As the overall evidence for the effectiveness of teaching of evidence based medicine (EBM) is not strong, and the impact of cultural and societal influences on teaching method is poorly understood, we undertook a randomised-controlled trial to test the effectiveness and learning satisfaction with two different EBM teaching methods ) for undergraduate medical students.A mixed methods study that included a randomised-controlled crossover trial with two intervention arms  and a nested qualitative study with focus groups to explore student perceptions of learning and to assess the effectiveness and utility of the two teaching methods.All 129 second-year medical students at the University of Hong Kong in 2007.The main outcomes measures were attitudes towards EBM; personal application and current use of EBM; EBM knowledge; future use of EBM.PBL was less effective at imparting knowledge than usual teaching consisting of a lecture followed by a group tutorial. After usual teaching students showed improvement in scores for 'attitudes towards EBM', 'personal application and current use of EBM' and 'EBM knowledge, which were not evident after PBL. In contrast to the usual teaching, students found PBL difficult as they lacked the statistical knowledge necessary to support discussion, failed to understand core concepts, and lost direction.The evidence presented here would suggest that the teaching of EBM within an Asian environment should adopt a format that facilitates both the acquisition of knowledge and encourages enquiry. Evidence-based medicine (EBM) has been defined as the "conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients". While tThe PBL constructivist model holds that students do not passively absorb new information but build new ideas and concepts on pre-existing knowledge . HoweverIn contrast to other students, medical students express a more dualistic view of knowledge acquisition, i.e. perceived largely as right or wrong or as true or false . Such beEducational research to support teaching and learning is important, both for its practical relevance but also to bring teaching and learning in line with current pedagogy including lifelong self-regulated learning and social learning theory . As the All second-year medical students (N = 129) attending the University of Hong Kong's five-year undergraduate medical program were included in the study. The trial was explained in detail to the year group. Students were assured of the anonymity and confidentiality of personal information for all responses throughout the study period.We undertook a mixed methods study that included a randomised-controlled crossover trial with two intervention arms, usual teaching and problem based learning, and a nested qualitative study with focus groups to explore student perceptions of learning. To ensure that all students had the same overall learning experience, we used a cross-over design, where students either had usual teaching followed by PBL or PBL followed by usual teaching.A two-stage randomisation strategy was adopted. First, students were divided into 13 standard learning groups of approximately equal size (9-10 students each) by the Faculty of Medicine's Medical Education Unit, independent of the research team. Assignment of students and the randomisation process were concealed from both the participants and investigators. Seven such learning groups were randomly assigned to the usual teaching intervention arm in the first half of the trial followed by the PBL intervention in the second half and six groups were randomly assigned to the PBL intervention arm followed by usual teaching intervention.Focus groups, using purposive sampling ,19 allowFigure The EBM curriculum spans five out of six body system blocks in the second year; four body system blocks were included in this trial. The teaching programme is self-contained within each body system block, and includes 2 two-hour sessions per body system block, so students had two sets of 2 two-hour sessions of one intervention arm in the first half of the trial and another two sets of 2 two-hour sessions of the other intervention in the second half of the trial. The EBM programme takes an integrated approach to applying the 5A's of EBM, i.e. assess the patient, ask an answerable clinical question, acquire the evidence, appraise the evidence, and apply the evidence back to the clinical situation, within each body system block.The usual teaching arm began with a 2-hour session for all assigned students. The students were first shown a video consultation where a doctor interviewed a real patient and took a clinical history. A faculty member then reviewed the salient points from the patient encounter on the video, identified learning points, collectively formulated a specific answerable clinical question, demonstrated a targeted literature search in real time online and found a suitable original article that would be critically appraised in the subsequent group tutorial (~20 students). Finally, the session concluded with a statistical primer on key concepts that would be useful in the critical appraisal exercise.In the PBL arm, using a paper case format, students in small groups (9-10 students each) met with a faculty tutor for the first 2-hour session and undertook essentially the same tasks as in the usual teaching arm. They watched the same video, identified learning points, formulated an answerable clinical question and carried out an online literature search for an original article.For both arms, the student's task between the first and second sessions was to read, understand and critically appraise the chosen paper as well as to complete a statistical worksheet.The content of the second 2-hour session was identical in both intervention arms. Students, following a set of guidance notes and facilitated by a faculty tutor, discussed and debated the relative merits and shortcomings of the paper, then they applied the discussion to the patient problem introduced in the video.Table a priori, outcome indicators most relevant to the learning needs and objectives of second-year medical students, as assessed by self-report through a locally validated, standardised knowledge, attitude and behaviour (KAB) questionnaire [We defined, ionnaire .The students completed the KAB questionnaire at baseline, at the first assessment, which was after the first two body system blocks  and at the second assessment, which was after two more body system blocks  Figure . KAB resThree focus groups were conducted,  Figure  to exploWe used a generalised linear model to compare questionnaire item scores by teaching method at each assessment adjusted for the baseline score in the first half of the trial and for the first assessment score in the second half of the trial, from which we report mean differences in the change in score with 95% confidence intervals. We present these differences with PBL teaching as the reference, so that a positive difference indicates a greater improvement for usual teaching. All analyses were performed using SPSS v. 16 .For the focus groups, all references to learning were coded by two independent coders to maximize the validity of the analysis, and the transcripts were explored to identify emerging themes, categories, and concepts until no new information was seen in the analysis.Ethical approval was received from the HKU/HA West Cluster Institutional Review Board.The distribution of measured baseline variables was balanced between the two interventions arms. Table Table Three main themes, 'learning skills and concepts', 'group process as an aid to learning' and 'role of the tutor' reflecting students attitudes towards and perceptions of learning in groups were identified in the focus groups. "Learning skills and concepts" reflects on the organisation and structure of the learning environment. In contrast to the usual teaching, students found the EBM PBL sessions difficult as they lacked the statistical knowledge necessary to support discussion, failed to understand the core concepts, and therefore lost direction. The limitations of minimally guided small groups  were ref"We need to learn something about the facts first - if we are not all prepared then efficiency in the small group is a disaster.""Without a whole class lecture we do not have enough knowledge for the small group discussion."In the theme 'group process as an aid to learning' in contrast to the 'usual teaching', the students as with others ,10 descr"Communications are easier in a small group. It is helpful to learn in a small group.""For PBL, we will talk about what was being taught during lectures, finding something in addition to what the teachers have told us."As reflected in the theme 'role of the tutor' students in EBM PBL defaulted to the tutor for in depth explanation and support more than in 'usual teaching', and were also more tutor dependent  than in "EBP PBL depends more on the tutor than normal PBL.""Tutors need to be more involved/interactive. It requires higher level of tutor skills.""In EBM PBL it is easy to loose direction. Many concepts are not understood."As medical school progress and assessment is very important to the students, they are highly motivated by their perceptions of efficient and effective learning. Overall students were frustrated by the EBM PBL learning process.We found that PBL (self-directed learning) was less effective at imparting knowledge than the usual teaching programme (directed learning) of a lecture followed by a group tutorial. In contrast to that found by others  the in dIn addition, clinical application and decision-making uncertainty further frustrated the students who sought defined answers to clinical questions. Such attitudes perhaps reflect a social and cultural learning environment that focuses on acquiring and transferring knowledge rather than generating hypotheses, new ideas or contradicting authority,14. ThisThe evidence presented here would suggest that the teaching of EBM within an Asian environment should adopt a format that facilitates both the acquisition of knowledge and encourages enquiry, comprising a prefacing lecture followed by a small group PBL session. In an Asian setting and perhaps elsewhere, small groups may need to reject the minimal guidance directive advocated for PBL curricula substituting instead small group tutors who have the necessary process and content expertise to identify misconceptions, identify problems, and provide immediate, content specific feedback to correct misunderstand or misconceptions . In addiOur study is one of a few randomised controlled trials of EBM learning in undergraduate students. We achieved an acceptable overall response rate for the questionnaire survey. Although non-response bias in the focus groups may have been a concern, purposive sampling was used to identify and recruit members to the focus groups and in the smallest groups representativeness was maintained. Although we were able to achieve allocation concealment, used validated and blinded outcome measures, standardized educational intervention, and had sufficient power to demonstrate a difference between the two study arms we were unable to blind the study participants and tutors, control for contamination between the study arms, or use the same tutor for all groups. While the crossover design may be seen as a weakness (as the effect of the intervention may not washout when crossing over) the study outcomes required comparison and were strengthened by this study design. This study only begins to investigate the challenge of assessing the impact of culture on evidence-based medicine in undergraduate medical students.Our randomised-controlled trial in an Asian setting indicated that undergraduate EBM teaching was most effectively implemented in a format that facilitates both the acquisition of knowledge and encourages enquiry. As such, our study also draws attention to the importance of locating teaching methods within their social and cultural context, so to take advantage of students existing epistemological beliefs.The authors declare that they have no competing interests.JJ carried out the study, contributed to the statistical analysis, shared in the writing of the manuscript, CMS performed the statistical analysis and shared in the writing of the manuscript, GML was responsible for the conceptual framework of the undergraduate EBM programme, advised on the analytic framework and shared in the writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Supplemental table S1Click here for file


