9) Decision making, mutual benefits and trade-offs; (10) Forecasting, future scenarios and risk; (11) Land use, practical applications and direct impacts; and (12) Change agents, financial systems/incentives and leverage points. These groupings are useful for helping participants compare similar questions by ensuring that questions that overlap or complement one another are placed together as they complete the prioritisation exercise.After categorisation, the subdivided list was emailed to workshop participants for an initial vote, for which individuals identified the most important four to six questions from those categories within their expertise. Twenty-three participants voted in all twelve categories, while eight participants voted on a subset of categories. These votes were then used to rank the questions within each category. Following this first round of voting, twenty-three participants from the research community and seventeen from the business community came together at a two-day workshop held in Cambridge, UK, during September 2015.During the second round of voting each category was discussed in its own session, attended by approximately ten participants. Each session had a chairperson and a facilitator chosen from the workshop participants and a note taker. If there was disagreement within the group, the group was polled and a majority decision was taken. During each session, the questions were discussed to identify, within each of the groups, a maximum of twenty-four questions to go through to the next stage. With the consensus of the group, questions could be edited, split, added or reformulated to improve them. These were allocated to ‘gold’, ‘silver’ and ‘bronze’ (eight in each) according to their relative merit, as judged by being important, answerable under the agreed research conditions, and relevant to business Table . The catReasons for eliminating questions from inclusion in the next round were recorded Table . Of thosOn the second day the twelve groups were merged to form four groups for the third round of voting, in which questions were initially ranked by their gold, silver or bronze classification of the previous day Table . Each grThis whole process was collegiate. Businesses in different aspects of the Nexus were able to identify their concerns and knowledge gaps, but the manner of the preparation and the design of the workshop enabled a collective approach. The twelve item template of key themes which emerged from the first round enabled focus for the subsequent stages. The outcome was a sense of commitment built on very differing but supportive expertise and experience.n = 706). Overall, there was a reasonable balance of “questions for business” and “questions about business” . The questions within these categories can be clustered around emergent themes, which are discussed below.Each of the twelve initial categories from the first and second rounds of voting was represented at least once in the final list. On average, the forty prioritised questions Table  receivedTools for decision-making: There is enormous potential for research and business communities to work together to apply new data and analyses to improve private-sector decision-making  tend to specialise on one or a few issues. The environmental agenda has a dearth of social scientists and a much more interdisciplinary approach is required across the board Stirling . Almost The commercial case around risk, investments and profitability: A primary concern that emerged prominently in the top-ranked questions was the need to examine the business case for increased sustainability of nexus resource use. Understanding how risks manifest around unsustainable management of food, energy, water and environmental systems is key for businesses operating under conditions of increased demand for natural resources . This includes issues such as how businesses can identify risks (including threats to reputation), demonstrating links between management of the nexus and supply chain security or price volatility, and how collaboration or cooperation can be enabled to address landscape level risks under situations of shared ownership and common goods. Risk is of fundamental importance to investors and this also emerged as a key concern in developing tools and metrics that allow financial institutions to incorporate concerns over nexus resources into their investment decisions .Understanding the conditions under which actions to enhance the management of food, energy, water and the environment contribute to company profits (economic sustainability) is vital to deciding when commercial drivers are sufficient to drive positive change versus situations where government regulation or further incentives will be required (Q21). It is not always in the interest of many businesses to encourage a reduction in consumption. Fundamental, then, is whether there are particular business models and particular conditions or contexts in which overconsumption is decoupled from growth and profits, such that commercial drivers enable businesses to benefit from a reduction in unnecessary natural resource depletion (Q16).Levers for behaviour change: Awareness and education about sustainability issues from a young age will have profound impacts on how society understands and responds to future challenges and goals . High priority questions in this area also focused on identifying the most effective types or classes of intervention; investigating, for example, the contribution of information, pricing, nudging and taxation on business and consumer behaviour (Q3). Likewise, several questions attempted to identify mechanisms by which businesses are incentivised to implement circular economies (Q20) and how incentives or financial instruments might be designed to enable investments in a volatile world .Closely aligned with understanding what motivates, enables or impedes businesses from managing the food-energy-water-environment nexus more sustainably are questions about the valuation of ecosystem services. Two questions in the top ten addressed the most effective ways to incentivise or regulate business to ensure that they recognise the value of ecosystems and manage the services that they provide more sustainably . If businesses were to increasingly value the environment by considering their dependencies upon it then there is an expectation amongst some that the commercial driver for sustainability will be recognised and acted upon, at least when that driver is shown to be positive. Equally, policy-makers need to be aware of businesses’ material impacts on resources and how these affect society more widely; this can help them to determine incentives and regulatory levers to change or mitigate negative impacts that affect the nexus.Governance and collaboration: Complex interactions between private and public goods highlight the importance of cooperation between the private and public sectors. A single business operating alone is unlikely to be motivated or able, under competitive market forces, to achieve sustainable management of nexus interactions. Effective governance and collaboration are therefore imperative. Three questions in the top half of the ranked priorities addressed the incorporation of nexus complexity into policy tools and governance systems; this demonstrates that these are key areas for future research. Specifically, the questions in this category focussed on: understanding what scales of governance are best suited to managing the nexus (Q15); developing policy tools capable of dealing with the complexity of interaction between important political agendas (Q18); and developing regulatory systems able to address the multiple and very different timescales of political, regulatory, natural and business cycles (Q19). A further two questions related to enabling collaboration between stakeholders at a landscape level . Such collaboration and engagement between actors is absolutely essential in managing nexus issues. More effective collaboration needs both research to investigate the most constructive processes for engagement as well as investment within the private sector to provide employees with the requisite skills and management frameworks to facilitate successful collaboration . Equally, investigating the factors that encourage particular businesses to take a leadership role that will have wider cross-sectoral benefits will also help practitioners identify mechanisms to scale up sustainable actions (Q33). However, exemplary leadership may not be enough when the challenges facing businesses are specific to the geography, politics and culture of the location of business operations. Therefore, identifying how best practice can be adapted to different geographies also emerged as important (Q39). Leadership from government is also essential, both in how governments can support improvements within the private sector (Q32) but also in how they can harness the enormous buying power of their direct procurement by supporting businesses and supply chains that optimise the sustainable use of nexus resources (Q27).Whilst many subject areas around which researchers and practitioners can collaborate to generate action-oriented research outputs were identified, a significant proportion of the submitted questions were assessed as already sufficiently well understood from a research perspective. This may, therefore, point to inadequate or slow communication of research results rather than inadequate research findings. It highlights again the importance of disseminating results effectively, quickly and widely to achieve maximum impact. Poor dissemination of research findings has been implicated in the research-implementation gap that has been identified in conservation science and other fields, such as health (Crosswaite and Curtice Research questions are rarely sought jointly from practitioners and researchers (Knight et al. The translation of research into transformative change is impeded because those best placed to judge the ‘actionable’ nature of the work are too often excluded from the project formulation stage (Lang et al.


