The problem of training human resources in health is a real concern in public health in Central Africa. What can be changed in order to train more competent health professionals? This is of utmost importance in primary health care.Taking into account the level of training of secondary-level nurses in the Democratic Republic of the Congo (DRC), a systemic approach, based on the PRECEDE PROCEED model of analysis, led to a better understanding of the educational determinants and of the factors favourable to a better match between training in health sciences and the expected competences of the health professionals. This article must be read on two complementary levels: one reading, focused on the methodological process, should allow our findings to be transferred to other problems . The other reading, revolving around the specific theme and results, should provide a frame of reference and specific avenues for action to improve human resources in the health field .The results show that it is important to start this training with a global and integrated approach shared by all the actors. The strategies of action entail the need for an approach taking into account all the aspects, i.e. sociological, educational, medical and public health.The analysis of the results shows that one cannot bring any change without integrated strategies of action and a multidisciplinary approach that includes all the complex determinants of health behaviour, and to do it within the organization of local structures and institutions in the ministry of health in the DRC. A partnership of the Ministry of Health of the Democratic Republic of the Congo (DRC) – more specifically, the directorate that is in charge of health science education – the French-speaking community of Belgium and various education and training associations made it possible to set up and carry out a teaching innovation project to bolster human resources in the health sector. One of the major public health challenges in Africa is to find efficient ways to enhance human resources in the health sector.The goal of the medical policy in the DRC is to promote the health of the population by providing high-quality health care that is complete and integrated and in which the community participates, within the general context of the fight against poverty . With th• restructuring the health system according to political, legislative and administrative orientations as well as updating standards of services;• increasing the availability of resources by implementing an adequate administrative process;• establishing an integrated system of preventive and curative care and health promotion for the target groups;• strengthening the programmes of support to health activities;• coordinating, promoting intrasectoral and intersectoral collaboration and partnership for health;• promoting a suitable environmental framework for health.The Ministry attaches priority importance to delivering high-quality care and health services and by: reaffirming the strategy of primary health care (PHC) as a fundamental option of the national policy on health; reaffirming the health zones' or districts' achieving a minimum package of activities as an operational unit; and regular procurement of essential drugs, including biological products and other laboratory reagents.Training of nurses in the DRC is organized in all provinces of the country and conducted through technical medical institutes (ITM), medical educational institutes (IEM) for the secondary level and higher institutes of medical technology (ISTM) for the higher level. In 1998 there were 308 ITM and ISTM in the country; to date there are 254 schools of nurses  at the secondary level in the country recognized by the DRC government.According to the type of management, these institutions are classified as public, officially agreed and private schools. However, the autonomy of management for all these schools is extensive, given the near inexistence of government subsidy. The infrastructure and quality of training differ widely from one ITM to another.As a rule, the solutions that one observes in public health are located in the area of further training for health professionals. To the extent that further training is important, it is disappointing to ascertain the low yields that these various training courses have in improving the quality of health care and services . There iWhen it comes to basic training, the young nurses who have just graduated from secondary school and make up the critical mass of health professionals in primary health care are commonly required by specific private or church employers to enrol in a full year of training after their academic studies in order to try to fill the gaps between their basic training and health professionals' actual training needs. Confusion on the part of the ministries and other forces involved is attested to by the absence of vision and lack of expertise in educational research and the lack of reforms of the educational curricula in order to keep pace with the fundamental changes that have resulted from the decentralization of primary health care.In addition, several other problems make this succinct analysis more complex. The unemployment rate for the country's nursing school graduates is extremely high, even for those who graduate from the best schools in Kinshasa, the capital. The situation is compounded by the almost total absence of quality management mechanisms, especially when it comes to taking stock of the health workforce that exists. The situation is a complex one in which the expected changes are not clearly discerned.The problem of health sector human resources is vast. The context that interests us in this article is that of the human resources who are in the front line when it comes to grappling with the various communities' health problems. These are the nurses who have completed  secondary school courses. They are the main primary health care professionals in Central Africa, especially in the DRC . This chThe question of research is at three levels: on the theoretical level, this article proposes importing a theoretical model from one field to another; on the methodological level, it uses the action research-like mode of data collection to better establish results; on the empirical level, in the DRC research is unusual ground from which to introduce an innovation.This qualitative research pursues two objectives: to present a methodology  and to study the results of its application . The qualitative hypothesis that subtends this thinking is that using an analytical, systemic planning and strategic synthesis model based on a systemic and participatory approach on various strategic and operational levels will procure the necessary vision for changing the basic education and training of nurses in  secondary schools in the DRC.There is a lack of literature on experiments and experiences that use analytical or planning methods to understand complex social realities and consequently the adoption of strategic plans of action that should result from such experiences. It is thus important for all the players in the process to use the outcomes of the various stages of analysis and planning to produce an appropriate and adapted logical framework. It is necessary, however, to be able to set down on paper the methodology that is used to be able to construct a model that by the end of the process appears obvious for the actors involved, i.e. teachers, internship supervisors and school management, personnel within the ministry's Directorate for Health Education and project officers.Generally speaking, three relatively distinct stages were necessary, as follows:In the DRC, the Ministry of Health and more specifically its directorate in charge of health science education, is responsible for the basic training of secondary school nursing students and further training of health professionals Figure .The situation sometimes varies in neighbouring contexts. Thus in Rwanda, for example, the Ministry of Education is in charge of basic education and training in the technical schools and for the medical professions. In France and Belgium, the general choice was to have the Ministry of Education responsible for most of the training and education of health professionals.We shall not discuss in this article the relevance of the place of oversight for this type of brief for training health professionals. We shall limit our remarks to the need to choose the best place for systemic planning and strategic vision in the existing context. So it is that in the 6th Directorate of the Ministry of Health of the DRC, which is in charge of paramedical secondary education, the need was perceived to develop systemic planning tools that would give a comprehensive, consistent vision of the sector's needs. While the 6th Directorate is indeed the strategic level, there was early involvement of an operational level, in the form of a sample of schools and teachers, and creation of a management unit to guide the implementation of the plans by the 6th Directorate and teachers from the grassroots.It is important to remember that we are talking about systemic and operational planning, not just strategic planning . For theThis article follows on from the given that organizations and human beings are complex, and one way to have public health actions that heed this complexity is to use a systemic approach to analyse them .Various models for a systemic approach exist. The approach that we chose to develop a logical framework for analysing, planning, and synthesising the work of the ministry's directorate in charge of health science education in the DRC is Green and Kreuter's PRECEDE PROCEED model ,7. The PThe PRECEDE PROCEED planning model is welcome because of its multidisciplinary approach, based on the fields of epidemiology, social sciences, behavioural sciences, education and health administration. In a nutshell, the fundamental principles that gave rise to this approach come from the multifactoral nature of all problems. Once this has been posited, all efforts made to act upon behaviour, the environment and social factors must necessarily be multidimensional and multisectoral.The acronym PRECEDE means "Predisposing, Reinforcing and Enabling Constructs in Educational/Environment Diagnosis and Evaluation", while the acronym PROCEED means "Policy, Regulatory and Organizational Constructs in Educational and Environmental Development".The PRECEDE-PROCEED model emphasizes planning interventions by focusing on the expected outcomes of actions based on epidemiological, social, behavioural, environmental, educational, organizational, administrative and political diagnoses of a socio-health and/or educational situation. The stages in the construction of a systemic model for analysing the problem that interests us – health science teaching – are adapted as the process unfolds. One of this method's great potentials is its great flexibility, or its ability to adapt to the specific analyses' needs.A systemic analysis and planning model is built dynamically, in a process that calls for continuous assessment. The model that the ministry's 6th Directorate came up with, and that is presented below, must change with changing knowledge in the area.It is important to stress the qualitative process of continual exchanges