In vivo this discrepancy could be due to the difficulty in identifying failures of transmission.The giant synapses of Held play an important role in high-fidelity auditory processing and provide a model system for synaptic transmission at central synapses. Whether transmission of action potentials can fail at these synapses has been investigated in recent studies. At the endbulbs of Held in the anteroventral cochlear nucleus (AVCN) a consistent picture emerged, whereas at the calyx of Held in the medial nucleus of the trapezoid body (MNTB) results on the reliability of transmission remain inconsistent. in vivo. Based on the temporal relationship between a cells' waveform and other potentials in the recordings, a statistical test is developed that provides a balanced decision between the presence and the absence of failures. Its performance is quantified using simulated voltage recordings and found to exhibit a high level of accuracy. The method was applied to extracellular recordings from the synapses of Held in vivo. At the calyces of Held failures of transmission were found only rarely. By contrast, at the endbulbs of Held in the AVCN failures were found under spontaneous, excited, and suppressed conditions. In accordance with previous studies, failures occurred most abundantly in the suppressed condition, suggesting a role for inhibition.We introduce a novel method for detecting unreliable transmission in vivo recording conditions.Under the investigated activity conditions/anesthesia, transmission seems to remain largely unimpeded in the MNTB, whereas in the AVCN the occurrence of failures is related to inhibition and could be the basis/result of computational mechanisms for temporal processing. More generally, our approach provides a formal tool for studying the reliability of transmission with high statistical accuracy under typical  In the AVCN, The shortcomings pertain to the possibility of mistaking signals from other cells as incidences of failed transmission. This possibility arises due to the summation of signals in the extracellular fields and has been known to experimenters for a long time. It also arises in the task of distinguishing spikes emitted from different sources (spike-sorting) where single and multiunit activity need to be distinguished. The refractory period of neuronal spiking is a basic criterion to distinguish these alternatives. To detect incidences of failed transmission, a similar approach can be used. McLaughlin et al. The presently developed test also constructs a specific interspike interval histogram, but statistically compares the distribution within one refractory period to the distribution at greater distances. In this way it becomes robust to noise and a significance level of the decision can be computed. The properties of the test are first investigated and adjusted using simulated voltage recordings. It is then applied to a (comparably) wide range of recordings from the endbulbs and the calyces of Held. In summary, we suggest that under the studied activity conditions  and employed anesthesia (ketamine/xylazine) failures of transmission occur commonly in the AVCN but only rarely in the MNTB. The present results have partly been presented at the ARO midwinter meeting 2008 (Abstract #845).All animals were handled in strict accordance with good animal practice as defined by the relevant regional animal welfare body, and all animal work was approved by the appropriate committee .Forty adult pigmented Mongolian gerbils  were used in this study. They were anesthetized with a xylazine-hydrochloride/ketamine-hydrochloride mixture  0.007 mg/g body weight injected intraperitoneally; ketamine  0.18 mg/g body weight initial dose by intraperitoneal injection). Subcutaneous, hourly injections of one third of the initial dose assured a constant level of anesthesia. Animals were placed in a sound-attenuated booth  on a vibration-isolated table and fixed in a stereotaxic device using a metal bolt glued to the bone on bregma of the skull. The nuclei were approached dorsally with the animal tilted at 27–34 degrees (AVCN) and 4–10 degrees (MNTB) to the midsagittal plane. Further details of the preparation are given in Stereotaxic coordinates of the AVCN  which distinguish them from other cell types and fibers in these nuclei. Only units exhibiting such CWs were included in the analysis. In the AVCN, Pfeiffer 0.5 ms . A numbeThe interpretation of the present results will depend on the origin of the components of the CW. Especially in the AVCN this origin is not fully understood. Possible origins for the individual components include the presynaptic AP, the postsynaptic EPSP, the postsynaptic axon hillock AP and the postsynaptic, retrograde AP The signal-to-noise ratio (Reliability of transmission was investigated under three response conditions: (1) spontaneous, (2) excited, and (3) suppressed activity. For the latter two, specific acoustic stimuli were presented to the animal which achieved the desired activity level for a given cell (see below). During condition (1) no acoustic stimulus was presented. Spontaneous activity was recorded for 30 s, in units with low spontaneous activity for up to 150 s. For condition (2), the combinations of level and frequency leading to significant increases in firing rate were selected from the (stimulus frequency - stimulus intensity) tuning recordings (next paragraph). The length of these recordings varied with the response area of each unit, amounting to 40 s on average. In condition (3), either a single or a two-tone paradigm  was chosen depending on the spontaneous rate of the unit (next paragraph).The excitatory response area of a neuron was measured by monaural presentation of short pure tones  in pre-defined intensity and frequency arrays. Combinations of 20 frequencies (logarithmic spacing) and 10 intensity levels (linear spacing in decibel) were presented in a randomized order. In spontaneously active units this single tone paradigm sufficed to determine significant reductions of (spontaneous) firing rate (Wilcoxon signed ranks test). In spontaneously inactive units a two-tone paradigm was used to detect frequency/intensity combinations evoking reductions in response rate relative to the response rate of a single excitatory tone  with a duration of 100 ms; second pure tone: varying frequency/intensity combinations with a duration of 40 ms starting at a 30 ms delay with respect to the first tone within an overall stimulus period of 200 ms). All tones were modified multiplicatively at their start and end with cosine shaped rise/fall ramps (5 ms each). After the rate-reducing frequency/intensity combinations were located, they were resampled with longer stimuli to increase the spike count for this condition .Stimulus waveforms were generated at a sampling rate of 97.7 kHz on a standard PC using custom written Matlab 7  software. Stimuli were then transferred to a real-time processor , D/A converted and further sent to two custom made earphones . These were fitted with plastic tubes  which were inserted into the outer ear canal at a distance of The goal of the following analysis is to decide whether an MNTB/AVCN recording contains failures of spike transmission .If such potentials are present address next question:Do they originate from a source which is connected to the CW's source?This is tested by determining whether the potentials obey the refractory period with respect to the CWs.If this is the case, it is likely that these potentials originate from the presynaptic ending (if certain shape criteria match).If the refractory period is not obeyed, the potentials have to originate from a different source, e.g. neighboring cells.No Failures and Failures, we chose the terminology no dependent potentials and dependent potentials, which will be abbreviated No Dep and Dep, respectively. This terminology reflects the basic fact that an analysis based on the waveforms can only determine whether the CWs and the candidate potentials are statistically independent or dependent. Their origin needs to be addressed later, based on additional analytical and biological considerations, which we postpone to the discussion.Recordings are divided into two classes, based on where they fall along these conditions: Failures of transmission are unlikely if either no candidate potentials exist or if the candidates stem from a different source. Failures of transmission are likely if the potentials stem from the same source. We decided to use a more cautious terminology. Rather than naming these classes Next, we describe the details of what constitutes a candidate potential , how these were collected, and how statistical dependence between them and the CWs was tested. This analysis, termed Independence Assessment of Potentials (IAP), can be divided into four steps:Detection of complex waveforms  histogram of the CWs. In this case the iPs are termed dependent (with respect to the TP of the CWs).In the latter case the histogram will be lower within one refractory period of the TP  than further from the TP (reference window: RefWin). Since the histogram will not always be densely sampled, a statistical test is needed that judges whether the rate of iPs in CritWin tion see .To account for sparsely sampled histograms, we divided RefWin into subwindows Hence, While nt S see . The sigIn essence, this method implements a test for potentials approaching each other closely in time, yet with the advantage that this method also works under noisy conditions. If noise reaches the trigger level, the histogram baseline is just shifted but the difference between the number of triggers within CritWin and RefWin is preserved. Although this reduces the power of the test, it still allows a distinction between dependent and independent iPs. Further it provides a statistical test to the researcher in place of visual judgement.The ability of the IAP to distinguish whether potentials stem from dependent or independent sources was validated by applying it to a range of simulated voltage traces see . Here thDep) and the absence of dependent iPs (No Dep). This task differs from the classical