Due largely to the use of improvised explosive devices (IEDs) and other explosives in recent military conflicts, blast-related TBI has emerged as a prominent injury sustained by warfighters. In the recent wars in Iraq and Afghanistan, traumatic brain injury (TBI) has been one of the most common types of injury sustained by soldiers and military personnel; of the ~380,000 TBIs reported in service members from 2000 to 2017, 82.3% were classified as mild (mTBI). While mTBI is associated with normal structural imaging, brief or no loss of consciousness, and rapid recovery of mental state, mTBI can nevertheless lead to persistent behavioral and cognitive effects. As in other cases of mTBI, exposure to low-level blast often does not cause immediate overt neurological effects, but may similarly lead to persistent behavioral and cognitive deficits. These effects are likely to be compounded when multiple exposures to blast and/or impact are sustained, since there is increasing evidence that multiple mTBIs can lead to chronic neurodegeneration. One common form of this deleterious outcome is frontotemporal lobar degeneration (FTLD), which is a progressive neurodegenerative process marked by atrophy of the frontal and temporal lobes, leading to frontotemporal dementia, a common form of dementia affecting behavior, cognition and language. About half of all cases of FTLD are marked by TAR-DNA binding protein (TDP-43)-positive protein inclusions. TDP-43, a DNA/RNA binding protein, controls the expression of thousands of genes and is associated with several neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, and chronic traumatic encephalopathy. TDP-43 abnormalities have also been associated with traumatic brain injury in both pre-clinical and clinical studies. The role of TDP-43 in the manifestation of FTLD pathology in military TBI cases is currently unclear, and to date there has been only a limited number of pre-clinical studies addressing the effects of repeated blast-related mild TBI (rbTBI) in relation to FTLD and TDP-43. This review will summarize some of these findings and address the concerns and critical knowledge gaps associated with FTLD manifestation with military populations, as well as clinical findings on other forms of mTBI. Traumatic brain injury (TBI) is one of the most common types of injuries sustained by military personnel. Between 2000 and 2017, TBI was reported in more than 380,000 service members, and more than 82% of these TBIs were classified as mild (mTBI) , 5. In aThough the acute symptoms of mTBI generally resolve quickly, mTBI can lead to persistent behavioral and cognitive deficits, which are thought to be compounded cumulatively following multiple mTBIs, with a “dose”-response relationship between number of injuries and symptoms –9. IncreTARDBP gene, controls the expression of thousands of different genes. In its pathological form, TDP-43 is hyperphosphorylated, ubiquitinated, cleaved into 25 and 35 kDa fragments, and mislocalized to cytoplasmic protein inclusions , the clinical manifestation of FTLD, has been reported in military Veterans. Various epidemiological studies have found that FTD is one of the most common forms of dementia in individuals under 65 years of age, after Alzheimer's disease and vascular dementia –25. It iBP genes . The remBP genes . Though adic FTD .Several preclinical studies have examined TDP-43 pathology, using various impact TBI models. These impact studies identified alterations in TDP-43 including proteolysis, phosphorylation, and formation of cytoplasmic TDP-43 granules even after only one impact. This has been shown in several studies using fluid percussion injury (FPI) or cortical contusion injury (CCI) in rodents, where TDP-43 was phosphorylated, mislocalized, and cleaved into TDP-25 and TDP-35 following injury, leading to neuronal loss –32. The A315T transgenic mice had significantly impaired cognition as measured by the Y maze following FPI  blast exhibited impaired memory, as measured by the novel object recognition task . Our resThere has been limited clinical research associating chronic TBI, TDP-43 and neurodegeneration, but there is some preliminary evidence that links these factors. The clinical behavioral symptoms associated with bvFTD, such as apathy and social dysfunction, are also frequently reported in cases of TBI and CTE  and therA common link between repetitive impact TBI and FTD is mislocalization and aggregation of TDP-43 in neurons and glial cells. In a study of human subjects who sustained a single moderate/severe TBI, TDP-43 expression was increased in the cytoplasm, though no increase in TDP-43 phosphorylation was observed . A case In cases of CTE with severe TDP-43 pathology, the pattern of TDP-43 expression mimics the pattern of expression seen in FTLD-TDP, with TDP-43 proteinopathy found in all layers of the cortex, particularly in layer II, and in the dentate fascia of the hippocampus , 16. AddLongitudinal studies which comprehensively monitor blast overpressure exposure and resultant chronic debilitations are currently lacking in the clinical literature. In a study of individuals with a history of repetitive mTBI, 16 of 21 military Veterans were diagnosed with CTE, three of whom were exposed to blast from IEDs . Of thosWhile there is ample evidence that multiple impact head traumas resulting in TBIs can lead to neurodegeneration, longitudinal studies addressing Warfighter needs in this domain are currently lacking, despite the emergence of evidence in Veteran populations that FTD is a primary concern. Studies targeting mechanisms underlying the pathogenesis of neurodegeneration using pre-clinical models addressing this Warfighter population are largely non-existent in the literature. LH wrote the manuscript. VS and LH structured the manuscript. VS and JL edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N = 195). Women applied to participate in the contest by recording their answers to several questions in a video interview. In the contest, they were assigned to teams of about ten women each and worked on several assessment-center-like tasks. After each task, each member of each team nominated the three women they believed showed the best leadership potential in their group. We operationalized women’s leadership emergence as the number of nominations received. We measured participants’ facial attractiveness, babyfacedness, social competence, and naïveté by having raters follow a specifically developed rating manual to rate the answers the women gave in the video interviews. In both studies, the results indicated that women with higher ascribed facial attractiveness had higher ascribed social competence, which significantly predicted leadership emergence in the contest. Likewise, women with higher ascribed babyfacedness had higher ascribed naïveté, which significantly, albeit only slightly, negatively predicted leadership emergence. We discuss the implications of the results for personnel selection.The underrepresentation of women in top positions has been in the spotlight of research for decades. Prejudice toward female leaders, which decreases women’s chances of emerging as leaders, has been discussed as a potential reason. Aiming to investigate the underlying mechanisms of this prejudice, we focused on the question of how facial characteristics might influence women’s leadership emergence. Because other research has related ascribed social competence and ascribed naïveté to attractiveness and babyfacedness, respectively, we hypothesized that ascribed social competence would mediate the impact of ascribed attractiveness on leadership emergence and that ascribed naïveté would mediate the impact of ascribed babyfacedness on leadership emergence. In a pilot study, we analyzed data from 101 participants of a women’s leadership contest held in 2015 in Germany. We then confirmed these results in a methodologically improved main study on other women who participated in the contest in one of two other years: 2016 and 2017 ( In modern Western societies, sex-related barriers to occupational success still exist and are reflected in economic data. Women hold only 28% of the CEO positions in the United States  and 16% Scientists have formulated several theories to explain differences in perceptions of the leadership potential of women and men and differences in their leadership emergence: As a basic principle, social role theory  suggestsAnother related, albeit more general theory that has contributed to explaining inequities in society on the basis of social group processes is the expectation states theory . This thNevertheless, several studies have provided empirical support for the validity of these theoretical frameworks in explaining differences in the performances of women and men with reference to leadership: For example, A more complex and new model combining aspects of personality traits with behavioral mechanisms was recently proposed along with a meta-analysis on leadership emergence: ascribed facial attractiveness would positively predict leadership emergence (Hypothesis 1). Moreover, we hypothesized that social competence would positively mediate the impact of facial attractiveness on leadership emergence (Hypothesis 2) because social competence represents a determinant of leadership skills . Moreover and analogous to Hypothesis 2, we investigated whether these babyface-specific attributions could explain this negative association: Therefore, we hypothesized that ascribed naïveté would negatively mediate the impact of ascribed babyfacedness on leadership emergence (Hypothesis 4).It seems plausible that a person with a high level of ascribed naïveté  due to his or her facial appearance would find it difficult to meet leader role expectations. Women may be particularly inclined to suffer from this misperception because they are more likely to encounter prejudice due to role incongruence in the first place . In addiTo summarize the aims of the present study, we intended to investigate the impact of facial characteristics on women’s chances of emerging as a leader, and further, whether the ascription of other attributes that are based on facial characteristics would mediate this effect in the setting of a women’s leadership contest. On the one hand, we hypothesized that ascribed attractiveness would be a positive predictor of leadership emergence and that ascribed social competence would mediate this relation. On the other hand, we hypothesized that ascribed babyfacedness would be a negative predictor of leadership emergence and that the ascription of naïveté would mediate this relation. Data collected during contests that were held exclusively for women in three consecutive years provided a basis of the analyses: 2015–2017. The predictor variables (ascribed attractiveness and ascribed babyfacedness) and the mediator variables  comprised raters’ evaluations of women’s appearance