and constant observations among the players  that made it possible to fill the gaps in the information-gathering process. All these elements are much more difficult to organize in one well-defined stage, but are indeed part of a process that stresses the participatory approach and comes under the third strand of the methodology being discussed. The development of the first model proceeded during the workshop held in Kinshasa at the starting of the project, with the participation of personnel from three pilot schools and the Ministry of Health in October 2002. The three-day workshop, with 40 participants from various institutional levels, permitted the establishment in December 2003 of strategic orientations and guidelines for the continued broadening of the programme.All PRECEDE PROCEED models are built upon the players' actual experiences of the problem to solve. The clarification of the problem itself, which is part of the epidemiological and social diagnosis, comes out of a debate that must be conducted with all the parties concerned. This problem will gradually become more and more clear as its statement shuttles back and forth among the parties until it eventually satisfies the strategic and institutional level that is in charge of the programme and that the problem concerns directly. If, thanks to a resolutely participatory approach, all of the players adopt the use of a systemic approach on a real strategic, managerial and operational level, it will become a solid tool for the entire teaching body concerned.The presentation of results is at two levels: PRECEDE results and PROCEED results. The first are mainly descriptive . PROCEED results are more normative, leading to certain recommendations for practitioners and other actors.To facilitate presentation of the results and understanding of this coherent, overall vision of the interrelated elements, it was considered pertinent to retranscribe the full model as it exists for the 6th Directorate of the DRC's Ministry of Health.To structure the results' presentation, we shall follow the order in which the model's construction progressed. The table must be read from right to left, starting with the epidemiological and social diagnosis, then going on to the behavioural diagnosis and from there to the analysis of the educational and environmental determinants of these behaviours, and then to end with the analysis of the institutional diagnosis see .In the Ministry of Health, all the players are concerned by the mortality and morbidity indicators in the country. For health professionals, the lack of quality of the service provision and care provided by their health system is an obvious cause of the people's lack of confidence in their health system . HoweverWho are these players and what behaviour can explain, through a direct link, the diagnosis of inadequacy? In answering these questions with the players themselves, we discover that there are groups of players that are never clearly identified yet are clearly related to this problem of inadequacy. This is the case, for example, of the donors and nongovernmental organizations (NGOs).Revealing all the groups of players makes it possible to see more easily why importance should be given to a multisectoral approach, especially one that covers teachers and medical and paramedical professionals. If the population is considered a group of players that is separate from the problem at hand, it will not be possible to take it into account in setting up action strategies, to the extent that the aim of such work is to better define people's expectations in terms of the quality of care and arrive at a better understanding of their behaviour.During the discussions, the teachers felt that priority had to be given to separating the group of teachers from that of intern supervisors in order to better highlight the particularities and role of the field training. The school managements revealed their specific role in this problem of mismatches. Indeed, the teachers' and supervisors' behaviour is strongly linked to their own behaviour in dealing with changes . We haveThis diagnosis allows for the factors that are linked to the environment and are direct causes of the epidemiological and social diagnosis. In a context such as that of the DRC, geopolitical and socioeconomic factors head the list, along with the health structures' inaccessibility. To take a more constructive approach without denying reality, it is necessary to focus the analysis of this diagnosis on the more targeted problem of the inadequacies in the training sector. This reveals variables that are more controllable for the levels that are concerned and that everyone agrees are connected to the problem. These are: the learning environment, teaching environment, class hours that facilitate or hamper certain types of learning, etc.The educational diagnosis enables one to home in on the educational and motivational determinants, which must not be overlooked when one goes on to an interventional phase. To the extent that the systemic approach gives significance to each group of players  as well, as is the case in the DRC, it is fully possible to set up a frame of analysis, assessment and action-research that presents the variables and determinants in a PRECEDE model that are specific to each specific group (action-research framework). This is what was done in the DRC to follow the changes in teaching practices, in conjunction with each intervention that was identified, that were made in the specific group of teachers. The results show that it is relatively easy to separate the educational determinants from each other in order to facilitate subsequent reflection about the strategic action to take.The predisposing factors that concern knowledge, experience, attitudes, perceptions and representations have a key place in relation to the behaviours of the players of interest to the Directorate for Education. This construction shows clearly that the training given is usually concerned with knowledge only and generally does not make use of the learners' life experiences.The other important result is to be able to visualize the place of representations in a conceptual framework that will likewise be used for the action. For example, there are the various representations of learning theories when it comes to teaching methods or unfounded beliefs about the quality of care. Specific models exist that enable one to delve much deeper into perceptions and beliefs ,11. ThesThe enabling factors in terms of actual competences  are too often disregarded and underestimated in interventions. Incorporating them in this model thus enables the directorate in charge of this branch of education to check to what extent the projects, programmes and other support measures consider this priority strand in terms of development independence.The reinforcing factors, which are sometimes also referred to as facilitating factors, are the determinants that act upon the positive feedback loops. The importance that all the players give to this type of variable in constructing the model confirmed the need that the directorate had already felt to find means to set up long-range monitoring mechanisms for the various activities engendered by the programme or by some more specific projects.The model contains a certain number of variables. It is clear that it can be enriched in the course of the process through its use and the players' better discovery and gradual appropriation of its features.In terms of results, the institutional diagnosis requires analysing the situation at the organizational level that corresponds to the level of the model's application. This is a national health science education programme under the Ministry of Health. As such, the institutional diagnosis stresses essential strategic variables if one wants to work on a well-knit, comprehensive set of changes. It thus entails the need to analyse the institutional standards when it comes to inspections and assessment, but also those governing health system management and health sector human resource quality management . This is also the level on which we shall discuss how the programme dovetails with other variables and determinants.Before strategic thinking can be put into place based on this situational analysis, it is possible to go on to a more dynamic reading of the relations between determinants and variables. So it is that the DRC's Ministry of Health directorate in charge of health science education foresees a certain number of strategic axes for action. The aim of the action is the problem's translation into an objective form. The directorate thus considered its main goal to be to improve the match between what is learnt in schools and health professionals' needs and the population's expectations.To better understand how the reading of the conceptual model brought us to the action strategies, it is useful to stress an intermediate step that is summarized in Figure A natural adaptation of the PRECEDE model was to define the groups of players by their behaviour. In this way, we obtained a better picture of the division of responsibilities to achieve a common goal and evidence of the need for interdisciplinary work . A comprThe results bring to the fore a number of behaviours that attest to a lack of independence, absenteeism, lack of collaboration, failure to connect theory and practice, a lack of communication, ignorance of the teacher's role, etc., depending on the group. Examination of these results prompts us to stress the importance that must be given to the learning environment and, when it comes to action strategies, the importance to give to a learning environment that is in tune with the strategic axes that are selected, in this part just as in the other parts of the situation's analysis.Given this finding and the need to link the educational and institutional diagnosis with the health problem (seen as an appropriate form for education), one proposed strategic hypothesis is to favour learning techniques that are based on active teaching methods .In going consistently through the various diagnoses and organizational levels, this choice led the education directorate to think about changing its programmes and standards so as to base them on novel teaching concepts such as skills-based learning  and settThe reading of these results in terms of strategic action reveals the need to bolster the analytical and planning process that already exists within the directorate to pay more attention to the educational and environmental determinants for all the target populations concerned. To our mind, the success of the expected changes in terms of narrowing the gap between the "supply" and the "demand" hinges on this.The following diagram summarizes the strategic axes that the Directorate for Health Science Education chose to achieve this objective on the basis of the PRECEDE analysis Figure .This figure reveals four axes to be reinforced:• to reinforce communication and coordination in conjunction with the other reinforcing factors: the pilot schools' teaching method committees, teaching monitoring and feedback, the setting-up of networks, etc.;• to develop methods to enhance the learner's autonomy: active teaching, constructivist approach, interdisciplinary, critical spirit, etc.;• to foster a