To improve health-care delivery, care providers must base their services on community health needs and create a seamless continuum of care in which these needs can be met. Though, it is not obvious that providers apply this vision. Experiments with regulated competition in the health systems of many industrialized countries trigger providers to optimize individual organizational goals rather than improve population health from a community perspective. Thus, a tension exists between the need to collaborate and the need to compete. Despite or because of this tension, community health partnerships are being promoted, and this should enforce a needs-based and integrated care delivery.In this single case study, we retrospectively explored how local health-care providers in Amsterdam collaborated for more than 30 years, interacting with the changes to the national health-care system. In-depth analysis of interviews, documents and literature focused on the complex relationship between the activities of this health partnership, its nature and its changing context.The findings revealed that the partnership itself was successful and sustainable over time, although the partnership lost its initial broad explorative nature and narrowed its strategic focus towards care of the elderly. Furthermore, the realized projects – although they enforced integrated care – lost their community-based character. This declining scope of community-based integrated care seems to have been influenced by the incremental introduction of regulated competition in Dutch health care. This casts doubts on the ability of health partnerships to apply a vision of community-based integrated care within the context of competition.Collaborating health-care providers can build seamless continuums of care in a competitive environment, although these will not automatically maximize community health with limited resources. Active policies with regard to health system design, incentive structures and population-based performance measures are warranted in order to insure that community-based integrated care through health partnerships will be more than just policy rhetoric. To improve performance in health care, providers should target their services to the health needs, beliefs and values of the populations they are intended to serve. This orientation enables them to offer an appropriate set of services that maximizes population health given the available resources -3. AccorIn practice, it is not self-evident that health-care providers will join collaborative programmes aimed at improving community health within the context of limited resources. These programmes may imply a loss of power, control or income for individual providers, because they realign health-care delivery in favour of more preventive, social and primary care services . NotwithDespite or perhaps because of this tension, health partnerships are being developed and promoted in various countries such as the United Kingdom ,15, the However, there is limited empirical evidence that health partnerships can be effective in this way -24. MoreIn this paper, we present a single case study in which the aforementioned topic has been addressed. We studied how health-care providers in 'South-eastern Amsterdam' (Amsterdam Zuidoost) have been collaborating for more than 30 years. The partnership known as the SGZ  and later the Zizo  was founded in 1973 with the aim of shaping the local health system in this urban district, which at that time had just been built. Its initial activities came out of a vision of 'community-based integrated care' ,28. SincWe addressed the following research questions: 1) Has there been a community-based integrated care vision to the partnership's activities throughout the period of more than 30 years? 2) How did the nature of the partnership change over this period? 3) How did the partnership balance its nature and activities with the changing context over this period? In the discussion, we elaborate on the findings and draw lessons for adapting and sustaining a community-based integrated care vision in local health systems through health partnerships.A single case study design was considered the most appropriate to retrospectively explore the development of the partnership in South-eastern Amsterdam over time. Semi-structured interviews and document analysis were used to describe the nature of the partnership and its collaborative activities. Literature was reviewed to describe the transformation of the Dutch health policy context and the South-eastern Amsterdam community.In 2003, we interviewed 17 key players selected on the basis of two sampling strategies  projects be initiated and implemented based on prioritized community health needs; 2) projects be responsive to the beliefs, preferences and societal values of residents; and 3) projects be aimed at creating a seamless continuum of care – the implementation of mechanisms that would facilitate and coordinate service delivery at the right time and in the most appropriate setting . In thisTo answer the second question, we analysed documents and transcripts using the Janssen typology . This tyTo address the third question, we used an open approach to explore the balancing process between the partnership and its context. The focal points were the crises underlying the formation and transformations of the partnership. We expected that balancing processes would be most visible during these periods. Because of this, we took a 'systems perspective', as the literature shows that factors that influence the effectiveness and sustainability of health partnerships are highly interdependent and cannot easily be isolated from each other .peer review), who critically appraised an earlier draft of the manuscript. Fourth, there were regular meetings of the co-authors throughout the entire study period. During these meetings, the data collection and analysis were monitored and discussed.We used several strategies to monitor and enhance the rigour of the data as well as to rule out validity threats. First, we tried to cross-validate key findings by triangulation of data. The data collected from different sources  were simultaneously analysed and reported. Second, we presented our preliminary findings to most of the respondents during a partnership meeting in December 2003. Furthermore, we sent Respondents 1 and 7 a draft of our manuscript and asked them to check the analysis and interpretations. Respondent 1 is the only one who has been involved with the partnership throughout the entire time period; Respondent 7 is the current chair and our principal contact. We used both ways of checking our analysis to validate the findings. Third, we solicited feedback from senior and other researchers , as strategic functions such as planning services, innovation, regional needs assessments, care commissioning, resource allocation and information management were centralized at the partnership level. This was done because member institutions could not initiate, develop and realize the projects themselves. Realizing successful projects required influence on national and local health policy-making for which joint strategic action was needed. In the later projects (9–12), strategic functions were more decentralized. Member institutions initiated and developed innovative projects themselves or in small coalitions. Projects were put on the agenda and discussed, but primarily to inform member institutions.The integration at the operational level in the projects was initially modest, but later increased and stabilized. In the first projects (1–4), operational activities were predominately focused on creating the prerequisites for integrated health-care delivery. For example, the partnership arranged accommodation for the centres and set up meetings for exchanging experiences among professionals on how to set up a primary care centre [d]. However, the partnership was not engaged in standardizing the processes in terms of responsibilities, protocols, information transfer, monitoring and feedback mechanisms. Nor did it have a role in the actual health-care delivery to individual patients.This changed in the later projects (5–11), in which the partnership executed various operational activities. For example, in the stroke service project (8), the partnership's employees chaired weekly steering committee meetings, assessed health-care needs, commissioned health care or mediated admission to an institution, monitored waiting lists in the participating institutions, registered all relevant information including transfers in an integrated patient record, communicated with patients and their carers, and could be consulted for advice . These operational activities could also be identified in the other projects except for project 12 [x].Based on differences in the typology of the partnership over time, three time periods were identified: the SGZ period (1973–1989), the Zizo I period (1989–1997), and the Zizo II period (1997-present). In the SGZ period, the focus of the partnership was on service development – that is, building a community-based integrated care system with its emphasis on primary care . The governance of the partnership was focused on serving the collective interests of the participating organizations, which essentially implied the realization of primary care centres in the area. An example of this are the efforts to realize a primary care centre in a neighbourhood where there were already individual general practitioners (GPs). The partnership firmly supported this initiative, thereby ignoring the opposition of some GPs with individual practices who were also participating in the partnership [d]. The structure was flexible, informal and with no hierarchies. It featured an executive committee supported by a small self-supporting executive office that supervised, managed, facilitated and coordinated the partnership's activities . There wIn the Zizo I period (1989–1997) the partnership focused both on service development and improving the existing services. Innovations featured new ways of integrated care delivery (project 5) as well as streamlining existing care processes – that is, coordinating and aligning the input of all professionals and institutions involved (projects 6–8). Governance concentrated on balancing the collective interests with the individual interests of participants. As formulated in the minutes of committee discussions in 1990, transparency and clarity were considered essential to nurturing fruitful negotiations, compromises, and experiments [j]. In this regard, respondents  also referred to a common remark used to express the unwritten rule of transparency:"'Well understood own interests" was a comment we often used.' (Respondent 4)The structure of the partnership was more formal and binding. The partnership's organization featured a two-person management team (i.e. a director and a secretary) and an executive office known as 'circuit management' that coordinated, executed and supported the collaborative activities. This organization was paid for in part by the participants themselves. The strategic fit changed as the public health office, community organizations and financiers  withdrew from the partnership (see Table In the Zizo II period (1997-present) the focus was on innovative activities, which mostly elaborated on the expertise of the circuit managers and the existing infrastructure. This is illustrated by the most recent projects (9–12), which build on the expertise acquired during the earlier projects. Documents  and respondents  substantiated this finding. Governance concentrated on seeking coalitions based on shared interests rather than on collective interests. This was explicitly stated: 'New projects are initiated by two or more health-care providers. One of these initiators is now responsible, not the Zizo.' [p]. Even so, the partnership culture was less dominant; each representative propagated the interests of their own organization rather than the collective ones.'You are allowed to have your own interests. You don't have to hide them.' (Respondent 8)The partnership structure was downsized by removing the management team. The circuit management (staffed by the three circuit managers) was maintained. Furthermore, an additional type of meeting was organized. What were known as 'health-care provider meetings' were held occasionally to prevent individual patients falling through the cracks . The partnership management was minimal, because the executive committee only prepared the meetings and supported the self-directed circuit managers. Finally, the strategic fit between the member institutions weakened, because the institutions for care of the elderly increasingly offered similar services.Figure The formation of the partnership in the 1970s went well. The health-care infrastructure in South-eastern Amsterdam had to be built from scratch, and this offered unique opportunities for experiments and innovation. At that time, the segregated health-care system and its uncontrolled expansion were considered problematic . This waIts ideas became influential through the support of stakeholders such as the municipality of Amsterdam, the sickness funds in Amsterdam and local community organizations. Moreover, in the early 1970s the SCAB attracted young health-care professionals, brought together in the 'Primary Health Care Working Group' (Werkgroep Eerstelijnszorg). Respondents  said that this group of people was willing to implement the SCAB's vision and work accordingly. The partnership became successful against this backdrop.'Yes, the partnership [during the first period] has been very successful. (...) There was an integrated vision. There were people who wanted to work according to this vision. There was the power to implement this. Even so, the circumstances were good: an easy geographical area and the support of financiers.' (Respondent 3)In the mid-1980s, the spirit dissipated for several reasons. First, respondents  noticed that the partnership's mission had been accomplished with the successful establishment of the primary care centres and the new ambulatory mental health-care organization. Second, the planned urban development turned out to be disastrous ,28. Due 'The financing of these types of organizations was stopped. (...) We said to each other – especially the former director of the AMC – that if we want to extend our planning work towards future issues in health care and maintain the collaboration, we have to fund the partnership ourselves.' (Respondent 1)The partnership continued at this crossroads, although it was radically transformed into a collaboration of health-care providers only. Visionary key players anticipated new opportunities for collaboration, as articulated in the concept of integrated care that was introduced at that time . They co'Everyone needed – everyone? no, not everyone – a great many needed that kind of binding structure, in which parties were a bit more committed to each other than in the SGZ. That was also the spirit of the times. If you want to organize a chain of care, you can't do this based on noncommittal agreements.' (Respondent 5)Political pressure was needed to keep the social care agencies in, as they were merging and reluctant to give away autonomy. For a similar reason, the home care agency timely suspended its membership. Representatives were discontented by the intensive home care project (project 5) that they considered to be their exclusive jurisdiction. The public health office and community organizations left for other reasons. The municipal health service Amsterdam simply left as a natural outgrowth of the withdraw of the municipality of Amsterdam. The community organizations were dropped out, because of their own declining vitality as well as the disinterest amongst the participating care providers.' [The dropout of community organizations] went too easy. We did not think it over very well. The idea was, we cannot ask them to pay a contribution and the new norm was: he who pays the piper dictates the tune.' (Respondent 1)Respondent 5 acknowledged this, but also underscored that there was no other option for the partnership. The core-business was to collaborate and set up integrated care arrangements among care providers. A systematic discussion with the community was not the primary aim anymore. Due to the dropout of all these organizations, the partnership got a health-care orientation with no link to public health and a weak one to social care.After the final decision to continue the partnership in 1989, the partnership was converted into an association funded by contributions by the participating institutions themselves. Soon, funding for project 5 was realized. Respondents  emphasized the enormous spin-off of this project, which was also substantiated by documents [j-l]. First, the regional sickness fund contracted the partnership to control the entire budget of intensive home care in South-eastern Amsterdam, thus giving the partnership a strategic position in the local health system and showing its usefulness. Second, in order to adequately run this project, the partnership had to set up and organize an administrative bureaucracy. This circuit management was staffed by professionals who soon became the experts on integrated health-care arrangements at the operational level.'We started with the intensive home care project, which in my view has been an important stimulus. (...) If you can show your member organizations it's profitable to collaborate on successful projects, then you start a motor that encourages collaboration in the broadest sense, also at the strategic level.' (Respondent 4)'It gave us a tremendous opportunity, but our organization was totally unprepared. It meant that you had to control financial flows, that you needed to have an administrative system, and that you needed to have people to do the job.' (Respondent 5)The expansion resulted in a partnership that had the expertise, the capacity and the support to initiate, develop and realize innovative and successful projects.In the mid-1990s new tensions arose. The incremental shift towards regulated competition resulted in defensive organizational behaviour. To reduce uncertainty, various member institutions merged , becoming part of large enterprises. Furthermore, one ad hoc intervention by government torpedoed core project 7. Legislation imposed the installation of 'Regional Individual Needs Assessment Agencies' (RIOs), which had to be organized independently of health-care providers . Lastly,These developments brought about a new crisis. First, the partnership's organization had to be dismantled. The staff that had been doing health-care needs assessments had to leave and/or work for the independent RIO in Amsterdam known as 'Tot & Met'. Because the operational activities were funded primarily on the basis of this project, the partnership ran out of financial resources. First and foremost, the crisis triggered a discussion on sustaining the partnership. The merged institutions had an orientation that exceeded the local health system, and they strategically aimed to offer seamless continuums of care services within their own institutional borders. Still, they remained committed to the partnership. By chance, all four merged institutions active in the entire Amsterdam area were represented in South-eastern Amsterdam, which inhibited the dominant market position of one of them (Respondent 15). Apart from that, they highly valued the work of the circuit managers, whose expertise and experience should be maintained and used for future innovations [k].'The situation of the partnership was quite desperate. Why do we still have a ZIZO? (...) I thought, if we want to maintain the partnership, we have to keep the circuit managers operational.' (Respondent 6)This ultimately resulted in a new vision that promoted the improvement of existing collaborative activities and capabilities in the domain of care for the elderly and the chronically ill .Several European governments promote health-care partnerships as appropriate vehicles for dealing with the tension between collaboration and competition ,15,20. TThe study presented in this paper challenges this view. However, due to the explorative and retrospective nature of our study, the data sources used and its contextual imperative, the transferability of the findings to other settings is limited. Still, we believe the findings have a broader meaning. First, validity threats were ruled out by the comprehensive data gathered and methodological approach followed. So, the study has provided a credible exploration of the development of a health-care partnership in Amsterdam over more than 30 years. Moreover, the long time horizon of the study was favourable: 1) It gave us the opportunity to explore the outcomes of a health-care partnership over time, as manifested in the projects; and 2) We could track the influence of competitive elements on the nature and activities of a health-care partnership. Given the existing knowledge gap and the political rhetoric concerning health partnerships , the finFirst, the 30-year history of the South-eastern Amsterdam partnership shows that the partnership successfully realized 12 collaborative projects and was sustained by changing its strategic goals over time. Its mission shifted from shaping a community-based integrated health system with its emphasis on primary care, by way of substituting integrated care arrangements for hospital care, towards elaborating on successful integrated care arrangements in care for the elderly. However, with this shift the partnership's activities were no longer planned on the basis of prioritized community health needs, and the community links fell away. This finding casts doubt on health partnerships' ability to implement programmes on the basis of a community-based integrated care vision. It indicates that the link between population health and health service delivery cannot be made by a health partnership of competing health-care providers only. Governing bodies and financiers should encourage a population-health focus and ensure that partnerships not only do things right but also do the right things.This brings us to the second notion: External forces had an unmistakable impact on the partnership and its activities. Known contextual factors from the literature  were all evident -25. TheiConversely, the role of the sickness fund increased. It became the most crucial stakeholder of the partnership. At first, this was beneficial. Later, the governance of the sickness fund was more ambiguous. This had to do with the increased financial risks concerning quality-based purchasing and the financial risks taken by the major health-care insurer in Amsterdam. This relates to the international debate on incorporating population-health considerations in health system governance. Governments currently delegate governance roles to competing health-care providers and/or financiers. However, incentives that trigger the uptake of a population-health perspective are often ignored ,13. TherApart from influences of national policies and local governance practices, the development of the partnership was also the natural outgrowth of building a local health care system from the scratch. Lewis et al. (2000) argue that rearranging health care in favour of more preventive and social interventions rearranges resources and incomes, creates winners and losers in a finite world . At the Despite the confinement of the room for discretion by external forces, the partnership was able and flexible enough to adapt and to survive. There is a broad range of literature discussing the critical capabilities and competencies necessary for sustaining health-care partnerships. In this paper we do not pinpoint specific capabilities and competencies that were decisive in Amsterdam. Rather, from a more abstract level our analysis indicates that the major driving force underlying the sustainability of the partnership has been its formation in the first place. Once the partnership was institutionalized and operational, it kept on nurturing its own survival. The people involved in and employed by the partnership interchangeably celebrated the partnership itself or successful projects or both in order to survive. In these processes, known factors such as personal factors, leadership, trust and culture were indeed evident.Taking these three basic notions together suggests that encouraging health-care providers to collaborate and compete at the same time will not lead towards a needs-based and integrated health system. Seamless continuums of care will be realized, but these will not necessarily maximize community health. Governing bodies have to govern partnerships through active policies related to health system design, incentive structures and population-based performance measurement in order to ensure that community-based integrated care is more than just policy rhetoric.Through partnerships, health-care providers can build seamless continuums of care. However, these will not automatically maximize community health within a context of limited resources. Active policies with regard to health system design, incentive structures and population-based performance measures are warranted to ensure that community-based integrated care through health partnerships is more than just policy rhetoric.The author(s) declare they have no competing interests.TP designed the study, conducted semi-structured interviews, analysed the data and primarily wrote the paper.DMJD designed the entire study, reviewed earlier drafts of the paper and supervised the research activities.NPCH conducted semi-structured interviews, helped analyse the data and critically reviewed earlier drafts of the paper.NSK was responsible for completing and supervising the study, designed the entire study and critically reviewed earlier drafts of the paper.The pre-publication history for this paper can be accessed here:Topic list.Click here for file


