Rhodnius prolixus is the main vector in Colombia. Control strategies in this region have shown poor outcomes due to the insect’s ability to disperse between the sylvatic and the domestic habitat. Because insect migration to houses is responsible to sustain contact rates between vectors and humans, understanding the risk factors that promote migration could be important in designing control strategies. In this respect, it has been reported that adult triatomines have the ability to move over long ranges at night attracted by artificial light. Thus, light bulbs could be playing a critical role in house invasion. The main objective of this study is to understand the role of artificial light, or simply light, in house infestation by R. prolixus.Chagas disease is the most important vector-borne disease in Latin America and Rhodnius prolixus dynamics. The model allowed us to simulate insect mobility and distribution in the village based on field results. We created 11 scenarios representing different amounts of light in the village  with 100 simulations each for a time of 1000 days (2.7 years) and compare the results between the scenarios.To investigate the role of light, we combined fieldwork in the village of Chavinave, Casanare, Colombia and a mathematical model of light allows for insect spreading and migration to previously un-infested areas.None of the Gomez-Nuñez traps were positive at any stage of the study, suggesting that insects do not colonize houses. The model predicts that with current village connections the proportion of houses that have visiting insects should be around 98 %. Additionally we showed that an increase in light could increase the contact rates between vectors and humans; a two-fold increase in human cases for a 30 % increase in the use and visibility of light on this particular village was estimated with the model.Increments in  Trypanosoma cruzi and is transmitted to humans by insects of the subfamily Triatominae (Hemiptera: Reduviidae). It is a major public health problem in Latin America and recent estimates suggest that 6–8,000 000 people are infected and 65,000 000 are at risk of contracting the disease [Rhodnius prolixus [T. cruzi and high mobility between the sylvatic  and domestic habitat [Chagas disease is caused by the parasite  disease . In Colo disease , 3. The prolixus , which iHouse infestation control efforts have focused mainly on fumigation. In Colombia, however, spraying has been of limited success  because Unfortunately, the eco-epidemiology of the disease is complex and human infection could be determined by several risk factors. These fall in two main categories: i) factors related to house infestation and ii) factors related to insect migration from palm trees to human dwellings. House infestation has been studied relatively well; it has been reported that insects like hiding in houses with adobe walls, palm leaves roofs and unfinished floors –16. In aR. prolixus and Triatoma infestans, studies have found that their feeding patterns and sensitivity to host signals peaks in the early evening [Recent studies demonstrated that adult Triatomines have the capability to move long distances at night –26 and i evening , 34, 35  evening , 36, 37.R. prolixus is a difficult task. The main challenge could be ethical considerations that impede setting up experiments within occupied dwellings, but also such an experiment would require a complicated and expensive system to track insect movement.Therefore, understanding the role that artificial light could play in house invasion can have important consequences for disease prevention , 38–40. R. prolixus. We collected data in the village of Chavinave, located in Mani (Casanare), where our group recently reported densities and natural infection with R. prolixus in native palms of Attalea butyracea [In this study we investigate the role of artificial light in the distribution and abundance of insects in a real village, and its possible consequences in terms of contact rates with utyracea . We obseT. cruzi in R. prolixus [The department of Casanare is known as an area of high-risk level of Chagas transmission  and repoprolixus . Our worprolixus  for addiTwo or three Gomez-Nuñez (1965)  boxes, dAttalea buttyracea palms are ubiquitous and have high densities of insects [Insects are also found in palm trees and in this region,  insects , 45. We  insects ). In addAccording to the Act 134 of 2011 of the Ethical Committee for Research of the University of Los Andes, the project accomplished all scientific, technique and administrative rules for health research established in Resolution 008430 of 1993 of the Ministry of Health.R. prolixus population dynamics within every patch (meta-populations) and migration of adults between patches. Insect dynamics are based on an age-structured population where Ei(t), Ni(t) and Ai(t) denote number of eggs, nymphs and adult respectively in patch i at time t.We constructed a mathematical model to simulate insect mobility and distribution in the village based on field study results. In the model we consider houses and palms from the Chavinave village to be habitats suitable for insect populations. We called these habitats “patches” and assumed that flying insects connect them. We included Thus, the rate of change of every age-group is described as follows:λ is the per-female egg production, τis the average hatching time, 1/γthe average nymph to adult maturity rate, Kis a patch’s carrying capacity and, δE, δN and δA are egg, nymph and adult per-capita mortality, respectively. Biological parameters with their values and units are summarized in Table where βiAi accounts for insects moving from patch i to any other patch and i. Both fluxes are modelled using a per-capita migration rate βi assumed to have saturation kinetics described by the following first order hill-equationMovement between patches is included in the model by the two last terms of equation 3. − σ is the maximum per-capita migration rate and η is the number of individuals at which half of the maximum per capita migration rate occurs.where j to destination patch i is captured by the adjacency matrix αji where every entry is defined by:Decision to fly from origin patch Dji is the Euclidean distance between patch j and patch i, and Lji is a matrix with 1 at entry j, i if patch i has a light that can be seen from patch j and 0 otherwise. Lji = 0 for every jwhen i is a palm . Equation 5 assumes that the maximum distance an insect can overcome is 200 mts [αji ' s for every i are normalized to produce an adjacency matrix with entries between 0 and 1, where 1 means maximum attractiveness.where  200 mts  and alsoThe village contains 30 houses and 71 palms for a total of 101 patches. The system of 303 coupled differential equations was solved numerically using Matlab. All the runs started with the same initial conditions: 20 insects per palm  , from 0 to 100 %. In other words, we varied the proportion of links that connect houses with palms and houses with houses in the village from 0 %  to 100 % . The scenarios were run 100 times and for each run we randomly assigned the entries in L that were equal to 1.To investigate the role of light on insect distribution and movement we developed 11 scenarios that differ in the amount of links that are equal to 1  as the average number of houses visited by insects divided by the total number of houses at steady state. Second, because it is not only the percentage of houses visited but also the number of insects moving into houses that determines transmission risk, we calculated a visiting index (VI) as the average number of insects per house divided by the number of visited houses per unit of time. Finally, we recorded the flux of insects from palms to houses (FPH), houses to houses (FHH) and house to palms (FHP).Lji changes the network configuration (i.e. what is connected and how strong), for every simulation we computed three more indices that reflect network metrics: First, we computed the number of groups of connected nodes (e.g. number of clusters - NC). This measurement gives the number of disconnected units in the village; as more clusters appear, fragmentation increases. Second, we calculated the proportion of patches in the biggest cluster (PPBC) which gives a measurement of how many patches are disconnected from the main network [The village with all its patches can be regarded as a directed network where connection between patches are represented by bidirectional links that have their weights given by equation 5. Insects can move between patches that have link weights above zero. Because manipulating  network , 48. Thi network , 48.L that can take the value of 1. When both distance (habitats that are close enough for insects to fly) and visibility (light is visible from source patch) are equal to 1, only 274 (64 %) connections remained present. 160 of those connections are from house to house and 114 are from palm to house. Out of the 7100 possible connections where L can be only 0 , we found that 474 are within flying distance. Thus, patches are connected via two possible links: those that are based on proximity and those that are based on light and proximity. For Chavinave we had a total of 748 links, which is shown in Fig. Figure R. prolixus were captured during a 1 week period and stored in plastic containers.The Gomez-Nunez traps  were insL that are equal to 1 reflects the presence of light in the village, and often in this manuscript we will refer to this variable simply as light  to Lji = 1 for every j, i (100 %). Because the entries equal to 1 were chosen randomly and repeated 100 times we could compute standard deviations, which are shown as shadows for every curve. Figures L. The current situation for Chavinave is described by the dotted line and corresponds to 64 %.We assumed that the proportion of entries in L are equal to 1) the proportion of houses that have visiting insects should be around 98 %. Figure Figure PPBC and NC show an opposite behavior: as the proportion of houses that have light increases the village has a higher PPBC while NC diminishes. As light goes to 100 %, NC is down to 5, but 90 % of the patches are all contained in one node. Thus, the village moves from 36 clusters at 0 % light to 5 where the village almost behaves like a single unit. We found that houses with the higher VI are all contained within the cluster that contains most of the nodes. In fact the other clusters are composed only by palm trees and one lonely house.APL can determine whether a cluster is within reach for insects. At a low proportion of houses with light (0–10 %), APL is small because one has many clusters with small number of patches each. As the proportion of light increases, APL increases to a maximum of around 4 patches at 30 % light, and at this stage the number of clusters is close to the minimum but contains patches that are weakly connected. As light increases even further, APL decreases because patches increased their connection within roughly the same number of clusters. VI and PIH could be potentially linked to APL, however we found that the increase observed in APL is not at a level that impacts insect movement in the network.FPH increases with a saturation behavior as the proportion of houses with light increases, reaching a maximum of 4 insects per day consistent with the connectivity created by light. On the other hand, FHH is always above FPH and shows a more linear behavior that reaches a maximum of 7.6 insects per day when all nodes are connected. This result is a consequence of the village organization: houses are close to each other with few palms within and the sylvatic habitat is in the periphery. At low light densities insects mostly see houses from palms but as lights start to increase the house network becomes predominant.One of the most striking findings from this work was the fact that insects do not domiciliate in this small village in a highly endemic region. The paradigm for Chagas disease transmission in Colombia usually assumes insect domiciliation and the coexistence of a sylvatic and a domestic cycle , 49, 50.Because we wanted to explore the role of light in insect distribution and mobility, we developed a model that captured what we learned in the field coupled with published information about insect biology in order to account for population dynamics. Light is an attractor as long as the house is within reach and as such it is expected that light and geographical position have interacting effects. Thus, to investigate the role of light, it is important to understand that results highly depend on the configuration of the village. Incidentally, Chavinave is constructed such that all houses are grouped together at the center of the network and they are surrounded by Gallery Forest that contains palm trees. Caution must be taken when extrapolating the results from this study.R. prolixus migration. R. prolixus tends to fly towards light in the early hours of the night, usually around 7 pm. This time coincides with human activities that involve use of light bulbs at home, like cooking or watching TV. In a recent study with T. dimidiata, researchers showed that this vector has peak activity between 1 and 4 am and thus it tends to infest the dwellings that are closer to public streetlights [Before exploring the results of our model, is important to mention why house lights as opposed to streetlights are relevant for etlights . We did The results obtained with this model suggest two interesting observations: i) light distribution and abundance affect VI and PIH because of a network effect and ii) once insects move to a house they do not return to the sylvatic habitat ; instead they stay circulating between houses due to a network effect. We explore these two interesting findings in some detail below.The behavior of PIH and VI suggest that contact rates between insects and humans will increase as light increases. This