learning environment that enables the learners to acquire knowledge: library, teaching materials, computer learning, computerized documentation centre, etc.;• to provide institutional and structural support: standards and curricula in tune with teaching and organizational innovations and skills targets that fit health professionals' needs and meet the community's expectations.The discussion will take place on two levels – the operational and the conceptual. On the operational level, we feel it is interesting not to dwell on the presentation of the model as it could have been applied, but on its actual application. The results are presented so as to allow the reader to understand how to organize the problems that are felt to exist in health science education in the DRC.Even if the Directorate for Health Science Education is well aware of its problems, the systemic modelling of the interconnected variables and populations seems to give it a conceptual and operational tool that is useful on various levels, as follows:• tool for dynamic analysis of the situation with regular updates;• tool for systemic planning that also enables the directorate to put forward arguments in dealing with donors and NGOs in the sector;• assessment tool that gives more importance to assessment criteria such as cohesiveness, consistency, relevance, appropriation, and comprehensiveness, i.e., process criteria;• research and evaluation tool that can also promote a more quantitative approach to analysing the relations between variables and various diagnoses or within the same diagnosis;• a dialogue-enhancing tool, for it gives the groups of players involved a vision of the planned change and a common objective.To sum up, this is a tool that provides a certain guarantee that the strategy development process is informed, meets the needs and is complete .The list of these advantages is obviously based on some baseline conditions: a participatory process in which the model is developed and operates and the appropriation of the concepts that subtend the model . Even thOn the conceptual level, the discussion will revolve around Figure We observed through the PRECEDE analysis and then the PROCEED strategic reflection phase that many disciplines converged in order to lead us to this hypothesis and a common objective of needs-matching. Indeed, when action is carried out it will be a matter of achieving a gradual advance that occurs along the  strategic axes defined earlier in this article. Moreover, we are confronted with strategic choices that involve at least three dimensions: a public health approach, an education approach and a sociological approach.These three dimensions are part of the data collection process's success, as well as the success of the strategic choices that follow. This reinforces the fact that the PRECEDE PROCEED model comes from the development of an approach aimed at meeting the need for education and health promotion tools and methods. So it is that we see numerous applications of this process in technical health education establishments that spring from a true systemic analysis of the problems with full mastery of a structuring capacity, unlike some other models such as causal analysis (17).Similarly, we can consider that defining a problem at an institutional and organizational level also requires the identification and involvement of all the parties concerned. We can also consider that the tools that help to understand the relations between elements and insist on a better search for behavioural determinants are prerequisites for organizational learning that has groups of players interact with each other. This is all the more true if the change that is ultimately expected (a match between two sides of the equation) is contingent on changes in the players' behaviour and practices, as is the case of health education.In terms of limits of this research, it targets the analysis of an inadequacy within human resources' management in health, which is that of training of nurses from professional technical levels. Other levels of inadequacies are worthy to be analysed in a complementary way relating to other health professionals, the sectors of health and education planning. The Green model is complementary to the use of methodological dynamic references much as the management of the project cycle focuses on managing interventions or projects whose aim is to contribute to changing a situation from unsatisfactory to satisfactory. Its use within the framework of the project could obtain more means while enabling developments relating to action research. In this context, the contribution from other disciplines, such as psychology, could be reinforced.With regard to the three levels of starting research – the theoretical, methodological and empirical – PRECEDE PROCEED analysis is a model that can be applied to varied situations and problems, although it must be used participatively and proactively in order to enhance its utility in specific circumstances as a personal transfer tool. On the empirical level, the will of all actors – and the Ministry of Health in particular – to have a clear vision of the projected change and manner of reaching that point, while integrating the complexity, was the element carrying the process. We advance the hypothesis that L. Green's systemic approach may become one of a set of active methods, such as problem-based learning, cooperative learning, or even project-based learning, to transfer to learners in nursing schools and sections in the DRC. Indeed, the ability to analyse and synthesize, but also to carry out education and health promotion actions, is essential.DRC: Democratic Republic of CongoPRECEDE: Predisposing, Reinforcing and Enabling Constructs in Educational/Environment Diagnosis and EvaluationPROCEED: Policy, Regulatory and Organizational Constructs in Educational and Environmental DevelopmentThe authors declare that they have no competing interests.FP is responsible for this research. She initiated the project in DRC. She is a specialist in public health and pedagogy. She participated in the design and coordination of the study and drafted the manuscript.JK and DB are two teachers in charge of the reform of the nursing programme in DRC. They set up the collection of data for this study and finalized the analysis.YC and AL participated in the design of the study and the adaptation of de Green's model in the field of nursing training. YC participated in writing the manuscript.MG specializes in management and pedagogy. She conceived the study with FP and participated in its design. DP took part in the elaboration of the methodology. She brought an expertise in health promotion and wrote part of the manuscript.All authors read and approved the final manuscript.Table 1. PRECEDE model for health science teaching in the DRC oversized tableClick here for file


Drosophila has emerged as an invaluable model organism for studying this regulation. Activation of the NF-κB family member Relish by the caspase-8 homolog Dredd is a central, but still poorly understood, signaling module in the response to gram-negative bacteria. To identify the genes contributing to this regulation, we produced double-stranded RNAs corresponding to the conserved genes in the Drosophila genome and used this resource in genome-wide RNA interference screens. We identified numerous inhibitors and activators of immune reporters in a cell culture model. Epistatic interactions and phenotypes defined a hierarchy of gene action and demonstrated that the conserved gene sickie is required for activation of Relish. We also showed that a second gene, defense repressor 1, encodes a product with characteristics of an inhibitor of apoptosis protein that inhibits the Dredd caspase to maintain quiescence of the signaling pathway. Molecular analysis revealed that Defense repressor 1 is upregulated by Dredd in a feedback loop. We propose that interruption of this feedback loop contributes to signal transduction.The innate immune system is ancient and highly conserved. It is the first line of defense and the only recognizable immune system in the vast majority of metazoans. Signaling events that convert pathogen detection into a defense response are central to innate immunity.  Drosphila, Foley and O'Farrell have identified numerous new regulators of the innate signaling response to gram-negative bacteriaBy silencing all conserved genes in As a typical metazoan suffers numerous microbial assaults during its lifespan, survival depends on robust defense strategies. Metazoan defenses are classified as either innate or adaptive. Adaptive immunity is characterized by elaborate genetic rearrangements and clonal selection events that produce an extraordinary diversity of antibodies and T-cell receptors that recognize invaders as nonself. While of profound importance, the adaptive responses are slow and limited to higher vertebrates. In contrast, the machinery of innate immunity is germ-line encoded and includes phylogenetically conserved signaling modules that rapidly detect and destroy invading pathogens . Model oSignaling in innate immunity consists of three steps: detection of pathogens, activation of signal transduction pathways, and mounting of appropriate defenses. The first step is triggered by the detection of pathogen-associated molecular patterns by host pattern recognition receptors . TypicalDrosophila as a model for analyzing innate immune signal transduction had a serendipitous origin. The Toll signaling pathway was discovered and characterized in Drosophila because of its role in specification of the embryonic dorsal ventral axis  pathway, mediates responses to gram-negative bacterial infection in osophila . Althougosophila . Althougosophila . The N-tDrosophila. We developed a cell culture assay that allowed application of this library to a high-throughput RNAi evaluation of Imd pathway activity. This screen identified numerous components of signal transduction , defined a hierarchy of gene action, and identified a novel gene, sickie (sick), required for activation of Relish. Focusing on regulation of the Dredd caspase, we identified a novel inhibitor of Dredd, Defense repressor 1 (Dnr1), which is upregulated by Dredd in a feedback loop that maintains quiescence. We propose that interruption of this feedback loop contributes to signal transduction.Unlike the Toll pathway, which was thoroughly studied in its developmental capacities, analysis of the Imd pathway is relatively recent. Its more complete genetic dissection may well define another conserved and fundamental pathway of immune signaling. Of particular interest, a pivotal step in the Imd pathway—the regulation of Dredd-mediated cleavage of Relish—is not understood. To begin to address this, we developed a powerful RNA interference (RNAi)–based approach to functionally dissect the Imd pathway. In collaboration with others at the University of California, San Francisco, we produced a library of 7,216 double-stranded RNAs (dsRNAs) representing most of the phylogenetically conserved genes of Dipt, that encodes an antimicrobial peptide, Dipt-lacZ. Commercial preparations of LPS contain bacterial cell wall material capable of activating the receptor PGRP-LC and act as gratuitous inducers of antimicrobial peptide genes in Drosophila tissue culture cells  