Brochopleural fistula following lung resection is a therapeuric challenge for thoracic surgeons.We describe a case of late bronchopleural fistula after right extrapleural pneumonectomy for malignant mesothelioma. Bronchoscopic attempts to repair it were unsuccessful.The use of a modified Y Dumon stent associated with glue apposition on the bronchial stump allowed us to close the fistula without the need of any surgical repair. The appearance of a bronchopleural fistula is a well-known and frightening complication of pulmonary surgery. Different methods have been used to solve this problem conservatively, from bronchial gluing  to stentA 64-year old man was referred to our department for right recurrent pleural effusion. A CT scan showed thickened right parietal pleura with a minimal pleural effusion, but no pulmonary nodules or lymphadenopathies or distant metastases. Because of negative cytology malignancy, the patient underwent a thoracoscopic pleural biopsy on December 2004 that showed an epithelioid monophasic pleural mesothelioma, followed by talc pleurodesis at the end of the procedure.Three weeks later the patient underwent a right extrapleural pneumonectomy with coverage of the bronchial stump with a flap of thymic tissue. The pathological examination confirmed a monophasic epithelioid pleural mesothelioma, with lung and pericardial fat infiltration, but no invasion of the diaphragm or pericardium (pT3 N0 M0). The postoperative period was uneventful.Ten months later the patient complained of a fever and cough. The diagnosis of broncho-pleural fistula was made immediately after the first appearance of cough and fever by bronchoscopy that clearly showed a 2-mm fistula on the mediastinal side of the stump confirmed by the methilene blue passage from bronchus to the chest tube. Rapid fluid drainage and antibiotic therapy allowed us to cure the infection of the pleural space avoiding the formation of empyema. After chest tube drainage, the attempt to conservatively close the fistula using biological glue was unsuccessful. So we decided to use a modified Y Dumon stent  to exclude the right stump. The right arm of the Y was shortened and the open end was closed with a silicone material from which the stent was fabricated, and then inserted in the tracheobronchial tree trough a rigid bronchoscope. The tracheal part was 18 mm in width and 4 cm in length, while the left bronchial arm was 16 mm in width and 2 cm in length. Under rigid bronchoscopy, we first introduced the biological glue  all over the bronchial stump that was of 1 cm lenght, followed by the insertion of the modified Y Dumon stent  until he died 4 months later for pulmonary emboli. The post-mortem examination showed a massive pulmonary thrombus in the left main pulmonary artery which explain the sudden respiratory insufficiency and death at home and an initial relapse of mesothelioma in the posterior part of chest wall, without signs of infections.Biologic glue has been used to treat bronchopleural fistulas, and its efficacy has been demonstrated . When noUsually, it is more difficult to treat a fistula on a main bronchus, both for the presence of a single lung and the challenge of inserting the appropriate stent for the changed anatomy following a pneumonectomy. A self-expandable covered tracheobronchial stent could solve this problem for its ability to adequately fit the tracheal and bronchial size as well described in a previous report .The use of a modified Y Dumon stent has some other advantages. First, it can be removed when the patient does not tolerate a foreign body in the bronchial tree; second, it does not give frightening complications such as erosion of the mucosa, with the possibility of perforation and/or severe bleeding.The only points for careful consideration in this kind of stent are to adequately evaluate the size of the trachea and bronchus, to avoid air or fluid filtration and then the possibility to keep the fistula patent. This has already been well described in other papers where the same kind of modified Dumon stent was used ,5. We usIt is our opinion that this kind of procedure could be useful in the treatment of main bronchus pleural fistulas, and it could be even used in larger ones, eventually associated with apposition of several devices . We also believe that this stent could be performed as a first choice when the fistula appears, thereby avoiding any surgical procedure.The author(s) declare that they have no competing interests.GMF, AT, MG enrolled the patient for clinical and surgical aspects, drafting the article.UC, MDS. MMC drafting the article, critical revision of the article.AT, MA, GR performed surgical operations, critical revision of the article, final approval of the version.


Existing literature to date suggests a relationship between cognitive attention and working memory (WM), but the relationship between overt inattentive behaviour and WM is less clear. This study examined the relationship between WM and parent-rated inattentive behaviour in a community sample of 140 children aged 7–12 years.Children completed 2 clinical (laboratory-based) measures of WM  and a measure of real-life WM, designed specifically for this study, while their parents completed questionnaires about their child's inattentive behaviour and other areas of functioning.Findings indicated that poorer performance on WM tasks predicted inattentive behaviour.These results are consistent with previous research linking WM deficits and poor attention in ADHD and normal populations. The present findings support a controlled attention model of WM. Working memory (WM) is a cognitive processing resource of limited capacity that allows the temporary storage of information while simultaneously processing the same or other information. Performance on WM measures correlates with performance on higher-order cognitive tasks involving reading comprehension, reasoning, and complex learning . A well-in the presence of interference as a critical control function of WM, and hence as the primary mechanism linking WM capacity with higher-order cognition Studies of individual differences in WM provide support for the conceptualization of WM as controlled attention. For example, studies that compared high- and low-WM span individuals in interference resistance and processing load found that individuals with high WM ability are better able to resist interference during encoding and retrieval than individuals with poor WM ability ,8. Moreocognitive attention and WM, but is unclear whether WM would relate to overt inattentive behaviour . Examples of overt inattentive behaviour are: difficulty sustaining attention in tasks or play activities, being easily distracted by irrelevant stimuli, and frequently interrupting ongoing tasks to attend to trivial noises or events that are usually ignored by others [Collectively, previous research supports a relationship between controlled y others . Such a y others . For exay others . Howevery others -15.The primary purpose of the present study was to examine the relationship between WM performance and inattentive behaviour in a community sample of school-aged children. According to the controlled attention model, poor/overloaded WM should lead to more interference, and hence, inattention/distraction. In line with this, we examined the correspondence between caregiver reports of inattentive behaviour and performance on two types of WM measures: clinical, laboratory-based measures of WM  and a real-life measure of WM. The real-life measure of WM was included in this study to obtain a broad assessment of different aspects of WM. Because most studies of WM take place in a highly controlled laboratory setting in which interference from external sources is reduced and rigorous constraints are placed on the child's behaviour, performance may either exaggerate, or conversely, mask the true magnitude of a child's WM capacity . In addiThe hypotheses for the present study were derived from the controlled attention view which argues that WM capacity is a valid predictor of (covert) attentional control, and that high- and low-span individuals allocate attention differently, with high-WM span individuals being more adept at resisting interference than their low-span counterparts . The preA secondary goal was to test the more specific hypothesis that visual-spatial WM, but not auditory-verbal WM, would be inversely related to inattentive behaviour ratings, such that better performance on visual-spatial WM tasks would be associated with lower ratings of inattentive behaviour. This hypothesis was based on Chhabildas et. al's findings  that ADHA total of 140 children aged 7 to 12 years participated in this study. The mean age of the sample was 10.03 years (SD = 1.72), and approximately half of the sample were females (n = 72). Information from the demographic questionnaire completed by caregivers indicated that approximately 62% of the participants were Caucasian, and the remaining participants were relatively evenly distributed across African-Americans (6%), Asians (6%), South Asian (3%), and other (20%), consistent with the ethnic representation of the large metropolitan city. Of the total sample, caregivers reported 98% of the children's primary language was English, 88% reported no learning or developmental disability, and 93% reported no significant hearing or vision problems. All participants were included in the study, as there were no inclusion/exclusion criteria, other than age, English spoken as primary language at home, and independence in mobility .The sample was recruited during a two-week period in the summer from visitors to a large water park just outside an urban city. A water park was chosen as the site of research data collection because it attracted many children in the appropriate age-range for the present study. As a thank-you gift, all participants received a pencil and a voucher for free items available from food stations on the park grounds.The measures for this study included clinical measures of auditory-verbal and visual-spatial WM , and a real-life measure of WM for the children, and questionnaires for the parents.The clinical measures of WM consisted of the Digit Span subtest of the Wechsler Intelligence Scale for Children, Third Edition (WISC-III)  and the The Digit Span subtest is a clinical measure of auditory-verbal WM. Children heard a sequence of digits  at a rate of one digit per second, and were asked to repeat the sequence of digits in the exact order it was presented. The length of the sequence started with two digits, and became increasingly more difficult (up to a maximum of nine digits) until the children obtained the required number of errors for discontinuation. The raw score for Digit Span Forward (DSF) is the number of correct trials . Digit Span Forward is a measure of verbal storage-only component of WM , children heard a sequence of digits  that they were required repeat in reverse order. The sequences became increasingly more difficult (from a sequence of two digits to a maximum of nine digits) until the children obtained the required number of errors for discontinuation. The raw score of DSB is the number of correct trials . Digit Span Backward is a measure of the storage and processing components of verbal WM , the researcher tapped the cubes (one cube per second) in a specified sequence that the children were asked to replicate. The length of the sequence started with two blocks, and became increasingly more difficult (to a maximum sequence of eight blocks) until they obtained the required number of errors for discontinuation. The raw score for SSF is the number of correct trials . Spatial Span Forward is a measure of the visual-spatial storage component of WM  .For Spatial Span Backward (SSB), the researcher tapped the cubes in a specified sequence that the children were required to repeat in reverse order. Like SSF, the length of the sequence became increasingly more difficult (from a sequence of 2 blocks to a maximum sequence of 8 blocks) until they obtained the required number of errors for discontinuation. The raw score for SSB is the number of correct trials . Spatial Span Backward is a measure of the storage and processing components of visual-spatial WM  .In all statistical analyses that follow, only raw scores were used for DSF, DSB, SSF, and SSB. Scaled scores are not reported here.The Key Search task is an experimental measure of real-life WM, designed for the purpose of the present study. This task was the real-life version of Digit Span and Spatial Span, and was individually administered. For Key Search Forward (KSF), children were instructed to visit a certain number of places in the water park . Verbatim instructions given to each participant are presented in Appendix A. While giving verbal instructions as to which places to visit on a given trial, the researcher simultaneously pointed to each place on a laminated map of the water park. This step, along with an initial screening of children's familiarity with the layout of the park (described below), was included to address the possible impact of way-finding ability. Children were instructed to visit each place (with a researcher) in the given order. For example, in trial 1 of KSF, the participant was instructed to go to the hot tub, the lazy river, and the lockers. Note that the places were not all visible from the start point and the Park map was not available to the child after the initial instructions. Thus, participants had to maintain and update that information as they walked from one place to the other. A score of 1 was assigned if the child visited all the places in correct order within a trial . The raw score for KSF is the number of correct forward trials . Because the children may have either engaged in verbal rehearsal of the places and/or constructed an internal visual-spatial map, KSF was thought to be a real-life measure of both auditory-verbal and visual-spatial STM, or storage component of WM.When the children completed a KSF trial , the accompanying researcher asked them to imagine that somewhere on the way to the final destination, the researcher lost his/her locker key. For Key Search Backward (KSB), the researcher asked the children to help find the key by re-tracing their steps. Appendix A displays the exact instructions given to each participant. The children verbally reported all the places they visited in backward order. A score of 1 was assigned if they correctly recalled all the places they visited in reverse order . The raw score for KSB is the total number of correct backward trials . Key Search Backward is thought to be a naturalistic measure of both auditory-verbal and visual-spatial WM. All Key Search scores described in the Results section below are raw scores.Unlike Digit Span and Spatial Span which both require the administration of all trials of the forward task until the discontinuation criteria had been met, after which all trials of the backward task are administered , the Key Search task required the forward and backward trials to be administered in alternating order. That is, after trial 1 of KSF was completed, trial 1 of KSB was administered. If a child received a score of 1 on either of the first trials of KSF or KSB, the child proceeded onto the second trials of KSF and KSB. Children started with a sequence of three places to visit . The sequences became increasingly more difficult  until they received a score of zero on both KSF and KSB in a given trial, at which time the Key Search task was discontinued. Finally, in order to discount the confound of familiarity with the layout of the water park, researchers screened children's knowledge of the water park before commencing the Key Search task. To do this, researchers asked the children to point in the direction of prominent landmarks (or indicate their proximity to visible landmarks) within the water park. All landmarks in the screening were included as places to visit in the actual Key Search task. If they pointed incorrectly, the researcher scored the item as zero, and corrected them by pointing in the correct direction of the landmark. A total score of zero on the screen indicates poor knowledge of the water park layout, while a perfect score of 6 indicates very good knowledge of the water park layout. Performance on the Key Search Screen was entered as a covariate in the relevant analyses to control statistically for way-finding abilities.The real-life Key Search task and two clinical tasks differed in several important ways. One important difference lies in the sequence of administration of the forward and backward tasks as discussed in the Methods section. Another difference between the clinical WM tasks and the real-life WM task is the time it took to administer. Specifically, while the duration for the Digit Span and Spatial Span tasks is typically under a minute for each trial, the duration for the Key Search task was approximately a few minutes for each trial . Given this difference in administration time, it could be argued that episodic or long-term memory was used in the Key Search task. However, the Key Search task was developed to be analogous to everyday tasks that tap into WM processes, and not identical to laboratory-based WM tasks. Real-life tasks that tap into WM typically have longer durations than Digit Span or Spatial Span. For example, a teacher may instruct a student to first copy task instructions from the board, take out the necessary workbooks and work materials, and then to start the necessary task. Another example would be reading comprehension, where the student would be required to read the text, understand the material, continually update information that he/she just read, and then to answer content or inferential questions based on the text. Thus the Key Search Task approximates the distracting conditions and duration under which children are typically required to engage working memory abilities in everyday activities. In short, the Key Search task was developed to be analogous to the clinical WM tasks (storage and manipulation of information), yet also to be similar to WM tasks that children may encounter in their daily lives. More research assessing the ecological validity of neuropsychological measures (such as WM tasks) is needed to evaluate the generalizability of such tasks.Parents completed three questionnaires about their children in three areas of interest: demographic information, a measure of inattention and hyperactivity/impulsivity, and a measure of psychological adjustment.The demographic questionnaire contained questions about the parent and child's background, such as parent education, child health , known learning disabilities or developmental disabilities, primary language, and ethnicity.The Strengths and Weakness of ADHD-symptoms and Normal-behaviour (SWAN) scale was used to assess inattention and hyperactivity/impulsivity . This scTo ascertain the psychological adjustment of the participants, as well as to explore the extent to which WM is associated with other types of difficulties other than hyperactivity-inattention (Hypothesis 1), parents were asked to complete a short questionnaire to screen for emotional and behavioural difficulties. The Strengths and Difficulties Questionnaire (SDQ)  is a briA booth was set up in a highly visible area of the water park and signage was posted throughout the park. The signs contained a fun and interest-provoking logo inviting park guests to participate in the study. All research staff involved with this study wore special T-shirts for identification purposes. Research staff attended the booth at all times to provide information about the study and to complete consent procedures. Parents provided written informed consent for their child to participate and children provided informed verbal assent. The study was approved by Ethical Review Boards at the University and Hospital Research Institute as well as by the Water Park. All test activities were conducted on dry land outside of the water areas. Children were tested individually and were accompanied by a researcher throughout the whole test session.Clinical WM testing took place in a partitioned part of the water park, in a tented area to simulate a quiet laboratory testing space. There were two partitioned "laboratories" with a table and two chairs in each. To reduce possible interference from noise from the water park during the verbal memory task, children listened to a pre-recorded audiotape of the verbal stimuli  which were presented through headphones. Thus, testing procedures for the clinical WM tasks were slightly modified to accommodate the naturalistic setting of the study. While the children completed the clinical and real-life WM tasks, parents were asked to complete some questionnaires about their child. The Digit Span, Spatial Span, and Key Search tasks were administered in counter-balanced order.In the first step, we screened the sample for ethnicity, parent-reported health problems , and psychological adjustment in the children and then investigated whether these problems influenced the findings. A total of 27 children were reported by caregivers to have health problems. To examine whether these self-reported problems had any effects on the results, all analyses were run with and without the 27 children. Virtually all results remained unchanged ; therefore, in the absence of clear evidence that these 27 participants were not part of a normal population, the following analyses included all 140 participants. In addition to screening for the difficulties stated above, the present study also screened for psychological adjustment in this community sample of children. The SDQ (North American version) yielded scores for five areas of emotional and behavioural difficulties. The proportion of caregivers reporting emotional and behavioural problems falling within the clinical range of each SDQ scale were as follows: 11% emotional symptoms; 9% conduct problems; 8% hyperactivity/inattention; 13% peer relationship problems; and 4% prosocial behaviour. The SDQ parent ratings for each scale approximated the proportion of children in the clinical range according to the SDQ norms .The second step was to examine the factor structure of the SWAN ADHD scale to determine the robustness of the inattention and hyperactivity/impulsivity scales, since this is a relatively new instrument and one that has not been investigated in a Canadian sample of children. A factor analysis, using a varimax rotation, was performed on the SWAN scale to check whether the data would yield the two proposed dimensions . The results confirmed two separate factors with Eigen values greater than 1: the Eigen value for the hyperactive/impulsive factor was 9.36, which accounted for 52.0% of the variance, and the Eigen value for the inattentive factor was 2.40, which accounted for 13.3% of the variance. Results confirmed that all nine inattentive items have significant loadings with the inattentive factor, while none of the nine hyperactive/impulsive items have significant loadings. Likewise, all nine hyperactive/impulsive items have significant loadings with the hyperactive/impulsive factor, while none of the inattentive items have significant loadings  to investigate age and gender differences in the caregiver behavioural ratings. The fourth step addressed our primary objectives. We first conducted correlational analysis and a factor analysis to determine whether the six measures of WM we had selected did in fact index the purportedly separable components of WM and then used regression techniques to test the hypotheses concerning the relationship between WM and inattentive behaviour. In view of the multiple comparisons to be made, alpha was set at .01 in all analyses to reduce the likelihood of Type I errors.Zero-order correlations revealed that age correlated significantly with all WM raw scores  such that WM performance on all tasks improved significantly with age . To examine the effects of gender , a one-way multivariate analysis of variance (MANOVA) was conducted on all WM measures . Table While age was not significantly correlated with SWAN Inattention , age was significantly correlated with SWAN Hyperactivity/Impulsivity  suggesting that as children's age increased, parent ratings of hyperactivity/impulsivity decreased significantly. A one-way MANOVA was conducted to determine the effect of gender  on the two dependent variables . Gender differences approached significance on the SWAN scales, Wilks's Λ = .94, F = 4.70, p = .011. Follow-up ANOVAs revealed a significant difference between males and females on SWAN Inattention only, F = 9.45, p < .01, such that parents rated boys as more behaviourally inattentive than girls . To examine the effects of gender , a one-way MANOVA was conducted on SDQ ratings . Although no significant differences were found among male and females on the SDQ scales in the multivariate analysis, Wilks's Λ = .93, F = 1.96, p = .09, we proceeded with follow-up ANOVAs to test for expected gender differences in hyperactivity/inattention and emotional symptoms. Univariate analyses revealed that the hyperactivity-inattention scale was the only SDQ scale that showed a significant difference between males and females, F = 6.94, p < .01, such that parents rated boys as more hyperactive-inattentive than girls , as well as with SWAN Inattention and Hyperactivity/Impulsivity scores. As can be seen in Table To test the main hypothesis , the first multiple regression analysis had SWAN Inattention scores as the dependent variable, and examined the significance of the unique variance added to the equation by WM performance over and above that which could be accounted for by age and gender. Age and gender were entered as predictors in step 1 and WM composite score as a predictor in step 2. Table Next, we tested the corollary hypothesis that WM would not be associated with parent ratings of hyperactivity/impulsivity. Therefore, the second multiple regression analysis had SWAN Hyperactive/Impulsive ratings as the dependent variable to determine whether WM performance was a valid predictor over and above age and gender. As shown in Table To test the second corollary hypothesis that WM would be associated only with inattentive behaviour and not with other psychological difficulties, we conducted a zero-order correlation analysis between the composite WM score and the five SDQ scales . It is important to note that the hyperactivity subscale includes 2 items for inattentive behaviour as well as three hyperactivity items. As predicted, there were no significant correlations between WM and SDQ Emotional Symptoms scale , SDQ Conduct Problems scale , SDQ Hyperactivity scale , SDQ Peer Problems scale , and SDQ Prosocial scale .Our primary goal was to test the hypothesis that poor WM would lead to susceptibility to interference, and thus be associated with higher behavioural ratings of inattention. Assuming that both WM and inattentive behaviour are continuously distributed traits in the normal population, we investigated this relationship in a community sample of school-aged children. Consistent with this hypothesis, we found that the children's WM performance predicted significant and unique variance in behavioural inattention as rated by their caregivers, over and above the contribution of age and gender. The finding that WM performance did not contribute significantly to parent ratings of hyperactivity/impulsivity and other behavioural/emotional problems, adds additional support to the hypothesis. Our findings are consistent with the controlled attention view of WM, which argues that the critical issue is not how much information WM can hold, but rather it is the extent to which executive control protects the information being processed from interference by irrelevant distractors. Findings from the present study also extend the controlled attention model of working memory by testing the predictions using parent ratings of inattentive behaviour . In the present study, children who exhibited weaker WM executive control were thought to have weaker ability resisting interference, which manifested as inattentive behaviour, as viewed by their caregivers.This finding converges with previous demonstrations that increased WM load results in greater distractor interference in Stroop-like tasks ,28, whicnot associated with ratings of hyperactivity/impulsivity or with other emotional/behavioural difficulties. A follow-up multiple regression analysis revealed that the composite WM score did not significantly predict SWAN ratings of hyperactivity/impulsivity. Moreover, a zero-order correlational analysis revealed that the WM composite score did not correlate significantly with any of the five SDQ behavioural/emotional scales at the set alpha level.Our primary hypothesis regarding the link between inattention and WM also required evidence that that WM performance was Another aspect of this study was to evaluate the relationship between experimental measures of real-life WM  and behavioural inattention. Given that the factor analysis failed to extract a separate factor for our real-life WM measure, this relationship could not be explored statistically. However, this finding of one factor may be an indication of ecological validity of the clinical measures of WM. Further investigation  into the ecological validity of the clinical measures of WM used in this study is recommended for future studies.Other findings from this study concerned age and gender effects of behavioural ratings. One analysis revealed that as children's age increased, parent ratings of SWAN Hyperactivity/Impulsivity decreased. An examination of gender effects revealed that parents rated boys as more hyperactive-inattentive than girls on the SDQ and more behaviourally inattentive on the SWAN. These findings are consistent with epidemiological studies of ADHD indicating that the ratio of males to females in non-referred samples ranges from 2.5:1 to 5.1:1 .Another limitation of this study is that our measure of behavioural inattention came from parent report only. Because children spend a large part of their day in school, teachers often provide valuable information regarding children's level of attention and behaviour. However, it was not feasible to obtain teacher reports of inattention for this study given that it took place in the summer when children do not attend school. Although studies have shown that parent-teacher concordance rate for ADHD is relatively poor , cross-iFinally, the main finding from this study revealed that WM is associated with caregiver reports of inattentive behaviour. However, it should be noted that other cognitive causes of inattentive behaviour should also be investigated. For example, research has revealed that groups of ADHD children demonstrate high degrees of between-subject variation in performance almost irrespective of task or setting . Such grbehavioural inattention in a community sample of children. Other studies have found that covert cognitive attention, or inattention, can predict individual differences in WM [A major finding of this study was a link between WM and es in WM -9. The pOur finding of a cognitive correlate of inattention in a community sample of children has important clinical implications. Mainly, problems with inattention in children are considered to be a developmental risk factor and are associated with poor achievement in reading and general cognitive delays ,36. Our The present study provides empirical evidence of a significant link between WM performance and inattention in a community sample of school-aged children. Specifically, the results suggest that children manifesting poorer performance on WM tasks are rated by their parents as more inattentive behaviourally than are children with better WM. Directions for future research include obtaining teacher reports of children's behavioural inattention, as children's ability to sustain and focus attention is highly relevant within the classroom environment.The author(s) declare that they have no competing interests.This study was part of ML's master's thesis project. ML conceived of and designed the study, coordinated data collection, performed statistical analyses, and drafted the manuscript. RT is the thesis supervisor of ML, and made substantial contributions to the conception and design of the study, interpretation of the data, and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.Key Search InstructionsKey Search Forward take me to each place in the same order I tell you. For example, if I say "hot dog cart and ice cream stand," I want you to walk me first to the hot dog cart and then the ice cream stand. Do you understand? So, if I say "showers and hot tub," where would you take me first? Second?For this task, I'm going to tell you some places in the park, and show you where they are on this map. Listen carefully because I will say the places only once. When I'm done, I want you to Key Search Backward You did a great job taking me to the places I told you. But, let's imagine I lost my keys somewhere along the way and we would have to search for them in the all places we just went to. I want you to help me re-trace our steps by telling me all the places we went to in BACKWARDS order, starting from here, then the second last place, and so on.Prompt: Now, where were we just before here?Key Search Forward same order I tell you. Ready?OK, we're going to do the same thing: I'm going to tell you some places in the park, and show you where they are on this map. Listen carefully because I will say the places only once. When I'm done, I want you to take me to each place in the Key Search Backward Once again, you did a great job taking me to the places I told you. As before, I want you to imagine that I lost my keys, and we must search for them in all the places we just went to. I want you to help me re-trace our steps by telling me all the places we went to in BACKWARDS order, starting from here, then the second last place, and so on.Prompt: Now, where were we just before here?Special Instructions- Do not lead the child. Stay either beside or a little behind the child.- Children are not to go in the water when doing this task. Tell the child to walk around the water.Factor Loadings for Each SWAN Scale Item. The data provided represent the factor loadings for the Inattention scale and the Hyperactive/Impulsive scale on the SWAN Scale.Click here for file


All theories about the origin and evolution of membrane bound cells necessarily have to cope with the nature of the last common ancestor of cellular life. One of the most important aspect of this ancestor, whether it had a closed biological membrane or not, has recently been intensely debated. Having a consensus about it would be an important step towards an eventual (though probably still remote) synthesis of the best elements of the current multitude of cell evolution models. Here I analyse the structural and functional conservation of the few universally distributed proteins that were undoubtedly present in the last common ancestor and that carry out membrane-associated functions. These include the SecY subunit of the protein-conducting channel, the signal recognition particle, the signal recognition particle receptor, the signal peptidase, and the proton ATPase. The conserved structural and functional aspects of these proteins indicate that the last common ancestor was associated with a hydrophobic layer with two hydrophilic sides (an inside and an outside) that had a full-fledged and asymmetric protein insertion and translocation machinery and served as a permeability barrier for protons and other small molecules. It is difficult to escape the conclusion that the last common ancestor had a closed biological membrane from which all cellular membranes evolved. Yet, beyond these universal features the nature of the last common ancestor of cellular life (or LUCA) is still intensely debated -5. The vund cell . In thisThe idea of a membrane-less, minerally compartmentalised universal ancestor has been proposed because archaebacteria and eubacteria have membrane lipids of different chemical composition and chirality  and because these different lipids are synthesized by mostly non-homologous enzymes ,8. If onHere I discuss what properties can we assign to the membranes or hydrophobic layers of the universal ancestor by carefully analysing the structural and function aspects of the universal membrane-associated cellular machineries.In all cells the translocation of proteins across the plasmamembrane (or ER in eukaryotes) and the insertion of most transmembrane proteins are mediated by a transmembrane protein complex, the protein-conducting channel  ,11. ProtComparative genomic surveys revealed that the central components of the translation, protein insertion and translocation machineries are present in all forms of cellular life ,13. The The conserved topology and sequence features of SecY reveal that the PCC is ancestrally membrane associated. SecY has an extracellular and an intracellular hydrophilic part, a conserved inner pore and ten conserved transmembrane segments ,15 1). This There is a clear asymmetry in the arrangement of the secretory machinery: signal peptide containing proteins bind to the PCC at one side, are translocated across the PCC, and are cleaved at the other side by the signal peptidase. The asymmetric arrangement of this machinery is self-maintained: the asymmetric SecY channel inserts new copies of itself and translocates new copies of the signal peptidase to the outside. This asymmetric arrangement is universally conserved indicating that it already existed in the universal ancestor. This means that the hydrophobic layers of the universal ancestor were asymmetric with an 'inside' and an 'outside' and this asymmetry was maintained autocatalytically. The active translocation of secreted proteins by the ribosome-signal peptide-SRP-PCC machinery also means that the hydrophobic layers of the universal ancestor must have presented a permeability barrier (i.e. were closed). Otherwise where and why would signal-peptide containing proteins have been translocated?0F1-ATPase. F0F1-ATPases generate a transmembrane electrochemical gradient at the expense of ATP or catalyze the synthesis of ATP using an electrochemical gradient [0 c subunit of the complex and the conserved function of the proton ATPase indicate that the last common ancestor was associated with a hydrophobic layer able to maintain a proton gradient, i.e. it was necessarily closed. A hydrophobic layer by itself, if not closed, is not enough for proton ATPase function. Importantly, the PCC also uses a proton gradient to drive the insertion of transmembrane proteins [There is additional evidence that the universal ancestor had a closed membrane that presented a permeability barrier to small molecules. As shown by comparative genomics, the last common ancestor carried in its membrane the ancestor of the universal membrane protein complex, the Fgradient . Althougproteins  highlighBy the comparative genomic and functional reconstruction of membrane-associated functions the following characteristics can be attributed to the hydrophobic layers of the last universal ancestor. These layers had a (i) hydrophobic core with two hydrophilic sides (ii) had a protein insertion and translocation machinery (iii) had a clear asymmetry (iiii) represented a permeability barrier to proteins and small molecules  numbers for the gene content of the universal ancestor . If thisNot accepting the cellular nature of the last common ancestor would mean that membrane-bound cells originated twice independently. The origin of cellular life where genes, membranes and catalysts are integrated in a replicating whole is probably the most difficult problem in cell evolution. The theory of the independent origin of eu- and archaebacterial cells has to solve this problem twice and therefore is clearly most unparsimonious. Given the evidence summarised here it is hard not to conclude that the universal ancestor was membrane bound. In this case it is time to start seriously considering different scenarios of lipid segregation or replacement ,8,9 to aThe author(s) declares that he has no competing interests.


A benchmarking of the most popular orthologous identification methods using functional genomics data identifies the two best methods. The transfer of functional annotations from model organism proteins to human proteins is one of the main applications of comparative genomics. Various methods are used to analyze cross-species orthologous relationships according to an operational definition of orthology. Often the definition of orthology is incorrectly interpreted as a prediction of proteins that are functionally equivalent across species, while in fact it only defines the existence of a common ancestor for a gene in different species. However, it has been demonstrated that orthologs often reveal significant functional similarity. Therefore, the quality of the orthology prediction is an important factor in the transfer of functional annotations (and other related information). To identify protein pairs with the highest possible functional similarity, it is important to qualify ortholog identification methods.To measure the similarity in function of proteins from different species we used functional genomics data, such as expression data and protein interaction data. We tested several of the most popular ortholog identification methods. In general, we observed a sensitivity/selectivity trade-off: the functional similarity scores per orthologous pair of sequences become higher when the number of proteins included in the ortholog groups decreases.By combining the sensitivity and the selectivity into an overall score, we show that the InParanoid program is the best ortholog identification method in terms of identifying functionally equivalent proteins. Orthology is one of the central concepts of comparative genome analysis, but is often misused as a description of functionally equivalent genes in different species. By definition, the term describes the evolutionary relationship between homologous genes whose independent evolution reflects a speciation event, whereas paralogy refers to genes that have diverged from a common ancestor through a gene duplication event . OrtholoSo far, the genomes from more than 200 organisms have been fully sequenced. Of particular interest for medical research are the full genome sequences of human and model organisms, such as fruit fly, worm, mouse, rat, and chicken. Genome sequencing projects on other model organisms, such as the chimpanzee , are alsHomo sapiens. The reasons for this difference in inclusiveness are twofold. Firstly, there are differences between the algorithms being employed, such as bidirectional best hits, the triangular best-bidirectional hits scheme of the COGs 6] and MCThe 'best bidirectional hit' (BBH) method is the most frequently applied method to determine orthologous pairs. It assumes that a cross-species protein pair in which each protein gives back the other protein as being the best hit in the whole other proteome is an orthologous pair. In this research, the best bidirectional hits were determined based on Z-values of the Protein World human-mouse and human-worm set, without a sequence similarity cutoff. In total, 12,817 human-mouse and 5,714 human-worm orthologous pairs were identified. Although the BBH method theoretically can give some many-to-many orthologs, it practically gives only one-to-one orthologous pairs.In the INP method , all posThe KOG database  is the eThe MCL algorithm  starts wWithin the Z1H method, all cross-species protein pairs that have a Z-score of 100 or higher are considered to be orthologs. The Z-value estimates the statistical significance of a Smith-Waterman dynamic alignment score (SW-score) through the use of a Monte-Carlo process . In thisThe PGT method uses the output generated by multiple alignments and subsequent tree calculation  to definBelow are a description and the workflow of the used benchmarks. The first two benchmarks measure 'direct conservation of functional parameters', that is, they examine only one protein in human and one protein in mouse/worm. The last three methods compare the relationship between two proteins in human with the relationship of their two orthologs in mouse/worm .The results of the group orthology methods were analyzed in two ways: we determined the average score for all pairwise orthology relationships within an orthologous group; and we only considered the best scoring pair within an orthologous group. The latter option obviously leads to a much higher score for the many-to-many orthology relationships. However, by including only one pair of orthologous sequences per orthologous group, that high score is balanced by a reduction in the total number of orthologous relationships (one per orthologous group). Both the number of orthologous relationships and the quality of these relationships are taken into account in the final assessment of the ortholog identification algorithms.et al. The 'best hit' of the worm tissue samples for each human tissue sample was seen as corresponding tissue. These ten corresponding tissues were then used to calculate the Pearson correlation coefficients between the human and worm proteins, from which only the positive correlations were used. Proteome sizes were corrected for this by multiplying them by two, before calculating the average proteome size. For visualization reasons we displayed error bars of only one-eighth of the SD. Because of the differences between the human-mouse and human-worm expression data analyses, we emphasize that the two figures  database  was usedet al. [et al. [We defined a list of 'true ortholog pairs', for both human-mouse and human-worm, as a reference set. We chose the Hox cluster proteins and hemoglobins as a human-mouse reference set because of its well-studied evolution in vertebrates. We determined the homeobox orthologs using Figure et al. , resulti [et al. , resulti [et al. , which h [et al. , was anaFor each of these parts of our reference set and for each of the six ortholog identification methods, we determined how many of these orthologous pairs were covered, together with the number of false positives (pairs having only the human protein or the mouse/worm protein from a reference pair). Finally, to have a fair comparison between the several ortholog identification methods, we calculated the number of orthologous pairs divided by the average proteome size.The following additional data are available with the online version of this paper. Additional data file 1 contains all end data used to create the figures. Additional data file 2 contains all of the protein pairs that are considered to be 'true orthologs' within our ortholog reference set, consisting of several protein families. The first column contains the name of the protein family, the second the human gene names and the third the mouse/worm gene names. The fourth column contains the corresponding human 'Protein World' entries, whereas the fifth column contains the mouse/worm entries. The last columns contain the orthologous protein pairs.All end data used to create the figures.Click here for fileAll of the protein pairs that are considered to be 'true orthologs' within our ortholog reference set, consisting of several protein families. The first column contains the name of the protein family, the second the human gene names and the third the mouse/worm gene names. The fourth column contains the corresponding human 'Protein World' entries, whereas the fifth column contains the mouse/worm entries. The last columns contain the orthologous protein pairs.Click here for file


The concept of mapping rhythmic activation of the heart dates back to the beginning of last century, with initial descriptions of reentry in turtle hearts , to the Despite these recent advances, multisite contact mapping suffers from several limitations, including the technical problems associated with amplification, gains, sampling rates, signal-to-noise ratio, and the inability to see signals during high-voltage shocks. In addition, an intrinsic limitation of current mapping techniques is their inability to provide information about repolarization characteristics of electrically active cells, thereby limiting our ability to study entire action potentials. In fact, intracellular microelectrode recordings are still considered the gold standard for the study of action potential characteristics in whole tissue. Microelectrode techniques are limited however, by an inability to record action potentials from several sites simultaneously, thereby precluding their use in high-density activation mapping.In part due to the above-mentioned limitations, the last few years have seen the development and use of voltage-sensitive dyes as a means to map not only activation, but repolarization as well. Voltage-sensitive dyes, when excited, provide an optical signal that mimics an action potential and thus allows the visualization of both activation and recovery processes in any region under view. This allows one to precisely evaluate the propagation of a wave of excitation and to measure its wavelength visually.Optical mapping techniques use imaging devices such as a photodiode array or a charge-coupled device video camera with the heart being illuminated and either continuously or spatially scanned. The basis for these techniques is the use of voltage-sensitive dyes that bind to or interact with cell membranes.Voltage-sensitive dyes are molecules that bind to the cell membrane with high affinity. While bound to the cardiac cell membrane, the dye molecules fluoresce light in direct proportion to transmembrane voltage. Therefore, voltage-sensitive dyes function as highly localized transducers of membrane potential, transforming a change of membrane potential into a change in fluorescent intensity . For anyThe styryl dye, Di-4-ANNEPS, is now the probe of choice for a number of laboratories that are studying complex patterns of activity in the heart. This dye works by the principle of electrochromism, whereby when provoked by excitation light, the molecule undergoes a charge shift from the ground state to the excited state; fluorescence occurs due the emission of a photon during transition from the excited to the ground state. The wavelength of the emitted photon is determined by the change in molecular energy when passing from the excited state to the ground state, less the energy lost in the process. Di-4-ANNEPS possesses several advantages, compared to most other optically sensitive dyes; it exhibits large fractional fluorescence changes during an action potential 8-15%) with low toxicity and photobleaching, and its signal amplitude and kinetics are stable for 2 to 4 hours without restaining the preparation . Th. Th24]. In this study, we also demonstrated focal activity within the pulmonary veins, the underlying mechanism of which appeared to be triggered activity. Both the epicardium as well as the endocardium of the pulmonary vein were studied; while reentry was seen only on the epicardial aspect of the vein, focal activity was seen only on the endocardium, close to the ostium of the vein.Reentry is the underlying mechanism for post-infarction ventricular tachycardia (VT) and the critical element of the reentrant circuit is the protected isthmus. Both anatomic and functional reentry have been proposed as potential models for such reentrant tachyarrhythmias. In anatomic reentry, the isthmus is fixed and located between two infarction scars or between an infarction scar and an anatomic obstacle such as the mitral valve annulus. In contrast, the isthmus in functional reentry is not fixed and is bounded by two functional lines of block located in the infarction border zone.Using optical mapping in isolated canine left ventricular preparation, the reentrant circuit of monomorphic VT after acute myocardial infarction can be directly visualized . The reeAs mentioned above, optical mapping was developed as a part of a quest to simultaneously map the activation as well as the recovery of electrically active tissues. The use of voltage-sensitive dyes, photodiodes, laser scanning and CCD cameras have made it possible to record electrical activity in tissue preparations as well as in a beating heart -28. SeveIn addition to the insights that have been gained into the understanding of specific arrhythmias, the role of optical mapping has been recently extended by Garrigue and colleagues to study the effects of pacing in intact hearts. They studied the role of voltage output, interventricular delay, and pacing sites in ventricular arrhythmia occurrence during biventricular pacing ; they utilized optical mapping in Langendorff-perfused guinea pig hearts to measure ventricular activation time and to examine conduction patterns during multisite pacing from 4 left ventricular and 3 right ventricular sites . Myocard


It is well know that siRNA efficiency is crucial in the RNAi pathway, and the siRNA efficiency for targeting different sites of a specific gene varies greatly. Therefore, there is high demand for reliable siRNAs prediction tools and for the design methods able to pick up high silencing potential siRNAs.In this paper, two systems have been established for the prediction of functional siRNAs: (1) a statistical model based on sequence information and (2) a machine learning model based on three features of siRNA sequences, namely binary description, thermodynamic profile and nucleotide composition. Both of the two methods show high performance on the two datasets we have constructed for training the model.Both of the two methods studied in this paper emphasize the importance of sequence information for the prediction of functional siRNAs. The way of denoting a bio-sequence by binary system in mathematical language might be helpful in other analysis work associated with fixed-length bio-sequence. RNA interference (RNAi) is a biological mechanism by which double stranded molecules inhibit gene expression by mediating sequence-specific mRNA degradation . The proMany efforts have been made trying to develop computational methods which can provide improved prediction of potentially functional siRNA -6. TheseIn the field of machine learning, siRNA prediction can be considered as a typical pattern recognition or classification problem. Based on this consideration we developed two kinds of algorithms, both of which achieved high performence. The first one is a sequence based statistical model that has been successfully used in signal peptide prediction . In thisAs we have known, to objectively assess a prediction method, a homogeneous and sufficiently large dataset is of high importance. It should also be very careful to combine datasets from different resources, because the efficiency of a siRNA changes variously under different biological and experimental conditions. Fortunately, the recently published Dieter's dataset from a high-throughput assay makes it possible to break the bottleneck of directly comparing different source datasets . BesidesWhether a siRNA can be denoted as functional or non-functional depends on its ability of silencing a target gene, which is often measured by the value of siRNA inhibitory activity. Thus we generated the positive and negative subsets according to the level of inhibitory activity. During our research, we took three cut-off values, 0.5, 0.6 and 0.7 to generate six combinations of positive and negative subsets for the two datasets table . That isFor each of the six combinations, both of the self-consistency and the jackknife test were performed. Especially, during the training processes of SVM algorithm, to compare the contributions of each of the three attributes, all seven combinations have been performed independently, which are "binary, thermodynamic and composition", "binary and thermodynamic", "thermodynamic and composition", "binary and composition", "binary only", "thermodynamic only", "composition only". All the results from the two methods have been listed from table Those results in table From table To compare with Dieter's work , we conset al . For the terminal four bases of the 3'end of the oligos, the targeting sequences would be extended for calculation. If extension is impossible (for example when the targeting sites on the targeted gene is before the third base of the gene), the thermodynamic value of the terminal four bases are set to be zero. The third attribute defines the composition features of siRNA sequences including 4 single nucleotide and 16 di-nucleotide compositions.To construct the vector space, we introduced three kinds of attributes, representing three aspects of a siRNA oligo. The first one is the binary system by which the 5 nucleotides of The parameters for the training process by SVM were set to be default values. The software used to implement SVM was SVM_light by Joachims [22]. The training process was carried out on the computer we used (Dell OptiPlex GX270 computer with an Intel Pentium4 2.80 GHz CPU).To objectively assess our prediction system, we employed the following measurements for sensitivity, specificity, accuracy and receiver operating characteristics (ROC):ROC (receiver operating characteristics)TP, true positive; TN: true negative; FP, false positive; FN, false negative.Peilin Jia conceived of the study, carried out the algorithm design and realization and helped to draft the manuscript. Yudong Cai and Yixue Li conceived of the study, helped to improve the algorithm and to draft the manuscript. Tieliu Shi, Yixue Li and Yudong Cai helped to revise the manuscript.Results by the method of SeqSta.Click here for fileResults by the method of SVM. All results, including those tested on the whole dataset and those gotten by the ten randomly chosen processes, are listed in this file.Click here for fileComparison results between our methods and Dieter's method.Click here for fileThe two datasets we used are described in this file, including all the sequences of the siRNAs and the density distribution of inhibitory activities of each dataset. We also described which siRNAs are selected during the ten random processes under different cut-off values in this file.Click here for file


Herpes simplex virus (HSV) type 1 and 2 are old viruses, with a history of evolution shared with humans. Thus, it is generally well-adapted viruses, infecting many of us without doing much harm, and with the capacity to hide in our neurons for life. In rare situations, however, the primary infection becomes generalized or involves the brain.Normally, the primary HSV infection is asymptomatic, and a crucial element in the early restriction of virus replication and thus avoidance of symptoms from the infection is the concerted action of different arms of the innate immune response. An early and light struggle inhibiting some HSV replication will spare the host from the real war against huge amounts of virus later in infection. As far as such a war will jeopardize the life of the host, it will be in both interests, including the virus, to settle the conflict amicably. Some important weapons of the unspecific defence and the early strikes and beginning battle during the first days of a HSV infection are discussed in this review.Generally, macrophages are orchestrating a multitude of anti-herpetic actions during the first hours of the attack. In a first wave of responses, cytokines, primarily type I interferons (IFN) and tumour necrosis factor are produced and exert a direct antiviral effect and activate the macrophages themselves. In the next wave, interleukin (IL)-12 together with the above and other cytokines induce production of IFN-γ in mainly NK cells. Many positive feed-back mechanisms and synergistic interactions intensify these systems and give rise to heavy antiviral weapons such as reactive oxygen species and nitric oxide. This results in the generation of an alliance against the viral enemy.However, these heavy weapons have to be controlled to avoid too much harm to the host. By IL-4 and others, these reactions are hampered, but they are still allowed in foci of HSV replication, thus focusing the activity to only relevant sites. So, no hero does it alone. Rather, an alliance of cytokines, macrophages and other cells seems to play a central role. Implications of this for future treatment modalities are shortly considered. Virus-host interactions are crucial for the outcome of infections. Several strategies have been utilized by viruses to overcome the host defence. For the virus to be successful, these evasive strategies have to be balanced with the pathology induced and the possibilities of transmission to new susceptible individuals. The mammalian host utilizes ubiquitous and redundant antiviral defence mechanisms. In different viral infections, different parts of the host defence seem to be crucial. However, the redundancy ensures that other systems are ready to take over, if one of them fails. The final outcome of a viral infection depends on a delicate regulation and timing of these antiviral effector mechanisms in response to the invading virus.A viral infection of an individual thus involves a conflict between the virus and the host, which could conceptually be viewed upon as a human controversy escalating to invasion and armed struggle. To understand the resulting course of events it is important to know each party of the conflict and to conduct an analysis of the powerful weapons held by each of the combatants. The present review analyzes the early non-specific events in the conflict upon herpes simplex virus (HSV) infection. Initially, each participant of the conflict, the infecting HSV and the non-specific antiviral weapons of the host, are described. Subsequently, the early events of the conflict, the armament, early strikes and the opening battle between HSV and the host are discussed. Insight into the early non-specific defence mechanisms are important for our understanding of the conflict and may indicate how to intervene during serious systemic infections.Herpesviruses are ubiquitous viruses generally infecting humans early in life. The majority of humans has had a primary infection with one or more herpesviruses and harbour these viruses in a latent state for the rest of their lives. The initial infection is most often asymptomatic, but can be symptomatic depending on the herpesvirus in question and the age and immune status of the host. The viruses are phylogenetically old and humans and herpesviruses have evolved together . This coAlphaherpesvirinae together with varicella-zoster virus. These alphaherpesviruses all show neurotropic latency, and mucosal or skin lesions are frequently seen as a result of viral reactivation from sensory nerves. The two types of herpes simplex virus confer the genera Simplexvirus 1 and -2, which were formally designated by the International Committee on Taxonomy of Viruses as Human herpesvirus (HHV) 1 and 2 . T. T411]. In treatment of HSV infections, we have for many years had a very powerful tool in the antiherpetic drug acyclovir and related compounds. But there are still therapeutic problems in the group of patients with generalized or CNS infections, and therefore it is tempting and timely to hypothesize on possible future treatment strategies. As described, it is clear that relatively discrete but early actions of the non-specific defence systems are crucial for the long term outcome of the infection. The same holds for the antiviral therapy, and early presumptive therapy and rapid diagnostics could thus potentially improve the final outcome. In the seeking for improved antiviral treatment, adjuvant therapy with anti-HSV antibodies could potentially accelerate the clearance of viral particles, and block viremic dissemination in patients, who are still seronegative at the time of treatment.Immunomodulatory treatment modalities imitating the early non-specific antiviral defence, working as described in this review, could be considered. The key players exhibiting the least toxicity by themselves could be used, taking advantage of potential synergy with other cytokines in the foci of HSV infection. In the future, molecules with affinity for various receptors are expected to be produced, and when we know the signalling mechanisms in detail and all the potential interactions, molecular signalling could be addressed directly by pharmaceuticals.In consequence of the crucial position of the type I IFNs in innate response to HSV, future analogues of IFN-α/β seem obvious as candidates for adjuvant treatment of severe HSV infections. This could be supplemented with IL-12, which would give the highest IFN-γ production in foci of HSV infection because of other cytokines such as IFN-α/β, TNF and IL-18 being present there. With focussed production of IFN-γ at sites of active viral replication and treatment with IFN type I analogues the focal antiviral activity could be increased markedly, without too much activity in areas without infection. To hamper systemic consequences of the enhanced proinflammatory reactions, such pro-inflammatory treatment could perhaps benefit from concomitant treatment with IL-4 or other STAT6-activating therapeutics in the future. This would further focus the activity to sites of active HSV replication. In situations with massive viral replication in nearly all organs, high-dose aciclovir should perhaps only be supplemented with anti-inflammatory medications and inhibitors of TNF, since many of these individuals risk to die from septic reactions.The author(s) declare that they have no competing interests.


Naegleria group I intron  and the PRP8 inteins in ascomycetes  provide important stepping stones towards integrated studies on how these parasitic elements evolve through time together with, or despite, their hosts.Self splicing introns and inteins that rely on a homing endonuclease for propagation are parasitic genetic elements. Their life-cycle and evolutionary fate has been described through the homing cycle. According to this model the homing endonuclease is selected for function only during the spreading phase of the parasite. This phase ends when the parasitic element is fixed in the population. Upon fixation the homing endonuclease is no longer under selection, and its activity is lost through random processes. Recent analyses of these parasitic elements with functional homing endonucleases suggest that this model in its most simple form is not always applicable. Apparently, functioning homing endonuclease can persist over long evolutionary times in populations and species that are thought to be asexual or nearly asexual. Here we review these recent findings and discuss their implications. Reasons for the long-term persistence of a functional homing endonuclease include: More recombination  than previously assumed for these organisms; complex population structures that prevent the element from being fixed; a balance between active spreading of the homing endonuclease and a decrease in fitness caused by the parasite in the host organism; or a function of the homing endonuclease that increases the fitness of the host organism and results in purifying selection for the homing endonuclease activity, even after fixation in a local population. In the future, more detailed studies of the population dynamics of the activity and regulation of homing endonucleases are needed to decide between these possibilities, and to determine their relative contributions to the long term survival of parasitic genes within a population. Two outstanding publications on the amoeba  In the organism centered view of evolution, the individual is considered the unit of selection. By being fit, the individual's genes have a greater chance to become fixed in the population. Challenging this view, Dawkins introduced a gene centered view of evolution  where thSelf splicing introns and inteins are remarkable examples of parasitic elements -7. TheseThe fitness of the molecular parasite and the fitness of the organismal host walk separate roads ,13. For Conceptually, the homing cycle should be formulated for interbreeding populations , i.e., tNaegleria, an apparently asexual amoeba, an intron with HE was reported to be of ancient origin, frequently lost in different lineages, but persisting as a functional enzyme in others [Cryptococcus [Several recent findings challenge the general applicability of the homing cycle: in n others . And in n others , all instococcus  but no etococcus . Accorditococcus , the groWe discuss several possible explanations, not all complementary, for these findings:(A) Sex and genetic exchange occur more frequently than assumed. Rare events of genetic exchange that bring HE containing alleles together with intron/intein free alleles might be sufficient to maintain the HE under purifying selection.Bacillus phages, introns with HE were reported to increase the fitness of their respective host by destroying the genomes of competing phages, containing a different HE, that co-infected the same host [Saccharomyces cerevisiae an endonuclease that triggers recombination events leading to mating type changes is a homolog of the HE containing intein in the vacuolar ATPase catalytic subunit [Thermococcus kodakaraensis [(B) The HE might have a function beneficial to the host. In case of ame host . Howeverame host . Many seame host , and somame host . Again same host : The org subunit ,26,27; aaraensis . The acqaraensis .(C) The homing endonuclease might be maintained by balancing selection. Butler et al  suggeste(D) The homing cycle might operate in subpopulations only. A low homing frequency might be sufficient to provide purifying selection for the HE function, but insufficient to fix the parasite in the whole population simultaneously. The process of fixation might be further delayed by a complex population structure. Decay and loss of the parasites' fitness already might occur in one subpopulation, whereas other subpopulations might be only at the beginning of the invasion phase. The resulting situation is comparable to waves in an excitable medium that run in circles, as in a fibrillating heart-muscle . In vma-1 intein in Saccharomyces cerevisiae [Naegleria isolates lost the intron. The same argument applies to the PRP8 intein: The acquisition of a new function is at odds with the presence of several PRP8 genes in euascomycetes that lost either the HE domain or the whole intein altogether. If the HE acquired a function beneficial to the host, why was it lost repeatedly during evolution?Hypothesis A is compatible with the other three scenarios, and the presence of a HE containing parasitic gene already suggests a low level of gene flow occurring in these presumably clonal organisms. It will be interesting to learn how the homing process in these organisms begins – in the best studied example, the revisiae -34, homirevisiae ,36. Hyporevisiae  acting iHypothesis C is unlikely to be a sufficient explanation on its own. It would require an exact balance between forces that increase and those that decrease the HE frequency. A slight shift away from the equilibrium would mean either fixation or extinction for the HE containing parasite. However, an approximate balance between decreased fitness of the host and active spreading of the parasitic element, combined with a complex population structure could lead to long persistence times without the homing cycle operating, especially when the selective disadvantage to the carrier and the mobility of the element were not the same in different subpopulations. This hypothesis on the surface appears similar to hypothesis D; however, in the absence of the homing cycle the empty target sites would not result from decay and loss of the parasitic element, but from the faster growth rate of the organisms that never possessed the element.The asynchronous homing cycle (hypothesis D) is a likely contributor to the long term persistence of functional HE in a single species. To corroborate this hypothesis, and to differentiate it from hypothesis C, more detailed population studies need to be performed, including sampling of isolated subpopulations with limited migration between them, and thorough sampling of well mixed isolated populations. In addition, a sampling of related species is also necessary since the detection of interspecies transfers depends on a sufficient sampling of taxa. At present it cannot be excluded that some interspecies transfer of the HE went undetected.The unexpected complexity in the life cycle of inteins reported by Butler and colleagues reinforces the need for detailed epidemiological studies of HE containing parasitic genes. These studies should be combined with attempts to better understand the biochemical and physiological regulation of the parasitic genes, with measurements of their effect on the fitness of their host, with determination of the transmission efficiency of the parasite, and with phylogenetic analyses that might reveal the frequency of HE loss form the parasitic gene. In case of introns the study of the epidemiological dynamics can be further complicated by several factors (see  for a re