in video interviews from self-recorded videos from the contest application phase. Peer nominations provided by group members after participating in assessment-center-like tasks at the contests served as the dependent variable leadership emergence. Therefore, we present a pilot study, which uses data from the 2015 contest, and then the main study containing a larger sample (data from the 2016 and 2017 contests) and a revised method.1 Data collection took place in two phases: In the application phase, women applied to participate in the contests by submitting their curriculum vitae and letters of recommendation as well as by using a professional software platform to record a video interview consisting of their answers to five or six questions. For each question, applicants could prepare their answer within 2 min. Moreover, participants were instructed to record each of their responses in a maximal duration of 3 min. The women who passed this application phase on the basis of the quality and persuasive power of all of their application documents were invited to participate in the actual leadership contest. At the leadership contest – between attendance at keynote presentations, workshops, and networking slots – contest participants took part in 2 to 3 assessment-center-like tasks in which they engaged in group tasks or group discussions or prepared speeches in groups of 7 to 12 women each. After each task, the women nominated the three group members who had been most convincing as leaders. Contest participants who received the most nominations by their peers were rewarded with vouchers, for example, for travel tickets or career coaching. Participants’ data were included in our study only when they agreed to the anonymous analysis of their data at the time when they applied to participate in the contest.The analyses based on data collected across three consecutive years of a women’s leadership contest conducted in Germany.Contest participants competed for peer nominations in each group task in short-term work groups. After each task, women completed a questionnaire to (a) nominate and (b) describe the three group members who had convinced them the most with respect to the group member’s potential to act as a leader. Leadership emergence was operationalized as the total number of nominations each woman received from her team members across the entire contest. The more nominations a woman received in the contest, the higher her leadership emergence index. However, we controlled for group size, that is, peer nominations possible in the contest, in the main study.Raters  evaluated women’s faces by watching their application videos. Raters watched a 25-s cut of one interview question without sound in order to focus solely on the participants’ outward appearance and to avoid any undue influence of other aspects . The length of the video sequence was determined on the basis of pretests as well as literature suggesting that a rating of appearance was possible after this amount of time see .ascribed facial attractiveness. The predictor variable ascribed babyfacedness resulted of the mean ratings of the four babyfacedness items  averaged across all raters.After the video sequence had finished, raters assessed the women’s facial attractiveness and babyfacedness. For each babyfacedness item, a rating manual depicted example pictures for the extreme values in advance .Before raters gave their evaluations, they were trained on the basis of a rating manual. In the pilot study, this manual contained black-and-white pictures for the extreme values of the babyfacedness items  a second time, uncut and with the sound on, and estimated the women’s social competence and naïveté. The mean of all items per scale averaged across all raters served as variables further used to test for mediation effects. Ascribed social competence consisted of six items in the pilot study  and of five items in the main study . Ascribed naïveté consisted of three items in the pilot study , and of four items in the main study .Age, professional experience, and leadership experience in years were obtained from the CVs the women submitted with their applications.M = 31.96, SD = 6.68). On average, these women had 9.73 years (SD = 6.04) of professional experience and 6.22 years (SD = 4.54) of leadership experience. Furthermore, 79.2% were German citizens, 5.0% other, and 15.8% did not provide information about their nationality.In sum, 109 women who participated in the contest in 2015  gave their consent to use information from their CVs and interviews for research purpose and were included. Videos of eight contest participants were excluded due to insufficient picture or sound quality. The ages of the final sample of 101 women ranged from 22 to 53 years  to engage in a 50-min group discussion about the skills needed for successful leadership, (b) to take 80 min to build a wooden construction, including several predefined intermediate goals, and (c) to take 50 min to prepare a political speech for a female executive politician. Each participant gave written nominations of the three most convincing group peers after each task (see section “Leadership Emergence”). Leadership emergence was calculated on the basis of the General Information (see section “Leadership Emergence”).The ascribed facial attractiveness scale (α = 0.98), the ascribed babyfacedness scale (α = 0.78), the ascribed social competence scale (α = 0.85), and the ascribed naïveté scale (α = 0.88) all demonstrated sufficient internal consistency. The ICC values were acceptable, ranging from 0.63 to 0.82 .4).To test Hypotheses 1–4, we calculated mediation analysis using the “lavaan” package  and that ascribed social competence was a significant predictor of leadership emergence . A bootstrapping estimation approach with 1,000 bootstrapped samples indicated a significant indirect effect . Yet, ascribed facial attractiveness did not significantly predict leadership emergence before  or after  adding the mediator, ascribed social competence. Because there was a significant indirect effect , we concluded that ascribed social competence acted as a mediator between ascribed facial attractiveness and leadership emergence, even though the analysis did not reveal a significant total effect.5Descriptive statistics for the dependent and independent variables are presented in Table SE = 0.10, p = 0.008) and that ascribed naïveté was a significant predictor of leadership emergence . The bootstrapping estimation approach with 1,000 bootstrapped samples indicated a significant indirect effect . Ascribed babyfacedness did not significantly predict leadership emergence before  or after adding the mediator, ascribed naïveté . The results indicated a mediating effect of ascribed naïveté, which means that the higher a participant’s ascribed babyfacedness, the higher her ascribed naïveté; and the higher her ascribed naïveté, the lower her leadership emergence, as Figure The results of the mediation analysis for Hypotheses 3 and 4 indicated that ascribed babyfacedness was a significant predictor of ascribed naïveté . On average, these women had 9.61 years (SD = 6.12) of professional experience and 4.69 years (SD = 4.33) of leadership experience  and 2017 (84 of the 114 participants gave consent) in our main study. The ages of the contest participants ranged from 21 to 52  to engage in a 45-min group discussion and (b) to spend 40 min preparing an industrial lobby presentation. In 2017, the women were asked (c) to spend 60 min preparing a huge event and (d) to spend 60 min preparing for a public debate on the gender issue. Each participant provided written nominations of the three most convincing group peers after each task (see section “Leadership Emergence”). We summed the number of nominations for each participant. For the analyses, we divided this sum of nominations by the number of peer nominations possible  in order to control for contaminations due to effects of group size.In the main study, the ascribed facial attractiveness scale (α = 0.98), the ascribed babyfacedness scale (α = 0.82), the ascribed social competence scale (α = 0.93), and the ascribed naïveté scale (α = 0.90) all demonstrated sufficient internal consistency. ICC values for all ratings in the main study ranged from 0.88 to 0.89 for the ascribed facial attractiveness items, from 0.81 to 0.89 for the ascribed babyfacedness items, from 0.71 to 0.84 for the ascribed social competence items, and from 0.68 to 0.83 for the ascribed naïveté items .Before we calculated the mediation analyses in a manner that was similar to the analyses used in the pilot study, we centered the predictor, mediator, and dependent variables on the mean of the respective contest year in order to enhance comparability for the total sample in the main study. For example, a participant’s ascribed facial attractiveness may have been high in comparison with the entire female population but not in comparison with her competitors in the contest, if all contest participants were also rated as highly attractive. By centering the variables on the respective sample means, we corrected for such potential group characteristics.7.As we did in the pilot study, we ran mediation analyses using the “lavaan” package in R. Seven participants had to be excluded from these analyses due to missing information on group size. We further controlled for possible issues of non-normality by, again, using robust ULS estimators. To control for the high correlations between the predictor variables ascribed facial attractiveness and ascribed babyfacedness as well as between the mediator variables, ascribed social competence and ascribed naïveté, revealed by the a priori analyses, we ran further analyses in which we included all variables simultaneously in one path model. As postulated by SD = 3.62) across the two tasks. On average (and before the variables were centered), the mean of ascribed facial attractiveness was 5.96 (SD = 1.31), and the mean of ascribed social competence was 4.80 (SD = 0.61). The mean of ascribed babyfacedness was 3.97 (SD = 0.85), and the mean of ascribed naïveté was 3.41 (SD = 0.63). See Table Contest participants in the main study received an average of 5.58 nominations , and the higher her ascribed social competence, the higher her leadership emergence . The bootstrapping estimation approach with 1,000 bootstrapped samples indicated a significant indirect effect . However, ascribed facial attractiveness did not significantly predict leadership emergence, neither before  nor after adding the mediator, ascribed social competence . Further, ascribed naïveté significantly predicted leadership emergence , supporting the results of the pilot study as well. In other words, the higher a participant’s ascribed babyfacedness, the higher her ascribed naïveté. However, the higher her ascribed naïveté, the lower her leadership emergence. The bootstrapping