genes, enhanced defense by RNAi (EDRi) genes, and constitutive defense by RNAi (CDRi) genes  genes .In an initial visual screen, dsRNAs that altered the induced or constitutive expression of β-galactosidase were selected as candidate innate immunity genes. We subjected all the initial positives to a more stringent retest where we resynthesized the candidate dsRNAs, retested these under identical conditions, and counted the number of β-galactosidase-positive cells. We defined DDRi dsRNAs as reducing the frequency of Dipt-lacZ-expressing cells to below 40% of LPS-treated controls, EDRi dsRNAs as increasing the frequency of Dipt-lacZ-expressing cells more than 2-fold, and CDRi dsRNAs as inducing Dipt-lacZ-expressing cells to a level equal to or higher than that induced by LPS. About 50% of the initial positives met these criteria, yielding 49 DDRi dsRNAs, 46 EDRi dsRNAs, and 26 CDRi dsRNAs . The entC. EDRiC genes are listed as both EDRi and CDRi . Inactivation of either actin with dsRNA directed to the actin UTRs demonstrated that both actin transcripts must be inactivated for an observable EDRi or CDRi phenotype C.Caenorhabditis elegans, several genes can be inactivated by RNAi in Drosophila without an obvious drop in the efficiency of gene inactivation –tagged Relish . We inactivated sick by RNAi and subsequently tested dsRNAs representing the five CDRi epistatic subgroupings for their ability to activate Dipt-lacZ expression in the absence of Sick  and those that downregulate an ongoing response (EDRis).Interestingly, groups of inhibitors implicate distinct pathways in immune regulation. For example, of the 17 genes that had the strongest EDRi phenotype, four encode splicing factors and four encode products that appear to interact with RNA. This functional cluster suggests that disruptions to some aspect of RNA processing/metabolism can substantially increase the number of S2 cells that activate expression of the Dipt-lacZ reporter in response to LPS exposure. While we do not know how RNA metabolism contributes to this phenotype, the repeated independent isolation of genes lying in a functional cluster reinforces a conclusion that the process is involved. Several other functional clusters were picked up in our screens. Three genes involved in Ras signaling  were identified as EDRi genes. In addition, we noted weak EDRi phenotypes with three additional Ras signaling components . These findings argue that Ras signaling downregulates responses to LPS. This might represent a negative feedback circuit. However, the finding that MESR4 also has a CDRi phenotype suggests that the Ras/MAPK pathway may also impinge on the maintenance of quiescence.(α-Tub84D), a kinesin motor (Klp10A), and microtubule-severing function (CG4448/katanin) were isolated as EDRi genes. Perhaps an event involving microtubule structures helps limit immune responses. The two cellular actin genes (Act5C and Act42A) were individually dispensable, but their joint inactivation produced both EDRi and CDRi phenotypes. A regulator of actin function, SCAR, was also identified as a CDRi, and both actin and SCAR CDRi phenotypes fell into epistasis Group II. This suggests that disruption of the actin cytoskeleton in quiescent cells can activate the immune response in a Relish-dependent fashion. Since S2 cells are induced to phagocytose bacteria, and changes in cell shape are induced in response to LPS, it would not be surprising if cytoskeletal functions contribute to immune responses. Indeed, microarray studies showed induction of numerous cytoskeletal components in S2 cells upon incubation with LPS . This and other tentative indications of involvement of this pathway were either not reproduced or fell below the threshold in retesting. We are left uncertain about SCF contributions to immune induction in our system.A previous conventional genetic screen for mutations leading to constitutive action of the Imd pathway in d Relish . A Skp1/As in the case of the CDRi and EDRi phenotypes, our screen for DDRi phenotypes identified numerous genes falling into functional categories. One potential limitation of our approach for identification of DDRi is that some genes required for ecdysone maturation may be selected as immune deficient. Additionally, one of the largest functional categories was genes involved in translation and included four ribosomal proteins, three initiation factors, two amino acyl-t-RNA synthases, and an elongation factor. It seems likely that RNAi of genes in this category affects translation of the Dipt-LacZ reporter, as opposed to affecting modulation of signaling events. To cull our collection of DDRis of such indirect modulators of the response, we developed a secondary screen that does not rely on de novo gene expression. Based on the previously described phenotypes of Imd pathway members, we reasoned that inactivation of the core components transducing the signal would compromise activation of the Relish transcription factor. To identify DDRi dsRNAs that prevented Relish activation, we prepared a GFP-Relish reporter cell line and rescreened DDRi dsRNAs for loss of GFP nuclear translocation in response to LPS.sick as involved in Relish nuclear translocation in response to LPS. Although cells treated with sick dsRNA failed to mount an immune response, the cells were otherwise healthy through the course of the experiment. Dox-A2 RNAi reduced the survival of cells and was effectively lethal within a few days of the scoring of the immune response. We conclude from this that Sick and Dox-A2 contribute to the central signal transduction process, but it is presently unclear whether Dox-A2 has a significant specific input or if its effects are secondary to a global effect on cell viability.In addition to confirming a requirement for Dredd and PGRP-LC in Relish activation, we implicated a proteosomal regulatory subunit Dox-A2 and identified a novel gene It is notable that only two DDRi genes passed our secondary screen based on GFP-Relish localization. Does this mean that all the other DDRis are not really involved? While we have not yet analyzed all these genes, we suspect that many of them will modify the Imd pathway, either impinging on the pathway at a point beyond Relish translocation, or quantitatively or kinetically modifying Relish translocation in a manner that we did not detect in our screens. Insight into this issue is likely to be derived from further epistasis tests that might place some of these DDRis in the signaling pathway.We identified an unprecedented large number of immune response inhibitors (CDRi genes) in our screens. As there are diverse steps within and potentially outside the Imd signaling pathway at which the CDRi inhibitors might act, we sought to position their actions with respect to known Imd pathway functions by RNAi epistasis tests. By sequential inactivation of known Imd pathway components and CDRi gene products, we tested whether constitutive activation of immune reporters by CDRi dsRNAs depends on steps in the signal transduction pathway. In this way, we defined five distinct epistatic categories of CDRi gene products. The four CDRi genes that continue to activate immune responses despite inactivation of Imd, Dredd, or Relish are likely to act on signal-transduction-independent factors that maintain transcriptional quiescence of Dipt. The largest group of CDRis (12) depends on Relish function but not on upstream activators of Relish. These are likely to include two types of regulators: one type that sets the threshold of response so that basal activity of Relish does not trigger pathway activity, and a second type that contributes to suppression of Relish activity. The latter type of regulator might include inhibitors that impinge on the late steps in the signal transduction cascade. For example, genes whose normal function inhibits the activity of the full-length Relish transcription factor might be required to make the pathway activator dependent, and these would be found in this category. The remaining upstream epistasis groups that rely on additional signal transduction components are strongly implicated as significant contributors to the immune induction pathway. As all of the CDRis induced robust immune responses in the absence of ecdysone (unpublished data), we propose that the CDRis have their input into the Imd pathway at a level that is the same or lower than the level of the input from ecdysone. Given that this is true for all five epistatic groups of CDRis, the result suggests that ecdysone has its input at an early level of the Imd pathway.sick is required for constitutive activation of the responses by inactivation of CDRi genes in Groups III, IV, and V genes but not for the action of CDRi Group II or Group I genes. If we assume a simple linear pathway, this would indicate that Sick functions upstream of Relish and downstream of Imd and Dredd. It is noteworthy that the epistatic data are consistent with molecular data indicating that Sick is required for Dipt-lacZ induction and the nuclear translocation of Relish in response to LPS. This combination of phenotypic, epistatic, and molecular data argues for participation of Sick in the regulated activation of the Relish transcription factor.The identification of five epistasis groups of inhibitors also provides reference points for a second round of epistasis tests that position novel DDRi genes within the Imd pathway. We used this approach to show that the novel DDRi One epistatic group struck us as particularly interesting. While Dnr1 inactivation caused ectopic Dipt-lacZ expression, simultaneous loss of Dredd or Relish restored cells to their resting state. These data indicate that the wild-type function of Dnr1 is to prevent Dredd-dependent activation of Relish. Consistent with this hypothesis, we identified a C-terminal RING finger in Dnr1 with greatest similarity to the RING finger motifs observed in the C-terminus of IAP proteins. In addition to regulating caspase activity, IAPs also regulate their own stability through ubiquitin-mediated proteolysis. Similarly, we observed that mutation of a critical RING finger residue greatly stabilized Dnr1. These features suggest that Dnr1 is a caspase inhibitor, suggesting that it might act directly to inhibit Dredd activity.We observed that exposure of cells to LPS transiently stabilized Dnr1 and that this stabilization directly paralleled the period of Dredd-dependent Relish processing. This suggested to us that Dnr1 stability and accumulation might be regulated by its target, Dredd, a regulatory connection that could establish a negative feedback loop. We confirmed that Dredd activity is required for accumulation of Dnr1. These results suggest that Dredd modulates a RING-finger-dependent Dnr1 destruction pathway .Our results are consistent with a feedback inhibitory loop where Dredd activity promotes accumulation of its own inhibitor ; howeverIn summary, a new and powerful screening approach has provided many candidate regulators of the Imd pathway of the innate immune response, and we suggest that the newly identified contributors Dnr1 and Sick will govern central steps in the regulatory cascade that activates the Relish transcription factor. While our analysis has led to a focus on these two regulators, we suspect that other genes among those isolated will