is mainly due to an increase in VI because PIH is almost saturated at 50 %. This result suggests that every house in the village is exposed and the number of new cases will be dependent on the contacts between insects and humans. For the particular case of Chavinave we found that, on average, a house with a light bulb can be seen from 64 % of all available patches; at this level our model predicts a PIH of 98 % with a VI of approximately 2 visiting insects per house. We could not corroborate this result in the field because residents were not very diligent at capturing insects.R. prolixus feeds from humans half of the time  and an average of 2 insects per house per year, 98 % infested houses would result in 1 new case of Chagas every 18 months. Figure In theory, with this information, it is possible to estimate the number of human cases for Chavinave. First, we know from previous studies in the same area that the natural infection of Triatomines in palms is about 60.3 % . Assuminorted by ), and thd by [−4 , we obtaThe second interesting finding relates to the observation that insects are trapped in the network of houses and do not return to the sylvatic environment. In other words we found that the flux from house to palms is 0. Another study showed that Triatomines that feed from humans in houses never return to the palm trees . It is oTo understand this pattern of movement we can use network metrics. Evidently, light increments lead to a decrease in the number of clusters and an increase in the proportion of patches in the biggest cluster accompanied by a vast reduction in the average path length above 30 %. This is due to the fact that the biggest cluster has almost all the patches connected and the number of links between them increases with light presence. This situation allows for insect spreading and migration to new un-infested areas , 57. In It is important to explore limitations of the model to highlight assumptions that naturally impose constrains to the results. First, we set insect migration to be entirely deterministic: insects choose the most attractive patch among a set of possible targets. This could be too simple as there might be some stochasticity involved. However, it has been suggested that Triatomines are strongly attracted by incandescent lamps , 28, 29 T. dimidiata and modeled by Slimi et al. (2009) [foci of transmission in the village. This result can be used to spatially target houses for control strategies (i.e. insecticide spraying) [From a public health perspective, understanding how light shapes the movement patterns of insects in a village allows the development of precise risk maps , 58, 60.. (2009)  . A riskpraying) . Moreovepraying)  or turnifoci of transmission in the village and target specific houses for control strategies (i.e. insecticide spraying).Our model coupled with the field work suggests: i) a two-fold increase in cases for a 30 % increase in the use and visibility of light on this particular village, ii) once populations of insects are trapped in the domestic environment they do not return to the sylvatic, iii) insect movement is strongly influenced by the presence of light because light increases network connectivity. In addition the network analysis showed that infestation risk for a particular house depends not only on its own status but also on its neighbor’s, and iv) developing risk maps that consider not only the proximity to the sylvatic area but also the possible connections among habitats, where light is involved, could help identify


Blood clot formation is important to prevent blood loss in case of a vascular injury but disastrous when it occludes the vessel. As the mechanical properties of the clot are reported to be related to many diseases, it is important to have a good understanding of their characteristics. In this study, a constitutive model is presented that describes the nonlinear viscoelastic properties of the fibrin network, the main structural component of blood clots. The model is developed using results of experiments in which the fibrin network is subjected to a large amplitude oscillatory shear (LAOS) deformation. The results show three dominating nonlinear features: softening over multiple deformation cycles, strain stiffening and increasing viscous dissipation during a deformation cycle. These features are incorporated in a constitutive model based on the Kelvin–Voigt model. A network state parameter is introduced that takes into account the influence of the deformation history of the network. Furthermore, in the period following the LAOS deformation, the stiffness of the networks increases which is also incorporated in the model. The influence of cross-links created by factor XIII is investigated by comparing fibrin networks that have polymerized for 1 and 2 h. A sensitivity analysis provides insights into the influence of the eight fit parameters. The model developed is able to describe the rich, time-dependent, nonlinear behavior of the fibrin network. The model is relatively simple which makes it suitable for computational simulations of blood clot formation and is general enough to be used for other materials showing similar behavior. Blood clots form to prevent blood loss after a vascular injury but can also lead to undesired complications such as thrombosis when forming intravascularly. The mechanical properties of the blood clot are of major of importance for its functioning and have been related to many diseases Weisel  but remaThe fibrin network forms in multiple steps after the conversion of fibrinogen to fibrin monomers, enzymatically catalyzed by thrombin. These monomers aggregate to form two-stranded structures known as protofibrils which subsequently polymerize into fibers that eventually form the fibrin network  after which the sample is quickly transferred to the titanium cone-plate geometry  of an ARES rheometer . To follow the network formation, a SAOS deformation with a frequency of 1 Hz and strain amplitude of 0.01 is imposed for 2 h. This deformation is within the linear viscoelastic regime and does not interfere with the network formation  Wilhelm  and convThe data are analyzed in terms of Lissajous–Bowditch plots in which the strain is plotted versus the stress, showing closed loops that illustrate the nonlinear viscoelastic behavior of the fibrin network . First, values for the (linear) shear modulus Using the obtained NSP and the values for lsqnonlin with a trust-region-reflective algorithm as implemented in the Global Optimization Toolbox of MATLAB. The fitting procedures are performed multiple times with initial parameter values chosen randomly from a broad interval using the Multistart algorithm. In this way, it is avoided that local minima are found.The three fitting procedures are performed using the nonlinear least-squares solver The constitutive model developed contains eight parameters that are estimated using experimental data. To get a deeper insight into the sensitivity of the model to these parameters, a sensitivity analysis is performed. A global variance-based method Sobol  is appliThe output quantities, on which the influence of the variance for each parameter is obtained, are inspired by the three main features observed in the nonlinear viscoelastic response of the fibrin network. The first output quantifies the softening effect, and is defined as the relative decrease of the stress at maximal strain during the strain interval with amplitude, Strain stiffening is quantified using the maximal stress during this interval, which occurs at the maximum strain amplitude of the first full cycle, The outputs defined above are obtained using a parameter set drawn from a specified uncertainty range using Latin hypercube sampling Saltelli . The parSaltelli .In this section, results are presented to illustrate the performance of the model. Representative results are shown for one sample and discussed in detail. Parameter values of multiple samples are shown to illustrate the variation between samples. The model is first used to describe the first LAOS sequence and the recovery that takes place during the following 2 h. Subsequently, the parameter set obtained is used to predict the outcome of the second LAOS sequence. Finally, the model is used to describe the response for networks that have polymerized for 1 h instead of 2 h, to study the influence of the presence of cross-links created by fXIIIa.The experimental and numerical results for the LAOS sequence are shown in Fig. After the LAOS sequence, a SAOS deformation is imposed for 2 h to be able to observe the recovery of the network during this period. The results of this measurement are shown in terms of the NSP, The parameter set found by fitting the model to the first LAOS sequence and the subsequent recovery, as described in the previous section, is used to predict the outcome of the second LAOS sequence. The results shown in Fig. To study the influence of cross-links created by fXIIIa, the same LAOS sequence as used before is applied to networks that have polymerized for 1 h instead of 2 h.Qualitatively, the results are the same as for the networks that polymerized for 2 h, and the model describes this well Fig. . A diffeThe recovery after the LAOS sequence, also visible in the network that has polymerized for 1 h, shows similar behavior as discussed before but increases faster Fig. b.The observations that the networks that have polymerized for 1 h show more strain stiffening and recover faster also follows from the parameter values found by the model. Figure The faster increase after the LAOS sequence is described by the parameter A third difference that is visible from the parameter values is the nonlinear viscous dissipation, described by A sensitivity analysis is performed to assess how the parameters influence the output uncertainty of the three defined outputs. For every output, the contribution of the parameters to the variance of the outputs is determined. These contributions are shown as a main effect, mentclasspt{minimaThe variance of the output related to softening, mentclasspt{minimaAs expected, the variance of the output for the strain stiffening behavior, The variance of the output related to the nonlinear viscous dissipation, The constitutive model proposed in this study describes the behavior of the fibrin networks during a LAOS deformation. Using the Lissajous–Bowditch plots, three nonlinear viscoelastic features have been distinguished and are subsequently incorporated in a Kelvin–Voigt model. Although the features are modeled in a phenomenological way, it is possible to relate them to structural changes in the network. The softening observed during the LAOS sequence originates from the semi-permanent elongation of fibers due to the imposed deformation (Münster et al. During the SAOS experiment that follows the LAOS sequence,the stiffness of the network increases. Experimentally, two time scales are observed. A fast initial rise in stiffness is followed by a slower increase for a longer time. The latter is attributed to the creation of cross-links by fXIIIa (Ryan et al. Fibrin networks show a remarkable amount of strain stiffening (Storm et al. The relation between mechanical and structural properties of the fibrin network has been studied under various conditions (Brown and Barker The constitutive model is used to describe the behavior of the fibrin network during a LAOS deformation and the increasing stiffness observed afterward. The results show that during these 2 h, a relatively fast increase of the stiffness is followed by a more gradual increase. These two effects, attributed to fiber remodeling and creation of cross-links, respectively, are not described by the model explicitly, since a single time constant is used. This explains why the description of the stiffness during this phase is relatively poor compared with the description during the LAOS deformation. However, the model is such that it can easily be extended to better describe this phase, but this has not been done here to focus on the nonlinear mechanical behavior of the fibrin network during the LAOS sequence.The model developed contains eight fitting parameters. The values for these parameters are found in three separate fitting procedures, using the data of the entire LAOS deformation. Therefore, an obtained parameter set describes the LAOS experiment as a whole and is not necessarily the best parameter set to describe a single deformation cycle. However, as shown in Fig. A sensitivity analysis has been performed to study the influence of variations in parameter values. For three outputs, related to the three observed nonlinear features, the contribution of every parameter to the variance of this output is determined. The expectation that the parameters used to model one of the effects have the largest influence on the output of this effect is confirmed. An example is that the parameter mentclasspt{minimaIn this study, a LAOS deformation is used to study the nonlinear viscoelastic properties of fibrin. Other protocols could have been used, such as strain ramps (Schmoller et al. The model developed works well in describing the nonlinear behavior of the fibrin networks. The model is flexible and can easily be adapted to describe the mechanical behavior of other biopolymers that show similar behavior such as collagen (Kurniawan et al. A constitutive model has been developed to describe the time-dependent, nonlinear viscoelastic behavior of fibrin networks. The model is developed using experimental results of a LAOS deformation and describes the observed softening, strain stiffening and increasing viscous dissipation that occur during multiple deformation cycles. Furthermore, an increasing stiffness that takes place after the LAOS deformation is captured. The model is able to describe all these features, and its generality makes it suitable to be applied to other materials showing similar behavior.


Euthanasia and physician assisted suicide (PAS) are highly debated upon particularly in the light of medical advancement and an aging society. Little is known about the professionals’ willingness to perform these practices particularly among those engaged in the field of palliative care and pain management. Thus a study was performed among those professionals.An anonymous questionnaire was handed out to all participants of a palliative care congress and a pain symposium in 2013. The questionnaire consisted of 8 questions regarding end of life decisions. Proposed patient vignettes were used.A total of 470 eligible questionnaires were returned, 198 by physicians, 272 by nurses. The response rate was 64 %. The majority of professionals were reluctant to perform euthanasia or PAS: 5.3 % of the respondents would be willing to perform euthanasia on a patient with a terminal illness if asked to do so. The reluctance grew in case of a patient with a non-terminal illness. The respondents were more willing to perform PAS than euthanasia. Nurses were more reluctant to take action as opposed to the physicians. The majority of the respondents would attempt to treat the patient’s symptoms first before considering life-ending measures. As regards any decision making process the majority would consult with a colleague.This is the first German study to ask about the willingness of professionals to take action as regards euthanasia and PAS without biased phrasing. As opposed to the general acceptance that is respectively high, the actual willingness to perform life-ending measures is low. The German debate on physician assisted suicide and its possible legalization should also incorporate clarifications regarding the responsibility who should eventually perform these acts. Euthanasia and physician assisted suicide (PAS) are highly debated upon particularly in the light of medical advancement and an aging society . In thisBut even in those countries that have legalized euthanasia or PAS there is an ongoing debate about the boundaries of these practices and questions regarding the responsibilities of physicians and nurses. Data from the Netherlands have shown that it is mostly general practitioners who perform euthanasia or PAS . Those pVarious surveys regarding euthanasia and PAS have been performed –12. HoweThe study is based on an anonymous questionnaire handed out to all participants of a palliative care conference and a pain symposium in 2013. Attendants were physicians and nurses. The concept of palliative care is an interdisciplinary one. Ethical decisions take place in a team approach thus acknowledging the role of the nurses in particular. Furthermore data from e.g. Belgium or the Netherlands has shown the central role of nurses in end of life practices such as assisted dying –30. In 1The first part of the questionnaire regarded personal data asking about gender, age, occupation and special qualification in palliative care or pain medicine.The questionnaire consisted of 8 questions on end-of-life (EOL) decisions: Concrete patient scenarios were used. They were adapted from a questionnaire by Seale , which wThe first two questions focused on a patient with a terminal illness, questions 3 and 4 focused on a patient with a non-terminal illness. Additionally the illness was described as “painful” in order to hint at clear physical suffering . The lasStatistical analysis was performed using SPSS Version 22.0 . The significance level was set to p ≤0.05. Chi Square and exact Fisher tests were used to analyze bivariate relationships. For the dichotomous feature of a terminal or non-terminal illness the non-parametric McNemar test was used.The study was approved by the ethics committee of the Ruhr University Bochum (Reg. no.: 4502/2012). Consent was given by the participants in completing the questionnaire.A total of 470 eligible questionnaires were returned, 198 by physicians, 272 by nurses .The willingness to perform PAS in non-terminal illness was 3 %.Concerning a terminal illness physicians were significantly more prepared to take action in both euthanasia and PAS (p ≤ 0.05), see Table As regards a patient with a non-terminal illness again the physicians were more prepared to take action. However, the differences between physicians and nurses were not statistically significant.Physicians with a special qualification in palliative care were more reluctant to hasten a patient’s death through euthanasia or PAS both in terminal and non-terminal illness Table . Only 3.Physicians with a special qualification in pain medicine on the other hand were more prone to euthanasia or PAS in case of a terminal illness than physicians without this special qualification: 8.3 % were willing to perform euthanasia vs. 6.5 % among physicians without this qualification. In case of a non-terminal illness it was the other way around: physicians with special qualification in pain medicine were more reluctant to hasten a patient’s death through euthanasia or PAS.Thirty-three physicians had the special qualification in both palliative care and pain medicine. Overall the differences between this group and the physicians that did not have both special qualifications were not statistically significant.Physicians without any special qualification in either palliative care or pain medicine were more willing to perform euthanasia in both terminal and non-terminal illness. However, the differences were not statistically significant.The majority of the respondents would not make the decision about euthanasia or PAS by themselves 64.5 %) but would consult with a colleague first, 59.6 %. This was true both for physicians and nurses . 21.7 % of the nurses were undecided and chose “I don’t know”.58.8 % of the physicians that would first attempt to treat the symptoms would estimate 4 weeks for the attempt to discover the conclusive patient’s wish. Among the nurses it was 30.9 % who would chose 4 weeks for the treatment attempt.Given that the study focused on attendants of a palliative care and pain conference the reference group holds a certain bias. However, it was the aim of the survey to explore the attitudes of professionals engaged in the field of palliative care and pain management specifically. Because of this bias the results obtained do not reflect the general opinion of German physicians and nurses but provide an insight on the point of view of those professionals faced with end-of-life decisions in daily practice. No control group was surveyed, because we were only interested in the possible focus group.Some of the practices the questionnaire asked about are currently illegal in Germany. This might prevent the respondents from giving honest answers. To reduce the reluctance to answer honestly the questionnaire was anonymous .Additionally the questionnaire at hand avoids terminology that provides a certain bias itself (e.g. the term “euthanasia”). The patient scenarios used are adapted from a questionnaire by Seale and were translated to German . AccordiRegarding the statistical analysis a post hoc performed logistic regression model concerning age, occupation and special qualification proved no significant results. This was not unexpected regarding the sample and its structure. Nevertheless this might point to possible deficits in the strength of the statistical significance of the data at hand.This is the first German study to ask about the willingness of professionals to take action as regards euthanasia and PAS without biased phrasing such as euthanasia from the Greek “help to die” in German “Sterbehilfe”. Only few studies focused on palliative care professionals in particular –40. ThisThe general support regarding the legalization of euthanasia and PAS is respectively high in Germany . HoweverThe differences in the attitudes of physicians and nurses are remarkable: Physicians are more willing to take action than nurses both in case of a terminal and a non-terminal illness. This result is surprising since former studies prompted to nurses being supportive of euthanasia and PAS , 42, 47.One reason for the nurses’ reluctance to take action might be that the nurses’ role in the performance of these end of life practices is still unclear even in countries that have legalized these practices . HoweverIn Belgium law requires the physicians to discuss the euthanasia request with the nursing team in charge , which shows that nurses do play an important role in end of life practices. This is particularly true since it is often the nurses that patients talk to about their wishes . The rolIn some cases euthanasia and PAS were even performed without request of the patient . This doA study among 1509 Dutch nurses showed that the majority of hospital nurses did not accept inserting an infusion needle to administer a fatal dose as a task for nurses, serving on a review-board for euthanasia cases was accepted only by 45 % of the nurses asked . HoweverIn line with previous studies we found physicians with a special qualification in palliative care to be more reluctant to end their patient’s life , 53–55. Little is known about factors influencing a physician’s willingness to perform EOL practices. A Canadian study among physicians of various specialties showed that “perceived behavioral control” was one of the main determinants for the willingness to perform euthanasia . MoreoveOur results show more willingness to perform PAS than euthanasia among the professionals, thus leaving the final responsibility to the patient himself. 13 % of the respondents willing to perform PAS on a patient with a terminal disease is a remarkable amount. This is in line with the emphasis put on the patient autonomy . HoweverThese diverse interpretations of the performance of euthanasia and PAS can also be found in the Netherlands, where both practices are legal: PAS is perceived to leave more responsibility to the patient himself, safeguarding his autonomy while at the same time leaving less burden to the physician .One aspect that has not been in the focus of research yet is the final decision making process. Our results show that the majority of the health care professionals asked would interact with a colleague before granting a request to hasten the patient’s death: 59.6 % of the respondents would consult a colleague first before coming to a decision. The need for a second opinion is also demanded in countries that have legalized euthanasia or PAS, e. g. Belgium or the Netherlands, where the consultation of a second physicians or a euthanasia review committee is a procedural requirement .Facing these legal regulations and European guidelines on palliative sedation, merely 60 % consulting others before life-ending decisions is a low figure . HoweverRequests for euthanasia or PAS also occur in palliative care settings , 64. SomA caveat is represented by studies demonstrating that the patients’ wish to die often changes over time and euthanasia requests are withdrawn , 63, 69.Regarding the length of the treatment attempt the majority of physicians estimated 4 weeks to be needed. Keeping in mind that depression can be a problem in patients at the end of their life  and thatOur results demonstrate a profound problem in the actual discussion about PAS in Germany: if PAS were allowed, who should perform it. General practitioners are not trained to perform any form of life-ending procedures. This has been documented extensively by studies from Belgium and the Netherlands, where false drugs were selected, euthanasia was mistaken for palliative sedation and vice versa , 72–75. Thereby our study should stimulate an ongoing discussion before opening political discussions to allow or prohibit PAS for physicians in Germany.


We consider data from a time course microarray experiment that was conducted on grapevines over the development cycle of the grape berries at two different vineyards in South Australia. Although the underlying biological process of berry development is the same at both vineyards, there are differences in the timing of the development due to local conditions. We aim to align the data from the two vineyards to enable an integrated analysis of the gene expression and use the alignment of the expression profiles to classify likely developmental function.We present a novel alignment method based on hidden Markov models (HMMs) and use the method to align the motivating grapevine data. We show that our alignment method is robust against subsets of profiles that are not suitable for alignment, investigate alignment diagnostics under the model and demonstrate the classification of developmentally driven genes.The classification of developmentally driven genes both validates that the alignment we obtain is meaningful and also gives new evidence that can be used to identify the role of genes with unknown function. Using our alignment methodology, we find at least 1279 grapevine probe sets with no current annotated function that are likely to be controlled in a developmental manner.The online version of this article (doi:10.1186/s12859-015-0634-9) contains supplementary material, which is available to authorized users. Alignment of time course gene expression data is an important problem since, ‘biological processes have the property that multiple instances of a single process may unfold at different and possibly non-uniform rates in different organisms, strains, individuals, or conditions’ . Such diVitis vinifera L., Cabernet Sauvignon) at the ‘Willunga’ and ‘Clare’ vineyards in South Australia. The experiment was run over the duration of the development cycle of the grape berries, from the closed-flower to ripe-red stage of the berries themselves. For each gene, we have a pair of expression profiles, one from each of the Willunga and Clare vineyards. Pairs of expression profiles for four example genes can be seen in Fig. We consider a time course microarray experiment conducted on grapevines  have been used for alignment of time course gene expression data . HoweverIn order to work with the typical sparsity of the grapevine data, as well as to provide a principled way to obtain a common alignment across both vineyards, we turn to hidden Markov model (HMM) based alignment methods.et al.  . We discard the expression profiles not differentially expressed in time at the 0.001 % significance level using LIMMA , as wellW1:19 and C1:17 (indexed by time) for the Willunga and Clare vineyards respectively. The alignment is obtained based on the assumption that both emission sequences arise from a single state sequence S1:19. The time points for the Willunga sequence W1:19 correspond directly to those of the common state sequence S1:19, while the time points for the Clare sequence C1:17 are obtained via ‘gap positions’ 1<g1<g2≤19. In our approach the gap positions are treated as parameters of the model to be estimated from the data.We present our alignment methodology based on an HMM for the scaled time course gene expression grapevine data. The conditional independence graph of the alignment model is given in Fig. For a single pair of expression profiles, there is usually insufficient information to identify optimal gap positions. However, since the grapevine data have been scaled so that all observed expression levels lie in the interval , the Markov state space and conditional emission distributions can be considered common for all genes. This allows us to estimate a single set of gap positions by pooling the data from all pairs of profiles.S1:19 that form the Markov chain component of the alignment HMM are discrete valued and take values in a common state space ΩS={1,2,…,N}. For convenience we use p(x) to symbolise both a probability density function and a probability mass function, in addition to using the event ‘ X=x’ as an argument.The state random variables a=T be the N×1 vector of initial state probabilities and A={aij} be the N×N state transition matrix of the Markov chain state sequence whereLet i=1,2,…,N andfor i,j=1,2,…,N.for Let wherej=1,2,…,N.for kth gene, we consider an underlying state sequence,L expression profiles for each gene as the emission sequencesTl≤T for l=1,2,…,L. The alignment of the lth expression sequence to the underlying common state sequence is defined by valuesIn the general case for the Taking the set of HMM parameters to beand the set of alignments to beℓ with respect to the HMM parameters λ and the alignment points τ. The model underlying this likelihood allows each gene its own unique state sequence but imposes a common alignment over all genes.the general alignment HMM log-likelihood can be written asK=8644, L=2 and T=19. Taking w and c to represent the expression data from Willunga and Clare respectively , we have T1=19, T2=17 andg1<g2 in the sequence 2,3…,19. That is,τ1,1=τ2,1=1). The log-likelihood of the alignment model for the grapevine data is thenFor the grapevine data, g1 and g2 are given. Note that our alignment HMM is a special case of a hidden semi-Markov model [There are well established methods for efficient calculation of the likelihood, finding the Viterbi paths and estimating the model parameters for a standard HMM . These mov model .ℓ with respect to the HMM parameters λ and the gap positions g1 and g2. A profile likelihood approach could be implemented by applying the Baum-Welch algorithm [g1,g2) and then maximising the profile likelihood g1 and g2.We fit the alignment HMM to the grapevine data by maximising the log-likelihood lgorithm  to obtaig1,g2) and the maximum likelihood estimates are selected from the enumeration. The estimate λ apply to both vineyards. Such a model is implied by  has a negative linear relationship to log-likelihood =10, the true positive rate is 85.3 % and the false positive rate is 21.9 %. This suggests that applying the same threshold is a potentially useful filter for the classification of developmentally controlled genes amongst a set of genes of unknown function.The left side of Fig. et al. [Grimplet et al.  surveyedet al. [H(k)≤ 10. That is, 1279 genes with no current annotated function are well aligned between the Willunga and Clare vineyards and therefore we now have additional information that these genes are likely to be controlled in a developmental manner Additional file By comparing the expression of genes under different growth conditions, as has been done in this paper, we are able to gain evidence regarding the reproducibility of gene expression patterns indicative of a role in development as opposed to a response to external signals. This information can be used as additional evidence in the further investigation of gene function. For example, using the annotation of Grimplet et al. , of the The proposed alignment method could be extended and refined in a number of ways. In particular, potential improvements may be obtained through more detailed modelling of the emission distributions in the HMM. In the present paper, we have applied Gaussian emission distributions to the expression profiles averaged over replicates within vineyards. This approach could be refined by considering the replicates as multivariate observations instead of averaging and also by considering alternatives to the Gaussian emission assumption. Autoregressive emissions as well as higher-order Markov components of HMMs have been investigated and found to improve performance in the identification of over-expressed genes . The incWe have presented a novel alignment method based on an HMM and demonstrated the alignment on the grapevine data. This is a model suitable for sparse time course data where interpolation is not appropriate. The estimated model parameters have simple interpretations and the estimated gap positions are well determined for the grapevine data. We have demonstrated that the estimates of the HMM parameters as well as the gap positions are robust against subsets of profiles that are not suitable for alignment. For pairs of expression profiles that are well aligned, the Viterbi paths or average profile representations can be used as the input to downstream analysis of the data. This allows for an integrated analysis of multiple site time course gene expression data such as the Willunga and Clare grapevine data.We have demonstrated the use of the Hamming distance and the log-likelihood as a measure of quality for the alignment of a pair of expression profiles. Pairs of profiles that are well aligned will have high log-likelihood and a small Hamming distance while the poorly aligned pairs will have low log-likelihood and a large Hamming distance. We have also shown, for a set of genes with known function, that classification of genes according to the Hamming distance has reasonable predictive power for the classification of developmentally driven genes. This both validates that the alignment we obtain is meaningful and also suggests the potential for helping to identify the role of genes with unknown function.The MATLAB code and grapevine data to obtain all of the output described in this paper are provided as Additional files. The raw gene expression data is stored at NCBI in the GEO database as GSE7677 (Willunga) and GSE8445 (Clare) Additional file


Cataphoric dependencies where a pronoun precedes its antecedent appear to call on different mechanisms in language comprehension from forward dependencies where the antecedent precedes the pronoun. Previous research has shown that the resolution of cataphoric dependencies involves predictive processes such as the active search mechanism, which hypothesizes the automatic search for an antecedent immediately after encountering a cataphoric pronoun. The current study employs gender mismatch to investigate whether the active search for an antecedent of a cataphoric pronoun is restricted only to grammatically licit positions. We present results from an event-related potential experiment on the reading comprehension of cataphoric dependencies in Dutch. Results show that gender mismatch gives rise to an anterior negativity at grammatically licit antecedent positions only. We hypothesize that this negativity reflects the prediction failure for an antecedent after encountering a pronoun, rather than a gender mismatch. We discuss the timing, topography and functionality of this negativity with respect to previous studies and how this relates to the ERPs elicited in the processing of structural constraints on pronoun resolution. The on-line interpretation of pronominal dependencies has raised several questions within theories of sentence comprehension. Forward pronominal dependencies – where the antecedent precedes the pronoun – and backward pronominal dependencies – where the pronoun precedes the antecedent – appear to call on different mechanisms in language comprehension. In the case of forward dependencies, their resolution requires retrieving the information about the antecedent at the position of the pronoun, which is closely connected with memory-retrieval processes . On the wh-gap dependencies (One of such predictive processes is the active search mechanism (ASM), found initially for the interpretation of ndencies . In the ndencies . This pandencies . We posti indicates that ihe and iPeter refer to the same person.Cataphoric pronouns are pronouns that occur linearly before their antecedent. In other words, they are instances of referential dependencies in which the antecedent follows the referentially dependent element, as illustrated in (1). The index (1)ihe had a broken arm, iPeter could not ride his bike. While he in (1) pose an interesting case for parsing theories. In order to resolve the interpretation of the pronoun with an antecedent in the same sentence, the parser needs to wait until the appearance of the antecedent. When the antecedent is found, the pronoun can establish a link with it for its own interpretation. However, this is only possible when the grammar allows the link between the cataphoric pronoun and the antecedent to be established. Consider the pronoun he in (2) and the pronoun his in (3). In contrast to the pronoun in (1), the pronoun in (2) cannot take the proper name Peter as its antecedent (as indicated by the starred index of j – he cannot have the same index/reference as Peter). However, Peter can be the antecedent of the pronoun his in (3).Pronouns such as (2)Hei/∗j said that Peterj is sick.(3)iHis brother said that iPeter is sick.himself vs. him), while Principle C restricts the distribution of Referential Expressions, including proper names such as Peter.The restriction of the pronominal reference in (1), (2), and (3) can be captured under the principles of the Binding Theory  that indbound (he in (1) does not bind the referential expression Peter, because the pronoun is embedded in an adverbial clause that does not contain Peter. Given that he does not bind Peter, the two can have the same reference. On the other hand, the pronoun he in (2) binds the referential expression Peter structurally and in such a case, coreference is excluded. His in (3) on the other hand is more deeply embedded in the structure , and therefore, it does not act as a binder of Peter. Thus, similar to (1), a cataphoric dependency can be established in (3). Referential expressions, such as John or the man, independently refer and select a referent from the domain of discourse. Given that Referential Expressions have independent reference, they do not need and in fact cannot tolerate a binder. The binder would act as an antecedent for the Referential Expression, which is in conflict with the referential status of the latter.We focus on Principle C, which prohibits a Referential Expression  from being bound . The proIn this study, we investigate whether Principle C of the Binding Theory is respected in cataphoric pronoun processing. As illustrated in (1), (2), and (3), whether a referential expression can be a potential antecedent for a cataphoric pronoun depends on the structural configuration. If a coreferential relation is established between a referential expression and a cataphoric pronoun and as a result, the referential expression is bound by the pronoun, Principle C of the Binding Theory would be violated. This paper examines how this type of violation affects parsing. In particular, it uses gender mismatch to investigate whether a search for an antecedent is restricted by structural constraints. Given that the parser respects structural constraints such as Principles B and C of the Binding Theory when interpreting pronouns on-line as shown by behavioral studies that have examined reading times e.g., , we expewh-dependencies, which demonstrate that (a) a search for a gap starts as soon as a wh-phrase is processed and (b) filling the gap position where the wh-word could be interpreted with an overt element (thus blocking the parser from interpreting the wh-phrase in that position) results in a longer processing time compared to a sentence where no wh-dependency was initiated No constraint/Match a.     iHis managers chatted amiably with some fans while ithe talented, young quarterback signed autographs for the kids, but Carol wished the children’s charity event would end soon so she could go home.    b. No constraint/Mismatch    iHer managers chatted amiably with some fans while the talented, young quarterback signed autographs for the kids, but iCarol wished the children’s charity event would end soon so she could go home.    c. Principle C/Match    iHe chatted amiably with some fans while the talented, young quarterback signed autographs for the kids, but iSteve wished the children’s charity event would end soon so he could go home.    d. Principle C/Mismatch    iShe chatted amiably with some fans while the talented, young quarterback signed autographs for the kids, but iCarol wished the children’s charity event would end soon so she could go home.his, being further embedded in the nominal structure, does not bind the referential expression young quarterback, allowing it to be a potential antecedent. In other words, in (4a), Principle C does not block the coreference relation between the pronoun his and the referential expression (the antecedent young quarterback), and these two elements match in gender. Therefore the cataphoric pronoun should be interpreted at the antecedent position. The no constraint mismatch condition in (4b) differs from the no constraint match condition in (4a), in that the gender of the pronoun her and that of the potential antecedent young quarterback do not match, creating a GMME. In the Principle C match condition in (4c), on the other hand, the pronoun he binds the referential expression young quarterback in the embedded clause. Thus, young quarterback is excluded as a potential antecedent of he due to a Principle C violation. Furthermore, both the pronoun he and the referential expression young quarterback match in gender, as both are masculine. Finally, in the Principle C mismatch condition in (4d) the pronoun she, binds the referential expression young quarterback in the embedded clause, just like in (4c); however, in this case, they mismatch in gender. Importantly, the GMME is expected to be absent in the Principle C mismatch condition (condition 4d) at the position of the referential expression young quarterback, relative to the Principle C match condition (4c), as the coreference relation is barred from being established due to Principle C, preventing the GMME to occur. Conversely, the GMME is expected to be present at the referential expression young quarterback position in the no constraint mismatch condition (4b), relative to the no constraint match condition (4a). The main findings of quarterback), whereas this reading time difference was absent at the referential expression in the Principle C conditions in (4c) and (4d). Furthermore, In the no constraint match condition in (4a), the possessive pronoun his/her can corefer with the referential expression Jeffrey Stewart , but in (5c) and (5d) coreference is not licit due to Principle C of the Binding Theory.(5)a. No Constraint/Match    iHis managers revealed that the studio that notified Jeffrey Stewarti about the new film selected a novel for the script, but Annie did not seem to be interested in this information.    b. No constraint/Mismatch    iHer managers revealed that the studio that notified Jeffrey Stewart about the new film selected a novel for the script, but Anniei did not seem to be interested in this information.    c. Principle C/Match    iHe revealed that the studio that notified Jeffrey Stewart about the new film selected a novel for the script, but Andyi did not know which one.    d. Principle C/Mismatch    iShe revealed that the studio that notified Jeffrey Stewart about the new film selected a novel for the script, but iAnnie did not know which one.Jeffrey Stewart  in (5b) relative to (5a), where the pronoun and the antecedent could corefer (the coreference does not violate Principle C). Moreover, the GMME or reading time difference occurs despite the fact that the potential antecedent is contained within a relative clause island. The GMME generated in the no constraint conditions (5a) and (5b) in the self-paced reading experiment by A GMME or reading slowdown is found at the antecedent position The current study aims to replicate the GMME results from previous studies ; a.o. usSince the current study examines gender agreement mismatches at the antecedent position in cataphoric configurations, a brief overview of the ERP studies that have tackled gender agreement issues is in order. Gender agreement mismatches have been examined in the ERP literature using different paradigms. In a different set of studies, gender agreement violations between a determiner and a noun, or between an adjective and a noun, showed a left anterior negativity (LAN) followed by a P600 at the noun position for Spanish, Italian, and German ; a.o., ahe that mismatched in gender with the previously encountered feminine antecedent the aunt. Note that coreference between he and the aunt is only blocked by the gender mismatch and not by the Binding Conditions, as pronouns, contrary to referential expressions, may be bound by their antecedent if the antecedent is located in a different clause (cf. Principle B of the binding theory).Finally, in a third set of studies, gender violations were tested in forward pronoun resolution dependencies, i.e., dependencies in which antecedents occur before pronouns. (6)The aunt heard that she/he had won the lottery.Similarly, studies that tested gender violations in comparable forward pronoun configurations in Dutch  and ChinTaking into consideration the results in these studies that have manipulated gender agreement, it is clear that a P600 component emerges constantly, regardless of whether the relation is one between (1) a determiner and a noun; (2) an adjective and a noun; or (3) an antecedent and a pronoun. While the P600 is preceded by a LAN or by a N400 in some cases in pure pronoun resolution cases more akin to the manipulation in the current study, only a P600 is obtained at the position of the gender-mismatched pronoun.cataphoric configuration, where the pronoun linearly precedes the antecedent. To summarize, the aim of this study is threefold. (i) First is to examine whether there is a GMME when the parser encounters the first potential antecedent of the cataphoric pronoun that does not match in gender. This would be an indication that the parser starts actively searching for a matching antecedent after encountering the cataphoric pronoun, even though the antecedent of the pronoun could, in principle, be found outside of the sentence. We predict the GMME to be present in the case of a mismatch, and absent in the matching condition. (ii) Second, we examine if the search mechanism is modulated by grammatical constraints such as Principle C of the Binding Theory. For cases where co-reference may lead to Principle C violations, we predict no difference between the match and the mismatch conditions. We predict that an ERP component is elicited only for referential expressions that can legitimately establish a co-reference relation with the cataphoric pronoun. (iii) Third, we examine if cataphoric pronoun dependencies generate the same kind of ERP components as forward pronoun dependencies. As discussed above, previous studies  The GMME effect has been quite reliable in the behavioral literature. Thus, we expect the size effect of the gender mismatch to be robust; (b) we would like to avoid reading fatigue as well as participant developing different processing strategies derived from the high number of proper names included in the items. Note that previous studies, which investigated the processing of coreference involving repeated nouns with the ERP technique, used a higher number of trials per condition for their experiments . Second, Principle C conditions, which contain a cataphoric nominative pronoun in masculine (7c) or feminine form (7d) that cannot co-refer with the referential expression Lodewijk in the embedded clause due to Principle C.We followed closely the set-up of the English word-by-word self-paced reading experiment by Mirjam in (7b) and (7d), and Thomas in (7c)]. Relevantly, even if pronouns could have co-reference with an antecedent outside of the sentence, the availability of an antecedent in the same sentence  guarantees that the pronoun-antecedent relation is resolved within the sentence. Feminine and masculine pronouns and referential expressions were counter-balanced. Previous reading time studies found effects at positions immediately following the antecedent (see Lodewijk Boer) in our data to ensure that there could be a region immediately following the proper name that was still connected to the antecedent position. However, considering the superior time accuracy of the ERP technique, our prediction was that the effect should be observable at the target position rather than at immediately following regions. Participants read 36 target stimuli such as those in  randomly interspersed with 35 unrelated fillers that were part of a different experiment that examined the processing of backward negative polarity item dependencies  and in Principle C conditions in (7c) and (7d), the pronouns corefer with an antecedent that appears toward the end of each sentence , and Position ; and two to examine effects between conditions: Constraint (No Constraint/Principle C), and Match (Match/Mismatch). Mean voltage-amplitude was considered as the dependent variable in the analysis, and p-values where corrected for sphericity where required.Previous studies that have examined gender mismatches consistently reported a P600 component. In order to evaluate the presence of a P600 in our experimental data, the 500–700ms time window was tested by means of a 4-way repeated-measure ANOVA, considering four within-subject factors. Two to evaluate the signal scalp distribution: M = 84.59%, SD = 5.44%). The accuracy scores were similar across conditions . The difference in mean values was not significant as shown by a 2 × 2 repeated-measures ANOVA randomized by subjects with Constraint and Match as independent factors and Response Accuracy as dependent variable .Average accuracy rates were high and no participants were rejected on the basis of accuracy  did not result in any significant main effect or interaction , as shown in the right most column of Table 1. However, visual comparison of the grand average time traces in the anterior electrodes for the No-Constraint Mismatch condition (7b) versus No-Constraint Matched (7a) condition shows an apparent sustained negativity in the 200–600 ms region . The anterior topography of the negativity can be observed in Figure 2. No such negativity is observed for Principle C Match/Mismatch conditions . The asymmetry observed in the No-Constraint with respect to the Principle C conditions supports the expectation of the experimental manipulation, therefore, an exploratory analysis was performed to investigate the reliability and nature of this apparent difference.We investigated ERPs at the subject position of the embedded clause, Constraint, Match, Hemisphere, and Position . Follow-up simple interaction analysis for each level of the Constraint factor reveals no significant interaction or main effect in Principle C conditions, while a significant 3-way interaction between Hemisphere × Match × Position is present in No-Constraint . A further breakdown of this interaction for every level of the Position condition shows a significant effect of Match factor at the Anterior sites , and no dependence on Hemisphere. The No-Constraint Mismatch condition (7b) waveform average amplitude is more negative than (7a) .An omnibus ANOVA performed in the complete 200–600 ms time window shows a significant 4-way interaction of Table 1 summarizes the omnibus ANOVAs and Table 2 provides the follow up simple interaction evaluation for those regions with significant interaction in the omnibus ANOVA. .The same analysis was repeated using sliding 200 ms long windows to localize the effect with respect to the onset time of the stimuli. Table 3 shows a summary of the main effects and post hoc pairwise comparisons observed in the two time windows  in the breakdown of the interactions observed in Table 2, which in all cases reflect a significant anterior negativity of the Mismatch condition for the No-Constraint case when compared with the matched counterpart.Finally, Table 1 should have a p-value lower than 0.05/4 = 0.0125. In addition, an individual 2 × 2 ANOVA – to verify the interaction of the Constraint and Match factors in the topographical regions of interest defined by the Position and Hemisphere factors considered in the above analysis – did not yield a significant interaction in neither of the time windows (p > 0.10). This result is very likely due to the low statistical power provided by the small number of electrodes in each region of interest, and the limited number of trials.However, the results of the exploratory analysis above present the multiple comparison problem (MCP). To limit the Family Wise Error Rate (FWER) to a 5% level, the individual comparisons reported in t-tests were performed on the difference wave of the Match and Mismatch conditions for both No-Constraint and Principle C factor levels. T-test included the original data and 2500 random within-subjects permutations. With this technique, we tested separately the null hypothesis that the Match and Mismatch position do not differ in the No-Constraint and Principle C conditions. The maximum cluster-level mass procedure in the No-Constraint Match versus Mismatch comparison returned a cluster at the central-frontal electrodes extending temporally from 300 to ∼420 ms with an alpha level p = 0.07 . In contrast, the procedure in the Principle C conditions did not reject the null hypothesis to any level of significance (p > 0.4).To address the problem of MCP and verify if the differences observed were reliable, the ERPs measured were analyzed with a repeated measures two-tailed cluster mass permutation test  using thMatch and Mismatch conditions in the No-Constraint cases only, with anterior topographic distribution over a window around 300–420 ms. The observed difference is both in the direction expected based on the theoretical predictions, and with a coherent spatial and temporal localization. This reinforces that the effect is reliable even with the aforementioned reduced confidence level, compared to traditional 5% values. The presence of a positive result in an experiment with a relatively low power in terms of the number of trials observed per subject and condition  suggests that the effect size is large and would be more prominent with an increased number of items , there is no need to search for a pronoun after encountering the antecedent  since this referential expression can be independently interpreted. In other words, we do not expect an active search for a pronoun in the case of forward dependencies. The P600 component in these cases, therefore, must correspond to a gender mismatch between the referential expression and the pronoun.In the current experiment, we focus on the relation between a Lodewijk in (7b) after encountering the feminine pronoun haar. We hypothesize that, in forward dependencies, the parser needs to retrieve the gender of the antecedent from memory and check for gender matching. The P600 could be a reflection of the gender mismatch alone. Conversely, in backward dependencies, the parser anticipates the appearance of an antecedent in the upcoming sentence as soon as it processes the pronoun. Thus, when the parser encounters the first potential antecedent position, it expects to find a matched antecedent. When it fails, there is a negativity generated instead of a P600 because the failure of finding a matching antecedent prevails over the GMME. With this claim we do not intend to imply that the gender mismatch does not occur at all or that it does not precede the expectation failure (since the failure of the prediction cannot occur before the mismatch is detected) rather that the failure of finding a matching antecedent veils the presence of a P600.In backward, cataphoric pronoun-antecedent dependencies, on the other hand, the processes underlying the generation and interpretation of these dependencies are different since the interpretation of the pronoun needs to be resolved. It is therefore reasonable to hypothesize that the parser prefers to start a search as soon as a pronoun is encountered. The anterior negativity in our experiment could be interpreted as related to the searching process itself, namely, a failure of a prediction and not so much to the gender mismatch. The GMME provides the evidence that the antecedent search is active in the no-constraint cases, but it might not be the primary reason for the generation of the anterior negativity. Nevertheless, after having examined previous literature on gender mismatches, we might still wonder why no P600 as well is generated for the gender mismatch at In the second experiment in One of the potential sources for the lack of P600 for the gender mismatch in our study might connect to issues that previous studies have discussed , such asPrevious studies that have elicited negativities have looked at agreement mismatches with personal pronouns and subject-verb agreement failures , at nounN = 4) who judged the sentence as grammatical (and thus considered that there was an antecedent outside the clause for the pronoun) showed a sustained negativity in frontal electrodes in the 500–800 ms. The referentially induced frontal negativity (Nref) elicited by All the negativities found in these studies reflect syntactic processes and in many cases they represent a response to syntactic violations. However, they do not always have the exact same scalp distribution or topography as the negativity in our study. The reporter who the senator harshly attacked admitted the error) in The reporter who harshly attacked the senator admitted the error), and in If we look at the studies with increased memory load, the sustained negativity in otro ‘another (MASC)’], which mismatches in gender with the antecedent camiseta [‘t-shirt (FEM)’] in cases of noun ellipsis in coordinated sentences. In their study, the gender mismatch results in an ungrammatical sentence  in our study where a second potential antecedent Mirjam can be used to resolve the interpretation of the pronoun haar) and the position in which the mismatch is detected is a determiner