question of whether a 0-hypothesis holds, where one specifies an acceptable level of correct classification only for the 0-hypothesis. Presently, the goal is to weigh the two hypotheses equally and choose a decision criterion that maximizes the overall percentage of correct classification.The test should distinguish between the presence (Dep blue and No Dep orange) are minimized to the same level by choosing the point where the two functions agree. This point corresponds to the desired balanced decision point This goal requires to minimize the Type II error of the test jointly for both hypotheses, i.e. maximize the power for both hypotheses. The power of a statistical test is usually defined as 1-lihoods  , purple hoods  . If a given recording accorded to these requirements, it was classified as Dep if the IAP result lay below the No Dep otherwise.From the averages of the cumulative densities  it is apn in Dep , blue bo  recording for each condition (if available for a given unit).A statistically sound analysis of signal transmission relies on trigger potentials (TP) well above the noise level and a sufficient number of complex waveforms (CW). Extracellular recordings from both nuclei studied exhibited a wide range of signal to noise ratios of the TP  iP rates with higher rates following the TP/CW than preceding it. In contrast, the recording depicted in Both No Dep, mostly because of the absence of iPs throughout the whole recording. An example of this kind, in this case a recording of spontaneous activity, is shown in No Dep case of the AVCN, no asymmetry of iP rates is observed. In Dep classification. While this demonstrates that IAP can detect (putative) failures in the MNTB as well, the interpretation of this particular case is involved. In contrast to the great majority this unit exhibited an unusually high variability in the delay from C1 to C2 .Dep result  of the AVCN recordings yielded a p result . For theDep result were reduced. In the AVCN, the percentage dropped to 50% (15/30), in the MNTB no recordings classified as Dep remained . In contrast, no MNTB recording was classified as Dep. Interestingly, in the AVCN the standard deviation of the baseline voltage during this response condition was not significantly increased  MNTB cases classified as Dep  do not provide sufficient information for a histogram, only the AVCN data are considered  exceeds the No Dep (0.36 SD 0.24) mean, the variability seen in the data prevents a statement of significant difference in mean. Generally, the iP proportion is quite variable for both Dep and No Dep with the exception of spontaneous No Dep recordings. This is consistent with previous findings For the recordings of spontaneous activity, the distributions of iP proportion for r values . For ther mean , . AlthougNo Dep cases is also high in the excited and suppressive/inhibitory conditions, since neighboring cells are likely to have similar activity levels under the same stimulation.It might not be surprising that the iP proportion for the Dep even if they actually originate from different sources. Although this classification is correct in the sense that both potentials depend on the stimulus, it would invalidate the study of signal transmission. Therefore, we addressed this concern by comparing the CFs of Dep and No Dep recordings separately for each condition . In the MNTB, the distribution of CFs has most of its weight above 3.5 kHz .The contrasting IAP results between AVCN and MNTB units could be related to their different distributions of CF. The CFs of AVCN units ranged from 0.3 to 3.3 kHz, CFs of MNTB units from 0.5 to 45 kHz . In the ondition . In the  3.5 kHz  correspoIn the excitatory condition a given unit usually emits thousands of CWs (mean: 2600 SD 1378) within approximately 60 s (depending on the number of repetitions), leading to an average firing rate of 80 Hz (SD 37 Hz). Due to the nature of auditory tuning curves in the MNTB, the response consists of episodes of very high firing rate (up to 500 Hz over 100 ms) alternating with periods of little activity.Dep cases) occur at the calyx of Held only for longer periods of more intense activity, another excitatory response condition was tested. A broadband, noise-like stimulus (reaching from two octaves below CF to one octave above CF) was presented for 100 s almost continuously . The loudness of the noise was adjusted to obtain a high firing rate (132 SD 38 Hz). This paradigm was employed in 22 cells with a high SNRDep, suggesting only a small increase in Dep cases due to the prolonged, higher frequency discharge.To test whether failures . It is hence important to assess, whether the collection of iPs in the windows is representative for the iPs in the whole recording.In both AVCN and MNTB, a considerable percentage of the recordings contained only few or no iPs in RefWin/CritWin, most prominent in spontaneous recordings from the MNTB, where approx. 70% had less than 10 iPs in RefWin/CritWin corresponding to low iP rates in the windows . Are theNo Dep: circles, Dep: pluses). This is indicated by the high r values together with slopes close to 1. In some cases the iP rate in RefWin even exceeded the overall iP rate, indicated by slopes greater than 1. This can be explained, assuming that all potentials, APs and iPs, are more likely to be emitted during stimulation than during silent periods. Note that low rates of iPs inside RefWin correlate extraordinarily well with low overall rates. Hence, the recordings with few or no iPs in RefWin/CritWin were correctly judged as No Dep since neither dependent nor independent iPs occurred in relevant numbers in the whole recording.In all response conditions  the rate of iPs in RefWin was a good predictor of the total iP rate  while indicating the need for reconsideration in the case of the calyces of Held (MNTB). In the MNTB only few recordings contained dependent iPs, thus rendering isolated prepotentials, i.e. failures of signal transmission, a rare event. By contrast, in the AVCN the majority of recordings contained dependent iPs suggesting failures of AP transmission, predominantly during the inhibitory/suppressive response conditions.The present study investigated the reliability of transmission at the giant synapses of Held The different results at the two synapses of Held emphasize the necessity for systematically assessing the origin of failure candidates before assuming a common origin with the complex waveforms. The decision as to whether potentials in a recording are dependent or not has to be based on extended stretches of the recording. Only a statistical approach can provide a reliable decision based on the probability with which the events occur in relation to the complex waveforms.In principle, a number of different constellations can lead to the observed dependence between iPs and complex waveforms. This ambiguity necessitated the present cautious choice of terminology, i.e. dependent iPs rather than failures. Based on the current biological understanding of the investigated nuclei, we will argue below that dependent iPs do most likely correspond to failures of synaptic transmission. Since dependent iPs were rare in the MNTB, we will focus on the AVCN.Aside from failures, a common cause can lead to dependence between iPs and the complex waveforms of a given unit: Most directly, dependence would be induced, if the iPs originate from a source which receives input via the same auditory nerve fiber. In this case, the second source would be another spherical bushy cell. Alternatively, dependence would also be present, if the given unit and the iPs are phase-locked to the same stimulus, which would also synchronize their refractory periods. Here, the second source could either be another spherical bushy cell or another auditory nerve fiber.Dep and No Dep was not CF dependent  differs from both the estimates in Functionally, the occurrence of failures of transmission does not necessarily imply imprecision, but can rather be a means of temporal sharpening. Phase-locking to pure tones has been shown to improve from auditory nerve fibers to the spherical bushy cells in vitro studies, the calyx of Held was long thought to provide reliable signal transmission in vitroBased on its elaborate morphology and a number of in vivo under electrical stimulation at high frequencies . Since ketamine, the anesthetic commonly used in in vivo experiments, interferes with the production of nitric oxide one might propose that failures are more commonly observed in awake animals which are exposed to extended periods of acoustic stimulation. Until further exploration of this issue we would conclude that failures of transmission are rare at the calyx of Held at least under the kind of acoustic stimulation, anesthesia, and species investigated in the present and the former in vivo studies However, it has recently been proposed that activity induces increases in the level of nitric oxide which in turn reduce the excitability of MNTB neurons and can lead to failures of signal transmission Although IAP was designed merely as an automated and statistically grounded procedure for spike-sorting and temporally comparing certain potentials, some discussion concerning its underlying assumptions, performance limits, and dependence on the shape of CW are provided.IAP relies on only few assumptions:Potentials transmitted via the same fiber are assumed to obey a refractory period. The duration of the refractory period was not critical for the performance of IAP and inter-spike intervals AP waveforms are assumed not to exhibit systematic variations in shape or size over the recording period . This requirement was checked visually for each recording prior to analysis.Dependent iPs are assumed to be of similar size as their corresponding TPs in CWs. This assumption bears the greatest biological relevance since its validity is determined by the cause of the dependent iPs. In the MNTB sizes are known to be similar at least under electrical stimulation Potentials from different sources are assumed to not be closely correlated in time. Phase-locking could have invalidated this assumption, but apparently did not play a significant role see . Note, hAside from a small number of CWs, IAP performance is mainly limited by the ratio between dependent and independent iPs/noise. If the rate of dependent iPs is far lower than the rate of independent iPs, the reduction in CritWin will only become significant after very long recording periods containing large numbers of CWs. Yet, it is debatable whether very low failure rates are of interest for the study of transmission reliability.Dep classification less likely. As mentioned in The shape differences of the CWs recorded in AVCN and MNTB could bias the results obtained in each nucleus . The CWs