also make important direct contributions to the Imd pathway. Furthermore, some of the groups of genes falling into functional clusters are likely to define physiologically relevant inputs into the induction pathway.A library of DNA templates bearing the T7 RNA polymerase promoter at each 5′ end was prepared from genomic DNA in a two-step PCR protocol. In the first round of PCR, targeted regions of DNA were amplified using gene-specific primers (18–22 nucleotides) with a 5′ GC-rich anchor (GGGCGGGT). Primers were designed to amplify a region of nonintronic genomic DNA between 250 and 800 bp with minimal sequence overlap to all other amplimers. Templates from the first step were amplified in a second round using a universal primer containing the T7 RNA polymerase promoter sequence followed by the GC-rich anchor TAATACGACTCACTATAGGGAGACCACGGGCGGGT. dsRNA was generated from templates in in-vitro transcription reactions for 6 h at 37 °C. In vitro transcription products were annealed by heating to 65 °C and slowly cooling to room temperature. All products were tested for yield and size by gel electrophoresis, with 97% giving satisfactory results.Drosophila Expression System Protocol using hygromycin B  as a selection marker. The Dipt-lacZ plasmid has been described previously  supplemented with 10% heat-inactivated fetal calf serum, penicillin, streptomycin, and hygromycin per well. dsRNA was added to each well at a final concentration of 10 μg/ml. Cells were cultured for 4 d at 25 °C and incubated an additional 24 h in 1 μM 20-hydroxyecdysone . LPS  was added at a final concentration of 50 μg/ml for 12 h. RNAi protocols were as previously described  with 40,000–50,000 cells in 200 μl of Schneider's escribed .2, 3.5 mM K3Fe(CN)6, 3.5 mM K4Fe(CN)6, and 0.2% X-Gal in DMF). β-galactosidase activity assays were performed as described previously  IMRB microscope. Immunofluorescent images were taken on an Olympus  IX70 driven with DeltaVision software . S2 cells were deposited on Superfrost Plus Gold slides  for immunofluorescence. Cells were fixed for 10 min in 4% formaldehyde (Sigma). Tubulin was detected with mouse anti-α-tubulin (Sigma). DNA was visualized with Hoechst 33258, and actin was visualized with rhodamine-coupled phalloidin . Images were processed with Adobe Photoshop 5.5, and figures were assembled with Adobe Illustrator 9.0.g for 3 min, washed in PBS and lysed on ice for 10 min in lysis buffer . Lysate was spun for 10 min at maximum speed, and the supernatant was added to sample loading buffer. Samples were separated by SDS-PAGE and analyzed by Western blotting. Anti-GFP antibody was purchased from BabCO , and HA and actin antibodies were purchased from Sigma.Dnr1-expressing vectors were prepared by cloning Dnr1 cDNA into pAc5/V5HisA (Invitrogen). The C365Y mutant form of Dnr1 was prepared with the Stratagene point mutation protocol using a TTCAATCCGTACTGTCACGTC sense primer and a GACGTGACAGTACGGATTGAA antisense primer. The mutation was confirmed by sequencing. For experiments with z-VAD-FMK, S2 cells were incubated in 100 μM z-VAD-FMK for 4 h at room temperature. Cells were harvested by centrifugation at 1,000


In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly—with no detectable lag—upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2–Arp3–actin nucleus. This actin monomer becomes the first monomer of the new daughter filament. This paper provides the biochemical and biophysical basis for actin filament formation, necessary for cell shape and motility The actin cytoskeleton determines the shape, mechanical properties, and motility of most eukaryotic cells. To change shape and to move, cells precisely control the location and timing of actin filament assembly by regulating the number of fast-growing (barbed) filament ends . The actThe Arp2/3 complex must be activated by both a member of the Wiskott–Aldrich syndrome protein (WASP) family and a preexisting actin filament before it will nucleate a new actin filament . The strBoth the Arp2 and Arp3 subunits of the complex bind ATP . HydrolyWe show here that the Arp2/3 complex rapidly hydrolyzes ATP on the Arp2 subunit upon filament nucleation. There are several events in the Arp2/3 nucleation reaction that might trigger ATP hydrolysis on Arp2: (1) binding of VCA to the Arp2/3 complex, (2) binding of VCA-Arp2/3 to the side of a preformed filament, (3) binding of a VCA-tethered actin monomer to the Arp2/3 complex, or (4) binding of a second or third actin monomer to form a stable daughter filament. We find that ATPase activity requires the combination of a preformed actin filament, a VCA domain, and an actin monomer, but does not require actin polymerization. This indicates that hydrolysis is triggered relatively early in the nucleation reaction—before completion of a stable daughter filament. Capping the pointed ends of actin filaments also stimulates Arp2 to rapidly hydrolyze ATP in the absence of monomeric actin and VCA and without branch formation. Thus, ATP hydrolysis on Arp2 is stimulated directly by interaction with conventional actin, presented to the complex either as a monomer attached to the VC domain of the WASP-family protein or as one of the subunits making up the pointed end of a preformed filament. To our knowledge this is the first direct evidence that the monomer supplied by the VCA domain is the first monomer of the new daughter filament. From these observations we propose a model for the mechanism of Arp2/3 complex activation by WASP-family proteins.32P-8-AzidoATP to the Arp2 and Arp3 subunits of the Arp2/3 complex  exposure to UV light. In the presence of γ-32P-AzidoATP at concentrations above the KD for ATP  to show that UV irradiation covalently crosslinks α- complex . Here we for ATP , γ-32P-Aficiency A. Additiaffected A. The ef32P-AzidoATP-Arp2/3 with 2 μM monomeric actin in polymerization buffer and initiated polymerization by adding 750 nM VCA, which activates rapid actin filament nucleation by the Arp2/3 complex .  We assayed timepoints both by SDS-PAGE and thin-layer chromatagraphy (TLC) during the same reaction to monitor remaining and cleaved 32P, respectively (32P cleavage) and cleavage has ceased by 90 s  (unpublished data). As a control, 32P-ATP hydrolysis is only seen when the Azido-ATP is covalently crosslinked to the Arp2/3 complex -bound Arp2 is inaccessible to the enzyme and remains unconjugated orthophosphate. We quantified hydrolyzed 32P-ATP and released phosphate by TLC  to calibrate the stoichiometry of ATP hydrolyzed by Arp2 , coincident with filament debranching , and nucleation is rapid (using N-WASP VCA), initiation of the polymerization reaction causes striking and near-complete ATP hydrolysis on Arp2  the Arp2/3 complex binds the sides of preformed actin filaments; (2) the Arp2/3 complex binds to the pointed ends of filaments, either by remaining associated with the daughter filament following nucleation or by capping preformed pointed ends; and (3) the Arp2/3 complex may interact with an actin monomer bound to the VCA domain of a WASP-family protein. There is abundant experimental evidence for filament side- and pointed-end binding by the complex , 2001b. Based on sequence conservation and biochemical similarities, ATP hydrolysis on Arp2 is probably driven by a mechanism similar to that which stimulates ATP hydrolysis on actin. The molecular details of how polymerization activates ATP hydrolysis on conventional actin, however, are not well understood. A leading hypothesis is that a “hydrophobic plug”—a loop between subdomains 3 and 4 of actin  and Arc4 (p20) subunits . The ArcATP release = 330 s; 32P signal is unchanged by the addition of free ATP. The 32P signal is only equivalent to hydrolyzed ATP in the absence of free ATP. The addition of free ATP should cause the excess of uncrosslinked Arp2/3 complex to compete with the small fraction of crosslinked 32P-ATP-Arp2/3 complex and thereby significantly reduce the 32P signal. The observation that the 32P signal is not reduced, rather than confirming that removal of free ATP has no effect, instead confirms that contaminating ATP is present for the latter part of the “ATP-free” condition, presumably released slowly from monomeric actin. The lag in the polymerization created by the initial absence of ATP would be present in the experimental ATP hydrolysis measurement, but may not have been present in the nucleation data presented because this was generated by a model-dependent computer simulation  are more than an order of magnitude faster than debranching of Arp2/3-generated dendritic networks (approximately 1000 s) . The kin2). This is consistent with the fact that in vitro the binding energy of this interface is sufficient to drive the interaction and promote the active conformation of the complex directly, even in the absence of VCA or a mother filament  and gel-filtered before use. Rat N-WASP VCA (398–502) and Human Scar1-VCA (489–559) with N-terminal 6His tags and TEV cleavage sites were bacterially expressed and purified by nickel affinity chromatography.We purified Arp2/3 from tography . We flas2, 1 mM EGTA, 10 mM Imidazole [pH 7.0]). We took care not to unintentionally shear the phalloidin-stabilized actin filaments by using wide-bore pipette tips.We prepared phalloidin-stabilized actin filaments by adding 1/10 volume of 10× KMEI to monomeric actin at room temperature for 20 min to initiate polymerization, then added twice the concentration of phalloidin and incubated for a further hour at room temperature  and added 6 μM γ-32P-labeled 8-AzidoATP . After a 2-min incubation to allow nucleotide exchange, we crosslinked for 9 s using a UV hand lamp , added 1 mM ATP and 1 mM DTT to quench the reaction and buffer exchanged into 1× KMEI plus 100 μM ATP, 1 mM DTT using a NAP5 column . We used the Arp2/3 for assays within 10 min of crosslinking. The same actin (including 7% pyrene–actin) was used for both ATP hydrolysis assays and correlative pyrene–fluorescence polymerization assays. We took ATPase time points by mixing 400 μl of the reaction mixture with premixed 400 μl of methanol and 100 μl of chloroform. We ran the precipitated protein on SDS-PAGE gel to separate the subunits and quantified 32P-labeling using a phosphoimager . For phosphate cleavage assays, we quenched timepoints into 1/10 volume 26 M formic acid, spotted on cellulose TLC plates, and separated the components in 0.4 M KH2PO4 (pH 3.4). We separately ran 32P-ATP and 32P-ATP treated with apyrase as standards to confirm the separation of 32P-ATP and cleaved 32P, respectively (unpublished data). As an alternative method of quantifying cleaved 32P, phosphomolybdate was extracted as in We diluted freshly thawed aliquots of Arp2/3 to 2.0 μM in 1 mM MgClAcanthamoeba actin with 7% pyrene–actin to monitor actin polymerization by fluorescence  (–1)  . We calc(–1) cf. . PolymerWe prepared filamentous actin as above and stabilized filaments with stoichiometric Alexa-488 phalloidin . We mixed 2 μM Alexa-488 phalloidin–F-actin with 20 nM Arp2/3, passed twice through a 30-gauge needle to shear the filaments, and incubated at room temperature. Timepoints were taken by diluting 500-fold and rapidly applying to poly-L-lysine–coated coverslips for visualization. Filament images were quantified for length distribution and branch frequency by a custom MATLAB  routine.