estimation approach with 1,000 bootstrapped samples indicated a significant indirect effect . Further, in line with the results of the pilot study, ascribed babyfacedness did not predict leadership emergence, neither before  nor after adding the mediator, ascribed naïveté  = 0.43, CFI = 1.00, RMSEA < 0.001, SRMR = 0.012, and is displayed in Figure indirect = 0.03, 95% CI , p = 0.271), nor the effect of ascribed facial babyfacedness on leadership emergence through ascribed naïveté  reached the conventional thresholds of significance. Furthermore, there were no unique direct effects of ascribed facial attractiveness , ascribed babyfacedness , ascribed social competence , or ascribed naïveté  on leadership emergence. However, when we combined the unique direct and indirect effects of ascribed facial attractiveness, we found a significant unique total effect on leadership emergence , suggesting that ascribed facial attractiveness had an effect on leadership emergence above and beyond ascribed babyfacedness and ascribed naïveté.We further tested the unique ability of each mediator to mediate the corresponding relation between the predictor and leadership emergence in one joint path model. In this model, we controlled for the correlations between the predictor variables and the mediator variables in order to identify the unique impact of each mediator. Overall, the model showed a very good fit, χAim of the present study was to increase the understanding of the effects of facial appearance and ascribed characteristics on leadership emergence by employing data collected at a women’s leadership contest. We captured characteristics of facial appearance by rating participants’ application videos as they responded to standardized interview questions. Peer nominations of the women from two exercises that were part of the contest served as a measure of leadership emergence. Analyses of data from both a pilot study and a main study revealed a significant indirect effect of ascribed facial attractiveness and babyfacedness ratings made by independent raters on leadership emergence. Ascribed social competence mediated the relation between ascribed facial attractiveness and leadership emergence, and ascribed naïveté mediated the relation between babyfacedness and leadership emergence. However, the results did not support Hypotheses 1 and 3 because neither ascribed facial attractiveness nor ascribed babyfacedness significantly predicted leadership emergence. When we tested the hypotheses separately, these findings held both, independent from the inclusion or exclusion of the mediators. Therefore, the analyses in this study revealed that both the women’s perceived attractiveness and their babyfacedness were not directly related to their leadership emergence but had an impact through ascribed aspects that were explicitly or implicitly related to these characteristics.Following the recommendations of a reviewer, we also tested the unique effects of our two mediators and predictors on leadership emergence. Analyses revealed a significant unique effect on leadership emergence only for ascribed facial attractiveness after controlling for ascribed babyfacedness and ascribed naïveté. This finding suggests that the previously reported indirect effects of ascribed social competence and ascribed naïveté showed a large overlap of variance and that the shared and not the unique portion of ascribed personality traits variance mediated the effect of ascribed facial appearance on leadership emergence. Therefore, this finding could signify that an underlying latent variable, namely, ascribed leadership personality, is mediating the effect between ascribed facial appearance and leadership emergence. However, our study was not equipped to handle such a latent variable model due to an insufficient sample size and number of indicators. Future research should investigate the unique and common portions of indirect effects of ascribed facial appearance on leadership emergence through ascribed personality traits by including more indicators for ascribed facial appearance and ascribed personality traits.These findings also suggest a higher relevance of facial attractiveness for leadership emergence because babyfaced women were perceived as more attractive. However, literature indicated babyfacedness to be related to attractiveness, especially in women . The modFurther bearing in mind the relation between babyfaced features and femininity , the queAscribed social competence, related to facial attractiveness, indicated an advantage for obtaining a large number of nominations in the women’s leadership contest. On the other hand, ascribed naïveté, related to babyfacedness, indicated a disadvantage. The socially enhancing effects of aspects of attractiveness were well in line with the literature e.g., . MoreoveThese results are particularly remarkable because the predictor and criterion variables were assessed with different methods. Although both kinds of variables were assessed with ratings, the ratings were made by different samples: The raters of the videos were students, whereas the raters in the contest were competing peers. Furthermore, the predictor variables consisted of ratings of recorded video interviews, whereas the criterion variables based upon real-life interactions in a competitive situation. As the two kinds of raters may have perceived the same targets of rating in different contexts, somewhat different processes – or at least differing on the level of awareness – may have taken place. For instance, the participants of the contest may have focused more on the competition and their own performance, whereas the raters in the lab explicitly concentrated on the features of facial appearance and personality they had to assess. The finding that ratings of video recordings were related to ratings from a real-life situation may hint at two possible mechanisms: First, the impressions that the raters got from the video recordings were similar to the impressions that the peers had during the contest, the latter of which may have shaped the interactions and ascriptions to a certain extent see . TherefoNevertheless, our results are well in line with previous research on the relevance of appearance for election outcome predictions in political leadership e.g., . In otheSecond and alternatively, these results may also indicate that consistency in first impressions based on facial appearance may lead people to treat other individuals in certain ways that subsequently shape these individuals’ outcomes and behaviors see . FurtherAnother advantage of this study is that the data collection was part of a real contest, and the sample consisted of real women who participated in the study to win the contest and enhance their reputations. This competitive setting could be interpreted as comparable to an actual personnel selection situation  where performance is required within a short period of time. However, the rather unconventional study setting somewhat restricts the interpretation with regard to generalizability: Peers from group tasks usually do not make hiring decisions in personnel selection, but rather, external observers or assessment experts do. Nevertheless, we aimed to contribute to knowledge about which variables are relevant for leadership emergence. Conclusions cannot be drawn with respect to leadership ascription or leadership performance in the medium or long term, especially in comparison with day-to-day teamwork without a competitive character, even though the contest participants had to balance competitiveness with cooperation in working toward a common goal during the tasks. However, our research revealed that appearance is especially important when no information or only a little other information is available . In lineAlthough the setting allowed obtaining an unusual sample of women, future research should include male participants as well as other ethnicities  to investigate interactions between sex and other personal attributes. For instance, previous research indicated that attractiveness judgments were better predictors of election success for women than for men . FurtherOur findings provide support for earlier studies e.g.,  that fouVisual appearance often influences the first impression a person makes, especially in personnel selection  and therefore affects the attributions made to an individual. Under a more practical perspective, we encourage organizations and hiring experts to enhance their awareness of such ascription- and stereotype-inducing mechanisms, helping women to overcome these gender-related obstacles to achieve leadership positions equally to their male counterparts. The use of standardized assessment criteria that are determined in advance (from the side of human resources) and the decision not to include a photo in a job application (from the side of an applicant) may be first steps to diminish such effects, at least with regard to aspects of  appearance.https://osf.io/kxm4w/).Datasets are in a publicly accessible repository: the datasets analyzed for this study can be found in the Open Science Framework , Ethic Committee of the Paris Lodron University of Salzburg. The protocol was approved by the Ethic Committee of the Paris Lodron University of Salzburg. All subjects gave written informed consent in accordance with the Declaration of Helsinki.FG collected the data for the pilot study, co-revised the rating manual, and supervised and organized the data collection and analyses for the main study. She mainly contributed to writing the Method, Results, and Discussion sections of the manuscript and revised earlier drafts of the entire manuscript. CV conceptualized the research, data collection and analyses of both the pilot study and the main study. She mainly contributed to writing the first versions of the Introduction and Discussion sections as well as the Results section of the pilot study. TO supervised the conceptualization of this research regarding the operationalization and methods. She revised earlier drafts of the manuscript, in particular providing theoretical input for the Introduction and Discussion sections.