The vestibular system of the inner ear provides information about head translation/rotation in space and about the orientation of the head with respect to the gravitoinertial vector. It also largely contributes to the control of posture through vestibulospinal pathways. Testing an individual severely deprived of somatosensory information below the nose, we investigated if equilibrium can be maintained while seated on the sole basis of this information.Although she was unstable, the deafferented subject (DS) was able to remain seated with the eyes closed in the absence of feet, arm and back supports. However, with the head unconsciously rotated towards the left or right shoulder, the DS's instability markedly increased. Small electrical stimulations of the vestibular apparatus produced large body tilts in the DS contrary to control subjects who did not show clear postural responses to the stimulations.The results of the present experiment show that in the lack of vision and somatosensory information, vestibular signal processing allows the maintenance of an active sitting posture (i.e. without back or side rests). When head orientation changes with respect to the trunk, in the absence of vision, the lack of cervical information prevents the transformation of the head-centered vestibular information into a trunk-centered frame of reference of body motion. For the normal subjects, this latter frame of reference enables proper postural adjustments through vestibular signal processing, irrespectively of the orientation of the head with respect to the trunk. The control of human upright and seated postures is based on information about body orientation and motion ,2. Neck-Supporting this view is the early seminal observation made by De Jong and colleagues  and CoheEvidence for cervical and vestibular neural integration is found in the rostral fastigial nuclei. In these deep cerebellar nuclei, on which massive vestibular and somatosensory signals converge, body-in-space motions are represented in a body frame of reference rather than in a head-based frame of reference ,10,11. THere we investigated if, alone, the vestibular signals, which contribute to the perception of head motion/orientation in space and which have connections with motoneurons of axial and proximal postural muscles, are sufficient to control sitting posture. This was done testing the capacity of a rare subject, with a large-fiber sensory neuropathy that resulted in a severe loss of position sense below the nose , to maintain a sitting posture. The contribution of vestibular information was specifically tested using two methods. One method consisted in creating a subliminal mismatch between vestibular information and actual body motion by slowly rotating the DS's head towards either shoulder in the dark. Controlling balance through vestibular information after undetected change of head-trunk configuration should lead to increase instability as the vestibular signals will no longer inform about the veridical body-in-space displacements.The second method employed in the present experiment consisted in externally stimulating the labyrinthine apparatus . GVS produces a pattern of irregularly firing vestibular afferents that resembles that of the natural response to linear or angular head acceleration ,14. WhenThe deafferented subject  suffered at the age of 31 from a loss of the large myelinated fibres from her whole body after a severe sensory polyneuropathy. Neurophysiological data of the DS have been reported elsewhere . In summThe experiment was carried out in a dim room such that the subjects could well see their trunk, legs and the experimental room with the eyes open but could not perceive change of luminance with the eyes closed when the experimenter rotated their head . The subjects sat on an 60 × 120 cm AMTI force platform placed on a 70 cm high plinth. No back or arm support was provided. Both legs were dangling with the hands resting on the subjects' thighs. Small spherical retroreflective markers were placed on both infraorbital margins, sternum, and at the center of the forehead. They allowed recording movements of the head using an automatic TV-image processor (E.L.I.T.E. system) at 100 hz. The center of pressure (CoP) exerted on the platform was recorded at 500 Hz. Subjects wore a light helmet on top of which was fixed a laser diode. This laser beam helped the experimenter to change the orientation of the subjects' head with respect to their trunk as will be specified below.All subjects were tested in three main conditions:The head was centered on the trunk. This condition was performed with the eyes closed or open.This condition was also performed with the eyes closed or open. For the trials with the eyes closed, prior to data acquisition, an experimenter rotated the subject's head very slowly in the horizontal plane (in about 15 s) until the laser beam centered on the head hit a 5 m distant target located 55° on either side of the subject. At the end of the trial, the DS kept the eyes closed and an experimenter grasped her shoulders while another brought her head back to the primary, central position rotating it back and forth a few times (3–4 rotations) at moderate speed (i.e. clearly perceptible by the DS). This procedure allowed the DS to re-open the eyes rapidly after the trials without having her realizing that her head had been previously rotated. The DS was simply told that these 3–4 passive head rotations were part of the experiment. To preserve homogeneity between conditions, similar procedures were taken for the Head centered condition (without vision). For the trials performed with the eyes open, the experimenter also slowly rotated the subject's head (in about 5 s here). With the eyes closed, the DS never reported perception of the slow (i.e. 15 s) head rotations introduced before the recordings. After the experimental session, when asked about the sensation she had during the experiment, the DS clearly expressed that she never perceived change of head orientation that occurred prior to data acquisition when she had the eyes closed. This confirms that, contrary to the patient tested by Day and Cole , the DS This condition was only performed with the eyes closed. The subject had the head centered on the trunk. Two seconds after the onset of recording, a rectangular, bipolar binaural direct electric current was delivered via 3 cm electrodes taped over the mastoid processes of the subject. The stimulations lasted 5 s and their magnitudes were either 0.75 or 1.5 mA. GVS produces a pattern of irregularly firing vestibular afferents that resembles that of the natural response to linear or angular head acceleration ,14. WhenThe trials lasted 10 s in all conditions. The Head centered and Head rotated conditions were first performed with the eyes closed and then repeated with the eyes open . Each subject performed eight trials per head orientation (randomly presented) for both viewing conditions . The GVS condition was ran after the Head centered and Head rotated conditions. Eight repetitions, randomly presented, were given by subject per type of stimulation . Rests of about 45 s were given after each trial during which the DS could lean against a portable back support put in place by the experimenter. The experiment was approved by the local ethics committee and was carried out in compliance with the Helsinki Declaration.For each trial, the length of CoP displacement, as measured over the 10 s recording periods, was used as an index of postural stability. The results obtained by the DS for each experimental condition are given in the additional tables (Table 1 and 2) which are too extensive to be presented herein. With the eyes open, the DS showed a stable posture as demonstrated by her CoP displacement  that was only slightly greater than that of the controls   to maintain an active sitting posture (i.e. without back or side rests) in the absence of vision. We argue that the DS controlled her posture through vestibular signal processing. Compelling evidence for a vestibular-based postural control comes from the marked increase of postural oscillations when the DS's head was unconsciously deviated from its primary, visually perceived, straight-ahead position. In this situation, and in the lack of head-on-trunk position information, the vestibular signals did not provide the DS with veridical information about her trunk displacements in space. Processing vestibular signals with such undetected head-trunk configuration is likely to have led to inappropriate postural adjustments with respect to the actual body oscillations. For instance, with the head unconsciously rotated to the left, the DS most likely detected (either consciously or unconsciously) backward displacements when she was actually moving leftward. Such craniotopic updating of body displacement with neck proprioception deficit presumably resulted in series of inappropriate postural adjustments in response to the vestibular stimulations.A great deal of the evidence for the contribution of vestibular signals to the control of posture comes from studies conducted with labyrinthine-defective subjects e.g. ,31] and  and 31]]Despite her severe loss of neck muscle proprioception, the DS's postural responses to GVS were still mainly oriented towards the anode side. For safety reason and in order to preserve the confidence of the DS regarding the experimental procedures, we did not deliver GVS when the DS had the head unconsciously deviated towards a shoulder. The large lateral CoP shifts of the DS following GVS support previous findings obtained in individuals with somatosensory deficits ,34. GVS Both the large CoP displacement observed when the DS had the head unconsciously deviated on the trunk and the large CoP shift after GVS onset suggest great sensitivity of the DS to vestibular stimulations. Such increased sensitivity has been reported when proprioceptive sense is deteriorated ,18,30 orAn unexpected result of the present experiment was the DS's increased body stability for the GVS condition as compared to the condition without GVS . Indeed, before and after the GVS-evoked large shift towards the anode side, the DS's CoP remained relatively stationary. Reasons for this improved balance in the GVS session remain uncertain. This experiment was the first attempt of the DS to sit without back or side supports since her deafferentation. It is then plausible that visual information, which was available in the conditions ran prior to the GVS condition (i.e. Head centered and Head rotated conditions with eyes open), allowed the DS to acquire a new strategy to control her sitting posture. This strategy may involve stiffening and freezing the trunk and legs, a strategy used by patients and elderly individuals with sensory impairments to control their balance while standing and walking -44. Of cAltogether, this case report demonstrates that, in the case of a severe loss of proprioception of the body and of the cervical region, and contact information with the surface of support, seated posture can be maintained without vision through vestibular signal processing. However, the optimization of this vestibular-based control of posture requires an accurate representation of the head orientation on the trunk.The author(s) declare that they have no competing interests.JB and LM conceived the study and collected the data. All authors evaluated the data, performed data analyses and wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Table 1. T-tests comparing the length of the CoP displacements of the deafferented subject in all experimental conditions.Click here for fileMovie 1. Dynamic representations of the deafferented subject's CoP displacements for representative single trials of the Head centered and Head rotated eyes closed conditions.Click here for fileTable 2. T-tests comparing the maximal CoP lateral shifts produced by the deafferented subject in the GVS conditions.Click here for fileMovie 2. Dynamic representations of the deafferented subject's CoP displacements for representative single trials of the GVS conditions.Click here for file