that allows nominal ellipsis within the second coordinated sentence. Both Among the ERP studies that have generated negativities, A sustained negativity (largest at anterior sites) has additionally been found in cases of referential ambiguity under the name of referentially induced frontal negativity Nref; , 2007, wIn short, we have argued that the anterior negativity in this study can be connected to negativities found in previous studies in that it involves (1) a gender mismatch; (2) a dependency that contains referential expressions in which coreference needs to be established, and (3) a dependency in which an expectation of the parser fails. Thus, even if the studies discussed thus far have looked at different phenomena, it seems that there are some common processes underlying all these negativities, such as building a referential dependency on-line and predicting a specific upcoming element in the sentence.In our ERP study on the processing of cataphoric pronoun dependencies in Dutch, we replicated earlier behavioral findings  supportiThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A micropositioning mechanism is described which utilises a single micromotor to radially position and then deploy a needle 1.5 mm outside the microrobot's body to deliver a 1 mL dose of medication to a targeted site. An analysis of the mechanics required to drive the holding mechanism is presented and an overview of microactuators and the state of the art in WCE is discussed. It is envisaged that this novel functionality will empower the next generation of WCE to help diagnose and treat pathologies of the GI tract.Wireless capsule endoscopy (WCE) enables the detection and diagnosis of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. However treatment of these pathologies can only be achieved through conventional means. This paper describes the next generation WCE with increased functionality to enable targeted drug delivery in the small intestinal tract. A prototype microrobot fabricated in Nylon 6 is presented which is capable of resisting peristaltic pressure through the deployment of an integrated holding mechanism and delivering targeted therapy. The holding action is achieved by extending an “anchor” spanning a 60.4 mm circumference, for an 11.0 mm diameter WCE. This function is achieved by a mechanism that occupies only 347.0 mm Inflammatory bowel disease (IBD) is a long-term condition of the GI tract. The most common forms of this inflammatory condition are Crohn's disease and ulcerative colitis which can affect the colon and small intestine. These autoimmune diseases can be treated with antibiotics; however diagnoses can be difficult and treatment can lead to side effects such as dysbacteriosis. An approach to diagnose conditions of IBD is wireless capsule endoscopy (WCE). WCE has become a valuable tool for the diagnosis of pathologies of the gastrointestinal (GI) tract . These sAn early example of a swallowable WCE is the M2A developed by Given Imaging Ltd. in 2000 , 4.WCE systems are generally restricted to diagnostic use as the availability of onboard space for surgical tools or medication limits the ability to treat pathologies such as ulcerative colitis . This inThe paper is organised as follows. Conventional WCE systems are passive medical devices which have been focused on imaging the GI tract; however for the next generation to be useful surgical tools they will require active mechanical parts to enable them to carry out tasks such as performing a biopsy or electrosurgery while resisting natural peristalsis.In order to overcome the limitations of conventional WCE researchers have been exploring methods of increasing a capsule's functionality such as methods of resisting natural peristalsis –8 or navμm diameter SMA wire which is housed in a trench that runs alongside the groove. The wire has been coiled to generate the large forces required to slide the clamper unit forwards to bring it into contact with the intestinal wall. The grasping unit comprises two arms made from SMA. The arms are normally in a closed position which is achieved by virtue of a flexure joint which biases the arms. The arms are activated by heating a 20.0 mm long by 50 μm diameter SMA wire which has been fixed into holes in the arms. The heating causes the wire to contract which opens the arms.An alternative clamping system for clamping to the wall of the GI tract for the purpose of long-term pH monitoring has been proposed by Menciassi et al. (2005) . The claCurrent WCE devices lack the ability to control their speed and direction relying on natural peristalsis to move them through the GI tract. A solution that overcomes this problem is the robotic legged locomotion device developed by Valdastri et al. (2009) . The 12-The procedure of inspecting the lining of the oesophagus using a flexible endoscope is routine; however it is also a very uncomfortable procedure for the patient. Tognarelli et al. (2009)  propose Increasing a capsule's functionality to include the means of delivering medication to a site of interest offers great advantages to patients. A commercial system developed by Phaeton Research attempts to deliver drugs to a specific location in the alimentary canal. The Enterion capsule  is adminThe InteliSite is a drug delivery system manufactured by Innovative Devices LLC . It openPhilips Research has developed IntelliCap , an inteThe telemetric capsule developed by Lambert et al. (1991)  has an iKarargyris and Koulaouzidis (2013)  propose It is challenging to diagnose pathologies of the small intestinal mucosa; however performing a biopsy can diagnose pathologies early such as in the diagnosis of small intestinal Crohn's disease . To thisPark et al. (2008)  have devKong et al. (2005)  have devA self-propelling endoscopic system with a miniature manipulator mounted at the front is proposed by Peirs et al. (2000)  for retrSMAs are increasingly being used by researchers as actuators to increase the functionality of WCEs as they can take advantage of their superelastic properties which allow large amounts of deformation to take place without permanently damaging the material.μm diameter SMA wires connected to a brass micropulley. The right-hand wire is wrapped clockwise around the pulley and the left-hand wire is wrapped anticlockwise around the pulley, with the ends of the wires fixed to the prismatic support. Applying a current of 360 mA and 10 V for a period of 4 s to the left-hand wire causes the wire to contract by 6% and rotate the pulley approximately 135 degrees. After a period of cooling the right-hand wire is activated bringing the leg back inside the prismatic support. The degree of leg rotation is dictated by the length of contraction of the 100.0 mm long SMA wire; however the geometry of the prismatic support limits the length of wire which can be used. This issue has been overcome by the addition of glass shafts mounted in the prismatic support which allow the wire to be wrapped around them without shortcutting the circuit. The SMA wire is 40.0 mm longer than required to fully open the leg as compensation for the resistance from the antagonist SMA wire during the rotation of the pulley is required.Gorini et al. (2006)  propose μm. The purpose of the clamps and hooks is to replicate the motion of insects such as the earthworm by mimicking its setae. The 180 μm diameter angled hooks are similar to those reported previously by Lee et al. (2004) [A two-way linear actuator mechanism driven by SMA springs is proposed by Kim et al. (2005)  for the . (2004) . The capTo perform a surgical task a degree of mechanical movement is required of the tools. For example, a needle must move forwards to pierce tissue or the jaw of a clamp must close to obtain a sample . The kinAn actuator solution for controlling a holding mechanism could be the use of a piezoelectric linear motor to drive the mechanism directly. These motors use the piezoelectric effect to drive the actuation. The piezoelectric effect is the change in a material's geometry due to an applied electric charge or a mechanical stress changing a solid's shape which will result in a proportional electrical charge . There a2) exhibit the piezoelectric effect and can be utilised in such applications as displacement transducers. The Squiggle piezoelectric linear motor manufactured by New Scale Technologies [Nonconductive materials such as quartz  of 0.5 N which makes it a good candidate for operating a holding mechanism.The Squiggle motor (SQL-RV-1.8-6-12) is one of the smallest piezoelectric motors in the world with a package size 6.0 mm long by 2.8 mm square and a leadscrew 12.0 mm long. The motor has a high positioning resolution of 0.5 An alternative to the piezoelectric Squiggle motor is a micromotor. Micromotors could potentially be used to drive the holding mechanisms directly. An examination of the latest available technology in micromotors shows that there are a limited number of micromotors on the market which could fit within the package size of the capsule and have a useful torque capability at a low RPM.Ø1.5 mm × 12.5 mm long geared micromotor manufactured by Namiki (number 10-010) [A micromotor which appears to have the required specifications is the  10-010)   [Ø1.9 mm × 10.82 mm long micromotor manufactured by Faulhaber (02/1) [The four-stage geared micromotor operates at 60 mW and runs at 76 RPM; this produces a stall torque of 1.6 mNm. The Namiki motor can be compared with the specifications of three alternative motors: a r (EC 6) , a Ø1.9 r (02/1) , and theThe Namiki micromotor can be evaluated for its ability to perform the task of moving a 0.5 g mass of stainless steel. The stainless steel would replicate the mass of the holding mechanism and represent 10% of the total weight of the microrobot.α is the angular acceleration and can be calculated at 265.3 rads/s2 from the motor's performance data and Io is the moment of inertia which can be expressed as mk2, where m is the mass concentrated at a radius, the Radius of Gyration (k), which would be 5.0 mm. This concentrated mass would be a worst case scenario as it assumes the total volume to be moved is positioned at the extreme limit of the microrobot.The torque required to move the mass of stainless steel can be calculated using the following equation:μNm would be required to operate the holding mechanism. Clearly the 1.6 mNm capacity of the Namiki micromotor would be sufficient to drive the mechanism; however consideration of losses due to the inherent system's friction would also be required.The results of the initial torque evaluation show that a torque of 3.32 SMAs can be used as an alternative to micromotors. The most commonly used SMA is superelastic nitinol which is an alloy of approximately 50% nickel and 50% titanium. Nitinol has superelastic properties which allow large amounts of deformation to take place without permanently damaging the lattice structure of the material. This allowable strain makes it possible to create super springs which can be triggered by the Joules effect. The disadvantages of using SMAs are that they require high power consumption and that they can affect their environment as a result of heating. Also they are unable to fully recover their original configuration after cooling due to twinning of the atomic crystal . This car) and the length (L) of wire required to rotate the pulley through an angle (θ) has been determined by Gorini et al. (2006) [SMA wire could be employed to drive a holding mechanism. The wire could be orientated in a pulley configuration minimising volume and attaining a mechanical advantage from the pulley. The approximate relationship between the pulley's radius (. (2006)  and is gr) of 0.9 mm and a required rotation (θ) of 90 degrees results in a theoretical length (L) of the wire being 35.3 mm. The actuator mechanism would require a longer wire to operate efficiently as the theoretical model does not take into account the friction in the system and the drop in performance of the wire due to multiple operations. Also the wire would need to overcome the resistance imposed by a second wire which would be required to return the pulley to its original configuration. A consideration of the difficulty in manufacturing and assembling such small and complex components would need to be taken into account when designing such a mechanism.The 0.04 represents a 4% contraction of the wire due to the Joules effect. Applying the formula to a pulley with a radius  [Ø11.0 mm × 32.0 mm long are capable of being swallowed by subjects aged 18 to 65 years. Therefore the increased volume will have little impact on the majority of patients being able to swallow the microrobot or on its ability to navigate the junctions of the small intestine such as the ileocolic valve. The detailed specifications for the microrobot's performance are outlined in Conventional WCEs have a volume of 2.0 cm. (2009)  have shoIn order to realise a WCE with added functionality such as the ability to resist natural peristalsis or targeted drug delivery it is important to consider how these mechanisms can be operated.Ø1.9 mm × 10.82 mm long allows the motor to be orientated perpendicular to the microrobot. The micromotor's novel configuration coupled with the bevel gear set allows rotation to be translated through 90 degrees and also a reduction in the micromotor's RPM while maximising the use of space (A single micromotor manufactured by Faulhaber (02/1)  has beenof space . The redDistending the GI tract wall in order to resist peristaltic contractions will result in forces being applied to the surface of the wall through the interaction of the legs with the wall. This interaction results in the possibility of the GI tract suffering from postoperative ileus. As a result of the GI tract being contacted the wall can cease to function. However postoperative ileus is usually associated with metabolic abnormalities or with the GI tract being handled such as in laparotomies or abdominal surgery and is not associated with endoscopy or with the use of WCE. The thin leg sections may also pose a potential risk of damaging the GI tract wall if external forces become too high. However a larger surface area could easily be integrated into the distal profile of the holding mechanism's legs to eliminate any potential trauma from operating the mechanism.The bevel gear set comprises a 13-toothed drive gear, connected to the micromotor, and a 48-toothed follower gear . The staThe gear train reduces the 1,538 RPM output from the micromotor to 8.4 RPM; this allows the holding mechanism to be fully deployed in approximately 1.8 s. Based on an average transit time through the small intestines of 23 mm/min  the capsThe overall geometry of the microrobot and the gear module, which is the ratio of the pitch diameter to the number of teeth on the gear, has a direct influence on the dimensions of the gears. For example, the 48-toothed bevel gear has a module of 0.2; this results in an overall diameter of 10.0 mm which represents the maximum diameter that could fit within the microrobot. A module of 0.1 has been selected for the gear train as this facilitates maximum speed reduction yet minimises the use of space. The design parameters for the complete gear train are specified in The 0.1 module chosen for the gear train results in a micrometre gear tooth profile . It is tFor the purposes of analysis the tooth can be modelled as a cantilever beam with an involute gear tooth profile. The contact angle between mating teeth is known as the pressure angle (Φ) and in this application it is set at an industry standard of 20 degrees for a spur gear.The stress figures determined by analysis are an important resource as they can be used to determine the material the gears are to be manufactured from and the manufacturing process.Ft is the load applied to the tooth, Kv is the velocity factor, bt is the face width, mn is the normal module, and y is the Lewis form factor.The bending stress for a spur gear tooth or a straight toothed bevel gear can be obtained by using the Lewis formula which has been modified to take into consideration the contact impact of the gears through the addition of a velocity factor:F) is ignored and also that the force is distributed evenly over the full face width of the tooth and that the stress concentration effect of the tooth fillet is also ignored.The assumptions made with the modified Lewis formula are that the full load is applied to a single tooth and the radial component force (Ft) acting at the circular pitchTf1 is the torque on the drive gear and d1 is the reference diameter of pinion and can be calculated by the number of teeth on the pinion (z) multiplied by the normal module (mn).To calculate the load  is a function of tooth shape and is independent of tooth size; it also does not take into consideration the stress raiser effect of the tooth fillet. It can be calculated as follows:z is the number of teeth on the gear.The Lewis form factor (mn) of a gear is the ratio of the pitch diameter to the number of teeth on the gear. The module has a direct relation to the geometry of the teeth and the overall diameter (OD); it can be calculated as follows:The normal module  is applied; therefore an FEA analysis has been performed to determine a more accurate level of stress distribution through the tooth  the FEA figures confirm that the 8 toothed gear must be manufactured from a metal rather than a polymer to ensure the teeth do not yield under load.The Von Mises FEA analysis assumes a worst case scenario; that is, at any one time only one pair of teeth are in contact with each other and they take the total load. Generally two pairs of teeth are in contact; however this may drop to 1.5 depending on the degree of tooth truncation and inaccuracies in tooth profile due to the manufacturing process. Increasing the value of the face width or increasing the module would reduce the stress in the tooth; however increasing the module would result in an increase in the overall diameter of each gear in the set and hence the overall size of the gear train would increase. Also increasing the tooth thickness will influence the overall length of the microrobot due to the stack-up of dimensions. However increasing the size of the components would make manufacturing generally easier.There are a number of process routes which can be used to produce the holding mechanism's gear train; for example, hobbing, rapid prototyping, and wire EDM are all methods which could be employed to produce the spur gear set which drives the legs in and out. However, feature geometry, material selection, and manufacturing cost dictated that CNC milling was chosen. Ø0.5 mm) to generate the tooth profiles and to achieve the tight root radii of the teeth. However the use of Nylon 6, which was selected for its mechanical properties, resulted in the gears having significant burrs owing to the manufacturing process. The burs can clearly be seen on the 8-tooth spur gear  suggests that the gear train would not run smoothly due to the reduced clearance; however on the larger gears the average thickness of the teeth was undersized (0.71 mm). This compensated for the thicker teeth and, once the burrs had been removed, resulted in a smooth running gear train. The slightly larger sized teeth on the smaller gears are an advantage in this instance as they can withstand a greater bending stress during operation.The prototype holding mechanism comprises the micromotor, gear train (bevel gears and spur gears), and two legs connected to the gear train which drive the tie bars and centre support in and out . The legDelivering medication to a target site of interest in the GI tract requires an ability to overcome peristalsis. A prototype microrobot with an integrated holding mechanism has been presented which achieves this goal through the deployment of an expandable set of legs that are driven by a micromotor and a novel gearbox configuration. The holding mechanism has been designed for ease of manufacture and also for ease of assembly. However there were a number of issues with individual component parts and with the assembly of the parts which required addressing before a working prototype could be realised. For example, the material and manufacturing process chosen to prototype the gears resulted in gears with significant burrs which required removal before the gear train could be operated. Changing the gear material to a stainless steel would overcome this issue and also open up alternative manufacturing methods such as hobbing or wire EDM.Manually operating the gearbox resulted in a smooth, free-running mechanism that took three and a half turns to fully expand the holding mechanism and three and a half turns to collapse it. However the calculated number of turns required to operate the mechanism is 2.6 turns. The difference in these two figures can be attributed to the backlash in the gear train which predominantly comprises the clearance between the gears, required for free running, and also the clearance between the gears' driveshaft and the housing. Reducing the backlash in future prototypes can be achieved through closer control of the manufacturing method chosen to produce the parts; for example, CNC milling and CNC turning could be used to produce the gears and gearbox. By applying tighter control over the dimensioning of the features the backlash in the gear train could be reduced. However the reduction in backlash would result in an increase in the manufacturing cost of the parts.Producing prototypes which meet intended requirements will generally involve a compromise between part accuracy, surface finish, size, and cost. The selected manufacturing method will satisfy the majority of requirements; however the designer will ultimately decide which parameters are the most important and therefore which can be compromised.In this paper we have presented the first holding mechanism of its kind for the purpose of resisting natural peristalsis in the GI tract. Exploitation of microactuators and conventional manufacturing techniques resulted in a holding mechanism which has been integrated into a WCE, occupying just 9% of the total available volume. The outcome of prototyping spur gears manufactured from Nylon 6 has highlighted the limitations with conventional CNC milling and material selection. This shows how adapting manufacturing methods to meet intended requirements can allow for radical changes in the capabilities of WCE systems in the future.