With the rapid expansion of scientific research, the ability to effectively find or integrate new domain knowledge in the sciences is proving increasingly difficult. Efforts to improve and speed up scientific discovery are being explored on a number of fronts. However, much of this work is based on traditional search and retrieval approaches and the bibliographic citation presentation format remains unchanged.Case study.The Telemakus KnowledgeBase System provides flexible new tools for creating knowledgebases to facilitate retrieval and review of scientific research reports. In formalizing the representation of the research methods and results of scientific reports, Telemakus offers a potential strategy to enhance the scientific discovery process. While other research has demonstrated that aggregating and analyzing research findings across domains augments knowledge discovery, the Telemakus system is unique in combining document surrogates with interactive concept maps of linked relationships across groups of research reports.research report schema, a document surrogate of extracted research methods and findings presented in a consistent and structured schema format which mimics the research process itself and provides a high-level surrogate to facilitate searching and rapid review of retrieved documents; (2) research findings, used to index the documents, allowing searchers to request, for example, research studies which have studied the relationship between neoplasms and vitamin E; and (3) visual exploration interface of linked relationships for interactive querying of research findings across the knowledgebase and graphical displays of what is known as well as, through gaps in the map, what is yet to be tested. The rationale and system architecture are described and plans for the future are discussed.Based on how scientists conduct research and read the literature, the Telemakus KnowledgeBase System brings together three innovations in analyzing, displaying and summarizing research reports across a domain: (1)  An unfortunate consequence of specialization in the sciences is poor communication across research domains – which can hamper the knowledge discovery process. Research findings in one area may be pertinent to another, researchers may be unaware of relevant work by others that could be integrated into their work and important findings just outside a researcher's focus can be overlooked. Compounding this problem is the difficulty of keeping current with new research findings that continue to grow at an exponential rate.Reliance on keywords and/or subject indexing to find relevant literature limits the researcher's ability to precisely search for and locate specific research findings. For example, a typical database query to locate all research articles reporting a statistically significant relationship between caloric restriction and cancer would retrieve articles reporting both concepts as represented by the indexing and keyword search – but not necessarily linked together as a research finding, with information regarding reported statistical significance of the finding, nor, perhaps most importantly, lacking representation of the linkages among the retrieved document sets.This lack of "interactivity" among retrieved citations is a critical limitation of traditional search and retrieval systems. As stated by Swanson (1986) in his examination of "mutually isolated literatures:""Knowledge can be public, yet undiscovered, if independently created fragments are logically related but never retrieved, brought together, and interpreted [..] This essential incompleteness of search and retrieval therefore makes possible, and plausible, the existence of undiscovered public knowledge ."In addition to this limitation of search and retrieval, there are questions about representing a set of documents: What format or display of the retrieval set most enhances users' ability to identify which documents need to be examined in more detail? How can users navigate across document sets  to enhance the discovery process? The bibliographic citation format is used by virtually all bibliographic databases today to report the results of database searches. However, it does not provide a way for the user to quickly review retrieved results for research methods and findings or to quickly view the relationships among the documents in the document set. Abstracts, even structured abstracts, simply do not provide a format conducive to rapid review of retrieved citations. In fact, the bibliographic citation format itself has changed little for the past two hundred years – even though it does not present an accurate representation of either the research methods or the research findings in a document .In spite of great improvements in document retrieval over the past twenty years, most information systems developed to promote scientific discovery e.g., -6), are , are 6])A comprehensive approach to these challenges is the goal of the Telemakus research program. Telemakus was named for the son of Odysseus who went searching for his father, the legendary Greek hero of Troy. Similarly, the Telemakus research program is developing approaches and tools for searching, knowledge discovery and mapping domain knowledge. The overall vision is to enhance the knowledge discovery process through retrieval, visual and interactive interfaces and tools. In close collaboration with researchers in the biology of aging, a working knowledgebase system has been designed to present aggregated citation information and research methods and findings for display in a conceptual schema.The Telemakus KnowledgeBase System provides the user with both a macro- and micro-view of domain knowledge. The macro-view facilitates identification of patterns – both expected and unexpected occurrences of relationships among research concepts – and permits visualization and dynamic navigation of scientific domains. The resulting maps are analogous to citation mapping work done by Small  but, insThis article describes the theories underlying the Telemakus KnowledgeBase System, provides an overview of its implementation, reports initial user feedback and explores future directions. Telemakus system builds on prior research in the areas of: (1) schema theory, (2) concept representation and (3) information visualization.Schemas are generalized mental models that provide a guide for structuring the process of production and comprehension of texts: "...at the simplest level, a schema is a description of a complex object, situation, process or structure. It is a collection of knowledge related to the concept ." Accord"The crucial fact is that the cognitive constraints on information processing which require the formation of semantic macro-structures and which organize acts and speech acts in global units, at the same time have social implications: they determine how individuals wish, decide, intend and plan, execute and control, "see" and understand their own and others' speaking and acting in the social context. Without them the individual would be lost among a myriad of detailed incoherent pieces of visual, actional and prepositional information ."Research on the application of schema theory to scientific research includes the schematic representation of psychological reports , clinicaUnderstanding a written text is a process of fitting it into a larger schema known to reader as part of their previous knowledge about the world. It is reasonable to expect that presenting written texts in familiar formats can enhance and potentially speed up an individual's ability to review and analyze large document sets rapidly.Fuller  investigThe predictability provided by schemas also applies to a document's metastructure. For example, Dillon  has propA second core component of the Telemakus system is based on concept representation.Concept representation is an important component in accurately representing facts from the document. Characterizing the location of concepts in a scientific document can greatly facilitate accurate document representation.Indexing a document – using a vocabulary or thesaurus of terms to represent the document – is a standard method employed to improve retrieval of relevant documents. Yet traditional approaches to indexing fall short of true document representation: reducing the words found in the abstract, title or full-text of the document may be suggestive of the content but are not truly representative of the methods and research findings. The indexing literature is replete with studies documenting interindexer inconsistency, even among experienced professionals using familiar well-documented systems . Studies"The information retrieval (IR) problem can be described as a quest to find the set of relevant information objects  corresponding to a given information need, represented by a query Q. The assumption is that the query Q is a good description of the information need N. An often used premise in IR is the following: if a given document D is about the request Q, then there is a high likelihood that D will be relevant with respect to the associated information need. Thus the information retrieval problem is reduced to deciding the aboutness relation between documents and queries ."Another problem with current indexing practice lies in the way the unique structure of the information elements in the document is obscured. Scientific research reports have a highly predictable structure, with an introduction, methods, results and conclusion. Concepts mentioned in the introduction or conclusion section of a scientific article may not be the primary focus of the research described within the document. For example, the Introduction may include discussion of research among several animal models whereas the target of the research study itself is a specific breed of mouse. However, current indexing processes (whether human or automated) rarely discriminate between locations of concepts in the document for indexing purposes. In addition, index terms do not represent the connections between the various elements in the document; thus, a significant amount of critical information for the scientist is lost.® or any other bibliographic database today that will answer the question: "Has anyone ever published data that supports a connection between cancer and caloric restriction? If so, what was the intervention, what type of experiments were done and what were the findings?" A successful response to a query of this type is extremely difficult or impossible in traditional information retrieval systems because: " [..] conventional IR systems that employ isolated term assignments seem inadequate for queries which are specific and empirical in nature. If, on the other hand, retrieval systems provide a link to represent the relationships between the variables of interest as reported in the documents, queries [..] would be better answered. That is, precision might be enhanced for specific and empirical queries when the relationships between the index terms were specified in retrieval systems [For example, it is not possible to unambiguously retrieve citations from PubMed systems ."In other words, the researcher asking the questions above can retrieve a set of citations that contain both topics but still must go through the full-text of each document to determine if the research specifically answers the question.Several research studies have explored the utility of relationships captured from data tables and figures in scientific research studies. Fuller, et. al.  describeThe importance of data tables for expert decision-making was underscored by Malogolowkin, et. al. who found that cancer researchers rely on ideas presented in numerical displays in published research studies for much of their design of new research protocols . MalogolOh ,29,30 inIdentifying semantic relationships in text involves looking for certain linguistic patterns in the text that indicate the presence of a particular relationship (or research finding) using pattern-matching to identify the segments of the text or the parts of the sentence that match with each pattern: "If semantic relationships can be identified accurately in the text, retrieval results can be improved by eliminating documents containing the required concepts but not the desired relationships between the concepts ."The third component of the Telemakus system is based on visual mapping of reported research findings.As previously mentioned, indexing strategies rely on "isolated term assignments." This approach leads to the loss of two important sources of information: (1) intra-document information, i.e., the research relationships studied and tested and (2) cross-document information, which captures and links research relationships across groups of documents or domains. This loss is the result of breaking apart the context of clearly linked in research findings in the data tables and figures, concepts typically linked together (the x-y axes of the tables and graphs).Once the research relationships have been extracted, concept mapping, a means of spatially representing knowledge in a visual format, provides a potential solution to the challenge of maintaining the inter-relationships between documents and reported research findings. Spatial representations can assist in understanding conceptual relationships across a domain. They can also assist in identifying previously overlooked potential research connections.Numerous approaches to visualizing an information retrieval space have been explored e.g., ,32,33), ,33, 32,3While a review of information visualization strategies is outside the scope of this paper, there is a growing body of work related to mapping metaphors and visualizing large document sets and database search results to provide the user with the ability to visualize relationships among documents and their contents ,39. In aConcept mapping represents knowledge graphically through networks of ideas. Such networks consist of nodes (points) and links (arcs/edges). Nodes represent concepts and links represent connections between concepts. Concept mapping has been used for a variety of purposes, including to communicate complicated ideas and, as in the Telemakus system, to demonstrate connections among research findings.How might one apply the theories previously described in developing a comprehensive "real world" information retrieval and knowledge discovery system? As reviewed in the previous section, the Telemakus system is built on and extends prior research in the areas of concept representation, schema theory and information visualization. Work on components of what has become the Telemakus system has been underway for many years with a particular emphasis on the importance and utility of relationships extracted from data tables and figures ,42-44. FBased on how scientists use and want to use the research literature, Telemakus brings together three innovations in analyzing, displaying and summarizing research reports across a domain:1. Research Report Schema: Research methods and findings are extracted and presented in a consistent, coherent and structured schema format which mimics the research process itself and provides a high-level research report surrogate to facilitate searching as well as rapid review of retrieved documents.2. Research Findings extracted from data tables and figures are used to index the documents, allowing searchers to request research studies which report a relationship between two concepts of interest.3. Visual Exploration Interface provides a dynamic map of extracted research findings to graphically display what is known as well as, through gaps in the map, what is yet to be tested.The Telemakus system consists of a database, research report schema and tools to create relationship maps among concepts across documents. The research report schema serves as a surrogate for the study, methods and research findings for each document as well as providing an interactive search interface. The schematic representations include standard bibliographic information , information about the research design and methods  and, most importantly, research findings derived from data tables and figures.