We present a novel method that uses a proteomic knowledge base to enhance the performance of a rule-learning algorithm in identifying putative biomarkers of disease from high-dimensional proteomic mass spectral data. In particular, we use the Empirical Proteomics Ontology Knowledge Base (EPO-KB) that contains previously identified and validated proteomic biomarkers to select We show that using EPO-KB as a pre-processing method, specifically selecting all biomarkers found only in the biofluid of the proteomic dataset, reduces the dimensionality by 95% and provides a statistically significantly greater increase in performance over no variable selection and random variable selection.Knowledge-based variable selection even with a sparsely-populated resource such as the EPO-KB increases overall performance of rule-learning for disease classification from high-dimensional proteomic mass spectra. While biological knowledge is typically used to validate the results obtained from the analysis of high-dimensional biomedical data, increasingly, it is being incorporated into the statistical analysis and modeling of such data. For example, the use of knowledge bases to help process and analyze biomedical data for markers of disease has been shown to produce better results than analyzing such data in isolation ,2. Biomem/zs) that are associated with disease ) AND (12282m/z = [0.50–0.65]) THEN (ALS = True),IF  for learning sets of IF-THEN rules from the three datasets described in the previous section. The rules learned by RL do not partition the input space as, for example, is done by the classification and regression tree (CART) algorithms; instead they cover overlapping regions in the input space. RL has been used in multiple domains including in novel biomarker discovery, and in parameter learning for protein crystallization  and is cSince RL cannot utilize continuous variables, we discretized the variables using the MDLPC discretization method developed by Fayyad and Irani . This meWe used balanced accuracy and Relative Classifier Information  to measuc) (Specificity(c)) refers to the sensitivity (specificity) of the target value c versus all other values of the target. TP(c|c) (True Positives) is the number of samples predicted to have the value c for the target variable given that the observed value is c, FN(¬c|c)  is the number of samples predicted to have a value other than c for the target variable given that the observed value is c, TN(¬c|¬c) (True Negatives) is the number of samples predicted to have a value other than c for the target variable given that the observed value is not c, and FP(c|¬c)  is the number of samples predicted to have the value c for the target variable given that the observed value is not c.where |C| is the cardinality of the target variable and Sensitivity(Relative Classifier Information (RCI) is an entropy-based performance measure that quantifies how much the uncertainty of a decision problem is reduced by a classifier relative to classifying using only the prior probability distribution of the values of the target variable uninformed by any predictor variable . The minFor comparing the performance of RL on the three variable selection methods, we used the Wilcoxon paired samples signed rank test and the paired samples t-test. The Wilcoxon paired samples signed rank test is a non-parametric procedure that tests whether there is sufficient evidence that the median of two probability distributions differ. The paired samples t-test is a parametric procedure used to determine whether there is a significant difference between the average values for two different sets of samples. The test assumes that the paired differences are independent and identically normally distributed.To evaluate the performance of RL on a dataset, we performed 10 runs of 10-fold cross-validation to generate a total of 100 folds. We computed the BACC for each fold and averaged over the 100 folds; we report the average BACC in the results. We computed the average RCI in a similar fashion. For the datasets derived using DSVS and BSVS, the BACC and RCI were validated using a randomization test. The randomization test consists of selecting randomly the same number of variables from the original datasets as selected by DSVS (or BSVS), applying RL to the dataset generated using the randomly selected variables, and evaluating its performance in terms of BACC and RCI using 10 runs of 10-fold cross-validation. To perform the randomization test, we generated 1000 datasets by applying random variable selection using the cardinality for each of the DSVS and BSVS methods. In all experiments including the randomization experiments, we utilized the same training and test folds for every dataset. Thus, each of the 100 folds consisted of the same samples (though different sets of variables), irrespective of the variable selection method that was employed.m/zs that were included in the antecedents of the rules learned by RL on each of the three datasets. For this analysis, we examined the rules generated by applying RL to the entire dataset containing all the samples.We also tabulated and compared the m/z range. Utilizing the EPO-KB, we were able to retrieve empirically generated m/z ranges , and we also included possible double charges when selecting the variables. The original ALS dataset contained 36,778 variables. With the DSVS, we generated 23 ranges (46 with double charges) translating to 1064 m/zs selected . For BSVS, since there was an increase in the number of proteins (32 proteins), we generated 64 m/z ranges including which lead to a total of 1852 m/zs selected . Of note, all of the variables selected by DSVS were also selected by BSVS.For both DSVS and BSVS, we have to correlate the protein to a The average BACCs for the different variable selection methods are given in Table The average RCI for the various variable selection methods are given in Table On the randomization test, performance of DSVS as measured by BACC and RCI was statisitically significant at the 0.10 level and the performance of BSVS on both BACC and RCI was statisitically significant at the 0.10 level Tables .m/zs present in the antecedents of the rules learned by RL on the entire dataset for the three variable selection methods are shown in Figure m/zs obtained by BSVS were also obtained by DSVS. Also, the rules produced by the None method had shorter antecedents  than either BSVS or DSVS (data not shown).The number of variables and the m/zs obtained by the three variables selection methods. Fifty percent of the variables obtained by None were different when compared to those obtained by BSVS, and DSVS selected no new variables when compared to those obtained by BSVS.Table m/z ranges derived from prior published knowledge to filter the large number of m/zs generated in a proteomic dataset using two strategies. First, we used a disease-specific strategy, in which we filtered the m/zs based on validated biomarkers associated with neurological diseases; this resulted in increased BACC that was statistically significant at the 5% significance level. Second, we used a biofluid-specific strategy, in which we filtered the m/zs by proteins that were present in the CSF; this resulted in increased BACC and RCI that was statistically significant at the 5% significance level. Our approach is similar to the that used in [m/zs that are theoretically predicted for proteins and peptides for several reasons. First, the theoretical m/z is often not predictive of the experimentally derived m/z, particularly for those proteins that fragment, cleave, or undergo post-translational modifications. Second, the same protein obtained from different biofluids differs slightly leading to different m/zs. Using a database like EPO-KB that contains empirically derived m/zs allows us to use m/zs that have been recorded in previous experiments, which are more reliable than theoretically derived m/zs.Proteomic datasets derived from mass spectrometry experiments are typically high-dimensional, noisy, and have small sample sizes. The incorporation of prior biological knowledge can help in statistical analysis and modelingof such datasets where the goal is to identify putative panels of biomarkers associated with disease. We used a database of previously validated biomarkers to perform knowledge-based variable selection to reduce the dimensionality of a proteomic dataset associated with ALS. Our results show that such knowledge-based variable selection can improve the performance of a rule learning algorithm for identifying biomarker panels. We used the  used in  except tOne limitation of our study is due to the limited data present in the EPO-KB. Thus, the number of variables selected by DSVS was more than 50% fewer than the number of variables selected by BSVS. In addition, the variables selected by DSVS was a subset of the variables selected by BSVS. The paucity of variables available for DSVS likely resulted in its performance being poorer compared to that of BSVS as well as on the randomization test. In future experiments, it would be interesting to examine if the "intersection" of the knowledge-based biomarker sets provide higher accuracy than the individual biomarker sets. In our current study, since the BSVS set is completely contained in the DSVS set, there is no advantage in using the "intersection" of the two variables sets. Another limitation of our study is that we did not examine the performance of other methods for learning predictive models in addition to the RL algorithm.m/zs for a putative biomarker panel, the m/zs have to be identified definitively with techniques such as peptide mass-fingerprinting or tandem mass spectral analyses. A resource like EPO-KB that contains experimentally verified m/z to protein associations, is valuable for assigning a small list of putative proteins to m/zs of interest in an experiment. While EPO-KB was originally developed for this purpose, this paper demonstrates an additional application of the knowledge in EPO-KB for biomarker analysis of mass spectral data.Regardless of how one selects In conclusion, we have presented an example of how even a sparsely-populated knowledge base such as the EPO-KB can be used to perform variable selection to increase classification performance on a high-dimensional proteomic dataset. As more information is added to the EPO-KB, it is expected that such knowledge-based variable selection methods would lead to more useful models. In the future, we will evaluate the combination of knowledge-based biomarkers with dataset specific markers for disease classification.The authors declare that they have no competing interests.JLL, SV, WRH, and VG contributed to designing the experimental design. JLL was responsible for performing the experiments and for writing the first draft of the manuscript. JLL, RPB, WRH contributed to the curation and maintenance of the EPO-KB. RPB generated and characterized the proteomic dataset. VG was responsible for the development of the Rule Learning program as well as oversight over the experiments.


Since the molecular cloning of the hepatitis C virus (HCV) genome for the first time in 1989, there has been tremendous progress in our understanding of the multiple facets of the replication cycle of this virus. Key to this progress has been the development of systems to propagate the virus in cell culture, which turned out to be a notoriously difficult task. A major breakthrough has been the construction of subgenomic replicons that self-amplify in cultured human hepatoma cells. These RNAs recapitulate the intracellular steps of the HCV replication cycle and have been instrumental to decipher details of the RNA amplification steps including the identification of key host cell factors. However, reproduction of the complete viral replication cycle only became possible with the advent of a particular molecular HCV clone designated JFH-1 that replicates to very high levels and supports the production of infectious virus particles. The availability of this new culture system raises the question, whether the use of replicons is still justified. In this review, we will discuss the pros and cons of both systems. Hepacivirus within the family Flaviviridae .,14.13,14infected .Inspite of great progress with HCV replicons, the lack of virus production was an obvious limitation of this system. Because of the assembly block imposed by adaptive mutations on one hand, and the very low (most often nondetectable) replication of nonadapted replicons on the other hand, a full-cycle culture system only became possible with the identification of an HCV isolate that was capable of high-level replication without requiring cell culture-adaptive mutations. This ‘holy grail’ was discovered by Kato et al. , who clo4 tissue culture infectious dose 50 (TCID50) per mL], much effort was made to improve the system. One major progress was the generation of virus chimeras consisting essentially of the JFH-1 replicase (NS3–NS5B) fused to the core to NS2 region of other HCV isolates  is perhaps the most important one ,42 Fig.. HCVpps TCP) that are composed of authentic virus particles that contain a subgenomic RNA. Upon infection of Huh-7 cells, these RNAs replicate to high levels, yet do not produce infectious progeny because of the deletion of the structural genes. This system is also well suited to study the early and late steps of infection, but does not require a biosafety level 3 laboratory as is the case for full-length HCV sequences.Very recently, Steinmann et al.  describeAnother system to study the HCV replication cycle in a more authentic setting is the infection of primary human hepatocytes (PHH). Successful infection could be demonstrated with serum- and cell culture-derived HCV particles ,45. Howein vitro, it has been a promising model system  that shares many features with HCV such as the genome organization and the overall replication cycle. As BVDV can easily be cultured  in Ref. ). An add in Ref. ).With the plethora of cell-based HCV replication systems developed in the past few years, most notably the HCVcc system, the question arises as to which role replicons will play in the future both in basic and applied research. At a first glance, the HCVcc system is superior because it covers the complete viral replication cycle . Howevercis-acting elements in RNA replication compared with other stages of the viral life cycle.When considering RNA replication , resultsProbably, the strongest impact the HCVcc system has deals with the late steps of the replication cycle, i.e. assembly and release. These aspects could not be addressed properly by any of the surrogate systems. Although the formation of virus-like particles had been described in insect cells upon infection with recombinant baculoviruses or in mammalian cells upon transfection with alphavirus vectors directing the expression of the HCV structural genes, none of these systems adequately reflects authentic HCV assembly ,57. ThisThe major limitation of the HCVcc system is its restriction to a single isolate (JFH-1) that supports efficient virus production. Although, production of infectious particles has also been described for a highly adapted H77 (genotype 1a) isolate, virus titres attained with this genome and infectivity of released particles appear to be very low . In thatBecause of the high ‘genetic flexibility’ of replicons, they can be designed with much more ease as compared with full length virus genomes. For instance, selectable replicons with a stably expressed reporter gene  have beeneo replicons) is exploited to preserve the replicon in the face of selective pressure imposed by an antiviral compound, resulting in the selection of cell clones with conserved mutations in the viral RNA. In contrast, when using the HCVcc system, this is not really possible because a strong antiviral compound with a high genetic barrier will rapidly eliminate the virus from the culture . Furthermore, if mutations at independent positions confer resistance, it will be hard to identify relevant mutations in the viral quasispecies. Although the construction of bicistronic selectable JFH-1 genomes that support the production of infectious virus particles is possible [Hepatitis C virus replicons have also been very instrumental to select for antiviral drug resistance. In most cases, selectable replicons have been used in which the selection marker  has now been achieved for a number of other cell lines such as human hepatoma cells , humThere is no doubt that the possibility to produce recombinant infectious HCV particles in the laboratory opens new avenues to study the viral replication cycle. The early and late steps can now be targeted by inhibitors of virus binding and fusion  or assem


One of the greatest challenges facing biomedical research is the integration and sharing of vast amounts of information, not only for individual researchers, but also for the community at large. Agent Based Modeling (ABM) can provide a means of addressing this challenge via a unifying translational architecture for dynamic knowledge representation. This paper presents a series of linked ABMs representing multiple levels of biological organization. They are intended to translate the knowledge derived from in vitro models of acute inflammation to clinically relevant phenomenon such as multiple organ failure.ABM development followed a sequence starting with relatively direct translation from in-vitro derived rules into a cell-as-agent level ABM, leading on to concatenated ABMs into multi-tissue models, eventually resulting in topologically linked aggregate multi-tissue ABMs modeling organ-organ crosstalk. As an underlying design principle organs were considered to be functionally composed of an epithelial surface, which determined organ integrity, and an endothelial/blood interface, representing the reaction surface for the initiation and propagation of inflammation. The development of the epithelial ABM derived from an in-vitro model of gut epithelial permeability is described. Next, the epithelial ABM was concatenated with the endothelial/inflammatory cell ABM to produce an organ model of the gut. This model was validated against in-vivo models of the inflammatory response of the gut to ischemia. Finally, the gut ABM was linked to a similarly constructed pulmonary ABM to simulate the gut-pulmonary axis in the pathogenesis of multiple organ failure. The behavior of this model was validated against in-vivo and clinical observations on the cross-talk between these two organ systemsA series of ABMs are presented extending from the level of intracellular mechanism to clinically observed behavior in the intensive care setting. The ABMs all utilize cell-level agents that encapsulate specific mechanistic knowledge extracted from in vitro experiments. The execution of the ABMs results in a dynamic representation of the multi-scale conceptual models derived from those experiments. These models represent a qualitative means of integrating basic scientific information on acute inflammation in a multi-scale, modular architecture as a means of conceptual model verification that can potentially be used to concatenate, communicate and advance community-wide knowledge. The sheer volume of biomedical research threatens to overwhelm the capacity of individuals to process this information effectively, a situation recognized by the National Institutes of Health Roadmap in its "New Pathways" statement with its call for advancing integrative and multi-disciplinary research. Effective translational methodologies for knowledge representation need to move both "vertically" from the bench to the bedside, and be able to link "horizontally" across multiple researchers focused on different diseases. The hierarchical structure of biological systems is well recognized. Information is generated by research endeavors at multiple scales and hierarchies of organization: gene => protein/enzyme => cell => tissue => organ => organism. The existence of these hierarchies presents significant challenges for the translation of mechanistic research results from one organizational level to another  and testing of these models.Agent Based Modeling (ABM) is a computational modeling technique that is object-oriented, rule-based, discrete-event and discrete-time. ABM has characteristics that make it well suited for the goal of dynamic knowledge representation and conceptual model verification. The structure of ABM facilitates the development of aggregated modular multi-scale models ,2. ABM aABM, however, is not without its limitations. Specifically, two major limitations affect its use as a multi-scale modeling platform. The first has to do with the "black box" quality of ABM. Since the models rely on an ill-defined principle of "emergence" in order to transcend the epistemological boundaries represented by the multiple hierarchies of system organization, their behavior is difficult to characterize analytically. Therefore, ABMs are not "mathematical models" per se, being able to be subjected to formal analysis and "solved." Rather, the use of ABM falls into the category of "simulation science," in which computational analogs of real world systems are produced and used in a fashion similar to traditional experimental preparations. As such, the sizes of the models, in terms of numbers of components and scope of their environment, must have the extensibility at least to approach the dimensions of their real-world reference systems, particularly when multi-scale phenomena are the goal. Analytical tasks such as parameter sensitivity analysis and behavior-space determination rely upon brute force computation to generate data sets dense enough for appropriately grained statistical analysis. This requirement leads to the second hurdle in the use of ABM in a multi-scale context: their relatively high computational requirements as compared to equation based models. Currently, in general, most ABM platforms run as emulated parallel processing systems based on a single threaded central processing unit. The execution of an ABM requires multiple iterated computations as each discrete event is carried out, many more than for equation-based simulations, resulting in significantly greater computational demands. Despite ongoing work on hardware and software configurations to increase the computational efficiency of running ABMs, currently computational costs constrain the size of feasible ABM implementation. There is ongoing work in the development of "hybrid" model systems intending to use equations to model those aspects of a system in which mean-field approximations are valid, and link these components to ABMs where spatial heterogeneity and it effects are significant ,15. AddiThese challenges notwithstanding, a modular multi-scale architecture using the agent-based paradigm is proposed in this paper. I believe the benefits of an agent-based architecture in terms of modularity, translational efficacy and structural/organization mapping to biological systems outweigh the current limitations of this technique. Furthermore, the case will be made that, in terms of effective knowledge representation, a qualitative approach may often suffice for the goal of conceptual model verification. Acute inflammation, as a ubiquitous multi-factorial example of biocomplexity, is used as the demonstration platform for a series of ABMs developed at multiple levels of resolution, extending from intracellular signaling leading up to simulated organ function and organ-organ interactions. Specifically, the model reference system is the clinical manifestation of multi-scale disordered acute inflammation, termed systemic inflammatory response syndrome (SIRS), multiple organ failure (MOF) and/or sepsis. These clinical entities form a continuum of disseminated disordered inflammation in response to severe levels of injury and/or infection, and represent one of the greatest clinical challenges in the current health care environment. The core of agent-based architecture is a "middle-out" approach that focuses on representing and modeling cellular behavior as the agent level. Cells form a natural choice for the agent level in an ABM architecture. Cells are categorized by type, based on discovered and hypothesized rules of behavior, and can, to a great degree, be treated as "input-output" devices acting within a local environment. Cells are structurally and functionally aggregated into tissues and organs, the overall behaviors of which are determined by the actions and interactions of their constituent cells. Furthermore, the bulk of ongoing biomedical research is aimed at affecting the behavior of specific cellular types by the manipulation of their internal rules, and it is exactly the translation of this type of information/knowledge beyond the realm of solitary cells that underlies the core need for a multi-scale modeling platform.Therefore, the initial design aspects of a multi-scale architecture for modeling acute inflammation hinge upon identifying the key actors involved, and determining existing hypotheses aimed at unifying the problem of disseminated disordered inflammation. Two such unifying hypotheses involve viewing disordered systemic inflammation as either a disease of the endothelium -18 or a The base endothelial/inflammatory cell ABM has been previously developed and described ,6. The f+) [The reference model for the EBABM is a well-described human cultured enterocyte model (Caco-2) and its responses to inflammatory mediators including nitric oxide (NO) and a pro-inflammatory cytokine mix ("cytomix") that includes tumor necrosis factor (TNF), interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) -26. Thes+)  both tho+) -27.The EBABM was constructed using the freeware software toolkit Netlogo . The arcThe topology of the EBABM is a 2-dimensional square grid. The grid has 21 × 21 cells, in each of which there is an epithelial cell agent ("epi-cell'). The size of this grid was chosen as a representative portion of a total cell culture surface for reasons of computational efficiency; the processes being modeled by the EBABM are proportional to the cell surface area and the model could be, if desired, scaled up to any size. There are also two additional simulation "spaces," one layer representing the apical extracellular space (from which the diffusate originates) and another layer representing the basal extracellular space (into which the diffusate flows if there is permeability failure). A screenshot of the EBABM during an experimental run can be seen in Figure Calibration of the model was done using three behavior patterns of the EBABM compared to observed phenomena in the reference experimental systems. The first calibration was for the basal diffusion rate. The diffusion coefficient in the unperturbed system was adjusted to match the rate of diffusion in the reference data set at times 12, 24 and 48 hours. This established the baseline control permeability. The second calibration was done to reproduce the levels of administered cytomix and NO. The reference data sets were the levels of measured NO in both the exogenous NO donor arm and the cytomix administration arm  addition of a NO scavenger , 2) addi+ . The NO hibition ,31 by thThe results of the in-silico runs of the EBABM can be seen in Figures + on permeability, with the data in Figure + at 0.1 mM dose. Figures + administration on TJ proteins, specifically ZO-1 and occludin  or sterile endothelial damage (simulating tissue trauma) as was the case in the base endothelial/inflammatory cell ABM, gut ischemia was modeled as a percentage of the total endothelial surface rendered "ischemic," a state defined in the rules for the endothelial cell agents as an "oxy" level < 60. The affected endothelial cell agents were randomly distributed across the endothelial surface. The degree (or percentage affected) of the initial "ischemia" was controlled with a slider in the Netlogo interface. Therefore "Percentage Gut ischemia" (= "%Isch") represents the independent variable as initial perturbation for this model. Other than the changes noted above, no other changes to the rules of either the endothelial/inflammatory ABM or the EBABM were made.To address the issue of modeling the production of post-ischemic, pro-inflammatory lymph, attention is focused on linking the knowledge that has been acquired regarding the characteristics of the substance, and relating this information to the components of the organ ABM. The known characteristics listed above are used to exclude potential candidate-substances/actors from consideration. Specifically, this group comprises any of the cellular agents and any of the included cytokines. Therefore, the search is limited to:1) An as-of-yet unidentified compound linked to cellular damage. An example of such a compound would be high-mobility box protein 1 (HMGB-1), which to date has not been looked for in post-ischemic mesenteric lymph. In the organ ABM this variable is termed "cell-damage-byproduct," and it is calculated as a function of total endothelial damage with a set decay rate consistent with that of other bioactive compounds associated with inflammation.2) A luminal compound that diffuses in response to TJ barrier failure. This would correspond to potential byproducts of gut bacterial metabolism, or bacterial toxins, or other soluble aspects of the gut luminal environment that would leak into the gut tissue by virtue of the loss of barrier function. This variable is represented by "gut-leak," which is equal to the "solute" (from the EBABM) that penetrates the failed barrier.3) A down-stream metabolite of compounds generated by the inflammatory process. These would most likely be compounds generated by superoxide and NO reactions. For purposes of these simulations, levels of NO will be used as a proxy for this possible candidate.Therefore, the goal of the organ ABM simulation runs will be to examine the time course levels of these three values and identify which one (if any) matches the reported time course effects of the post-ischemic mesenteric lymph.The initial goal with the organ ABM simulations was to determine the greatest non-lethal level for "Percentage Ischemia" (%Isch). It should be noted again that the name of this variable is descriptive for how it is implemented in the ABM, and not intended to match quantitatively, per se, with measured ischemia in vivo. Rather "%Isch" is representative of the initial conditions for the simulation that will produce a pattern of simulation behavior that matches that of the in vivo system . A paramThe initial experiments with the organ ABM examined the effect of gut ischemia on TJ protein metabolism and the consequent effect on epithelial barrier function. The primary purpose of these experiments was to confirm that the epithelial agents' TJ metabolism and inflammatory signaling rules, as transferred from the EBABM, retained time course validity when the initiating stimuli were generated from endothelial/inflammatory cell interaction instead of exogenously administered cytomix. Figure hypothesis that some "later" byproduct of damaged gut tissue, rather than a diffused material or direct metabolite of first-pass inflammatory mediators, is the responsible compound in post-ischemic mesenteric lymph. It is recognized that this is "guided speculation;" however, it also demonstrates how the construction and use of models in the ABM architecture is an evolving process that parallels the development and refinement of conceptual models. As will be seen in the next section, the next scale of biological organization to be addressed in the ABM architecture involves the extension and integration of this hypothesis.The output from a representative run of the organ ABM with %Isch = 35 is shown in Figure Organs do not exist in isolation; their mutually complementary functions interact to sustain the organism as a whole. Unfortunately, disease states can lead to a breakdown of these interactions, causing a cascade effect as single organ dysfunction can lead to multiple system failure. Sepsis and MOF are characterized by a progressive breakdown in these interactions, leading to recognizable patterns of linked organ failure . TherefoThus far emphasis has been on the development of the gut organ ABM, and in order to model gut-pulmonary interactions it is necessary to develop a pulmonary ABM as well. Drawing upon the endothelial-epithelial bilayer configuration for a "hollow" organ, the pulmonary ABM utilizes the same endothelial-inflammatory cell component as the gut ABM, predicated on the relative homogeneity in structure and function of capillary endothelial cells (the blood brain barrier being the notable exception). Furthermore, pulmonary epithelial cells behave very similarly to gut epithelial cells with respect to tight junction metabolism and epithelial barrier function . TherefoFollowing the development of both a gut ABM and a pulmonary ABM, the next step is to connect them in a linked model. The topology of this relationship consists of two parallel bilayer planes, each bilayer representing one of the organ ABMs Figure . This