Despite tremendous advances in artificial language synthesis, no machine has so far succeeded in deceiving a human. Most research focused on analyzing the behavior of “good” machine. We here choose an opposite strategy, by analyzing the behavior of “bad” humans, i.e., humans perceived as machine. The Loebner Prize in Artificial Intelligence features humans and artificial agents trying to convince judges on their humanness via computer-mediated communication. Using this setting as a model, we investigated here whether the linguistic behavior of human subjects perceived as non-human would enable us to identify some of the core parameters involved in the judgment of an agents' humanness. We analyzed descriptive and semantic aspects of dialogues in which subjects succeeded or failed to convince judges of their humanness. Using cognitive and emotional dimensions in a global behavioral characterization, we demonstrate important differences in the patterns of behavioral expressiveness of the judges whether they perceived their interlocutor as being human or machine. Furthermore, the indicators of interest displayed by the judges were predictive of the final judgment of humanness. Thus, we show that the judgment of an interlocutor's humanness during a social interaction depends not only on his behavior, but also on the judge himself. Our results thus demonstrate that the judgment of humanness is in the eye of the beholder. As the use of the Internet and virtual reality applications is largely spreading into everyday situations, the need for human-like autonomous agents is rapidly growing Technologies supporting the design of virtual spaces have now reached a level of maturity advanced enough to obtain highly convincing results regarding purely visual aspects of agents However, attempts to convince subjects of the humanness of an artificial agent have remained unsuccessful Since the early days of artificial intelligence research, language has been identified as the main output to test the human-likeliness of artificial agents' cognitive abilities in experimental settings. The pioneer of the artificial intelligence field, Alan Turing, proposed a now famous test in order to determine the capability of an agent to mimic humans The efforts of robotics or computer-mediated communication researchers have yet to be successful in creating an agent able to pass the Turing Test At the current state of knowledge, what the best software can teach us is still limited, due to inherent biases which have been led by the programming. Since the earliest work in computerized language, the vast majority of researchers studied the linguistic and interaction behavior of the best agents available To answer this crucial question, we selected as a model the Loebner Prize in Artificial Intelligence. The Loebner Prize is a recent version of the Turing Test, using linguistic production in the form of dialogue between programs, human subjects, and judges. Since 1991, this yearly contest gathers human participants and machines (linguistic software) that try to convince judges about their humanness by communicating through computer terminals without seeing each other. After the conversations, the judges decide which terminals were controlled by humans and which ones were controlled by programs.machines by the judges. While programs attempt to fool the judges so they believe in their humanness, human subjects should not have to make any particular effort to convince other humans that they are indeed who they claim to be. What could have happened during the interaction so that human beings were considered as non-humans? What parameters need to be present to guarantee a positive identification of a human within a social interaction narrowed down to computer-mediated communication?However, up to now, a highly interesting aspect of the Loebner Prize has been neglected. Throughout the years, some human participants have been perceived as Instead of studying good robots, this new approach of examining the linguistic behavior of “bad” human participants focused on how human beings could have been considered as robots during the course of a blinded social interaction. The Loebner Prize model enabled us to study semantic aspects of a dialogue that can deceive a judge on his interlocutor humanness, by examining which faulty events have created such a negative behavioral response toward the participant. Using cognitive and emotional dimensions in a global behavioral characterization, we suggest the importance of multimodal, emotional and cognitive parameters when analyzing complex social behaviors. Our results demonstrate that the judgment of an interlocutor' humanness during a social interaction not only depends on his behavior, but also on the behavior of the judge himself. Thus, we demonstrate that the judgment of humanness is in the eye of the beholder.Loebner Prize in Artificial Intelligence. During this annual contest, subjects and conversational programs try to convince judges of their human nature through computer-mediated communication. No conversational program has yet succeeded in fooling judges, but some human subjects failed to convince the judges of their human nature and were judged as robots by at least one of them. In the present study, we focused on the characteristics of the dialogues between human subjects who have been judged at least once as robots and their respective judges. The linguistic productions were thus divided into four groups: subjects judged as humans, the same subjects judged as robots, judges making a human judgment of their interlocutor, and judges making a robot judgment of their interlocutor.Dialogues were extracted from the Loebner Prize in Artificial Intelligence website database (http://www.loebner.net/Prizef/loebner-prize.html). Dialogues in which subjects were considered as robots by a minimum of one judge were selected. Over the years of the Loebner Prize, several ranking techniques were used to determine which contestants seemed more human than others. When judges were asked to separate the terminals that they believe were controlled by humans of those that they believed were controlled by machines , their judgment indicated which participants were considered as robots. When judges were asked to give each terminal a “humanness” score between “definitely a machine” to “definitely a human” on a five points Likert scale, we considered that the followings judgments “definitely a machine” and “probably a machine” meant the judges considered their interlocutor to be a robot . When judges were asked to rank terminals between 1 as being a human to 6 as being a robot, we considered that the followings ranks 4, 5 and 6 meant the judges considered their interlocutor to be a robot . Finally, when judges were asked to divide a percentage between 2 terminals according to their humanness , we decided that 60% was the breaking point from which a terminal would be considered as a robot .Dialogue samples were collected via transcriptions available on the Analyses could only be performed when the logs of all subjects' conversations and their detailed ratings were available. The dialogue transcripts of the subjects and their respective judges were used for further analyses. All transcripts were first saved in a text format compatible with Microsoft Word software and then normalized in order to carry out linguistic analyses.The parameters used to analyze the dialogues were gathered in three broad categories: descriptive parameters, cognitive parameters and indicators of interest. Descriptive parameters were: number of words, sentences, posts, mistakes, words per sentence, words per post, sentences per post, number of social words, long words (more than 6 letters), positive emotion words, negative emotions words, total emotions words, articles , greetings at beginning, greetings at end, and acknowledgments. Cognitive parameters were: number of self-references , references to relatives (family and friends), compliments, occurrence of aggressiveness, and occurrence of emotions . Indicators of interest were: number of questions, questions per post, and overall number of cognitive words used.Parameters were collected using classification grids or the Linguistic Inquiry and Word Count program (LIWC) Patterns of behavioral expressiveness were built using five dimensions selected accordingly to their relevance for inter-individual interactions. The five dimensions selected were occurrence of aggressiveness, self-references, references to relatives, compliments and occurrence of emotions. The data for each of the four groups were then normalized depending on their relative importance across the groups.Analysis of the different parameters was performed using the non-parametric Wilcoxon paired test or Student paired t-tests, when the normality of the distribution allowed it. Comparisons were made between two groups . Patterns of behavioral expressiveness were compared using the non-parametric distribution free Kolmogorov-Smirnov analysis of the parameters' distributions, in order to account for differences in the patterns of behavioral expressiveness between the groups. When applicable, results are presented as mean ± SEM.The main findings of our study were the evidence of important differences in the patterns of behavioral expressiveness of the judges whether they perceived their interlocutor as being human or machine. Subjects judged as robots used fewer words per post, fewer long words and fewer articles than those judged as humans. Furthermore, subjects judged as human made more posts and more compliments than did the judges perceived them as been human. They also used more words per post, more long words and more articles than the judges judging as human. Finally, judges judging as humans asked more questions, more questions per post and used more cognitive words than did subjects judged as human.All transcripts available since the first year of the Loebner contest in 1991 were collected. Data were available for years 1992, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2003, 2004, 2005, 2006, 2007, 2008, 2009, and 2010. However, several years of the contest had to be excluded from the sampling. Specifically, detailed ratings of the dialogues were not available for 5 years of the contest , and the dialogues of 2001 contest were not available. During 6 of the remaining years of the contest , no subject was considered as a machine. Our final sampling thus consisted of 4 years of the Loebner Prize .Among these 4 years, a total of 9 subjects were judged at least one time as a machine by at least one judge . These sp<.05 between the subjects judged as human and the judges judging as human, p<.05 between the subjects judged as human and the subjects judged as machine, Significant differences were observed for several structural parameters. In particular, in the number of posts , the number of questions per post (p<.05 between the subjects judged as human and the judges judging as human), and the overall number of cognitive words used (p<.05 between the subjects judged as human and the judges judging as human).When the subjects were judged as human, judges displayed significantly more indicators of interest toward the subjects . DiffereIn the present study, we investigated a large corpus of linguistic interactions in a controlled setting. Our innovative approach allowed us to demonstrate important differences in the patterns of behavioral expressiveness of the judges whether they perceived their interlocutor as being human or machine. In order to be perceived as human, robots should maintain a balanced response to their human interlocutor within each behavioral dimension. Furthermore, the indicators of interest displayed by the judges were predictive of the final judgment of humanness, suggesting that, at least to some extent, the judgment of humanness lies in the eye of the judge himself.While most researchers investigate the blurred area between perception of humanness and non-humanness – referred as the “Uncanny Valley” in the field of robotics and animation Due to the nature of the Loebner Prize, the fact that the subjects would talk more than the judges was expected. Length of statements is considered as crucial for reciprocal communication In this setting, subjects who used more articles and more sophisticated words in an overall lengthy dialogue tended to be evaluated more positively – meaning more human-like – by the judges. Accordingly, previous studies have demonstrated that large lexical diversity in speech was usually positively evaluated When taken individually, very few of the cognitive parameters analyzed here were significantly associated with a positive judgment of humanness. However, grouping the main cognitive and emotional dimensions in order to define global patterns of behavioral expressiveness pointed to a more complex picture. First, slight differences were assessed between the subjects whether they were judged as human vs. as machine. More importantly, this analysis strategy unveiled significant differences between the expressed behaviors of the judges whether they perceived the subject as human or as machine.A dialogue is usually characterized by collaborative interactions between the agents. Theoretical models endorse the notion that dialogue coherence is supported by cooperation and balance among agents at each step of the interaction When containing only necessary information or lacking conversational tone, computer-mediated communication can be interpreted as rude, and consequently affects perception of likability and friendliness of the interlocutor Our results show that self-disclosure (quantified in the present study by the two following parameters: self-references and references to relatives) was not directly associated with the judgment of humanness. However, this broader dimension clearly was one of the main factors responsible for the variability in the patterns of behavioral expressiveness. A balanced self-disclosure displayed between the partners about themselves and their relatives seemed to be associated with a positive response, while too much self-disclosure from an interlocutor led to a disproportional aggressive response. Although not statistically significant, we also observed a tendency to consider as a robot a partner that overused emoticons  or excessive laugh. These results are in line with some previous studies. For instance, reciprocity in self-disclosure, as calculated by a positive correlation between the amounts of self-disclosure from the partners, seems to be a significant aspect of interactions It is important to mention that demographic information of the participants whom behavior was analysed here was not available. Thus, in the present study, it was impossible to decipher whether the age or the gender of the participants may have impacted the perception of humanness. However, previous studies seem to indicate that gender has no effect on reciprocal communication in computer-mediated communication A key issue is to understand whether the variations of the patterns of behavior expressiveness observed originated from the judge himself, or were triggered by the behavior of the subject aiming to convince the judge of his humanness. If both mechanisms are involved, some of our results suggest that the judge's behavior impacts the evolution of the dialogue and thus the resulting judgment of humanness. Therefore, the quantification of the indicators of interest displayed by the judge – such as direct questioning – is of major importance.Direct questioning is known to stimulate interpersonal attraction If indicators of interest are central for interpersonal communication, their form may however vary depending of the experimental situation. Because the Loebner Prize setting favored the linguistic aspect of communication, it may have reinforced the impact of questioning over other indicators of interest.In conclusion, by focusing on the linguistic and meta-linguistic behavior of subjects judged as non-human in a computer-mediated communication situation, our results shed a new light on the mechanisms of perception of humanness. We demonstrated here striking differences in the patterns of behavioral expressiveness of the judges whether they perceived their interlocutor as being human or machine. Furthermore, the indicators of interest displayed by the judges were predictive of the final judgment of humanness. These results provide us with a better understanding of the general phenomena underlying the process of humanness judgment and interaction dynamics in computer-mediated communication. Furthermore, they also provide new avenues for optimizing artificial agents designed to communicate with humans. Our results emphasize the collaborative aspect of dialogue, as well as the multi-dimensional and multi-factorial nature of this process. While classical descriptive analysis can provide important data, we also demonstrated that a complete analysis can not rely only on strictly descriptive factors, but should integrate cognitive and emotional dimensions in an integrated behavioral characterization. When creating synthetic agents, designers should keep in mind that equilibrium in the reciprocity of the exchanges between humans and agents is one of the central factors in order to convince a human about the humanness of its interlocutor. Finally, when taken together, our results strongly demonstrate that, in an interaction situation, the judgment of the humanness of an interlocutor not only depends of his behavior, but also on the judge himself.


In Canada, workforce shortages in the health care sector constrain the ability of the health care system to meet the needs of its population and of its health care professionals. This issue is of particular importance in peripheral regions of Quebec, where significant inequalities in workforce distribution between regions has lead to acute nursing shortages and increased workloads. Information and communication technologies (ICTs) are innovative solutions that can be used to develop strategies to optimise the use of available resources and to design new nursing work practices. However, current knowledge is still limited about the real impact of ICTs on nursing recruitment and retention. Our aim is to better understand how work practice reorganization, supported by ICTs, and particularly by telehealth, may influence professional, educational, and organizational factors relating to Quebec nurses, notably those working in peripheral regions.First, we will conduct a descriptive study on the issue of nursing recruitment. Stratified sampling will be used to select approximately twenty innovative projects relating to the reorganization of work practices based upon ICTs. Semi-structured interviews with key informants will determine professional, educational, and organizational recruitment factors. The results will be used to create a questionnaire which, using a convenience sampling method, will be mailed to 600 third year students and recent graduates of two Quebec university nursing faculties. Descriptive, correlation, and hierarchical regression analyses will be performed to identify factors influencing nursing graduates' intentions to practice in peripheral regions. Secondly, we will conduct five case studies pertaining to the issue of nursing retention. Five ICT projects in semi-urban, rural, and isolated regions have been identified. Qualitative data will be collected through field observation and approximately fifty semi-structured interviews with key stakeholders.Data from both parts of this research project will be jointly analysed using triangulation of researchers, theoretical approaches, methods, and results. Continuous exchanges with decision makers and periodic knowledge transfer activities are planned to facilitate the dissemination and utilization of research results in policies regarding the nursing recruitment and retention. French acronym: MSSS) estimates a current deficit of 3000 nurses, with an expected deficit increase to 10 000 in 2014 and to 17 000 in 2019 [Workforce shortages in the health care sector are a widespread problem in Canada, as in many countries of the world . This me in 2019 . The MSS in 2019 . The useFrench acronym: CSSS). A CSSS is a multivocational institution operating local community service centres, residential and long-term care centres and, where applicable, a general and specialized hospital centre [very or quite problematic [In Quebec, the services provided by the health and social service providers in a local health and social services network are coordinated by a local authority, called a Health and Social Services Centre  involving nurses who had left their jobs had an impact on their return to work [ICTs, including telehealth, have the capacity to improve access at all levels of care for a large range of health conditions, offer patient-centred care at lower costs, increase the efficiency of clinical decision-making, and improve the efficacy of chronic health management . These t to work ,23. Whil to work ,14 show  to work ,24.In sum, with the increasing implementation of different ICT applications in the Canadian health care system, their impact on work satisfaction and staff retention in the health care sector should be evaluated in a more systematic manner in order to inform health care sector decision-makers ,24. ConsListening for Direction III on issues related to health services and policies [The training, planning and distribution of the workforce are among research priorities for health services in Canada . This prpolicies : the worpolicies ,26, partThe present project aims to better understand the influence of telehealth on professional, educational, and organizational factors concerning nurses working in peripheral regions. According to recent data, these factors could have an influence on nurse recruitment and retention. As well, the project hopes to support the CSSS partners in the evaluation of new work practices related to ICTs and their impact on nurses in Quebec and, more particularly, those working in peripheral regions. This project therefore has two objectives: 1) Study how the use of ICTs in the reorganization of nursing work practices can influence recruitment and retention factors in peripheral regions; 2) Explore how the use of ICTs influences the quality of life at work, the continuity of services as well as continuing education and knowledge transfer opportunities, focusing particularly on five projects that reorganize nursing work practices using ICTs.et al. [Table et al. . This frThe results collected in our previous work ,14,27,28As well, the information gathered by our previous work ,14,27,28et al. [Lastly, telehealth may have an effect on the three types of continuity of care described by Haggerty et al. : informaet al. , it is iA participatory research approach was chosen for this research project. This approach allows the issue of nursing workforce to be addressed from its internal social and historical contexts . As suchTo meet the first objective, aiming to better understand how new work practices involving telehealth can influence nursing recruitment, a descriptive study will be carried out of innovative projects in Quebec relating to the reorganization of work practices based upon ICTs. To do this, individual interviews with the Director of Nursing Services (DNS) and Director of Human Resources (DHR) of each CSSS will be undertaken. A general interview framework will guide the interview questions, however the questions may be adapted to the CSSS workforce situation and the nature of the work practice reorganization project. The factors that helped or hindered the recruitment of nurses will be explored. In total, approximately forty interviews will carried out.However, the number of interviews may vary according to data saturation criterion , that isBefore the interview, the participants must read and sign a consent form. The interviews will be approximately one hour in length and will be recorded with the participants' consent. The interviews will be transcribed verbatim and the content will be analysed using N*Vivo software. Thematic content analysis will be done using methods described by Huberman and Miles , includiThe results of this first step will allow development of a questionnaire to analyse the influence of telehealth on professional, educational, and organizational factors associated with nursing recruitment as well as factors determining nursing graduates' and newly graduated nurses' intentions to practice in peripheral regions. The study of the intention to practice in a peripheral region will be based upon Ajzen's Theory of Planned Behavior (TPB) ,37, a moThe questionnaire development will comply with methodology proposed by Gagné and Godin ,39. Adap2 equal to 0.10); the second group will be composed of five additional variables with an increase to R2 equal to 0.02 .The final questionnaire will be distributed to third year students as well as students who graduated during the previous year of from the nursing programs at Université Laval and Université de Montréal. As this is an exploratory study aiming to validate a theoretical model, the goal of the survey is not to generalize the results. As such, convenience sampling among nursing students will be done at the Faculties of Nursing, to which the researchers are affiliated. A letter will be sent to all students to notify them of the study and to solicit their participation by filling out the enclosed questionnaire. The letter will indicate that completing the questionnaire means implied consent to participate in the study. Approximately 600 questionnaires will be distributed and we estimate a response rate of 50%. Consequently, we plan to collect around 300 completed questionnaires, which will allow for adequate statistical significance. In fact, according to Cohen's formulas , a samplThe data will be compiled and analysed using SPSS software. First, descriptive analyses of the variables will be done for all variables. Then correlation analyses will be done for theoretical variables. Chi-square analyses will verify the association between the intention to practice in peripheral regions and socioeconomic and educational variables  derived from the literature on aspects related to health professional recruitment, forming the control variables. Finally, hierarchical regression analyses will be done to evaluate the influence of the TPB variables on the intention to practice in peripheral regions, as well as the impact of the control variables on this intention. More in-depth analyses  could be done according to the relationships discovered between the collected variables.We chose a multiple case study approach, selecting five CSSS in peripheral regions as the studied cases. A case study is an appropriate strategy as it allows us to study a complex phenomenon in a global manner, by situating it in its social, political and historical context . This meThe first case, at a semi-urban CSSS, has a work practice reorganization project aiming to improve the access to and frequency of services for clients with chronic obstructive pulmonary disease (COPD) by introducing a remote patient monitoring (RPM) project. More precisely, the project seeks to optimise the roles and responsibilities of participants in the continuum of care, in order to maximise work organization. 2- The second case, at another semi-urban CSSS, implanted a work practices reorganization project to support the mobility of home support nurses, including the introduction of a software program, in order to respond to an increased number of cancer patients and an acute shortage of nursing resources. The project, initiated in 2007, principally aimed to introduce a computerized tool for care planning (SyMO) which gives a general overview of clientele profiles, their needs and priority interventions, up to date in real time. This care tool enables the improvement of the quality, the accessibility and the continuity of services, facilitates communication between different professionals, and offers better coordination of interventions, taking into account the rapidly evolving clientele. The new tool facilitates work organization and allows for increased time with clients at home, owing to the streamlining of administrative and secretarial tasks performed by nurses as well as to the availability of an important source of care protocols, facilitating interventions. This pilot project conducted by the Oncology and Palliative Care Unit is now being extended to other sectors of home care within the CSSS. 3- The third case, at a different semi-urban CSSS, currently has a work reorganization project aiming to improve the access of home care services on the basis of the available human, financial, and technological resources available. This is a complex operation of work reorganization based upon the introduction of new computer tools better aligned with new nursing practices . In total, the project concerns more than 100 nurses spread across three establishments. 4- The fourth case study, at a rural CSSS, focuses on a work practices reorganization project aiming to improve the quality and accessibility of health care. The project offers, with the help of telehealth, distance primary health care to the 14 000 residents spread over an immense territory of 19 000 km. In this project, the doctor, who is many kilometres away from the patient, uses the "hands" of a nurse and the "eyes" of adapted instruments. 5- Finally, contacts were established with an isolated CSSS, which has a work practices reorganization project using information technologies to offer distance training for nurses.Semi-structured interviews with key stakeholders, including regional leaders, managers and nurses, as well as field observation will constitute the principal sources of data in order to well document each case. The respondents will be identified by the contact network method  and willThe collected data will be analysed qualitatively using the N*Vivo software. An iterative analysis method will be used as the categories for analysis will first be determined by concepts from our team's previous studies ,14,27, bThe data relating to the two study objectives will be jointly analysed using triangulation of researchers, theoretical approaches, methods, and results . The comKnowledge translation (KT) is defined as a continuous exchange process between knowledge producers and users to promote ethical knowledge use ,52. AccoWith the same goal of ensuring the relevance of this study and the use of its findings , the teaSix other meetings between researchers, decision-makers, and users are anticipated throughout the project . The resIt is important to note that the research team was established to ensure the relevance of the study and the use of its results . As suchThe end-of-grant knowledge translation of the project has three objectives: 1) Ensure that our findings and recommendations correspond to the needs of local decision-makers and to the realities of health professionals; 2) Allow for the use of our results and recommendations by decision-makers across the province of Quebec and eventually in other Canadian jurisdictions; 3) Have our results validated by the international scientific community.For the first objective, the intended audience will be the health care professional associations, the CEOs and the DHRs of health care organisations in the region. The participation of decision-makers in the dissemination of the results at this level is essential as many studies have shown the importance of key stakeholders in knowledge translation . The resFrench acronym: ASSS) of Quebec. A non-technical language report which follows the 1-3-25 format, considered an efficient way to reach decision-makers [For the second objective, involving decision-makers across the province of Quebec and of Canada, activities will first target the Health and Social Services Agencies (n-makers , will beLastly, for the third objective of the knowledge translation plan, the results will be published in scientific journals with high impact factors. The results will be presented at the annual congress of the Latin Association for Health Systems Analysis, where a session on the workforce and ICTs will be proposed. To ensure that the three KT objectives are reached, a short questionnaire on the quality of the presentations and the intentions to use the results will be distributed to attendees at the end of each presentation. This questionnaire will be adapted from psychosocial theories to explain knowledge use behaviour ,58. The The authors declare that they have no competing interests.MPG, HP, and JD drafted the study protocol. CAM translated and adapted the protocol for publication. All authors critically revised and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/11/27/prepub


Bipolar disorder (BD) is considered one of the most disabling mental conditions, with high rates of morbidity, disability, and premature death from suicide. Although BD is often misdiagnosed as major depressive disorder, some attention has recently been drawn to the possibility that BD could be overdiagnosed in some settings. The present paper focuses on a critical analysis of the overdiagnosis issue among bipolar patients. It includes a review of the available literature findings, followed by some recommendations aiming at optimizing the diagnosis of BD and increasing its reliability. Bipolar disorder (BD) is a chronic, recurrent mental illness first described by Jules Falret in 1854 as Folie Circulaire (circular insanity). Later, it was renamed as manic-depressive Psychosis. Nowadays, BD is considered one of the most disabling mental conditions, with high rates of morbidity, disability, and premature death from suicide [These discrepancies in prevalence point to a lack of consensus on the current definitions of BD, which may favor the overdiagnosis of major depressive disorder (MDD) in detriment of BD . Based oOver the last decade, however, BD has been progressively incorporated by culture. Patients with BD are now commonly displayed in the media, and allusions to this condition are frequently found not only in the scientific literature but also in autobiographical books and fictional writing. This trend has drawn attention to the possibility that, regardless of well-demonstrated rates of underdiagnosis, BD could be overdiagnosed in some settings.  The potential downsides of overdiagnosing BD are several. They include the negative effects of unnecessary labeling, the risk of harm related to unnecessary treatments, and the misuse of health care resources, with important human and financial implications –8. The present paper comprises a critical analysis of the overdiagnosis issue among bipolar patients. We begin with a review of the available literature findings, followed by some recommendations aiming at optimizing the diagnosis of BD and increasing its reliability.The database PubMed (1990–2013) was searched, using the MeSH terms “bipolar disorder” and “diagnosis.” In addition, we carried out a manual search of bibliographical cross-referencing. The papers obtained were manually screened, aiming at identifying original articles, reviews, and case reports focusing on the overdiagnosis of BD.  We identified seven studies that included data on the overdiagnosis of BD . The resTwo of those studies , 13 util The phenomenological distinction between BD and some personality disorders may be challenging, not only because of the overlap between some personality disorder features and the diagnostic criteria for mood episodes but also because of the lack of reliability of the time criteria for BD. For example grandiosity is a common feature in narcissistic personality disorder, and patients with histrionic personality traits can be mistakenly diagnosed with mania . Anger a Maybe the most important debate regarding the interface between BD and personality disorders is related to the issue of differential diagnosis versus comorbidity. That is particularly true when it involves patients with borderline personality disorder (BPD). While the cooccurrence of BPD and BD seems to be quite common , the sim On the other hand, the multiple similarities between the two disorders have raised the issue that BD and BPD actually belong to the same spectrum, therefore representing different presentations of the same basic condition . While t The rates of comorbidity between substance use disorders and BD are extremely high . On the  There is a marked trend toward increases in the diagnosis of mental disorders in children , 24. SpeDSM-IV criteria have been widely used for the diagnosis of psychiatric conditions over the last two decades. Although the reliability of its criteria for the diagnosis of BD, especially in the presence of mania, has been extensively demonstrated, there is evidence suggesting that some of its criteria may lack validity . A recenFurthermore, some of the criteria used to characterize manic or hypomanic episodes are unspecific, such as decreased concentration, racing thoughts, and psychomotor agitation. These symptoms can be found in several different conditions, such as anxiety disorders and attention-deficit disorder. Moreover, the distinction between hypomania and nonpathological behavior is often problematic, which can sometimes result in the overdiagnosis of BD type II.  Perhaps the most important limitation of the DSM-based diagnosis of BD comes from its inability to identify bipolar patients without a typical history of classic manic or hypomanic symptoms, that is, a patient whose index episode is depressive. Attempts have been made to minimize this limitation through the description of features that, if present in a patient with depression, are highly suggestive of bipolar disorder . Among t The concept of “bipolar spectrum” represents the antithesis of the issues exposed above in regard to the limitations of DSM-IV criteria for BD. Based on a dimensional approach, it expanded the diagnosis of BD beyond the categorical framework established by DSM-IV, based on the assumption that individuals with a strong genetic load for BD can experience different varieties of bipolarity. Consequently, many patients who do not meet criteria for BD based on the DSM-IV criteria may be diagnosed as suffering from “soft” forms of BD, which sometimes can overlap with the patient's temperament . Of noti Despite the controversy surrounding this concept, it has gained immense popularity over the last decades, and many clinicians adopt this model to formulate (or sometimes justify) their diagnosis of BD. Despite sometimes minimizing the diagnostic labeling represented by a categorical approach, a dimensional diagnostic model (such as the one represented by the bipolar spectrum concept) tends to blur the boundaries between different diagnostic concepts and normalities, decreasing even more the diagnostic reliability across different providers .There are several additional possible reasons for overdiagnosis. For example, disability awards especially due to mental illness are on the rise. Between 1987 and 2005, the share of Supplemental Security Income's adult caseload disabled due to a mental disorder rose from 24.1% to 35.9%. In a recent study, 172 patients on disability due to a diagnosis of BD were assessed, and only 47.6% of them actually met criteria for BD .  The recent publication of DSM-5 was expected by many as a promising step in improving the diagnostic accuracy of BD. Increase in energy and activity was included as a core symptom for the diagnosis of mania or hypomania, in addition to elated mood. Mixed episodes were removed from DSM-5. Instead, the presence of mixed features became a specifier, which may be present among patients with manic or depressive episodes . It is s On the other hand, the medical literature is scant in regard to interventions aiming at minimizing the overdiagnosis of BD. In a recent paper  it was a The “two-step diagnostic process” described above could also be accomplished through the systematic administration of a screening instrument for BD, such as the Mood Disorder Questionnaire  prior to Furthermore, over the last decades, considerable advances have been made in the better understanding of the pathophysiology of BD through neuroimaging studies, analysis of neuropsychological findings among bipolar patients, genetic analysis, and several other biological measures. Whereas it is expected that these progresses bring about increments in the diagnosis of BD in the long term, it is unlikely that a single biomarker might be able to identify patients with BD, given the biological and clinical heterogeneity of that condition . On the  Given the current status of BD as a construct, diagnostic errors and the mistaken identification of patients as suffering from that condition are likely to occur. Nevertheless, improvements in the nosological and psychopathological characterization of BD, as well as clinician education on the correct interpretation of the different factors involved in the detection of BD, may increase the reliability of that diagnosis. Similarly, the identification of biological markers for mood disorders represents a promising area of research and will likely bring about improvements in the precision of the BD diagnosis in the near future.


Dysalotosaurus lettowvorbecki is a small ornithopod dinosaur known from thousands of bones and several ontogenetic stages. It was found in a single locality within the Tendaguru Formation of southeastern Tanzania, possibly representing a single herd. Dysalotosaurus provides an excellent case study for examining variation in bone microstructure and life history and helps to unravel the still mysterious growth pattern of small ornithopods.Dysalotosaurus was carried out using regularly developed and alternating slow and fast growing zones. Dysalotosaurus was a precocial dinosaur, which experienced sexual maturity at ten years, had an indeterminate growth pattern, and maximum growth rates comparable to a large kangaroo.Five different skeletal elements were sampled, revealing microstructural variation between individuals, skeletal elements, cross sectional units, and ontogenetic stages. The bone wall consists of fibrolamellar bone with strong variability in vascularization and development of growth cycles. Larger bones with a high degree of utilization have high relative growth rates and seldom annuli/LAGs, whereas small and less intensively used bones have lower growth rates and a higher number of these resting lines. Due to the scarcity of annuli/LAGs, the reconstruction of the life history of Dysalotosaurus demonstrates the influence of size, utilization, and shape of bones on relative growth rates. Annuli/LAGs are not the only type of annual growth cycles that can be used to reconstruct the life history of fossil vertebrates, but the degree of development of these lines may be of importance for the reconstruction of paleobehavior. The regular development of annuli/LAGs in subadults and adults of large ornithopods therefore reflects higher seasonal stress due to higher food demands, migration, and altricial breeding behavior. Small ornithopods often lack regularly developed annuli/LAGs due to lower food demands, no need for migration, and precocial behavior.The variation in the bone histology of  Ever since scientists began to work with the remains of those extinct animals that lack direct living descendants, they dreamed of being able to accurately reconstruct life histories and, at least partially, social structures and behavior. Unfortunately, it is almost impossible to obtain such fundamental information using only morphological and/or statistical methods, because absolute ontogenetic dates of age or time of sexual maturity are not determinable. Size classes within a bonebed of a single species, surface texture of bones, or degree of suture closure are examples of tools often used to estimate relative age and ontogenetic status of fossil animals, but these methods are always highly imprecise e.g. . The stuDysalotosaurus lettowvorbecki was the subject of this study. Dysalotosaurus was found during the famous German Tendaguru expeditions of 1909 to 1913, which took place 60 km west of the seaport of Lindi, southeast Tanzania Kentrosaurus, Dysalotosaurus is known from only a single locality, but the two closely related monodominant bonebeds found in channel lag deposits Dysalotosaurus herd was probably trapped in one of the numerous tidal channels of that ancient coastal plain Dysalotosaurus quarry will be published in a subsequent paper.The basal iguanodontian ornithopod dinosaur Dysalotosaurus was previously studied by Anusuya Chinsamy-Turan Dryosaurus lettowvorbecki. The generic name Dysalotosaurus was made a synonym of Dryosaurus by Galton D. lettowvorbecki and D. altus. However, an ongoing revision of the anatomy of both taxa  is often hampered by the ontogenetic expansion of the marrow cavity and/or secondary remodeling. This problem was often solved by the back-calculation of the lost/obscured number of annuli/LAGs PlateosaurusMaiasauraHypacrosaurusThere is also a high variability in the number of annuli/LAGs between different individuals within a single population e.g. , betweenA last important point is the assumption that all annuli/LAGs counted in a bone are indeed true annual layers. These lines can also be generated as a result of environmental stress, such as scarcity of food, illness, or during seasons of pairing or reproduction Another actualistic method used to estimate relative age of extinct animals is ‘Amprino's Rule’ e.g. . AmprinoDysalotosaurus and its close relative Dryosaurus  from the generally distinct sequence of growth cycles internally to a much more uniform area externally. The latter resembles a very thick slow growing zone and only a very weak internal zonation is recognizable ; 6A–E.Secondary remodeling is very rare, which differs from the remarks of Chinsamy The comparison of longitudinal sections of a large femur and of the smallest sampled femur  reveals Due to the highly variable features within the shaft, between different femoral cross sections, and even within a single section, ontogenetic stages are difficult to distinguish. The use of most of the features, such as the degree of development of primary osteons, vascularization pattern, or secondary remodeling, was therefore limited, and there is often a smooth transition between successive ontogenetic stages. However, useful indicators of ontogenetic stage are, in addition to absolute size, the number of growth cycles and the degree of development of distinct areas, such as the Posterolateral Plug.Dysalotosaurus, and the overall size of other known specimens indicates that none of the preserved femora would fit into this stage.This stage, already described in some other ornithopods The marrow cavity is very large compared to the bone wall thickness see also . The intThe external circumferential profile is more pronounced and the Posterolateral Plug is well visible. The drift of the marrow cavity from approximately anterior to posterior is in progress, which is indicated by the well-compacted CCCB of a larger anterior wedge as well as a deeper incision into the posterior bone wall . The primary osteons are more numerous and there is a decrease in the proportion of knitted texture. There are the first occurrences of isolated secondary osteons. Growth cycles are well distinguishable and reach two to three in number .The development of the external cross-sectional profile as well as of distinct areas  is now complete . The antThe units of the cross sectional bone wall are strongly diversified . The antThe cross-sectional shape of the tibia is almost egg-like in distal sections and almost circular in proximal sections, but there is always a straight anterior wall, which opposes the fibula when in articulation . The shaAn endosteal layer is developed almost exclusively in medium to large sections , at the anterior edge of the marrow cavity, an unusual bone tissue is preserved ; 11. It The recognition of distinct ontogenetic stages in the tibiae is more difficult than in the femora, because there are fewer tibial sections available, and because most of the available specimens, belonging to a medium size range  belong to this stage. The cross-sectional units are strongly differentiated and the bone wall thickness is highly variable . The CCCThe shape of the cross sections varies from a lateromedially wide and flat oval outline distally to an almost circular oval shape more proximally . CCCB isThe bone matrix of the primary compact bone wall consists mainly of fibrolamellar bone tissue, although the anterolateral corner can be built by parallel-fibered tissue in some of the more proximal sections . HoweverPrimary osteons are numerous and dense, but there are high numbers of relatively smaller and longitudinal osteons with a strongly birefringent single ring of lamellar infilling . Such smAnnuli/LAGs are more abundant than in femora and tibiae, but their distribution is still very inconsistent .Secondary osteons are very rare. They are mainly located at the edge of the CCCB in the most distal or proximal sections, but they mainly occur close to the internal margin of the anterolateral corner along the edge of the short endosteal layer  or withiThe differentiation of humeral cross sections into ontogenetic stages is much more ambiguous than in the femora and tibiae. The only clear features are the size and the number of growth cycles.As in the other sectioned elements, this stage is not preserved.The smallest sections with not more than a single growth cycle belong to this stage . The sloAll remaining cross sections belong to this stage and a further subdivision is not possible. The number of growth cycles exceeds one and the laminar vascular pattern predominates .Due to the scarcity of preservation of fibulae, cross sections could only be produced from levels very close to or within their proximal metaphysis. Therefore, periosteal compact bone is, if at all, often present as a thin layer surrounding parts of the bone wall externally and it was impossible to get a truthful count of growth cycles.The overall shape of the cross sections is oval to kidney-like with very thick and strongly curved bone walls anteriorly and posteriorly. Most of the outer rim of the marrow cavity is poorly defined because of wide cavernous spaces surrounded by a loose network of trabeculae. An endosteal layer can only be observed along the thinner lateral and medial walls. This band of lamellar bone is very thick posteromedially .The thin layer of periosteal primary compact bone consists of fibrolamellar bone tissue, although the primary osteons are often relatively small and scattered. Endochondral bone tissue is often developed between this peripheral fibrolamellar bone and the internal CCCB.The medial wall differs strongly from the other units, because it is heavily altered by dense Sharpey's fibers, so that the area is strongly birefringent under polarized light . The bonSecondary osteons are very common in these metaphyseal cross sections. The CCCB is not involved, but its external border and most of the endochondral tissue is strongly remodeled. Internal and mid cortical areas of the posterior corner may even consist of dense haversian tissue of at least two generations of secondary osteons . The medIn the cross section of the large fibula GPIT/RE/5109, possible medullary bone is preserved internal to a part of the endosteal layer that fans out . The medThe sections cut directly at the maximum lateromedial width of the prepubic process have a wide oval shape . SectionThe periosteal compact bone wall is very thin compared to the overall diameter of the cross sections. There is no consistent internal margin, because a single large marrow cavity is absent. However, some of the internal cavities are quite large. These cavities are always of resorptive origin, because remnants of periosteal compact bone are often still preserved in some of the thicker trabeculae .This tissue consists of vascular fibrolamellar bone. Well-developed primary osteons are mainly visible in the dorsal and medial parts of the bone wall, but they are not very dense and mostly longitudinal in arrangement . Mainly Numerous small secondary osteons occur in the trabeculae between the large pseudo-cavities as well as in the internal areas of the periosteal compact bone wall, where they sometimes form haversian tissue. Their abundance decreases towards the medial side.The combination and correlation of the fractional values of the growth cycles for each group of cross sections resulted in a quite consistent number of years represented by these cycles. Thus, the combined growth cycles in femur group one  represent 11 years, those of femur group two (sections from the base of the fourth trochanter) represent 12 years , and thoTo calculate the respective body masses for the correlated growth cycles with the Developmental Mass Extrapolation method Dysalotosaurus are represented by the observed and correlated growth cycles in the femoral cross sections of groups one and two (By using the first nine (femur group one) to ten (femur group two) secured growth cycle values, the respective values of the MISM, and the maximum body mass, four sigmoidal growth curves were created. The remaining growth cycle values, representing unsecured growth cycles external to the MISM, were plotted into the curves subsequently . The man and two .Dysalotosaurus. The largest sampled femur  would therefore represent an age of 16.5 years . However, by plotting the respective values of the distal mediolateral width of femora or their midshaft circumference (representing body size) versus age, the MISM is then located very close to the inflection point of the curve  of see e.g. . Femora Dysalotosaurus, the femur and tibia are the largest and primary weight-bearing bones intensively used for locomotion. The humerus is comparatively much smaller  and was likely not used in weight bearing or locomotion. It is therefore not surprising to find it less densely vascularized within a relatively thinner bone wall. The sampled prepubic process is even more different than the femur and tibia in these characters, because it serves only as muscle attachment site and is not involved in active movements or in bearing weight.A possible explanation for growth rate changes may be the absolute size of the respective element within the skeleton combined with the degree of utilization, which includes two components: (1) the degree the element participates in weight bearing and (2) the functional demand on the bone. In the case of the biped Diictodon reached higher relative growth rates than its femur Talpa europea) by Enlow & Brown Allosaurus  have higher relative growth rates than the bones of the zeugo- and autopodium, because the latter are often smaller in overall size and share functions, such as weight bearing or muscle activity, among each other. The absolute forces acting on each of them are therefore smaller than in the stylopodium. This is the case for the less vascularized radii and ulnae compared to the humeri and femora in In the end, the relative size of a bone in a skeleton reveals its importance in weight bearing and/or movement and its relative growth rate compared to other elements is therefore predictable to a certain degree.Dryosaurus altusCross sections with very consistent outlines  or of endosteal lamellae (mainly humeri). The external periosteal regions possess here well organized primary osteons in a low density, osteocyte lacunae are also rarer than in other units, and the collagen fibrils are mainly transversely organized ; 13C. AlVaranus  and by the primary bone wall thickness. This can be seen between different elements of the skeleton. The thickest primary bone walls are developed in femora and tibiae with 12.5 and 11 mm, respectively. These elements preserve the highest number of growth cycles, which counts up to nine in the largest sections alone and up to 12 after ontogenetic correlations in all sections. Humeri, which have a maximum primary bone wall thickness of 5.3 mm in the samples, have only up to five cycles in a single section and up to ten after the correlation. The much thinner primary bone wall in the prepubic process can preserve only two cycles at maximum. The relative distances between growth cycles are also dependent on the cutting level within the shaft, because the average thickness of the periosteal bone wall is increasing towards the mid diaphysis and the portion of CCCB at the total bone wall thickness is here insignificant The number, relative distances, and developmental degree of growth cycles are highly variable in Dysalotosaurus, but they are rather rare, especially in femora  might be dominating over bone wall thickness. In elements with relatively high growth rates , only unfavorable environmental conditions (e.g. long draughts) or dramatic events in the individual's life history  may have resulted in the rare development of annuli/LAGs.Thus, the development of annuli/LAGs in the sampled skeletal elements of Dysalotosaurus. Especially large and derived sauropods have much weaker cycles, such as polished lines The relatively random and rare formation of clearly defined annuli/LAGs is in striking contrast to the pattern seen in many other dinosaurs. In theropods e.g. , mainly MaiasauraHypacrosaurusCoelophysisNone of these studies have mentioned the kind of growth cycles found here. Their identity as possible annual markers is now, however, unambiguously demonstrated. Despite the often relatively weak appearance ; 13F, thDysalotosaurus probably exists in a wider range of taxa, because the cyclicity between zones of oriented collagen fibrils is also mentioned in Alligator . Thus, this kind of growth cycles will probably be found in more tetrapods in the future and should provide age estimations especially in taxa with an otherwise poor record of annuli/LAGs.It is important to note that the type of growth cycles described for lligator  therein,Dysalotosaurus is only preliminary.Since all the sampled elements are isolated and microstructural details vary between different elements of a skeleton, the correlation of ontogenetic stages in femora, tibiae, and humeri of MaiasauraOrodromeusDryosaurusThe second ontogenetic stage of all three elements  as a sign for ceasing growth n humeri ; Tab. 3.n humeri ; 9E, numn humeri ; 9H, andDysalotosaurus is most similar to DryosaurusThe ontogeny of the bone histology in Orodromeus, on the other hand, reveals a vascularization pattern, which is usually found in skeletal elements of Dysalotosaurus with relatively lower growth rates, such as humeri or prepubic processes  with the largest femur of Dryosaurus . A mark or transition (MISM) is visible in these cross sections  This event is commonly combined by a slow-down of growth rate in many other tetrapods e.g. ; (2) Therus herd .AlbertosaurusThis gap shows the underrepresentation of individuals and is probably the result of banishment and/or increased mortality of this size class. In recent and at least temporarily gregarious ungulate mammals, young males predominantly suffer increased mortality around the time of sexual maturity, because they are driven out of the herd very early by prime-aged males  and Allosaurus (10 years) is located, as in Dysalotosaurus, within the lower third of the exponential growth phase and not at the curves point of inflection, where growth rate reaches its maximum Tyrannosaurus, the estimate of 18 years is close to the inflection point, which is similar to Albertosaurus , where the ratio would be 8∶1  within the growth curves is well below the estimated asymptote at approximately 16.4 years of age . AdditioDysalotosaurus individuals could be reproductively active for more than five years, but none of them obviously reached somatic maturity. Dysalotosaurus was highly vulnerable to most of the contemporaneous predators due to its relative small body size and the lack of any defensive structures (as in Kentrosaurus). This may be a reason, why sexual maturity was delayed until the ninth year of life. The cost of reproduction was too high for small individuals due to high vulnerability to predation.Many of the Dysalotosaurus.Another factor for the high mortality rate around time of sexual maturity and, especially, the prolonged exponential growth phase in sexually mature individuals might be intraspecific competition within a herd. Larger/stronger individuals surely had a more dominant role within the herd and a better chance for reproduction than smaller/weaker individuals. Fast and extended indeterminate growth could therefore be regarded as a survival advantage for Dysalotosaurus exhibits a growth pattern, where annuli/LAGs as representatives of a zonal bone tissue are rather scarce, completely absent, or are replaced by less obvious growth cycles. On the other hand, large ornithopods, other ornithischians, prosauropods, and all theropods more derived than Herrerasaurus  and environmental conditions . For the Tendaguru region with its reconstructed seasonal change of humidity The results for Orodromeus, Dysalotosaurus, Tenontosaurus, and Maiasaura at first in the late juvenile stage . The first LAG in Tenontosaurus is also not consistently developed in all specimens and is sometimes substituted by a band of differing oriented collagen fibrils Dryosaurus altus, LAGs were found in all three subadult femora, but at non-overlapping relative positions indicating at least three different growth cycles for the two smaller specimens and up to 15, if one includes the largest femur and calculates the number of LAGs by back counting Dryosaurus is indeed similar to Dysalotosaurus in its growth pattern, which is implicated by a similar vascularization pattern and the absence of an EFS, then the number of developed LAGs would be still rare in the large femora of Dryosaurus. In Dysalotosaurus, ten out of 14 femora (excluding the juvenile stages) bear one (in one case two) LAG or annulus  and the presence of an EFS in the largest individuals OrodromeusDysalotosaurus and Dryosaurus. However, Orodromeus seems to be rather an exception among small to medium-sized ornithopods regarding its growth pattern, although LAGs and annuli were recently also found in small ornithopods from high latitudes Orodromeus at the beginning of somatic maturity is estimated by Horner et al. Orodromeus  odromeus , to whicodromeus  demonstrTelmatosaurus and Zalmoxes, which are treated as secondarily downsized taxa due to their restricted island habitat In conclusion, higher food demands and seasonal migration of large ornithopods could be one reason for the much more consistent development of annuli/LAGs in their long bones compared to small ornithopods. Exceptions may be the ornithopods Breeding strategy and courtship/rut: Dysalotosaurus, Orodromeus, and other smaller ornithopods were probably precocial as hatchlings  tosaurus ; Fig. 24It is important to note that the remarks on the reasons for different growth patterns in ornithopods are tentative hypotheses. The variability of growth patterns, especially in smaller ornithopods, is striking and ontogenetic histological studies of more taxa are urgently needed to strengthen or disprove them. Nevertheless, the occurrence and/or consistency of annuli/LAGs in ornithopods is dependent on a mixture of absolute growth rates (which depends on maximum body size), relative growth rates , the degree of seasonality of the respective habitat, and the liability of the taxon to seasonal effects including temperature, humidity, food supply, migration, and behavior . Phylogeny plays a rather unimportant role, as already indicated by Werning Dysalotosaurus. For this purpose, up to 70 individual bones were sampled, comprising femora, tibiae, humeri, fibulae, and prepubic processes.The large amount of specimens, representing a wide range of ontogenetic stages, offered the unique opportunity to learn more about the modes and reasons of variation in bone tissues and allowed insight into the growth pattern and life history of the ornithopod dinosaur Dysalotosaurus is their increasing abundance in skeletal elements with lower relative growth rate. The number of growth cycles naturally increases during ontogeny, but this definitely is not the case for annuli/LAGs. The extraordinary variation in the development of annuli/LAGs in Dysalotosaurus eliminates prediction of their existence and relative number in skeletal elements of different ontogenetic stages.Variation within the bone tissue was mainly found between different skeletal elements and between different units of single cross sections. The former is the result of different relative growth rates, which are dependent on the individual size of a certain element and its degree of utilization within the skeleton. Skeletal elements with a large absolute size, with main weight bearing functions, and elements intensively used for movements (e.g. for locomotion) experience higher relative growth rates than other elements. Some elements have of course combined these characters, which explain the highest growth rates in the femur for instance. Accordingly, the only predictable model on the occurrence of annuli/LAGs in Intra-cortical variation in bone tissue is mainly the result of osseous drift and variation in bone wall thickness during growth. The relationship between osseous drift, bone wall thickness, bone tissue variation, and resulting relative growth rates, can now be better defined:A long bone with a bended long axis experiences osseous drift from the convex to the concave side of this long axis.Relative growth rates, derived from the organizational degree and the density of vascular canals, are lower on the convex side of the bended long axis and higher on its concave side.Growth rates are also relatively higher in thicker cross sectional units than in thinner units.Variation in bone tissue within a cross section decreases the more consistent and round the transverse shape of a bone is. A shaft with a triangular transverse outline contains more variation than a shaft with a circular transverse outline.In the case of partial sampling of a bended long bone, the part with the best potential record of ordinary bone tissue and growth cycles is the flat wall on its concave side.Dysalotosaurus is most similar to Dryosaurus altus in respect of ontogenetic stages, rarity of annuli/LAGs, variation of bone tissues, low degree of secondary remodeling, and the absence of an External Fundamental System. This confirms the close relationship and a similar growth pattern and general life style of these taxa.The bone histology of Dysalotosaurus, despite the scarcity and variability of annuli/LAGs. Growth curves of femora  revealed that Dysalotosaurus grew with a moderate rate in its juvenile stage until approximately six years of age, experienced accelerated growth during its sexually immature stage until reaching sexual maturity at approximately ten years of age, and had its exponential growth phase as sexually mature individual until the 14th year of life, where the maximum growth rate was reached. Afterwards, the growth rate decelerated and might have reached asymptotic growth well after 20 years. However, most likely none of the members of the Dysalotosaurus herd reached the growth plateau of somatic maturity.A new type of growth cycles was used to reconstruct the life history of The group of large individuals within the size-frequency distribution obviously consists of sexually mature individuals, because medullary bone was found in a tibia and a fibula of this size range. The time of initial sexual maturity was discovered as a transitional mark (MISM) in five large femora representing a slight slow-down of bone apposition rates.Dysalotosaurus to withstand intra-specific competition and its high liability for predation.Indeterminate growth, combined with delayed sexual maturity, is assumed to represent the optimal growth strategy of Dysalotosaurus were finally combined with a relationship between abundance and consistency of annuli/LAGs in recent mammals and their respective seasonal environment. Smaller species of ornithopods are less exposed to seasonal effects than the large species mainly based on differences in food demands, growth rates, and breeding strategy. In fact, the achievement of large size within Ornithopoda was probably linked to a change in breeding strategy from precocial to altricial behavior.The results of the bone histological study of The key literature for an introduction into bone histology, where also the here used terms are explained, comprises Castanet et al. The sections used by Chinsamy 30 femora, 12 tibiae, 13 humeri, seven fibulae, and eight prepubic processes were sampled, but not all of the obtained thin sections were well preserved. Thus, 11 femora, two tibiae, and four humeri were inappropriate to be considered for measurements and correlations and are therefore also not included in the Dysalotosaurus. It is important to note that it was impossible to take thin sections from a standard level, because only incomplete specimens were used. Furthermore, it was aimed to cause as less damage as possible, so that most of the cuts were carried out close to broken surfaces. Thus, the sections are standardized to a single interval along the bone shaft and not to a single level , the Staatliches Museum für Naturkunde, Stuttgart (SMNS), and the Institut und Museum für Geologie und Paläontologie, University of Tübingen (GPIT). Measurements of additional specimens were also made in the Museum für Naturkunde, Berlin (MB) and the Natural History Museum, London (R/NHMUK). All sampled bones  where already broken, lacking either the distal or proximal ends. In case of the femora, it was also possible to use isolated shafts, because the distal beginning of the fourth trochanter or the medial depression helped to clarify its orientation and the best position for the thin section. The prepubic process of the pubis represents the only non-long bone element and was chosen to highlight further variability within the skeleton of le level . DistincThe bones were cross cut with a diamond powder disk on a precision saw. Due to the brittle nature of many bones, they were temporally embedded in acetone dissolvable two-component epoxy-resin (Technovit 5071) during the sawing process. By the following well known process of grinding and cutting see e.g. , the proDysalotosaurus generalizes the observations for each of the elements. Some cross sectional units have very special and recognizable features, which helped to orientate them even without the actual bone. Distances are only measured along the anteroposterior axis or the mediolateral axis . It was the goal to correlate the cycles of all cross sections of one group of a single skeletal element, to count the final number of cycles, and to equalize them into years. Superimposition of sketches did not lead to a good correlation of growth cycles due to variation in cross sectional shape and the course and distances of growth cycles to each other. Hence, another way was chosen to get a correlation, which was also carried out by using Adobe Photoshop 7.0 software.The end of each growth cycle was marked in the sketches by a permanent line. A standard location within the cross sections, which usually revealed the best record of growth cycles, was determined for femora, tibiae, and humeri respectively. In tibiae, two fitting locations were found and the final growth cycle values were then averaged.The first step towards the correlation of cycles was the definition of an unambiguous and repeatable midpoint for every used cross section . FemoralThe cross sectional shape of tibiae and humeri were much more oval in shape. Here, the midpoints of two circles were used to determine the midpoint of the cross section. One circle was graphically scaled down as small as possible to enclose the cross section externally and just tangent the outer edge. The second circle was scaled up as large as possible to tangent the outer cross sectional edge internally. The midpoint of a straight line, which was drawn between the two obtained circle midpoints, was then determined as the midpoint of the cross section .During the next step, the distance between the cross sectional midpoint and each of the recorded growth cycles was measured and transformed into partial percentages of the distance between the midpoint and the external cross sectional edge. The reference measurement for each of the cross sections, representing 100% from midpoint to external edge, was already measured before at the respective sampled specimen. Since not the same reference measurement could be taken from each of the femora, tibiae, and humeri, regression equations were calculated with Microsoft Office Excel 2007 software to get the allometric relationships for these distances. In the end, all reference measurements were transformed into diaphyseal circumference and distal mediolateral width in femora, distal mediolateral width for tibiae, and mediolateral width at the deltopectoral crest for humeri . The datIt is important to note that each measurement from the cross sectional midpoint to a growth cycle was taken perpendicular to the course of the latter. In all femora, the best growth record was preserved in the lateral part of the posterior wall, close to the posterolateral corner. A straight line was drawn from the midpoint parallel to the course of the growth cycles and the measurement was then taken laterally from the midpoint and perpendicular to the course of the growth cycles . In all A special cycle, observed in five large femora and marking initial sexual maturity (MISM), was measured in the same way as the growth cycles.All measured percentages of growth cycles were then transformed, in a third step, into partial values of the reference distance of the respective cross section (representing 100%) and recorded in an Excel file. The values of each cross section were sorted in their respective group, one in humeri, two in tibiae, and four in femora. The following correlation of growth cycles was therefore done only within a single group. The still uncorrelated growth cycles of each group were related to age in years. A diagram was then created, were the x-axis represents age and the y-axis partial reference values of the growth cycles of each cross section of this group. The correlation of growth cycles to age in years started by fitting the lowest known value to an age of one year. This could be done, because the respective value was derived from the smallest sampled specimen , where tTwo out of four groups of sampled femora were chosen to convert their age related growth cycles into body mass estimates. The samples of the other two groups are not appropriate, because their location within the shaft is either too proximal or too distal, and their small number of recorded growth cycles only covers three to four years. In contrast, growth cycles of several samples in femoral group one and two were often placed within the same year of age during correlation. In this case, the average of all values of this year was used as the basis for the body mass calculation.Dysalotosaurus femora, W = 0.16 CF2.73, where W is the weight and CF is the circumference of the femur.Two methods of calculating body mass by skeletal elements were considered. The first method was derived by Anderson et al. Dysalotosaurus.The accuracy of this method was recently doubted Alligator ))+d describes the sigmoidal course of this type of growth curve . The variable a was derived from the largest known femur with a calculated body mass of 115.3 kilograms. Only the secured growth cycle values were integrated and all unsecured values, including the values externally to the MISM, were excluded. The latter values were entered afterwards into the curves to evaluate their significance and possible age correlation. The MISM itself was included with the corresponding age of 9.5 years in femur group one and 10.5 years in femur group two. A total of four curves were created, including the calculated body masses by the The calculated body mass of the averaged growth cycles was therefore plotted against their respective age in years. The equation To get yearly and daily growth rates, the calculated yearly body masses were derived by using the sigmoidal equations and the four parameters of each of the four growth curves. One version corresponds to the growth rate in a recent year (365 days) and the second version corresponds to a year in the Late Jurassic , which contained approximately 377.76 days x = ln ((a/(y−d)−1)/b+c Click here for additional data file.Table S1List of all specimens and measured data of humeri, tibiae, and femora, which were used for the allometric calculation of the reference values necessary for the correlation of growth cycles in the sampled specimens.(DOC)Click here for additional data file.Text S1This text comprises a more comprehensive description of the thin sections of all five skeletal elements of Dysalotosaurus and additionally includes notes on the modes of preservation of the bone microstructure as well as on the occurrence and distribution of osteocyte lacunae and Sharpey's fibers.(DOC)Click here for additional data file.


This is a lucid read on a complex virus, cytomegalovirus (CMV), that affects those in all ages of life from the unborn fetus to the elderly pensioner. Griffiths has used one of the many self-publishing, internet-based services to publish and distribute this book via the popular amazon.com and amazon.co.uk websites. All profits from its sale are being donated to the Royal Free Charity, which has supported his research on CMV for many years. The book is arranged chronologically; the 18 chapters are headed by a range of years, starting with 1910 (Chapter 1) and ending in 2012 (Chapter 18). An interesting approach is used in the Prologue (dated 180 million years ago), where the virus speaks to the reader in the first person. The virus’s voice summarizes the most important aspects of its virology and its clinical impact on a largely unsuspecting human population. Several important themes are introduced here: 1) the author’s clear views about the relative irrelevance of experiments on mice to the human situation; 2) the missed opportunities that studying CMV earlier could have created by helping the scientific community to understand and more effectively combat other viruses, such as HIV; and 3) how a vaccine approach would be the best way to control this viral pathogen.The book starts off by discussing other viruses of clinical importance , gradually setting the scene for the emergence of CMV. Interspersed between the history of the science and the emerging clinical importance of this virus are patient case histories. At least 1 case is described in each chapter , covering the most common clinical CMV scenarios. These include CMV infection and disease in newborns, pregnant women, transplant patients, persons with HIV/AIDS, and other immunocompromised patients. Other cases also demonstrate the virus’ impact on otherwise healthy teenagers studying for exams, retirees caring for their grandchildren, and recovering burn and gunshot victims . Such case histories provide clear examples of complications arising from both primary and reactivated CMV infection and disease.Most  of the relevant major scientific and medical advances related to CMV are noted, including the development of the various anti-CMV drugs, with due credit given to the investigating teams. Most passionately of all, Griffiths carefully charts gradually changing attitudes to the development of a CMV vaccine.Curiously, the author writes about his own role in the CMV story in the third person—perhaps to maintain a scientist’s natural objectivity. Notably, in the latter half of the book, he tends to use more technical jargon, which may cause problems for some readers. Yet, this does not necessarily detract from the book’s value; rather, it may provide reassurance to the readers that they are, indeed, in the hands of a master.The development of a CMV vaccine is now gaining support internationally, thanks in no small part to the efforts of the author. When the vaccine is eventually available, anyone with any doubts about its potential benefits can simply read this little book.


Biological samples are also required to improve the understanding the environmental mechanisms of heart disease . The goal of this paper is to focus on preanalytical issues  related to collection of biological samples for research purposes. In addition, the paper provides an overview of the efforts made recently by our Institute in designing and implementing a high-security liquid nitrogen storage system (-196°C). We described the implementations of reliable preservation technologies and appropriate quality control  in order to ensure maximum security for personnel as well as the quality and suitability of the stored samples.Biobanks play a crucial role in "-Omics" research providing well-annotated samples to study major diseases, their pathways and mechanisms. Accordingly, there are major efforts worldwide to professionalize biobanks in order to provide high quality preservation and storage of biological samples with potentially greater scientific impact. Biobanks are an important resource to elucidate relevant disease mechanisms as well as to improve the diagnosis, prognosis, and treatment of both pediatric and adult cardiovascular disease. High-quality biological sample collections housed in specialized bio-repositories are needed to discover new genetic factors and molecular mechanisms of congenital heart disease and inherited cardiomyopathies in order to prevent the potential risk of having a fatal cardiac condition as well as to facilitate rational drug design around molecular diseases ( In 2samples” .Indeed, the storage of human samples  represents a great opportunity for the medical science in order to discover biomarker and molecular signatures of most common diseases, such as cancer, cardiovascular diseases, neurodegenerative diseases, and diabetes.Clinically validated biomarkers may provide information to be used for the risk/predisposition, diagnosis, screening, monitoring of prognosis and prediction of response to treatment -4.Thus, there are currently major efforts worldwide in order to move from a “do-it-yourself” tissue collection- as is most frequent at present- to professionalized biobanks for providing high quality preservation and storage of biological samples with potentially greater scientific impact , 6. Unfortunately, despite the importance and complexity of this issues, there is very little published information concerning the suitable methods for a correct preservation and storage of biological samples as well on the design and development of a biobank.The aim of the paper is to give a view based on our recent experience in the design and development of a liquid nitrogen biobank as a key resource for clinical research.In spite of decades of clinical and epidemiological research, the etiology of many complex  diseases is still largely unknown.The ‘omics’ technologies, including transcriptomics, proteomics, and metabolomics, are revolutionizing our understanding of mammalian biology, promising the discovery of new biomarkers and raising the perspective of “personalised medicine” -10.Transcriptomics is the identification and characterization the mRNA transcripts that are present in a sample at the time of collection by using the tools of molecular biology and bioinformatics. Gene expression profiling can be performed by using high throughput techniques such as microarray technology in order to determine which genes are differently expressed as result of changes in diseased conditions or in environmental conditions -10.Proteomics is the study of all proteins present in specific cell types or tissues in order to understand their role in pathophysiological conditions. In contrast to the genome, the proteome is highly dynamic over time, between cell types and in response to the environment -10.Metabolomics is defined as the study of small metabolites involved in the energy transmission in cell by interacting with other biological molecules following metabolic pathways -10. MetaFurthermore, regulations of gene expression by epigenetic mechanisms has emerged as one of the most crucial determinants of cellular behaviour of common diseases , 12.Indeed, epigenetics describes the mechanisms that enable cells to respond quickly to environmental changes and provide a link between genes and the environment . Finally, the concept of the 'exposome'- reflecting the cumulative effect of exposure from the gestation onwards- has recently been introduced as a complement to the genome in studies of disease etiology , 14.The exposome concept promotes the application of "-omics" tools in environmental health by comparing biological samples from diseased and healthy subjects in order to better characterize intermediate pathways, potentially providing the 'missing links' among exposures, genes, and diseases , 16.In this light, the most important prerequisites for “omics” studies is the careful handling and storage of precious biological sample in order to preserve the quality of the final stored samples of most epidemiological and clinical studies.1).The biomolecules that are currently of major value in modern biobanking, retained in biofluids and tissues, are DNA, mRNA, proteins, peptides, phospholipids, and small metabolites  remains the leading cause of mortality and morbidity in both children and adults , 18. AthAdvances in cardiology research during the last years has allowed the discovery of numerous potential biomarkers on protein or even genetic level. These biomarkers are an exciting new tool for guiding diagnosis and treatment as well as for facilitating the individualization of adult patient care. Nevertheless, considerable uncertainty remains with regards to the incremental predictive value of a panel of biochemical and genetic biomarkers beyond conventional risk factors. Integrated approaches, combining circulating, imaging, and genetic biomarkers, are required to make a complete assessment of the potential clinical usefulness of biomarkers for risk prediction of CVD disease .Congenital heart malformations are the most prevalent and fatal of all birth defects, occurring in nearly 1 in 100 live births . Before Inherited cardiomyopathies, including ion channelopathies, are among the most common genetic cause of sudden cardiac death, especially in the young (including trained athletes). Important progress has recently been made in identifying the genetic causes of cardiomyopathies, which, in turn, has provided new opportunities for practitioners, patients, and families to use this genetic information . HoweverFurther research is, therefore, needed to discover new mutations and understand the role of the genetic factors underlying variable penetrance and variable expressivity as well as the function of genetic modifiers, common polymorphisms, multiple mutations, and epigenetic mechanisms. Specifically, in cases where no definitive cause is identified at postmortem (i.e. Sudden Unexpected Death), the “molecular autopsy” may be as a key process in the investigation of the cause of death . FurtherIn spite of the epidemiological evidence indicating that environmental toxins can heart disease, the mechanisms of the cardiovascular injury and heart effects are not fully understood. Clearly, additional studies are required in order to improve the understanding of environmental mechanisms of heart disease but also in developing specific preventive or therapeutic strategies for minimizing the environmental mechanisms of heart disease. Therefore, biobanks are an important resource for cardiovascular research to elucidate relevant disease mechanisms as well as to improve the diagnosis, prognosis, and treatment of CVD diseases. Indeed, storage of blood and tissue samples  from diagnosed CVD patients and from experimental models is of great importance to identify pathways involved in disease initiation or progression in order to prevent the potential risk of having a fatal cardiac condition as well as to facilitate rational drug design around molecular diseases . 2).When building a long-term archive of biological material for future research, many factors must be considered at the initial collection stage, and it is, therefore, recommended the strict adherence to standardized protocol in order to obtain high quality biobanks -30. The Besides methodological issues, a crucial prerequisite to collect samples for a biobank is the ethical approval from an Institutional Review Board or Institutional Ethics Committee .Universal Declaration on Bioethics and Human Rights (2005), UNESCO Universal Declaration on the Human Genome and Human Rights (1997), Declaration of Helsinki on Ethical Principles for Medical Research Involving Human Subjects , biological samples should be acquired only with the informed consent of participants, who, thus, transfer for free their powers on the biological samples to the biobank, also defining the limits within which they can be used [In accordance with applicable law and ethical principles pertaining to the protection of human subjects , whereas serum is collected into a tube that contains no additives or in many cases a form of micronized silica to promote clotting. For genetic analysis, the collection of EDTA-blood is good for DNA-based assay. ex vivo cellular injury, disintegration and cellular granule release, and action of proteases may occur after sampling. Indeed for some types of biomarkers it might be feasible to add a mixture of protease inhibitors in order to avoid extensive degradation of the proteins in the sample.Furthermore, time is another crucial factor throughout the process of sample collection and processing as Although blood samples are often kept at room temperature until processed, a general recommendation is that the sample is separated as rapid as possible into different components  and each component is kept at the appropriate temperature -31. The When the samples are not being analysed immediately, they should be maintained at low temperature because room temperature results in degradation of labile protein biomarkers (e.g. cytokines), anti-oxidants , and other analytes (such as folate and vitamin B12).Indeed, temperature control during the processing of samples and the long-term storage is essential and determines the usefulness of samples in future analyses -36.As general recommendation, storage at −80 °C over a prolonged time is adopted as a minimum for aliquots of serum and plasma which contain a large number of soluble molecules most require very low temperature to remain intact -35. AnywThe best method for preserving cells and biomolecules is their cryopreservation in liquid nitrogen which is becoming an increasingly common storage option for biomedical research -42. The However, the establishment and maintenance of liquid nitrogen biobanks require careful planning to ensure that personnel using the facility are working in safe condition and the samples are maintained in a suitable environment for long-term storage by using reliable preservation technologies and appropriate quality control.The most important safety consideration is the potential risk of asphyxiation when escaped nitrogen vapourises and displaces atmospheric oxygen, leading to oxygen depletion which, in turn, can very rapidly cause loss of consciousness. Consequently, liquid nitrogen refrigerators should be subject to periodically preventative maintenance and should have low oxygen alarm systems.1 summarizes the precautions for liquid nitrogen storage banks.In addition, it is important that staff are trained in the use of liquid nitrogen and individual protective equipments in order to minimize the risk of adverse incidents. Table Accordingly, our Institute at CNR Campus  for biologic specimen collections from current and previously funded projects.The core of the liquid nitrogen facility is an appropriate room containing the cryo-containers connected to a fixed external station for periodically refilling of liquid nitrogen.Access to biobank is controlled though electronic bagdes and all the processes are managed by a specially designed software system  that automatically and securely manages the supervision and the surveillance of cryobiological storage samples, including traceability of the storage conditions.3).Indeed, this system allows users to track biosamples from acquisition to analysis, to store annotations associated with those samples as well as to constantly monitor parameters and critical operating conditions such as oxygen level, management of the supply in liquid nitrogen, synchronization of the fillings of cryo-containers and management of the acoustics and visual alarms from environmental sensors. Any alarm that is identified as “critical” will be remotely sent by SMS, vocal announcement or by e-mail to operators in order to allow intervention in case of emergencies Fig.  diseases. Biobanks are a critical resource for such molecular-based biomedical research, playing a key role in the area of personalized medicine in which the treatment will no longer be “one size fits all”, but instead “tailored” to the molecular and genetic profile of each patient. Despite this potential, researchers often overlook the critical points which can affect the quality of biological samples. Therefore, we would highly encourage Researcher Centers to support and develop well-organized Biobanks which include a high degree of expertise in issues related to sample collection and processing, and implementations of reliable preservation technologies and appropriate quality control in order to ensure the quality and suitability of the stored samples. This strategy will offer to identify and validate new diagnostic and therapeutic approaches based on novel biomarkers and molecular targets, promoting their translation into clinical practice.