® (UMLS®) Metathesaurus® as the basis for creating a controlled vocabulary.The elements extracted by the Telemakus system from full-text documents are listed in Table The UMLS Metathesaurus is a rich database of information on concepts that appear in one or more of a number of different controlled vocabularies and classifications used in the field of biomedicine. It provides a uniform, integrated distribution format of over 95 biomedical vocabularies and classifications and contains syntactic information. All Metathesaurus concepts are assigned to specific types or categories – e.g., "Disease or Syndrome," "Virus" – and the Semantic Network contains information about the permissible relationships among these types – e.g., "Virus" causes "Disease or Syndrome" . The 200The thesauri are reviewed (curated by expert indexers) in order to create a consistent controlled vocabulary structure. As indicated in Table At present, data extraction utilizes systems with both manual and automated processes. An evolving thesauri-building and revising approach are important components of the Telemakus system to ensure that vocabulary identification and management reflect the specialized needs of the knowledge domain as new research concepts are identified and reported.®, etc.). Database elements are extracted and verified against the relevant thesaurus. As new concepts are identified the UMLS is checked for the preferred term and it is added to the appropriate Telemakus thesaurus – along with synonyms, narrower and broader terms.The knowledgebase construction process begins with an Internet search of a bibliographic database  by the researchers. Concentrating on the legends from data tables and figures focuses the extraction process and reduces the background noise of the full-text document, making the process tractable. In general, the information content of data tables and figures can be broken into two types: "facts" and "findings." Facts include reporting experimental design and comparative characteristics of animals in the study group . Findings are the results of the study (the research findings). Research findings are extracted from each of the "findings" data tables in a process described in Figure Table ®, (NLM®) is being tested as a means of automatically parsing the legends from the data tables and figures to identify preferred UMLS concepts for addition to the Telemakus thesauri. MetaMap maps arbitrary text to concepts in the UMLS Metathesaurus; or, equivalently, it discovers Metathesaurus concepts in text. With this software, text is processed through a series of modules. First it is parsed into components including sentences, paragraphs, phrases, lexical elements and tokens. Variants are generated from the resulting phrases. Candidate concepts from the UMLS Metathesaurus are retrieved and evaluated against the phrases. The best of the candidates are organized into a final mapping in such a way as to best cover the text [A current focus is the application of natural language processing (NLP) techniques to assist in the automation of concept extraction process. MetaMap, a program developed by the National Library of Medicinethe text .The Telemakus system architecture centers on: a relational database; a set of tools used to populate the knowledgebase with data extracted from bibliographic databases and full-text research reports; and several server side tools and programs responsible for delivering the content of the database to the public via the WWW. The entire system is built from open-source components, leveraging standard protocols and tools whenever possible.The document processing system is initiated by an analyst who runs, reviews and edits as necessary extractions from the document being processed. It currently consists of a number of discrete phases to download, extract and analyze each document. These services are built primarily in Java running behind Tomcat and Apache and accessed by the analyst through the browser.For the public Telemakus website interface, a number of open-source solutions have been selected and configured. An Apache web server intercepts all requests and delegates them to surrogate processes dedicated to each respective task. For requests to display the data from the database, the request is delegated to Zope, a content management service, for responding to the user's request. This typically includes running SQL queries against a PostgreSQL database and rendering the results in the conceptual schema that serves as a surrogate for each document. For tasks beyond simple queries and HTML requests, a Java Servlet™ is employed. As plain HTML is insufficient to effectively display and interact with the relationship map, a Java™ applet, TouchGraph, is used.TouchGraph is an open-source concept-mapping tool for creating and navigating links between information sources. The tool was chosen for Telemakus because of its flexibility, customizing capabilities, high quality source code and compatibility with most browsers and operating systems (OS). The TouchGraph visualization package serializes maps to and from XML. By using Java, HTTP and XML, TouchGraph makes it easy to dynamically feed content to generate interactive nodes-and-edges maps.Figure Figures The research report schema also serves as a convenient interface for searching for related research concepts, offering a rapid way of following research connections through the database. For instance, clicking on "killer cells, natural – ad libitum" would retrieve additional articles that present data tables linking those two concepts.The "map it" function, at the bottom of the retrieval set Figure  provides. Caloric restriction was an ideal starting point for Telemakus because it is an important and rapidly expanding specialized area of the biology of aging that is also highly interdisciplinary. Telemakus is a component of the Science of Aging (SAGE) project funded by the Ellison Medical Foundation. Other SAGE partners include the American Association for the Advancement of Science and Highwire Press, Stanford University (SAGE Knowledge Environment web site: ).The first completed Telemakus knowledgebase focuses on caloric restriction in aging and is freely available at Formal usability testing of Telemakus is underway and will be the subject of a future article. Because a major goal of the Telemakus research program is to study scientists' approaches and preferences for accessing and using the scientific literature in order to create models and approaches for user-centered knowledgebases, researchers have been involved in the iterative design and testing of the system from its inception. The primary goals of this evaluation are to:1. Determine scientists' preferences for working with the research literature.2. Model preferred features based on those preferences.3. Test the completeness of schema elements and structure as a document surrogate.4. Experiment with and identify optimal visual representations to meet user needs.5. Iteratively review/evaluate/test for improved performance in response to user feedback.6. Identify domain(s) for future knowledgebase creation.In general, response to each successive iteration of Telemakus has been positive and included constructive feedback for system enhancements and expansions. User feedback affirms that retrieval based on research findings is a unique and highly desirable core function. Further, the Telemakus schematic document surrogate has been enthusiastically received as a major improvement over the traditional citation format with abstract. As one researcher stated , "The strengths of Telemakus are doing what PubMed does not do, which is to give an outline of the main points and to allow searching off the figure/table legends, organisms/sources and outcome fields."Additional feedback relates to the labeling of concept relationships as "statistically significant." Some researchers are interested in knowing the level of reported significance  and asked for a detailed labeling to document this. In addition, there have been requests to consider labeling the relationships . Early testing of the mapping function resulted in the observation that color-blind individuals would not be able to see lines that were labeled with red or green, which led to a change in the mapping color scheme.There have been many additional suggestions for improving the visualization, including addition of three-dimensional representations and allowing more user control of the presentation itself. Some researchers have expressed interest in being able to build maps based on the date a particular research finding was reported. This functionality would create time sequence maps that show the progression of research over time and, perhaps, will demonstrate paths of research that have been discarded prematurely and may be worth re-visiting. A number of researchers have indicated the utility of this approach for teaching purposes – for a student to quickly get a sense of the research "facts" in a domain. There have also been requests for tools to support downloading subsets of the knowledgebases, as well as tools to allow individuals to manipulate maps and add their own research findings and ideas to the concept maps.While initial Telemakus development has focused on the research literature related to caloric restriction and the biology of aging, the goal is to expand into additional domains. For example, tables of genetic sequence information, which display reported relationships between gene sequences and diseases, are a natural area of expansion for Telemakus. There is great potential for building linkages between Telemakus knowledgebases and other factual databases, e.g., NCBI entrez resources. In addition, scientists from other domains beyond biomedicine  have indicated that a customized schematic representation of research findings could be very useful in their domains.Speeding up document processing so Telemakus can easily and efficiently scale for comprehensive treatment of domains is a key priority. As discussed previously, the UMLS Metathesaurus resources  are proving extremely useful. In addition, the Semantic Network will be tested for enhancing searching and visualization of research findings.We will continue to utilize an iterative development method so that results of usability evaluation can immediately inform development of additional features. In particular, we want to test our hypothesis that the mapping feature will promote knowledge discovery by showing graphically what is known as well as, through lack of links, what research linkages have not yet been tested.Since basic sciences researchers tend to initially focus on the data found within a report's tables and figures , extracting the headings and providing linked research concepts mimics a researcher's traditional approach to reading the research literature ,48. WhenOne of the long-term goals of the Telemakus system is not to build knowledgebases "ad infinitum" but rather to create flexible tools for users to quickly and efficiently locate and visualize aggregate research findings from any domain which reports research findings as data. As more and more full-text research reports are available on the Internet, we believe the tools we are developing will provide an important approach for focusing on research findings and providing visual cues for quick review and assimilation.The Telemakus KnowledgeBase System builds on a good deal of prior research in a variety of domains. It provides a flexible new approach for creating knowledgebases to facilitate retrieval and review of scientific research reports. In formalizing the representation of the research methods and results of scientific reports, Telemakus offers a potential strategy to enhance the scientific discovery process. While other research has demonstrated that aggregating and analyzing research findings across domains augments knowledge discovery, the Telemakus system is unique in combining informative document representations with interactive concept maps of linked relationships across groups of research reports. Telemakus presents a novel approach to creating useful and precise document surrogates and may re-conceptualize the way we currently represent, retrieve and assimilate research findings from the published literature.None declared.SF conceived the study and contributed to its design, coordination and evaluation. DR and PB participated in the design of the study. DR led the overall coordination and drafted the manuscript. PB led the technical implementation. GMM contributed to the design, coordination and evaluation. All authors read and approved the final manuscript.