isTwo clinical conditions were simulated to model organ-organ crosstalk along the gut-pulmonary axis of inflammation. The first has already been discussed at length: gut ischemia resulting from shock, demonstrating the effect that the gut has on the lung. The second will be a primary pulmonary process that will in turn affect the gut: pneumonia. "Pneumonia" is modeled as in infectious insult to the lung ABM using the same initial infectious insult rules as for the base endothelial-inflammatory ABM ,6. InfecFigure Therefore, to accomplish this goal, a very abstract means of organ support is modeled in the form of "supplementary oxygen." This function increases the amount of "oxy" that is able to be diffused through the pulmonary epithelial barrier and therefore available as systemic oxygenation. This is the qualitative equivalent of increasing the fraction of inspired oxygen, and therefore alveolar oxygen, and therefore can increase the partial pressure of oxygen diffused in the blood. It is qualitative in so much as there is no attempt to reproduce the dynamics of gas exchange, or the binding of hemoglobin to oxygen in the blood, or the effects of redistributed ventilation-perfusion matching in the lung as a result of hypoxia. This degree of detail is beyond the scope of this initial demonstration model; however the qualitative behavioral effects do show that this type of support, even abstractly modeled, increases the richness of the behavior of the model as a whole, and can extend the examinable behavior space of the model to situations that can approximate the effects of organ support in the ICU. The corresponding changes in outcome with this type of simulated organ support can be seen in Figure This sequence illustrates an important point in creating translational models of disease states. The tendency may be to attempt to model the pathological state being studied, i.e. creating a model of sepsis. However, it needs to be remembered that pathological states result from transitions from normal physiological behavior, and if the intent of a model is to facilitate the eventual transition from disease back to health, then "normal" mechanism must be the basis of a translational model. The need to capture transitions from one state to another takes on further importance when the pathological state results, as with sepsis, from medical/clinical interventions. Therefore, the architecture of a modeling structure needs to be flexible enough to accommodate the addition and integration of these factors, and it is hoped that the presented modular structure of the ABM architecture demonstrates this capability.The biomedical research community today faces a challenge that has paradoxically arisen from its own success: as greater amounts of information become available at increasingly finer levels of biological mechanism it becomes progressively more difficult for individual researchers to survey and integrate information effectively, even within their own area of expertise. Though technology, via tools like PubMED, the introduction of new publication formats like open-access journals and the development of a whole slew of bioinformatics tools, has aided the distribution and availability of biomedical information, it still falls upon the individual researcher to concatenate that information into a conceptual model that represents it. These mental models guide the direction of their individual research and, in aggregate, the form the components of the evolving structure of community knowledge. However, the formal expression of mental models remains poorly defined, leading to limitations in the ability to share, critique and evolve the knowledge represented in these conceptual models, particularly across disciplines. As a result it is increasingly difficult for both the individual researcher, and the community as a whole, to "know what it knows."so long as these hypotheses and assumptions are made explicit. This is necessary in any attempt to formalize the representation of conceptual models, as it will always be necessary to deal with the issue of incomplete knowledge. These models can aid in the scientific process by providing a transparent framework for this type of speculation, which can then be used as "jumping off" points for the planning and design of further wet lab experiments and measurements. I propose that ABM is a method well suited to fulfilling the goals of dynamic knowledge representation.These limitations can be overcome by developing methods of formal dynamic knowledge representation to allow researchers to express and communicate their mental models more effectively. Furthermore, in order to be able to "see" the consequences of a particular hypothesis-structure/mental model, the formally represented knowledge should be moved from a static depiction to a dynamic model in which the mechanistic consequences of each hypothesis can be observed and evaluated. In addition, as seen in the example of modeling the pro-inflammatory aspects of the post-ischemic mesenteric lymph, a method of formal dynamic knowledge representation also allows researchers to propose alternative solutions and generate hypotheses in the process of creating a model, This paper has presented a series of ABMs that are intended to introduce a multi-scale architecture that has the potential to serve as an overall unifying structure for representing biomedical knowledge. The "encapsulation" represented by the agent-based paradigm does not preclude the development of equation based or stochastic models; rather the modular, encapsulating structure is agnostic to the nature of the agent rule systems, and agnostic to the method of linkage to the various components. This is consistent with the "functional unit representation method" (FURM) concept developed by Hunt ,53) in wThe ABMs presented herein represent admittedly abstract representations of mechanistic hypotheses, but this need not be the case. Equations "encapsulate" knowledge as well, by providing mathematical abstractions of behavior that none-the-less must actually be implemented by some biological object. The extensive work on the mathematical characterization of intracellular processes in the systems biology field can form the basis of cell-level agent rules. In particular, the encapsulation offered by the ABM paradigm offers a method of meeting current challenges in the application of mathematical modeling techniques, such as parallel implementation of stochastic modeling with Gillespie algorithms, to reproduce population behavior and transcend biological scales of organization. The complexity and detail of these models is constrained only by the scope of that knowledge, and the ability to compute subsequently expressed rules. The former is the subject of the ongoing scientific process aimed at identifying mechanisms; the latter is the being addressed by a concurrent research community that seems to follow, at worst, the exponential progress represented by Moore's Law.© processor (as found in Sony's PS3) and graphical processing units (GPUs). However, despite the computational promise of these hardware platforms, there are still significant hurdles to the efficient implementation of ABM on these distributed systems. Central to these is the latency between intra-processor computational speed and that of node-to-node inter-processor speed. One approach is to improve the efficiency of the computational demands, such as reducing the number of agents that need to be treated individually via "dynamic agent compression" [Currently ABMs are severely limited by their computational requirements. For instance, the Netlogo models presented here are limited to a few thousand agents running abstract rules on a high-end desktop machine , with the result that a run of 7 days simulated time in the gut-lung axis ABM takes approximately 30 minutes. While scaling up pure ABM models is at this time not feasible, there is promise on the horizon. Advances in supercomputing have moved into implementation of distributed systems, including grid computing, massively parallel machines such as IBM's Blue Gene P, and the use of novel chip technologies such as the Cellression"  or strearession" . Anotherression" -60. ThatIn short, the agent-based paradigm, with its defining characteristics of encapsulation, modularity and parallelism, can provide an over-arching design architecture for the computational representation of biological systems. The examples presented herein are intended to be an introduction to this framework. For example, the detail of the molecular events can be represented at a finer grained level using ordinary differential equations, Gillespie-type algorithms or even particle-based signaling models. Cell behavior can be expressed as differential equation models derived from more detailed kinetic knowledge of their response curves. The physiological functions of individual organs can be represented using detailed physical system models detailing shear forces, stress response curves and contractility patterns. Every encapsulated object, at any hierarchy, can be represented in exhaustive detail using mathematical tools. However, two primary question exist: 1) is it even possible to exhaust the level of detail achievable to a pure reductionist's satisfaction? And 2) is it even necessary for the goal of conceptual model verification and representing knowledge? The modular, multi-scale agent-based architecture presented herein does not seek to answer those particular questions, but does hope to function as a seeming paradoxical solution to both questions by: 1) offering the opportunity to dig as deeply and with as much detail as desired, but also 2) to allow knowledge to be expressed effectively and usefully in the qualitative fashion that most researchers use to establish their conceptual models. This latter point cannot be over-emphasized, as ultimately the defining aspect of science is skepticism, the Popperian goal of hypothesis nullification.. Netlogo is a self-contained modeling toolkit, and is available for Windows, Macintosh and Linux. The Netlogo version of the innate immune response/endothelial model can be accessed at . The EBABM itself is available for download at: . The endothelial/inflammatory cell model can be downloaded at: . The Gut ABM and the Gut-Lung = Axis are available for download at: .The software used to create this model, Netlogo , is free+ : nicotinamide adenine dinucleotide; NF-kappa-B: nuclear factor-kappa B, NO: nitric oxide; NSF: N-ethylmaleimide-sensitive factor; SIRS: Systemic inflammatory disress syndrome; TJ: tight junction; TNF: tumor necrosis factorABM: Agent based modeling; ARDS: Adult respiratory distress syndrome; Caco-2: culture human enterocyte line; FURM: functional unit representation method; EBABM: epithelial barrier agent based model; ICU: Intensive care unit; IFN-gamma: interferon-gamma; IL-1: interleukin-1; iNOS: inducible nitric oxide synthetase; MOF: Multiple organ failure; NADThe author declares that they have no competing interests.