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The excellent optoelectronic properties of graphene give it great potential for applications in optical detection. Among the graphenes obtained through many synthetic methods, epitaxial graphene obtained by thermal decomposition on silicon carbide has remarkable advantages for preparing photodetectors. In this research, epitaxial graphene has been successfully prepared on a silicon surface (0001) of semi-insulating 4H-SiC substrate with a size of 10 mm × 10 mm and epitaxial graphene has been converted to quasi-free-standing graphene by hydrogen passivation. Two metal-graphene-metal photodetectors were fabricated using the two types of graphenes above and the photo-absorption properties of detectors have been investigated under 650-nm laser illumination with different illumination powers. From a comparison of the performances between the two detectors, it was found that a photodetector fabricated with quasi-free-standing graphene shows enhanced performance under a light power of 0.018 mW. Responsivity and external quantum efficiency reach maxima of 5.11 A/W and 9.74%, respectively. This dramatic improvement is mainly due to the disappearance of the buffer layer in epitaxial graphene, providing a new method to achieve optimization of graphene-based opto-electrical devices. Photodetectors, part of the technical foundation of our information society, are widely used in communications, imaging, sensing and other applications . Many di2-hybridized carbon atoms arranged in a hexagonal honeycomb lattice. Thanks to its excellent optical and electrical properties, including wavelength-independent absorption [In the idealistic case, graphene is a two-dimensional material formed by SPsorption , high thsorption , low dissorption  and the sorption ,16, thersorption ,18,19. Hsorption . HoweverThere are many ways to manipulate the band gap of graphene. In particular, three mechanisms have been proposed—(1) Quantum confinement. Graphene is prepared as a graphene nano-strip; then quantum effects and edge effects cause the band gap of the graphene to be opened ; (2) DopIn this research, metal-graphene-metal (MGM) photodetectors have been fabricated in which the two gold electrodes are the source and the drain, respectively, on the same side of the epitaxial graphene. Many detectors with various architectures have been reported, among which the MGM photodetector is the simplest. At the interface between graphene and metal, an electric field is generated due to carrier migration , so thatTwo kinds of epitaxial graphene for photodetector fabrication had been prepared. We named one of the graphenes, the one subjected to hydrogen passivation, quasi-free-standing graphene (QFSG). The other graphene, which was not subjected to hydrogen passivation, epitaxial graphene (EG). The main difference between these two graphenes is that there is a buffer layer in the EG and no buffer layer in the QFSG. Our experiments show that quasi-free state graphene on silicon carbide has better photoelectric conversion properties than epitaxial graphene. For photodetector fabricated with QFSG, a responsivity of 5.11 A/W and an external quantum efficiency of 9.74% at a wavelength of 650 nm are realized. These parameters are well-suited for graphene photodetectors.The epitaxial graphene (EG) for photodetector fabrication was obtained by thermal decomposition on the Si-terminated surface (0001) of semi-insulating 4H-SiC substrate with a size of 10 mm × 10 mm. The 4H-SiC substrate was single crystal and was supplied by State Key Laboratory of Crystal Materials, Shandong University. The substrate was processed using a chemical-mechanical polishing (CMP) technique in advance. Then a standard RCA cleaning method was performed. And RCA was a typical wet chemical cleaning method that was the most commonly used. The growth process of EG includes three principal steps—(1) The substrate is etched in order to obtain parallel and regular wide steps on the surface under a hydrogen atmosphere at a temperature of about 1600–1700 °C. (2) The graphene growth process starts when the temperature and chamber pressure reach about 1750 °C and 800 mbar, respectively. (3) The epitaxial graphene is cooled down to room temperature.QFSG is obtained by extra hydrogen passivation treatment of EG. The silicon-carbon covalent bond between the buffer layer and the SiC substrate is broken and the silicon dangling bonds are saturated when the hydrogen passivation process is carried out . This pr2, while the total area of the device sample is about 4 mm2. A semiconductor test system  was used in the performance measurements of the photodetectors to investigate the photodetecting properties of the two samples. The excitation light source came from a semiconductor laser diode system with a peak wavelength of 650 nm and a laser spot diameter of about 2 mm. A bias increase from 0 to 10 V was applied linearly to both electrodes. A photo response could be detected when the laser illuminated the device.The application of traditional lithography technology could significantly damage the photoelectric properties of graphene. Therefore, in this study, electrodes were made by a sputtering process on two different epitaxial graphenes with a hollow aluminum contact pattern mask. Before the fabrication of the photodetector, these two epitaxial graphenes were cleaned using acetone and ethanol solutions. Then the surfaces of EG and QFSG were covered by aluminum masks, following which the sputtering processes were carried out. At the same time, gold clusters were regularly deposited on the surface of the epitaxial graphene to form gold films as dictated by the shapes of the aluminum masks. A structural diagram of the device sample is shown in In this research, high-quality and micropipe-free SiC wafers were used to process the EG and QFSG. After the hydrogen etching was completed, we got a formation of regular steps morphology with micrometer-sized terrace width and elimination of surface defects on the SiC substrate. This is positive for nucleation and growth of high-quality epitaxial graphene. Subsequently, the carbon atoms preferentially nucleated at the edge of the step and then extended to the stepped platform. Large flat terraces on the SiC substrate can be seen in −1. The intensity of the D peak is related to the degree of defects in graphene, so the D peak is also called the defect peak. The G peak is induced by the stretching vibration of the SP2 carbon atoms, so the stress applied to the graphene can be judged by the shift of the G peak position. As to the 2D peak, its relative intensity and full width at half maximum (FWHM) are usually used to estimate the number of layers of stacked graphene [−1 and the FWHMs of QFSG ranged from 30 to 40 cm−1. By substituting the FWHMs of the 2D peak into the empirical formula.Additionally, Raman measurements were performed to characterize the layer thickness and the quality of the EG and QFSG. It is well known that there are three characteristic peaks in graphene’s Raman spectra, which are commonly called the D, G and, 2D peaks. They appear, respectively, around 1350, 1583 and 2700 cmgraphene . To estiWe can further find that the average number of layers of the EG and the QFSG are 1.38 and 0.38, respectively. The Raman data for two epitaxial graphenes show characteristics of the graphene structure with a buffer layer and with a graphene monolayer.phI is the photocurrent excited by the laser and darkI is the current observed without illumination. Here the net photocurrent curves of the QFSG sample are obtained by changing the bias voltage from 0 up to 10.0 V, as shown in −5 A at a laser power of 0.018 mW and a bias voltage of 10 V. When the laser power exceeded 0.036 mW, there were some different results—the photocurrent obtained under the 0.051 mW laser irradiation was larger than that obtained under the 0.075 mW laser irradiation. That can be related to the heat capacity of graphene and Seebeck effect, which needs to be studied more deeply  and external quantum efficiency (EQE) of the QFSG sample in this experiment were also calculated. R can be obtained by substituting the photocurrent value into the following formula:etectors ,33.It can be concluded from the above comparison that the net photocurrent of the QFSG sample is significantly higher than that of the EG sample. The Fermi level of graphene in the EG sample was increased by the underlying buffer layer, resulting in the conversion of graphene to n-doped graphene . That caIn summary, the buffer layer in epitaxial graphene on SiC severely limits its photo-absorption property. Here we used the hydrogen passivation method to get QFSG from EG and we manufactured an MGM photodetector using QFSG. Photoresponse testing for 650-nm-wavelength lasers was implemented on the QFSG sample, giving 5.11 A/W and 9.74%, respectively, for the maximum values of R and EQE at a light power of 0.018 mW. These results show that QFSG has great potential as a visible-light photodetector and deserves further development.


Anti-chlorpyrifos antibodies were immobilized successfully on the graphene surface. Under optimal conditions, graFET sensor showed an excellent response for chlorpyrifos detection in the linear range of 1 fM to 1 µM with a limit of detection up to 1.8 fM in spiked samples. The developed graFET biosensor is highly stable, sensitive, and specific for chlorpyrifos as confirmed by its significant ability to detect changes in electrostatic potential. These findings signify useful efficacy of immunobiosensors for the detection of chlorpyrifos and other organophosphates in fruits and vegetables.Chlorpyrifos is one of the most widely used pesticides that acts on the nervous system by inhibiting acetylcholinesterase. Prolonged use of chlorpyrifos causes severe neurological, autoimmune, and persistent developmental disorders in humans. Therefore, in this study, a highly sensitive and robust biosensor platform was devised by fabricating graphene field effect transistors (graFET) on Si/SiO Chlorpyrifos  is an organophosphate that is used worldwide to control pests on fruits, vegetables, including a variety of crops. It has high toxicity and long retention time that act on the nervous system and affects mainly neurological development, as well as causes cancer and reproductive disorders4.The widespread use of pesticides in agriculture has led to pesticide traces in the air, water and soil. The presence of pesticide traces in food and environment poses serious health issues8. However, these analytical techniques suffer from several drawbacks such as tedious sample preparation, sophisticated and expensive instrumentation with a consistent need of a trained operator, and are not on-site applicable. Compared to developed analytical methods, these charge sensitive electrochemical sensors offer a variety of advantages such as high sensitivity, quick response, ease of operation and on-site applicability that are promising alternatives for rapid detection of pesticides12. Enzyme-based sensors are gaining importance due to their high sensitivity and fast response but are limited to a certain class of pesticides14. Immunosensors work on the principle of antigen-antibody (Ag-Ab) interaction with high specificity and sensitivity that can be used for the detection of pesticides, narcotic drugs, bacteria, and viruses21. Field effect transistors (FET) are excellent charge sensitive sensors, which can be used for real-time monitoring to study the target-analyte interaction at the electrode surface, and an input signal can be translated into a readable output electrical signal. Liquid state measurements are more desirable than a dry state in FET-based sensors due to real-time detection of biological molecules at less than 1 V, as high temperature can degrade the native state of proteins22. FET-based on graphene as the biosensing platform has been used for the detection of target analyte larger than 30 kDa antigens, antibodies or charged molecules22. Graphene, a single layer of carbon atom arranged in honeycomb lattice, with a linear, gapless band-structure, shows a plethora of interesting properties like quantum hall effect, large Young’s modulus, thermal conductivity, etc. as well as offers several advantages for biological applications like high mobility, large surface to volume ratio due to its ability to absorb various aromatic biomolecules by π-π stacking and electrostatic interactions which makes it ideal for biosensor applications23. It has also shown immense potential for detecting bioactive molecules such as enzymes, DNA, aptamers, making it important for industrial use as well30.Currently, detection of these pesticide residues is based on various analytical methods such as liquid chromatography (LC), high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), capillary electrophoresis (CE), and enzyme-based assay2 substrate followed by thermal evaporation of Cr/Au (5/50 nm) electrodes after electron beam lithography and has been used for the detection of chlorpyrifos (Chl) with a limit of detection (LOD) of 1.8 fM in standard samples that is 10-fold (an order of magnitude) less than the previous reports31, making them one of the most sensitive detectors of chlorpyrifos till date. Along with ease of fabrication, the developed devices are highly sensitive, cost effective, can detect minute changes in antigen concentrations, and can be easily integrated into electronic chips for real-time sensing for on-site applications.Here, we report the detection of chlorpyrifos pesticide on functionalized graphene-FET-based immunosensor. The graphene-FET is fabricated using exfoliation technique on Si/SiOChl-Ab was labelled with graphene via carbodiimide chemistry. For this, 0.5 mg of Ab was added dropwise to 75 μM EDC (1-ethyl-3-(3-dimethylaminopropyl) and 75 μM NHS (N-hydroxysuccinimide) solution. The reaction was incubated at RT for 2 h and centrifuged at 10,000 rpm for 15 min at 4 °C and pellet was redissolved in 1 mL of phosphate buffer (PB), pH 7.4. 100 μg of chl-Abs were added dropwise to freshly activated graphene and incubated at RT for 30 min followed by incubation O/N at 4 °C. The graphene (gra) labelled Abs were further characterized to confirm the binding by spectroscopic and microscopic techniques.The morphological studies of graphene and gra-chl-Abs nanocomposites were done with scanning electron microscope (SEM) (Hitachi S-3400N). The samples for SEM imaging were made electroactive by coating with gold using Quorum SC7620 sputter coater. Atomic force microscopy (AFM) was performed on Bruker Dimension Icon. Fourier transformed Infra-red (FTIR) spectroscopy was carried out using Shimadzu 8700 FTIR. Optical characteristics were studied by UV-Vis spectrophotometer using (Shimadzu 2600).35. Exfoliation was performed on Si/SiO2 substrates using scotch tape (3 M magic tape) technique where 285 nm SiO2 forms back-gate dielectric. Suitable graphene flakes were identified by an optical microscope (Olympus BX51) followed by spin coating and baking of one layer each of PMMA 495 and PMMA 950 at 150 °C that formed the positive resist for e-beam lithography performed in Raith Pioneer to pattern the contact pads. MIBK and IPA (1:3) were used to develop polymer layer post e-beam lithography. Afterwards 5/50 nm of Cr/Au were thermally evaporated to form the source-drain electrodes. The sample was then mounted on ceramic chip carrier using silver epoxy. The contact pads were ball bonded in TPT HB05 wire bonder machine.To fabricate FET devices, we have used mechanically exfoliated graphene, which is known to perform at very low noise levelsAll electrical measurements were done using SR 830 Lock-In Amplifier. Keithley 2400 source meter was used to provide back gate voltage. The chl-Abs were immobilized via carbodiimide activation reaction that activates the carboxylic groups on graphene surface and binds via amine groups present on antibodies. The immobilization and hybridization of chl-Abs on FET electrode was studied at different stages by recording changes in resistance. The immobilization of chl-Abs (1 ng/mL) was done in 50 mM PB (pH 7.4). The washing with PB was done at each step to remove excess and unbound material. Chl-Ag was added at different concentrations (1 fM to 1 µM). Real-time sensing in liquid state was done by applying a constant current of 100 nA across the sample and monitoring the resistance change in the FET for various antigen (chl-Ag) concentrations.Functionalization of graphene was achieved by carbodiimide chemistry that resulted in the activation of carboxylic groups in graphene and helps in covalent bond formation with amine reactive chl-Abs. The fabrication of functionalized graphene for sensing chl-Ag was done with FET immunosensor. While blocking non-specific sites with BSA on the surface of graphene, chl-Abs was immobilized and specific Ag added on the electrode surface. Change in resistance (%R) was monitored w.r.t. increase in chl-Ag concentration Fig. .Figure 1−1 for C=O and C-C stretch, respectively. Presence of peak at 1372 cm−1 for C-N confirmed that binding occurred between graphene and chl-Abs. The resistance (R) vs gate voltage (Vg), measured in constant current mode, showed the characteristic bell-shaped curve with a maxima at ~8 V that corresponds to charge neutrality point or Dirac point  of SLG showed a clear graphene layer . UV-Vis spectra Fig.  showed tint Fig. . The tratra Fig.  displayeyer Fig. . Figure yer Fig. .Figure 216.Figure 22. Blocking unbound surface of graphene was done with BSA and the change in the resistance was monitored  is 2 g/kg, therefore the developed graphene-based ultrasensitive immunosensor can be used for detection of chlorpyrifos in fruits and vegetables.Supplementary Information


Thus far, attempts at reliably fabricating suspended graphene devices comprising metal contacts, have often been hampered by difficulties in exceeding sizes of 1 µm in diameter, if using UV lithography. In this work, area of suspended graphene large enough to be utilized in microelectronic devices, have been obtained by suspending a CVD graphene film over cavities, with top contacts defined through UV lithography with both wet and dry etching. An area of up to 160 µm2 can be fabricated as backgated devices. The suspended areas exhibit rippling of the surfaces which simultaneously introduces both tensile and compressive strain on the graphene film. Finally, the variations of the Fermi level in the suspended graphene areas can be modulated by applying a potential difference between the top contacts and the backgate. Having achieved large area suspended graphene, in a manner compatible with CMOS fabrication processes, together with enabling the modulation of the Fermi level, are substantial steps forward in demonstrating the potential of suspended graphene-based electronic devices and sensors.Freestanding (suspended) graphene films, with high electron mobility (up to ~200,000 cm  The incor SupG)  resultinIn this paper, a combined atomic force microscopy (AFM) and Raman spectroscopy (RS) approach was used to investigate the nature of backgated devices comprising SusG, in terms of both the in-built static strain inherently present and the additional dynamic strain that can be produced by applying a bias to the electrodes.2) and aluminium backgate; #2, SU-8 with rectangular cavities (20 × 8 µm2) and aluminium backgate; #3, SU-8 with circular cavities (5 × 5 µm2) with indium tin oxide (ITO) backgate; #4, SiO2 with multiple square cavities (5 µm diameter) and n-type silicon backgate.Four different devices were fabricated. For all samples, CVD graphene was used. The four devices mainly differ in the nature of the insulating material used to define the cavities, the shape and dimensions of the cavities, and the (semi)conductor used to form the backgate electrode. The four devices are as follows: #1, SU-8 with rectangular cavities  sputtering. After that, a 2.5 µm SU-8 (a negative photoresist) was spin-coated over the silicon dioxide layer. Next, reactive ion etching (RIE) was used to define the cavities. For device #3, the same process used for device #1 and #2 was repeated, except now an ITO layer with a thickness of 200 nm was grown on the Si surface instead of the aluminum layer. For device #4, a similar lithographic process as for devices #1 and #2 was followed but with a SiO2 layer of 1.3 µm thickness thermally grown directly on the Si surface.For devices #1 and #2, a thin film of aluminum with a thickness of 200 nm was evaporated on the Si surface to provide a backgate contact. A SiO2:The detailed process of fabrication of the cavities varies depending on whether these are fabricated in SU8 or SiO3PO4: HNO3: CH3COOH: H2O = 73%: 3.1%: 3.3%: 20.6%) solution. The ~1 μm deep cavities were etched in SU8 by reactive ion etching  power and gas flow rate of 50 sccm O2 and at an etching rate of ~50 nm/min). The Al hard mask was later removed by dipping the substrates in Al etchant solution.SU8 was spin coated onto the Si substrate, to a ~1 μm thick layer, then flood exposed in UV (λ = 365 nm) for 2 min and hard baked at 180°C for ~25 min. A 50 nm thick evaporated Al hard mask patterned with cavities was used to expose the 0.5 μm photoresist (PR) film. Solution development of the top PR (S1805) was followed by the wet-etching of the exposed Al in phosphoric nitric acetic acid  power and gas pressure of 22 mtorr and an etch rate of 25 ± 2 nm/minute was achieved to create ~1 μm deep cavities in ~35 ±4 min). The remaining Al hard mask was then removed by Al etchant.A 150 nm thick Al hard mask layer was evaporated directly on ~1.5 μm thick SiOFor all devices, CVD graphene was supplied by 2-DTech Ltd, grown on a 25 µm copper film. Graphene was covered by a sacrificial polymer layer and then separated from the copper by wet etching in ammonium persulfate (APS) until all the copper was etched away. The film was then transferred onto a pre-patterned silicon substrate.2 environment for 5 min to obtain Ohmic contacts.Top contacts were thermally evaporated as a thin film of gold (100 nm) using UV lithography. Even though the sacrificial layers were removed from the surface of the exposed graphene, the device still exhibited residual polymer contamination that to be entirely removed, required further steps. For devices #1 to #3, a large part of the removal of the protective polymer layer was done by exposing the surface to acetone in vapor form (acetone fog). This was done in order to make the polymer removal procedure more delicate towards the membranes, which tend to otherwise break rather easily. During this early part of the fabrication process, the cleaned device was, annealed at 300°C under NElectron microscopy studies of the SusG membranes were performed on samples #1 to #3 (more details are provided in section 1 of the Supplementary Information (SI)). The results are shown in In all three cases, the number of membranes that survived the fabrication process unbroken was limited, often around only 10% or less. Membranes tend to not always be completely broken and the graphene sheet in that case appears to be hanging by a ‘thread’ . In Comparison between the AFM data of SusG in 2, 160 µm2 and 25 µm2. The SusG being recessed is a result of both gravity and adhesion, most likely through van der Waals interaction, to the side wall of the cavity. Such adhesion is one of the origins of strain over the SusG surface, as will be shown later.Samples #1 to #3 have rather large areas of SusG: 120 µmThe edges of the cavities appear, in places, not to be atomically flat, as can be seen in the AFM micrograph in Raman spectroscopy can be used to determine both the doping and strain of the SusG compared to the SupG. Detailed information about the Raman system and parameters used are given in the SI, section 1.RS is often used to detect the number of graphene layers , their c−1 to 10 cm−1, compared to the values recorded for the SupG. Furthermore, there is a much larger spread of values for SusG compared to SupG. This would suggest a significant degree of local variations in either the film uniformity, strain status or doping levels over the surface of each individual SusG area.The average peak positions of the G and 2D mode in Differences between the G and 2D modes of SusG and SupG can help identify the type and level of doping present. For example, p-doping doping could originate from residual photoresist (PR) (in SusG) and n-doping could be due to SU-8 (in SupG) . On the Before we move onto the next section, it is worth pointing out that the large spread of peak positions for the 2D mode of sample # 1, can be explained by the presence of anisotropic strain. The 2D mode is activated by a second-order double resonance process near the Dirac point of the band structure of graphene. The presence of any external influence, such as strain, naturally leads to shifs in the electron momentum around the Dirac point, which directly affects the mechanism of generating the 2D mode. By looking carefully at the microscopy images of samples #1 and #2, in F) [F and doping levels  . The folne,h [F) ,51, defi−16 eV s, Fν is the Fermi velocity, 108 cm/s [where 108 cm/s , Dep is o 65 meV , dependio 65 meV ,47. Takis 33 meV .−1 and −1. While the former is needed in From the measured data, it is apparent that the average G and 2D peak positions, for unstrained/undoped graphene can be estimated as Sample #2 was measured before and after the PR was completely removed. A Raman line mapping (line length ~3.8 µm) was obtained, where Raman data was collected from 19 data points (200 nm apart) along the SusG surface. 12 cm−2 [Moreover, once the residual PR is further removed through laser annealing , the RS data is different: e.g. the I(2D)/I(G) ratio exhibits variations across the surface and these can also be modulated by the application of a backgate bias voltage from −10 V to −30 V, as shown in 12 cm−2 . This ruLocal variation in the RS data and resulting local strain can be associated with the existence of static ripples on the graphene surface, which are limited and constrained while the membranes are supported by a thin PR layer (associated with non-zero doping), but which become more pronounced and also tunable by an applied bias via the backgate, once the supporting action of the PR is removed (associated with zero doping). Already at 0 bias, such ripples result in mechanical deformation of the membranes, evidenced by substantial increases in the FWHM of the G bands (a well-known result of the splitting of the degeneracy of the G mode subbands) compared to a membrane with PR support. Some limited tunability can be further attained by applying a bias up to −30 V to the backgate with respect to the top contacts. Increasing the negative voltage reduces the FWHM, therefore one can conclude that either the strain decreases, changes in the Fermi energy are being triggered, or both. The apparent effect of mechanical deformation, i.e. strain, can, in fact, be linked to changes in the Fermi level of the graphene surface. According to Mohiuddin et al.  changes where  is 0.99 . The qua is 1.99  (1.24 [5 is 1.99 ). The quculation , which iF pinned at the Dirac point, i.e 0 eV. However, other sites have either compressive or tensile strain (blue and red areas), leading to a lower or higher Fermi level up to +70 meV and −110 meV with tensile and compressive strain, respectively. The Fermi level can shift positively/negatively giving rise to excess n/p carriers, therefore likely forming charge puddles in the adjacent area even at 0 V gate biasing.It can be seen, from F can be further tuned by operating at different backgate voltages. Upon gate biasing, the electrostatic force will pull and stretch the surface of graphene thereby modifying the amount of strain and hence EF, as shown in These variations of the ETo show this, sample #4 was used. The morphology of sample #4 is first studied using AFM and scanning electron microscopy (SEM), just like the other samples. Three locations were selected: L1, broken SusG; L2, intact SusG and; L3, intact SusG with thicker residual PR . Figure S,D, which was varied from −15 V to +15 V. The map in S,D equal to 0. In such case we notice how the EF oscillates between slightly positive and slightly negative values, corresponding to local tensile and negative strain. The maps in S,D to a positive or negative non zero value forces changes in the Fermi level. Worth pointing out that as a dielectric covers the backgate contact, then the charges (positive) that appear on the surface of the dielectric are opposite to those of the backgate contact (negative). The average value of the Fermi level over the SusG , and why it is very difficult to directly extract such parameters for SusG. The simple reason is that much of the electrical behavior of the devices in this paper is dominated by SupG rather than SusG, as the membranes occupy a limited surface area in between the metal contacts . This means that, e.g., the series resistance between SusG and SupG is always dominated by that of SupG, and any, e.g., increase in mobility or carrier concentration (or conductivity) becomes undetectable. Therefore, a completely different device layout, with limited SupG compared to SusG would be required to extract such parameters quantitively, which is the reason such values cannot be given in this work.2. We have studied a number of specimens and showed that large areas of up to 160 µm2 can be fabricated as functional backgated devices. There are significant hurdles in suspending arbitrarily large sizes, as the larger the area of suspended graphene, the deeper the cavity needs to be, to avoid the membrane sinking and touching the bottom of the cavity. We observed that static ripples are created on the suspended graphene and that such ripples introduce both tensile and compressive strain, simultaneously.Areas of suspended graphene have been obtained by transferring a CVD graphene film over cavities in either SU-8 or SiOWe have analyzed variations in the Fermi level in the suspended graphene areas, as a function of bias of the top contacts while having fixed the voltage of the backgate. We identify that the areas of suspended graphene mechanically react to being attracted/repelled to/from the backgate electrode. This mechanical deformation adds tensile strain to the film, and the Fermi level becomes less negative. We estimated that the tensile strain introduced in this way results in a change of Fermi energy of up to 50 meV, an appreciably large tuning. This effect can be exploited to detect different types of adsorbates sitting on the graphene surface. When the graphene is exposed to adsorbates, these will typically make a chemical bond with the surface. Our results show that strain and Fermi level can be effectively controlled by external voltage. Therefore, we postulate that: firstly, a voltage controllable shift in the Fermi level could be exploited to increase the adsorbate binding strength with the graphene surface; secondly, unlike supported graphene, suspended graphene will likely exhibit a substantial localized strain field when chemically bound to an absorbate. Such strain fields can in turn shift the Fermi level upwards or downwards relative to the Dirac point. These suggest that suspended graphene can form the basis of an electronic sensor with increased sensitivity.


Accumulation of these MCHR1 PET-tracers in rat brown adipose tissue (BAT) in vivo provided first indication of MCHR1 expression in rodent BAT. To rule out off-target binding, affinity of both MCHR1 ligands toward adrenergic beta-3 receptors (ADRB3) was examined. Further, specific binding of [11C]SNAP-7941 to brown adipocytes and effects of MCHR1 ligands on brown adipocyte activation were investigated. SNAP-7941 and FE@SNAP evinced to be highly selective toward MCHR1. [11C]SNAP-7941 binding to brown adipocytes was shown to be mainly MCHR1-specific. This data strongly indicates MCHR1 expression in rodent BAT and moreover, a peripheral, anti-obesity effect of MCHR1 antagonists directly exerted in BAT is proposed. Moreover, MCHR1 expression in murine brown adipocytes was confirmed by protein and mRNA analysis. We conclude that MCHR1 PET imaging contributes to basic research in endocrinology by elucidating the involvement of the MCH system in peripheral tissues, such as BAT.[ The fact that BAT activity, quantified as [18F]FDG uptake, was negatively correlated to body mass index and percent body fat indicated a potential role of BAT in energy expenditure in humans -O-[methyl-11C] methylreboxetine thereby enhancing the understanding of BAT activation, control, and metabolism  was underestimated for a long time, it nowadays attracts scientific interest as a therapeutic target for obesity and related metabolic diseases such as type 2 diabetes –3. The gn humans –6. PET itabolism –9.via uncoupling protein 1 (UCP-1) resulting in increased fatty acid oxidation and dissipation of excess energy as heat. Thereby, BAT regulates fuel metabolism by increasing glucose and triglyceride uptake, decreasing blood glucose and lipids, and by serving as glycogen storage  in brown adipocytes. Subsequent lipolysis or increased uptake of glucose and its conversion into fatty acids finally lead to the uncoupling of ATP synthesis  storage –14.via BAT  of unlabeled SNAP-7941 for displacement purposes caused uptake enhancement of both MCHR1 PET-tracers in interscapular BAT depots  was prepared in-house using a fully automated cassette-based synthesizer  within the clinical routine production at the Vienna General Hospital, Austria. [11C]SNAP-7941 was synthesized as reported elsewhere using an automated module  (3H](-)CGP-12177 and [125I](-)Iodocyanopindolol were purchased from PerkinElmer, Inc. . All other reagents and cell culture supplies were purchased from standard commercial sources.2-CGP-12177 as the respective radioligand. Test compounds  were added in rising concentrations, whereby the concentration of solvent [ethanol or dimethyl sulfoxide (DMSO)] in the final assay volume remained always 1%. Nonspecific binding was determined using 10 μM carazolol. Total binding was determined in the presence of 1% respective solvent. Incubation was performed for 90 min at 27°C. Filtration through GF/B filters  was performed using a cell harvester  and was followed by two washing steps using ice-cold wash buffer . Filter pieces containing receptor-bound radioactivity were shaken for 30 min in scintillation cocktail  before liquid scintillation counting . IC50 fitted binding curves were generated using the GraphPad Software 5.0  using nonlinear regression. Ki values were calculated using the Cheng and Prusoff equation assuming a Kd value of 109.2 nM for [3H]CGP-12177 according to the literature . Competitive binding assays were performed using 13.6 ng/μL ADRB3 expressing CHO-K1 cell membrane suspension (cf. 2.3) and 5 nM FDG (150 kBq per dish) for another 50 min. As glucose concentration is high in cell culture media thus interfering with [18F]FDG uptake, cells were starved to achieve reasonable signal. Experiments were performed on at least three different days.Kinetic measurements of [18F]FDG incubation, cells were pre-treated with the adrenergic beta-3 agonist , adrenergic beta antagonist (2 μM (S)-propranolol), the MCHR1 ligands (20 μM SNAP-7941 or FE@SNAP), or vehicle (baseline). [18F]FDG uptake was performed for 50 min at 37°C . Supernatant was taken off, cells were washed with ice-cold PBS and finally trypsinized. Radioactive cell fractions were gamma counted  and normalized to percentage uptake per well. [18F]FDG uptake under baseline conditions refers to 100% uptake. Three independent experiments were performed in triplicates. Statistical analysis was performed using an unpaired, two-tailed t-test.Brown pre-adipocytes were seeded, grown to confluence and differentiated in 6-well plates as described in section General. Cell starving was performed for 1 h using unsupplemented, glucose-free DMEM. Thirty minutes prior to [18F]FDG uptake measurements, was not indicated for [11C]SNAP-7941 binding experiments. Cells were pre-treated for 30 min with ADRB3 ligands -propranolol, 2 μM each) or MCHR1 ligands . Baseline was determined using DMSO or ethanol, respectively. Sample processing, data analysis and statistical testing were performed as described in [18F]FDG Uptake Experiments: Effects of MCHR1 Ligands on Brown Adipocyte Activation.Brown pre-adipocytes were seeded, grown to confluence and differentiated in six-well plates as described above. Thirty minutes prior to the experiment, maintenance medium (cf. 2.1) was discarded and replaced with respective serum- and additive-free medium to avoid plasma protein binding of the PET-tracer. Cell starving, as performed for Iodocyanopindolol association phase did not affect binding to CHO-K1-ADRB3 cells. A second application of 20 μM MCHR1 ligand after 120 min led to a decrease in cell-associated radioactive signal, which was more pronounced after a third addition of unlabeled compound  showed no effect on [125I]Iodocyanopindolol binding to CHO-K1-ADRB3 cells.Additionally to the conventional affinity determination using cell membrane preparations, displacement experiments using FDG uptake to 137.7 ± 12.2% referring to baseline, whereas the non-selective adrenergic beta receptor antagonist (S)-propranolol did not alter [18F]FDG uptake. The opposite effect was observed for MCHR1 ligands, as [18F]FDG uptake was significantly reduced, when SNAP-7941 (63.24 ± 2.6%) or FE@SNAP (73.03 ± 13.6%) were applied . Moreover, competition of [11C]SNAP-7941 using ADRB3 ligands resulted in 95.8 ± 8.6% binding/well for (S)-propranolol (not significant) and 91.1 ± 3.6% binding/well for CL 316,243, respectively , whereas low expression was detected in mouse lung (mean Ct 35.5) and murine brown adipocytes (mean Ct 33.1). No Mchr1 mRNA expression was found in mouse spleen (mean Ct 38.5) that served as a negative control -propranolol and pindolol differed slightly from literature due to different experimental settings , which emphasizes the importance of testing reference compounds for distinct experimental settings. Obtained Ki values for FE@SNAP and SNAP-7941 suggested intermediate affinity toward ADRB3 and thus potential ADRB3 mediated effects when pharmacological doses of unlabeled compounds are applied . However, the affinity of both ligands toward MCHR1 is several thousand times higher than toward ADRB3. Due to the applied trace amounts of PET ligands , we conclude that [18F]FE@SNAP and [11C]SNAP-7941 are selective PET-tracers for MCHR1 imaging. Additionally performed real-time kinetic measurements using CHO-K1-ADRB3 cells and the yet rarely employed LigandTracer® technology provide a cell binding technique which is closer to in vivo imaging techniques like PET, as high resolution time-activity curves are generated by applying a single tracer concentration. Displacement of [125I]Iodocyanopindolol binding to CHO-K1-ADRB3 was achieved when concentrations ≥40 μM of unlabeled MCHR1 ligands were applied. This finding is in accordance with the Ki values in double digit μM-range obtained from competitive binding studies using cell membrane preparations and [3H]CGP-12177 as the respective radioligand.For affinity determination toward ADRB3, a competitive binding assay using CHO-K1-ADRB3 cell membranes was established and tested using known ADRB3 ligands. The obtained K18F]FDG uptake measurements) characteristics of the BAT phenotype were achieved 4–6 days after induction, thus, experiments using brown adipocytes were performed in this time frame. [18F]FDG uptake by mature brown adipocytes followed linear kinetics mirroring the unique trapping mechanism of [18F]FDG: it is transported via glucose transporters and intracellularly phosphorylated by hexokinase leading to trapping of the respective metabolite [18F]FDG-6-phosphate, as further metabolism is hindered  and metabolic  are observed for pharmacological doses of SNAP-7941 and FE@SNAP. The role of MCHR1 in brown adipocytes, especially the involvement in glycolytic activity and glucose transporter expression is yet unexplored.ADRB3 stimulation by the agonist CL 316,243 in brown adipocytes led to enhanced SNAP-7941 accumulation evincing specific binding to brown adipocytes. This finding points at MCHR1 expression in brown adipocytes and a subsequent involvement of the melanin-concentrating hormone system in brown fat depots. Western Blot analysis and qPCR finally showed the expression of the MCHR1 in murine brown adipocytes. In addition, as MCHR1 expression in lung is often discussed, we included mouse lung into our analysis. Our qPCR confirmed the abundance of Mchr1 mRNA in mouse lung, which is in accordance with Kokkotou et al. -propranolol, demonstrating the superior affinity of [11C]SNAP-7941 toward MCHR1 compared to ADRB3. As (S)-propranolol displays even higher affinity for the adrenergic beta-1 and beta-2 receptor, [11C]SNAP-7941 binding to adrenergic beta receptor subtypes other than beta-3 can additionally be excluded (In general, our findings support the initial theory of Ito et al. assuming that the anti-obesity effect of MCHR1 antagonists is not limited to a central action, but MCHR1 antagonists may also act peripherally in BAT . Expressin vitro results are in accordance with the previously performed in vivo experiments administering [18F]FE@SNAP to conscious animals without the use of anesthesia, where significant blocking was achieved by a pharmacological dose SNAP-7941 (11C]SNAP-7941 in BAT was shown after administration of a pharmacological dose of the unlabeled compound. PET imaging in small animals requires anesthesia in order to prevent laboratory animals from moving during the examination. However, anesthesia bears the disadvantage of possible molecular alterations, which may affect the examination's outcome, as it is already well-established for brain PET imaging (in vivo study, on ADRB3 and/or MCHR1 expression are not described, though potential effects cannot be excluded.Moreover, these NAP-7941 . Contrad imaging . Effects11C]SNAP-7941, as extensive in vitro and in vivo investigations evinced the superior properties of [11C]SNAP-7941 compared to the fluoro-ethylated analog regarding radiochemical yield, metabolic stability, target affinity, selectivity, and imaging contrast (18F]FE@SNAP was not further evaluated in this context.Investigation of specific binding to brown adipocytes was solely performed for FDG was introduced for imaging and quantification of BAT activation. Although primarily perceived as a clinical tool, PET employing specific tracers can also add to basic research. In this way, MCHR1 PET imaging provided first evidence for MCHR1 expression in BAT and will contribute to the elucidation of the MCHR1 axis in BAT.The value of PET for BAT research was ultimately recognized when the radiolabeled glucose-analog, [11C]SNAP-7941 and [18F]FE@SNAP in rat BAT gave the initial hint for MCHR1 expression in BAT. The herein performed in vitro binding studies, demonstrating specific [11C]SNAP-7941 binding to brown adipocytes and superior selectivity toward MCHR1 compared to ADRB3, provide first evidence for a direct involvement of the MCH/MCHR1 system in brown adipose tissue. Additionally, MCHR1 expression in murine brown adipocytes was demonstrated on mRNA and protein level for the first time.The present study aimed at enhancing the understanding of the interplay between MCHR1 ligands and the pharmacologically complex brown adipose tissue. Uptake of SNAP-7941 and [18F]FE@SNAP. OK and FK provided brown pre-adipocytes and established the induction protocol. GE designed the primers for qPCR. GE and LT helped with the establishment of the protocol for qPCR. KP and HS synthesized precursor compounds for radiolabeling and standard compounds. TB, CP, and EK analyzed the data and composed the draft. MM, GE, KW, WW, MH, and HV helped with outcome interpretation and reviewed the manuscript. All authors have given approval to the final version of the manuscript.TB, CP, and MM conceived the study and designed the experiments. TB, KB, EK, and CP performed the experiments. CP performed the radiosyntheses of [Without relevance to this work, author WW is a part-time employee at the Center for Biomarker Research in Medicine–CBmed Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.