Symptomatic hypogammaglobulinemia in infancy and childhood (SHIC), may be an early manifestation of a primary immunodeficiency or a maturational delay in the normal production of immunoglobulins (Ig). We aimed to evaluate the natural course of SHIC and correlate in vitro lymphoproliferative and secretory responses with recovery of immunoglobulin values and clinical resolution.Children, older than 1 year of age, referred to our specialist clinic because of recurrent infections and serum immunoglobulin (Ig) levels 2 SD below the mean for age, were followed for a period of 8 years. Patient with any known familial, clinical or laboratory evidence of cellular immunodeficiency or other immunodeficiency syndromes were excluded from this cohort. Evaluation at 6- to 12-months intervals continued up to 1 year after resolution of symptoms. In a subgroup of patients, in vitro lymphocyte proliferation and Ig secretion in response to mitogens was performed.32 children, 24 (75%) males, 8 (25%) females, mean age 3.4 years fulfilled the inclusion criteria. Clinical presentation: ENT infections 69%, respiratory 81%, diarrhea 12.5%. During follow-up, 17 (53%) normalized serum Ig levels and were diagnosed as transient hypogammaglobulinemia of infancy (THGI). THGI patients did not differ clinically or demographically from non-transient patients, both having a benign clinical outcome. In vitro Ig secretory responses, were lower in hypogammaglobulinemic, compared to normal children and did not normalize concomitantly with serum Ig's in THGI patients.The majority of children with SHIC in the first decade of life have THGI. Resolution of symptoms as well as normalization of Ig values may be delayed, but overall the clinical outcome is good and the clinical course benign. Streptococcus pneumonia, Haemophilus influenza and Staphilococcus aureus, but the infections may be of unusual severity, persistence, or frequency.Pediatric patients with "recurrent infections" within our area are referred to the pediatric immunology clinic in the Kaplan Medical Center. Few fulfill the clinical criteria of the immune deficiency "red flags", Table In the last 10 years, tremendous advances in the fields of molecular medicine and genetics, have made possible the definitive diagnosis of most combined immunodeficiency patients, agammaglobulinemia patients and clinical syndromes associated immunodeficiency patients, on the basis of a recognized genetic aberration leading to a protein product dysfunction ,4. NeverIn this study we aimed to evaluate the natural course of disease in symptomatic hypogammaglobulinemia of infancy and correlate in vitro lymphoproliferative and secretory responses to mitogens in this population with recovery of immunoglobulin values and clinical resolution.Children more than 1 year of age, with recurrent infections, defined as more than three episodes of acute otitis media and/or more than one episode of acute sinusitis and/or more than one episode of pneumonia or the presence of a severe deep seated infection  within the last 6 months, or fulfillment of one of the "red flags" of immunodeficiency, see Table All procedures were performed according to accepted ethical standards of the Institutional Review Board of Kaplan Medical Center. The parents of all the children were informed accordingly and gave their permission for participating in the study and blood sampling.Total serum Ig levels were measured by nephelometry  and serum IgG subclasses have been assayed with an immunodiffusion commercial kit . Specific antibody production was not evaluated.5 cells were stimulated for four days with 0.01% w/v SAC , 2.5 μg/ml PWM, 20 μg/ml E. coli: O55:B5 LPS or with 20 μg/ml PHA . The cells were then pulsed with 1 μCi/well of [3H]-Thymidine  and incorporated radioactivity was measured by a β scintillation counter. Proliferation was expressed as stimulation index (SI).Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood of patients and age-matched donors  on ficoll isopaque gradients . Patient and normal donor cells were cultured in microtiter plates with culture medium: RPMI medium supplemented with 10% FCS , 10 mM Hepes buffer, 100 U/ml penicilin 100 μg/ml streptomycin, 2 mM L-glutamine, and 100 μg/ml kanamycine (Sigma Israel), and were grown at 37°C with 7.5% CO2 in air. 5 × 10In parallel, cell culture supernatant aliquots were harvested and Ig isotype concentrations were measured in the culture supernatants, by a solid-phase immunoassay, in Nunc- Immunoplate Maxisorp 96 wells . The plates were coated with goat anti-human IgM, IgG or IgA antibodies . Biotinilated goat anti-human IgM, IgG or IgA  and streptavidin-alkaline phosphatase  were used for measurement of IgM, IgG and IgA respectively. Resulting yellow dye intensity was read by an ELISA reader . Dye units were converted to immunoglobulin concentrations by extrapolation from standard curves determined by using purified myeloma proteins of known concentration in every assay.We used non-parametric tests to compare the means  and a standard analysis of variance to compare between groups. Logistic regression analysis was used for comparison of distribution of dichotomous values between the groups. Analysis was performed using SPSS for windows ver 9.0.32 patients were included in the study, 24 males (75%) and 8 females (25%) with a mean age at diagnosis of 3.4 years (range 1.2 – 7.0). Clinical presentations included severe and recurrent Ear-Nose-Throat (ENT) infections – 22 patients (69%), pneumonia, bronchopneumonia or severe, recurrent upper respiratory infections – 26 patients (81%), diarrhea – 4 (12.5%) and atopy related complaints – 20 patients (63%). A positive family history of recurrent, unusual or severe infections was obtained in 7 patients (22%). Demographical and clinical data of patient cohort is summarized in Table Out of the initial 32 patients, 17 (53%) spontaneously corrected their Ig abnormalities. This group included 15 boys (88%) and 2 girls, mostly All defects were partial, no patient in this group, showing a complete absence of a given Ig isotype. The clinical course was benign, only 9.4% (3/32) patients requiring IVIg . 38% (12/32) of patients received prolonged antibiotic prophylaxis  and resolution of clinical symptoms occurred in 84% of patients (14/17 in the THGI and 13/15 in the non corrected group). All calculated p values, non significant for comparison between the 2 groups.Comparative analysis of serum Ig isotype levels at diagnosis showed no significant difference, between the transient and non-transient group.In vitro lymphocyte proliferation and Ig secretion were measured in 9 patients (5 patients with THGI and 4 with non-corrected IGD) on one or more occasions. Lymphocytes proliferative responses to SAC, PWM and LPS showed no differences between the groups and no significant differences from childhood norms. The proliferative response to PHA was significantly increased (p < 0.005) after the correction of Ig abnormalities, overshooting normal controls Fig .Quantification of the in-vitro immunoglobulin secretion in response to the various mitogens showed significant isotype and mitogen dependent variation. The IgM secretory response to PWM and LPS, was low in hypogammaglobulinemic patients. After normalization of serum Ig values, the IgM response to LPS stimulation increased, see table For both IgG and IgA, the response in normal children was significantly better than in either group of patients. The IgA secretion index to all 3 stimulatory mitogens was minimal in hypogammaglobulinemic patients, even after serum Ig correction and differed significantly from age matched controls , table Young patients with recurrent infections represent a sizeable portion of the daily practice of all primary care pediatricians and family physicians, with parents clamoring for a solution with the accumulation of lost daycare or school days. The physician is faced with the dilemma when parental assurance will suffice, rather than initiation of a costly immunological investigation. Often, the presenting clinical signs and symptoms are insufficient for an educated diagnosis, as well as Ig levels in infants below the age of one year. The present investigation was initiated in order to try to contribute additional understanding how to differentiate between cases of primary immune deficiency and those who are not.We prospectively studied the outcome of SHIC in 32 patients during a period of 8 years, mean follow up of 3.2 years. During this time more than half corrected their Ig abnormalities. The mean follow up of the non-corrected IGD group is slightly shorter  than that of the THGI group (2.5 years and 3.5 years respectively) which leads us to speculate that some patients in the "non-transient" group may eventually correct. This is consistent with previously published reports from Dalal et al. who, after a follow up of 10 years, found that 70% of patients had complete resolution of their Ig abnormalities .Interestingly, the majority of our patients were males (75%), and a higher proportion of males corrected their serum Ig . This finding of male preponderance is not uniquely ours. In the reports of Dalal et al. , 24/35  resolved their tendency for recurrent infections irrespective of their Ig values. Only a minority of patients required any medical intervention, 38% received antibiotic prophylaxis and only 9% intravenous Ig replacement therapy. We observed no difference in the clinical presentation or follow up of the transient group as compared to the non corrected IGD patients and resolution of serum Ig abnormality did not cause a complete clinical remission in all patients. This may be due to a residual inability to mount an adequate antibody response to specific antigen challenge, data that has not been evaluated in our series. The impaired IgG and IgA in vitro secretory responses, seen in hypogammaglobulinemic patients, even after serum Ig normalization, may be an expression of such an inability, which warrants further investigation.Atopy was a prominent associated complaint in 21/37 (57%) of our patients. Especially so in comparison with the prevalence of asthma in this age group in Israel – 7% ,23 and tThough no clinical evidence of T-cell functional impairment was observed in our patients , previous reports -28 suggeTHGI is a relatively common cause of symptomatic hypogammaglubulinemia in infancy in our area. Most children will spontaneously correct their Ig abnormalities during the first decade of life. Though tests of cellular or humoral stimulation index, are not as yet capable of differentiating the transient from the non-transient patients upon their presentation, significant isotype and mitogen specific variability is evident. The relative preservation of the in vitro IgM secretory response and the lack of IgA/IgG response in patients with hypogammaglobulinemia, argues for a delay in isotype switching as the molecular basis underlying the clinical entity of transient hypogammaglobulinemia of infancy.The author(s) declare that they have no competing interests.SHIC – Symptomatic Hypogammaglobulinemia in Infancy and ChildhoodTHGI – Transient Hypogammaglobulinemia of ChildhoodIg – ImmunoglobulinIGD – Imunoglobulin deficiencySAC – Staphylococcus aureus cowan IPWM – Pokeweed mitogenLPS – LipopolysaccharidePHA – PhytohemagglutininIVIg – Intravenous ImmunoglobulinENT – Ear, nose & throatMIK – carried out the patient care and follow-up, was responsible for the database organization, data analysis and for manuscript coordination and writingZT – is one of the research initiators, carried out the patient care and follow-up and contributed to the manuscript writingIA – carried out the in vitro cell proliferation and Ig secretion studiesRS – carried out the in vitro cell proliferation and Ig secretion studiesMS – carried out the patient care and follow-up, was responsible for the database initiationIZ – is one of the research initiators, carried out of the laboratory evaluation and contributed to the manuscript writingAll authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here: