Green tea is widely consumed in Asian countries and is becoming increasingly popular in Western countries. Epidemiologically, it has been suggested that green tea consumption prevents type 2 diabetes. The present study was aimed at providing evidence of improvement in glucose metabolism in diabetic mice and healthy humans upon green tea consumption.Green tea promoted glucose metabolism in healthy human volunteers at 1.5 g/body in oral glucose tolerance tests. Green tea also lowered blood glucose levels in diabetic db+/db+ mice and streptozotocin-diabetic mice 2–6 h after administration at 300 mg/kg without affecting serum insulin level, whereas no effect was observed in control mice . The serum protein profiles of db+/db+ and +m/+m mice were analyzed for the first time by SELDI (surface-enhanced laser desorption/ionization)-TOF (time-of-flight)-MS (mass spectrometry), and then compared to investigate any effects of oral green tea administration on serum proteins. The protein profiles in db+/db+ mice showed that the spectral peak intensities at the mass/charge ratios (m/z) of 4119, 4203, 4206, 4211, 4579, 9311 and 18691 were >3 times lower, and those of 13075, 17406, 17407, 17418, 17622, 18431 and 26100 were >3 times higher than respective peak intensities in +m/+m mice. When green tea was administered to db+/db+ mice, the peak intensities were markedly decreased at m/z 11651 and 11863, and slightly decreased at m/z 4212. The peak intensities at 7495, 7595, 7808, 14983, 15614, 31204 were markedly increased after the administration.The present study provides evidence that green tea has an antidiabetic effect. Although we could not find simple reversed effect of green tea on the diabetes-induced modifications of the levels of several serum proteins, we found that the 4211 (4212) Da protein level that was decreased in the diabetic state was further decreased after green tea administration. This is the first report demonstrating that a certain serum protein may be involved in the antihyperglycemic effect of green tea. The contribution of this protein should be further studied. Camellia sinensis, Theaceae) is a popular beverage in East Asia, and also used as a herbal remedy in Europe and North America. Green tea is considered to be antiinflammatory, antioxidative, antimutagenic, and anticarcinogenic [Green tea -TOF (time-of-flight)-MS (mass spectrometry), because the SELDI-TOF-MS enables the measurement of the relative abundance of proteins of various sizes in a blood sample. Here, we compared the proteomic patterns of sera from diabetic db+/db+ and wild-type mice, and further investigated the influence of green tea administration on the proteomic patterns of serum in diabetic mice. If the diabetic modifications of serum protein levels are reversed by green tea administration, these affected proteins can be used as therapeutic targets in diabetes.We investigated whether oral glucose tolerance is improved in healthy volunteers by drinking a suspension of green tea powder. Blood glucose levels were measured before and 30, 60, and 120 min after drinking 1.5 g of green tea. The effects of hot water without tea powder on blood glucose level in the same participants were also investigated. Fig. The crude extract of fresh tea leaves picked from either No.12 or No. 13 cultivar at 300 mg/kg significantly lowered BGL 2–6 h after peroral administration in streptozotocin (STZ)-diabetic ddY mice in the fasting state . These results suggest that both fresh green tea leaf extract and green tea suspension lower BGL without changing serum insulin concentration.2+ , were also used to fractionate proteins in the serum. Then, these arrays were analyzed using a ProteinChip system, and the amount of individual serum proteins was estimated from the peak intensity of the mass spectral signal and the mass/charge ratio (m/z) equivalent to the molecular weight of each protein. All the spectral peaks of serum proteins from diabetic mice, which were significantly different (P < 0.05, by unpaired t-test) in intensities from those of serum proteins from wild-type mice, are shown in Fig. To explore the mechanisms underlying the antihyperglycemic effect of green tea, proteomic patterns of sera from diabetic (db+/db+) and wild-type (+m/+m) mice were first investigated and compared to identify peaks specific to the diabetic state using SELDI-TOF-MS. Samples of sera from fasting +m/+m mice were loaded onto CM10 (a cationic exchanger, pH4) ProteinChip arrays Fig. . The otht-test) in terms of peak intensity by the green tea administration, but not by the saline administration, are described in Fig. Serum protein profiles of db+/db+ mice were investigated 2 h after green tea administration, and compared with those administered with saline (control). Again, we confirmed a significant decrease in BGL by green tea in the blood samples used in these proteomic analyses (data not shown). All the protein signals that were significantly changed  of biomarker proteins that were abnormally contained in the serum of diabetic mice, without identifying individual serum proteins. As a result, we found that the levels of several serum proteins were significantly altered in the diabetic state. Secondly, we investigated which marker proteins are affected by green tea administration. Despite the changes in the levels of several serum proteins after green tea administration, none of the protein peaks specific for diabetes were sensitive to the administration, except for a peak at m/z 4211(4212). The level of this 4211(4212) Da protein was reduced both in the diabetic state and by green tea administration. Thus, no simple reversed effect of green tea on the diabetes-induced modifications of serum protein levels was observed.The 42114212) Da protein has not yet been identified, but only two candidate groups of vertebrate proteins are found by scanning Swiss-Prot database : pancreatic polypeptide (PP) (primary accession numbers P13083, P37999, P41519, P38000, P11967) and antibacterial peptides [β-defensin C7 precursor (018815), 4 kDa defensin (P56686), cryptidine-5 precursor (P28312), antibacterial peptide BMAP-34 precursor (P56425)]. PP is primarily expressed in the endocrine cells of the pancreas, and the plasma PP concentrations are elevated by food intake [ Da proteThe changes in serum protein profiles by green tea also demonstrate the increase in the peak intensities of hemoglobin-related multi-MS signals, suggesting the adverse side effects of green tea, although blood samples from db+/db+ mice tended to exhibit a hemolytic feature compared with those from wild-type mice (data not shown). Interestingly, the hemoglobin-related multi-MS signals shown in Fig. We observed that green tea improved oral glucose tolerance in humans. It is therefore likely that green tea is prophylactic against diabetes and ameliorates diabetic hyperglycemia. Green tea consumption at moderate doses may be associated with a reduced risk of type 2 diabetes in apparently healthy individuals by controlling postprandial hyperglycemia.The control of postprandial hyperglycemia by green tea can help reduce the risk of type 2 diabetes. In the present study, we provide evidence showing that green tea promotes glucose metabolism in healthy humans, and produces an antihyperglycemic effect in diabetic mice. In addition, we analyzed the serum protein profiles of db+/db+ and +m/+m mice for the first time using SELDI-TOF-MS, and further investigated its association with any effects of oral green tea administration on serum proteins. Among the several proteins that were significantly lowered in the serum of diabetic mice, the 4211(4212) Da protein was significantly decreased after green tea administration. This is the first report demonstrating that a certain serum protein is involved in the antihyperglycemic effect of green tea. The contribution of this protein, therefore, should be further investigated in a future study.Moreover, we speculate that the observed effects of green tea on BGL are primarily due to the promotion of insulin action in peripheral tissues, such as skeletal muscles and adipocytes. Indeed, a recent paper showed that green tea supplementation for 12 weeks ameliorates insulin resistance and increases glucose transporter IV content in a fructose-fed rat model resembling the human type 2 diabetes mellitus . Since tC57BLKS/J db+/db+ mice  and its age-matched control C57BLKS/J +m/+m mice  were purchased from SLC (Shizuoka, Japan) and used as the type 2 diabetic mouse model. Male ddY mice (4 weeks old, purchased from SLC, Shizuoka, Japan) were singly injected with STZ (150 mg/kg, i.v.), and then used 4–6 weeks after the injection (28.8–38.5 g, BGL: 247–600 mg/dl). The age-matched normal ddY mice (34.7–38.6 g, BGL: 129–197 mg/dl) were also used. The animals were housed (3–5 per cage) under a daily cycle of 12 h light and 12 h darkness, with free access to food and water. Animals were treated as approved by the Toyama Medical and Pharmaceutical University Animal Research Committee, and according to the guidelines for animal experiments established by the Japanese Pharmacological Society.Mice were deprived of food for 11–14 h. Blood samples (20 μl) were collected from mouse tail veins under ether anesthesia. In experiments for proteomic analyses and serum insulin measurements, blood samples (100 μl) were collected from the orbital venous plexus of mice under ether anesthesia. The food deprivation was continued throughout the measurement of blood glucose levels (until 6 h after the administration with green tea). In the oral glucose tolerance test as described below, blood samples (0.3 μl) were obtained from human skin microvessels using FreeStyle (Nipro, Osaka, Japan).Healthy human volunteers (18–24 years old) were fasted 12 h before the starting point of experiments. The participants were perorally administered with either a suspension of green tea powder or hot water at 9:20 a.m. Ten min after the administration (at 9:30 a.m.), the participants were perorally administered with 225 ml of Trelan-G75  containing 75 g glucose. BGLs were measured before and 30, 60, and 120 min after the administration with Trelan-G75 (glucose). All the subjects enrolled in this study were ethnic Japanese. Before participation, the purpose and risks of the study were carefully explained, and written informed consent was obtained from all the participants. The protocol was approved by the Toyama Medical and Pharmaceutical University Ethics Committee regulating human research.g for 1 min at 4°C, and its supernatant was immediately separated from the pellet.BGLs were measured using ANTSENSE II (Horiba, Kyoto, Japan) in the mouse study and using FreeStyle (Nipro, Osaka, Japan) in the human study. Serum insulin levels were measured using an insulin-ELISA kit (Morinaga Seikagaku, Tokyo, Japan). To prepare the serum samples, blood samples from mice (100 μl) were kept on ice for 2 h, centrifuged at 16,000 × 2+  (Ciphergen Biosystems), were used to fractionate proteins in serum. For the proteomic analyses with Q10 ProteinChip array, samples were 10-times diluted with denaturation buffer (7 M urea/ 2 M Thiourea/ 4% CHAPS/ 1% dithiothreitol/ 2% ampholine) and incubated on ice for 10 min, and then 10-times more diluted with buffer of 50 mM Tris-HCl (pH8). When CM10 ProteinChip array was used, samples were 10-times diluted with the denaturation buffer and incubated on ice for 10 min, and then 10-times more diluted with buffer of 100 mM NaOAc (pH4) or 50 mM HEPES (pH7). When IMAC30 ProteinChip array was used, samples were 100-times diluted with phophate-buffered saline. Spots on the different arrays were equilibrated with the buffer used for sample dilution, e.g., Tris-HCl buffer for spots on Q10 array, and each sample solution (70 μl) was loaded onto two separate spots on the arrays. After incubation for 20 min with rotation spots were rinsed with water and air-dried completely. The spots were analyzed using the SELDI ProteinChip system (PBS-IIc, Ciphergen Biosystems). In each sample, data from the two spots were averaged (duplicate assay). If a signal/noise ratio was larger than 2, the peak was considered to reflect the amount of a protein. Quantitative nature of the instrument was confirmed as previously described [Quantitative serum proteomic profiles were measured with SELDI-TOF-MS (Ciphergen Biosystems, Yokohama, Japan). The mouse serum was prepared as described above. Several types of ProteinChip Array, i.e., Q10 (an anionic exchanger, pH8), CM10 (a cationic exchanger, pH4 and pH7) and IMAC30-Cuescribed . The m/zCamellia sinensis, Theaceae) (Cultivar No.12, Chinhsuan and No.13, Tzuiyu) were prepared at the Taiwan Tea Experiment Station, picked in May at Nan-Tou County, and immediately used without drying. The leaves were steeped in hot water (95°C) for 30 min and the filtrate was condensed under reduced pressure. The dry weight of the extracts was determined, and the extracts were dissolved in saline for administration into mice. On the other hand, green tea powder was donated by Fukuju-en (Kyoto, Japan). The particle size (median diameter) of the green tea powder was 2.9 μm, according to the measurement using a Centrifugal Particle Size Analyzer (Shimadzu, Kyoto, Japan). In the human study, the tea powder (1.5 g) was added to 150 ml of hot water (80°C) and then whipped with a bamboo whisk. In the mouse study, the tea powder was suspended in saline at room temperature using a sonicator. The contents of caffeine and catechins in these tea samples were determined by HPLC: the samples were injected into an HPLC column (TSK-GEL, ODS-80TM, Tosoh, Tokyo, Japan); the eluent was 10 mM phosphate buffer (pH2.6)/MeCN (gradient: 5 to 15%) at a flow rate of 1.3 ml/min. The data are shown in Table Fresh raw leaves of tea  followed by the Scheffé's multiple range test. Values of P less than 0.05 were considered to be significant. Especially, to determine the significance of the time-dependent effects of green tea  and the peak intensity averages at m/z indicated . The analyzed peak is indicated by arrows in the data of mass spectral signals. **P < 0.01; significantly different from the peak in wild-type mice, by unpaired t-test. Types of ProteinChip used were described in the Fig. 6 legend.Click here for fileChanges in serum protein profiles of db+/db+ mice after green tea administration MS spectra shows typical changes in the serum protein profiles of db+/db+ mice administered with saline (left) or green tea (right). Graph shows the peak intensity averages at m/z indicated, before (open column) and after (closed column) administration with saline (n = 4) or green tea (n = 4). The analyzed peak is indicated by arrows in the MS spectra. **P < 0.01; significantly different from the peak obtained before the administration, by unpaired t-test. Types of ProteinChip used were described in the Fig. 7 legend.Click here for file


We examine results of gain-of-function experiments on retinocollicular maps in knock-in mice [Brown et al. (2000) Cell 102:77]. In wild-type mice the temporal-nasal axis of retina is mapped to the rostral-caudal axis of superior colliculus. The established map is single-valued, which implies that each point in retina maps to a unique termination zone in superior colliculus. In homozygous Isl2/EphA3 knock-in mice the map is double-valued, which means that each point on retina maps to two termination zones in superior colliculus. This is because about 50 percent of cells in retina express Isl2, and two types of projections, wild-type and Isl2/EphA3 positive, form two branches of the map. In heterozygous Isl2/EphA3 knock-ins the map is intermediate between the homozygous and wild-type: it is single-valued in temporal and double-valued in the nasal parts of retina. In this study we address possible reasons for such a bifurcation of the map.We study the map formation using stochastic model based on Markov chains. In our model the map undergoes a series of reconstructions with probabilities dependent upon a set of chemical cues. Our model suggests that the map in heterozygotes is single-valued in temporal region of retina for two reasons. First, the inhomogeneous gradient of endogenous receptor in retina makes the impact of exogenous receptor less significant in temporal retina. Second, the gradient of ephrin in the corresponding region of superior colliculus is smaller, which reduces the chemical signal-to-noise ratio. We predict that if gradient of ephrin is reduced by a genetic manipulation, the single-valued region of the map should extend to a larger portion of temporal retina, i.e. the point of transition between single-and doulble-valued maps should move to a more nasal position in Isl2-EphA3 heterozygotes.We present a theoretical model for retinocollicular map development, which can account for intriguing behaviors observed in gain-of-function experiments by Brown et al., including bifurcation in heterozygous Isl2/EphA3 knock-ins. The model is based on known chemical labels, axonal repulsion/competition, and stochasticity. Possible mapping in Isl2/EphB knock-ins is also discussed. Topographic ordering is an important feature of the visual system, which is conserved among many visual areas . Thus, tThe mechanisms responsible for topographic ordering have been lately under thorough examination. Following the original suggestion by Sperry , it was In this study we address the results of gain-of-function experiments, in which the retinocollicular maps were modified by genetic manipulations . RGCs ofIn addition to the observation of the overall map doubling in homozygous knock-ins Figure , Brown eBrown et al.  suggest absolute values of Eph/ephrin expression levels, rather than relative differences. Our model manages to reproduce all the essential features of experiments described in Brown et al. [The point, which we make in this study, is that more experimental tests are needed to justify the suggestion about relative expression levels. To make our point clear we present a model for the retinocollicular map formation, which is based upon differences in the n et al. , includin et al. ,23.To test various hypotheses we use a model for retinocollicular map formation employing stochastic Markov chain process. Our model is based upon three principles: chemoaffinity, axonal competition, and stochasticity. Some features of our model are similar to arrow model of Hope, Hammond, and Gaze . The impRA(i), where i = 1...100 is the RGC index, which also determines a discrete position of the cell in the retina. We have verified that results presented below do not depend on the number of cells, as long as this number is large enough. Each RGC is attached by an axon to one and only terminal cell in SC, which has an expression level of ligand given by LA(k), where k = 1...100 is the index in SC, also describing the terminal's topographic position. The receptor density RA is an overall increasing function of its index i, while the ligand density LA is decreasing, when going from k = 1  to k = 100  positions  is positive and gradients run in the same directions, PEXCHANGE >PRETAIN by the same reasoning. The described process will tend to exchange the order of gradients and, therefore establish the correct order of topographic projections. By using probabilities described by Eqs. (1) and (2) we incorporate the chemoaffinity principle into our stochastic model. This step is then repeated for another nearest neighbor couple, chosen randomly, and so on, until a stationary distribution of projections is reached. Such a process belongs to the class of Markov chain processes, since transformations to the map are determined only by the present state of the mapping and are not otherwise affected by development history [Assume that the product of gradients in Eq. (1) is negative, i.e. the gradients run in the opposite directions, which corresponds to the correct order of axonal terminals in SC. Then  history .α = 0 and when α is very large. In the former case (α = 0) the information about chemical labels cannot affect the solution, since it is multiplied by 0 in Eq. (1). Hence, the map is completely random  the molecular cues are very strong. They eventually produce solution in which the axons are perfectly sorted in SC in the order of increasing density of receptor  can be solved exactly for at least two limiting cases: when m Figure . In the r Figure . An integ Figure . We conce Figure .α  axis on retina to lateral-medial axis in SC . EphB2/3RB(1), RB(2), LB(1), and LB(2) are EphB receptor and ephrin-B ligand densities at neighboring points 1 and 2 in SC. This probability is similar to Eq. (1). Notice a sign change compared to Eq. (1), which insures that PEXCHANGE >PRETAIN if the order of gradients is wrong, i.e. if the gradients of receptor and ligand are antiparallel. By choosing this sign we therefore ensure attraction between axons and ligands.Here The details of our simulations are described in Methods. Our model allows not only exploration of two-dimensional maps Figure  but alsoIn our model the map is formed through interaction of three factors: Eph/ephrin-based chemorepulsion/attraction, competition between axons for space, and noise. It is the latter that fuses two maps together in rostral SC Figures  in this As we have shown above both ligand and receptor distributions influence the range of single-valued portion of the map independently  are therefore spatially separated for the DV axis. To observe mapping in these conditions we performed a numerical "experiment" on the Isl2/Two bifurcations observed in Figure It is also possible that activity-dependent mechanisms drive blending of two maps. Activity leads to focusing of projections whereby axons with close locations in retina are effectively attracted to each other in SC. Activity-dependent attraction will blend axons positioned proximally in SC, therefore ventral bifurcation, described above, may be robust with respect to these factors. The dorsal bifurcation, on the other hand, may or may not be observable if activity-dependent focusing of projections takes place. These questions will be addressed in future studies.Brown et al.  demonstrabsolute values of chemical label, as seen from Eq. (1). Indeed, in our model adding a constant value to all densities does not change the resulting mapping, since (1) depends only on differences in expression levels. But this manipulation decreases the relative differences in the expression of EphAs between neighboring knock-in and wild-type cells. Hence, our model is not based on relative differences between receptor densities. Yet, we demonstrate that it can account for experimental results in detail. Thus, we suggest that existing experimental evidence is not sufficient to distinguish relative and absolute labeling in the narrower sense.Can we take this idea to the next level and hypothesize that relative differences between neighboring retinal cells represent the chemical signal? This suggestion was used  to explaOf course, our model also accounts for the caudal displacement of wild-type TZs, thus resulting in a relative labeling system in the broad sense. In the first approximation, this model performs a sorting procedure, understood mathematically, of the fibers based on the expression levels of EphA. Our procedure uses differences in absolute values of EphA densities rather than relative differences. We suggest that more quantitative evidence is needed to distinguish these two "relativity principles".α is a function of label densities. Thus, the condition α ∝ 1/(RA·LA) ensures the Weber's law for axonal "perceptual thresholds", since chemical signal is proportional to the relative differences.Relative labeling in the narrow sense can be incorporated in our model too, if coefficient α → ∞), when perfectly ordered map is formed, our 1D model with nearest neighbor exchanges is equivalent to the 1D version of the arrow model. However, in the stochastic regime, description of developmental noise is different here. In particular, we relate features of the map, to the distribution of chemical labels. We argue that this feature is important in understanding experiments [Theories based on chemoaffinity principle are reviewed in . Some feeriments , since deriments ,9. In thPrestige and Willshaw  suggesteIn a recent study Honda  considerWhen dealing with numerical simulations one always faces the question of the degree of realism with which to model the data. Does one have to model behaviors of individual atoms, or description on the level of axons is sufficient? In this work we choose the level of description on the basis of what is known about this system. We realize that our model does not capture many behaviors, but we argue that the mechanisms involved are unclear at the moment to be incorporated into a more detailed model. Our approach also fulfils its original goal, which is to reproduce the results of experiments  and to gModel presented here does not describe the difference between development along TN and DV axes. The former mapping is controlled by original axonal overshoot along the RC direction in SC, with subsequent elimination of topographically inappropriate projections ,9. In coWe present a model for retinocollicular map development, which can account intriguing behaviors observed in gain-of-function experiments by Brown et al. , includiTo find a stationary distribution of the RGC's axons in the SC, we use the following computational procedure. We consider a linear chain of 100 RCG that are connected to one and only terminal cell in SC each. The receptor and ligand expression level profiles used in the computations for wild-type, heterozygote and homozygote are shown at Figure 6 (nearest neighbor exchanges). The main parameter of our model is taken to be α = 30 throughout the paper. It is chosen to fit the experimental data from Brown et al. [α is roughly equal to inverse of the relative diameter of the TZ squared. Thus, in our results, TZ occupies, roughly, 20% of the entire SC, which corresponds to the value of α given above. The probability distribution and the position of the maximums shown at Figure 4 realizations of stationary solution separated in time by 103 iterations (nearest neighbor exchanges).The typical stationary solution for one realization is shown at Figure n et al. . We haveLA(x) = exp(-x) ].We base our choice of parameters for the distribution of molecular markers on experimental observations in mouse retina and SC. Thus, the distribution of ephrinA2 and A5 is obtained in . The tot) Figure , etc. No2, obtained in [RA(x) = exp(-x)  = exp(-2x)  Figure .LB(y) = exp(-y) and RB(y) = exp(-y). As with the EphA/ephrinA the non-unit overall factors in these distributions are absorbed in parameter β (see below).The profiles of expression of EphB/ephrinB pair are measured in  similar Here we describe our 2D model in more detail. We consider an array of 100 by 100 RGC, which are connected to 100 by 100 different points in colliculus. Each RGC is characterized by two levels of expression for two receptors, EphAs and EphBs, described in the text. The concentration profiles are taken to be the same for EphA and EphB receptors in the wild-type species. In the homozygote and heterozygote cases the concentration of EphA is taken as shown at Figure α = β = 30.The process of development is modeled as follows. We randomly choose a pair of axons in SC separated either in RC or in ML direction. We exchange their positions with the probability given by Eq. (1) or Eq. (3) respectively. We then repeat the process until a stationary distribution of probabilities is reached in the same manner as for 1D case. Note, that this time a chosen pair of axons, say in RC direction, may not be a neighboring pair, but consist of two axons separated by any distance in SC. This procedure dramatically decreases the convergence time to the stationary distribution, which is the same as in the case when we choose the neighboring axons only. The noise level is taken to be the same for both RC and ML directions, that is 6 iterations. The temporal evolution of the map for the label in the central retina is shown at the Figure The spatial 2D distribution of the axons corresponding to labeled RGCs is shown at Figure In both 1D and 2D cases the calculations were performed on Dell PowerEdge 1600SC server. The programs, written on Matlab (MathWorks, Inc.), are available for download in .Equations for the probability of switching of two axons (1) and (2) can yield probabilities, which are below 0 or larger than 1. Thus, in the numerical implementation of our model instead of (1) and (2) we use expressions with soft cutoff at 0 and 1, i.e.These probabilities are restricted to be between 0 and 1. In addition, when differences in ligand and receptor densities between neighboring points are not large, (4) and (5) are equivalent to (1) and (2).final probability distribution for connectivities does not depend on whether the swaps are local or global. This statement is true, for any distribution of chemical labels, in 1 or 2D. It is true however if Eq. (4) is used to calculate probabilities of transitions. In particular, we show that Eq. (4) leads to a Boltzmann distribution of the probabilities of connections, which does not depend on the locality/globality of transitions. Thus, we can present final maps for both local and global transitions interchangingly, since results pertaining to the final state of the map, such as in Figures One could use exchanges between nearest neighbors to implement map development, as described in text above and in . Alternar if Eq.  is used PA and PB. These probabilities satisfy the detailed equilibrium condition [Let us now derive the probability distribution of projections in the final map. We perform our derivation for the 1D case; in 2D it is similar. We proceed using the detailed equilibrium principle, frequently employed in statistical mechanics . Consideondition where the transition probabilities are given by equation (4). After some algebra, it is possible to show that the transition probabilities are given by a simpler than (4) formEA and EB are 'state' variables, depending on the current arrangement of axons in the targetwhere i, with L(i) being the ligand concentration and R(i) the receptor concentration. The latter, of course, depends on the arrangement of axons, corresponding to the state A. The transition probability PB → A is given by the same expression, with exchanged indexes A and B. The detailed equilibrium condition (6) leads then to Botzmann probability distribution of the states of the mapHere summation is assumed over all termination sites in SC, denoted by index not depend on the methods of reconstruction. Thus, both global and local transitions will lead to identical final arrangement of the map. This property is well-known in considering Metropolis Monte-Carlo procedures. What does depend on the methods of reconstruction is the time, which it takes to reach the final configuration. Thus, as it was mentioned above, global transitions lead to the final state much faster. With local transitions, on the other hand the map can freeze in the original state, and it may take an exponential time to reach the final configuration.Eq. (9) is instrumental in showing that the final distribution of projections in our approach does We thus conclude that our results presented in this study are universal in that they do not depend on the exact developmental mechanism, but only on the distribution of 'chemical' tags.


For more than 2,000 years, from the time of Aristotle onwards, it was thought that the complete body plan of human beings  was present in the fertilized egg. During pregnancy, a preformed miniature human being, or homunculus, grew bigger and bigger; development was simply a process of growth.Then, in the mid-18th century, Carl Friedrich Wolff described how the chick gut, basically a tube, forms from an initially flat sheet of cells, overthrowing at a stroke the preformation theory of embryology. We now know that development is a complex series of coordinated processes that transforms the amorphous ball of cells produced from the fertilized egg by cell division into an intricate body containing numerous specialized tissues and organs. And we are beginning to understand how a wide array of transcription factors—proteins that bind to regulatory sequences within genes to control their expression—guide the sequential stages involved in development. It seems that these factors form regulatory networks that control the temporal and spatial waves of gene expression that underlie and are required for organized body building.Susan Mango and her colleagues are studying the role of transcription factors in controlling organ development. The organ they are studying—the pharynx of the nematode worm—is relatively simple. This muscular tube, which passes bacteria  from the mouth to the midgut, contains fewer than 100 cells of only seven different types.To get an overall picture of the regulatory sequences within genes that are involved in the temporal control of pharyngeal development, the researchers identified 339 candidate pharyngeal genes by comparing gene expression profiles in mutant worm embryos that had excess pharyngeal cells with those in mutant embryos lacking pharyngeal cells. Then, by referring to a database that details gene expression patterns in nematode worms and embryos, the researchers classified 37 of their candidate genes as having early-onset expression and 34 as having late-onset expression.Next, the scientists carefully examined the DNA sequence of each gene for candidate regulatory regions that might contribute to its temporal regulation. Of nine candidate motifs revealed by this search, six functioned as regulatory sites in in vivo assays. The researchers estimated that these six elements, together with sites that bind PHA-4—a member of a family of transcription factors that are important in digestive tract development in many animals—account for the timing of onset of expression of about half of the nematode's pharyngeal genes. Finally, the researchers used combinations of the newly discovered temporal regulation sites and PHA-4 sites in a genome-wide search that predicted pharyngeal genes and their time of onset of expression with greater than 85% accuracy.From these results and those of previous studies, Mango and her colleagues propose a model to explain how the temporal control of pharyngeal gene expression needed for pharynx development is achieved. The earliest time for pharyngeal gene expression, they suggest, is determined by how well PHA-4 sticks to a particular gene's binding site. However, gene expression only occurs if other factors that bind to the regulatory sites are also present, and the exact combination of these factors determines which gene is active at any given time. The identity of these factors remains to be discovered. Nevertheless, at least for this simple organ, we now have a much better idea of how the complex process of organ formation is controlled at a molecular level, and it is likely that similar regulatory networks will underlie the formation of other organs as well.


Basic manufacturing principles are becoming increasingly important in high-throughput sequencing facilities where there is a constant drive to increase quality, increase efficiency, and decrease operating costs. While high-throughput centres report failure rates typically on the order of 10%, the causes of sporadic sequencing failures are seldom analyzed in detail and have not, in the past, been formally reported.Here we report the results of a failure mode analysis of our production sequencing facility based on detailed evaluation of 9,216 ESTs generated from two cDNA libraries. Two categories of failures are described; process-related failures (failures due to equipment or sample handling) and template-related failures .Preventative action based on a detailed understanding of failure modes is likely to improve the performance of other production sequencing pipelines. The average Q20 read length of data generated in the past 12 months (December 2003 to December 2004) from various library types and vector systems is 751 bases. The present study was undertaken to provide insight into the causes of sequencing failures and possible corrective actions. Although our pipeline uses exclusively ABI 3700 and 3730XL automated sequencers, these results should be applicable, in principle, to the improvement of other high throughput sequencing platforms.In the past decade, the demand for DNA sequence data has driven the transformation of sequencing from a research activity into a manufacturing process. High-throughput sequencing facilities are focused on establishing automated procedures that maintain long read length and high overall success rates. It is neither practical nor economical to test each and every DNA template before sequencing . Sequenc20 read length for the entire data set was 771 bases, average pass rate was 87% which was calculated as a percentage of sequencing reactions yielding a minimum of 600 Phred 20 bases. Figure 20 read lengths from the full data set. The analysis methodology employed to determine the failure mode of each trace is outlined in Figure 20 < 600) for further analysis to determine failure mode. The electropherograms from the 1,172 failed reads were evaluated and subsequently categorized into failure mode categories. 64 of these reads were yielded from sequencer capillaries that were clogged and therefore were removed from further analysis and categorized into the "Blocked capillary" failure mode. The remaining 1,108 traces were further classified into nine additional failure mode categories including Low signal strength, Mixed clone with vector sequence, Mixed clone- no vector sequence, Low signal to noise ratio, Excess dye peaks, Hardstop, Repetitive sequence, Homopolymer stretch, and Poly A tail. Results and trace characteristics used to classify each read are as described in Table We generated 9,216 reads from 2,304 clones selected randomly from two cDNA libraries. For each of the two libraries, 1,152 bacterial colonies containing cDNA inserts were picked and arrayed into 384-well microtiter plates  in the preliminary "Low signal strength" category in Table -3, 2.2 × 10-5, and 9.7 × 10-8, respectively. Thus, it is unlikely that more than one read failed per clone by chance.The 164 reads that appear to have sufficient template DNA are grouped by source clone for further analysis. Four possible reads may have failed for each unique clone: 5' replicate #1 (F1), 3' replicate #1 (R1), 5' replicate #2 (F2), and 3' replicate #2 (R2). There were 49 occurrences where two reads failed from one source clone, 2 instances where three reads from the same clone failed and 4 occurrences where all four reads failed from the same source clone. These results are shown in Table Groupings with two failed reads from the same end of the template, "F1/F2" and "R1/R2" showed trace data with noisy background or no signal trace therefore there was insufficient information to determine actual template characteristic leading to the problematic results. With the low probability of seeing two failed traces from one unique clone, we conclude that these failures are most likely a result of template DNA with 5' or 3' ends that are problematic for sequencing. Possibilities include secondary structure, mutated priming sites, long poly A tail, or other homopolymer stretches near the priming sites on the template.The "F1/R1" grouping contained failed reads from both ends of the same replicate but successful end reads from the second replicate. This would indicate that there were no clone related attributes on either end of the template to prevent good sequence thus the failed reads were assessed a process-related failure due to loss of DNA during the precipitation process.The "F1/F2/R1/R2" grouping indicates that no passing sequence data were obtained from 5' or 3' ends of both replicates from the original source clone, all 16 reads showed presence of DNA at each observational check in the process pipeline. The probability that 4 reaction products from a single clone were lost during precipitation is extremely improbable and it is more likely that the original clones had attributes that prevented successful sequencing from either ends. We therefore assign 5' and 3' template-related failure modes for the 16 reads in this grouping.The final groupings of "F1/F2/R1" and "F2/R1/R2" contain 6 reads in total. Each include failed reads from both ends of one replicate and a single failed read from the second replicate. We maintain the same logic for our failure mode evaluation by assessing both a template-related failure to the 2 failed reads from the same replicate and a process-related loss of DNA during precipitation failure for the single read from the second replicate.There were 44 reads in the singleton category where only a single read (F1, F2, R1, or R2) failed from each clone. These failures cannot be attributed to the template as three other reads were successfully sequenced from the same source clone. Failure mode was therefore likely to be process related and due to loss of DNA during the precipitation wash steps.20: 500–599 bp bin. This is likely the result of a presence in greater amount of one reaction product over the second leading to proportionately stronger signal strength.Having established a stringent pass criteria of 600 Phred 20 bases or greater, we observed a broad range of failure modes distributed among the failed reads. Reads less than 100 bp did not yield useable data and were usually a result of process-related failure modes. Process-related mishaps in the pipeline would include missed colony picks, blocked capillaries on sequencers, faulty tips on automated liquid handling devices, or lost DNA template during precipitation. The sequences generated have low average read lengths, or in cases where DNA is totally lost, zero read length. 88% of reads within the largest "process" related failure mode category, "low signal, no DNA", were a result of failed cultures at the beginning of the process pipeline. The absence of DNA was verified early on by failed growth in the source microtiter plates and further confirmed by failed growth in the stamped replicate agar plate. Failures are perhaps due to missed bacterial colony picks and adjustments such as recalibration or refining the morphology criteria of the colony picker should help. The second most prevalent failure mode in the "process related" category, "Mixed clone with vector sequence" (30% of reads in this classification), is likely a result of two side by side colonies being picked into the same well or through contamination originating from the use of automated liquid handlers. These problems might be alleviated by increasing the stringency of the proximity criteria on the colony picking device to ensure sufficient distance exists between two bacterial colonies before they are chosen for picking. As we use new tips on the instruments daily, increasing the length or number of wash cycles on the automated liquid handlers between volume transfers may help in reducing carry over contamination from one plate to another. This will need further investigation. Cross contamination between adjacent wells can also arise with the mishandling of plates, or when tips are plunged too deeply into the wells, displacing the volume into adjacent wells. Adjusting the liquid handler by reducing the depth and speed of tips entering each well may help prevent the occurrences of cross contamination within a plate. Despite the presence of mixed clones within one well, traces often yielded discernable sequence with read lengths broadly distributed up to the Q23N (N = A, G, C) anchored primer to resolve the 3' ends of cDNA inserts.Template-related failures often still yielded reads of several hundred base pairs. Template characteristics that might contribute to failed or truncated reads could be poly A tails, homopolymer stretches, or other highly repetitive regions. Problems in sequencing these types of regions are well cited in the literature and are dependent on chemistry and methodology used in sequencing. These problems are best avoided by using an anchored primer for first strand synthesis of cDNA as the first step in library construction. "Poly A tail" was the single largest cause of failed sequence in both the template-related failure category as well as the entire data set, yet the reads make up the majority in the higher failed read length bins. In our regular production pipeline, we often circumvent this problem by using a combination of a 3' primer to resolve sequence immediately following the poly A coupled with an oligo(dT)The FMA pipeline described here was tailored to our high-throughput 384-well automated sequencing pipeline but many of the components in this platform are shared within the high-throughput sequencing community such as the alkaline lysis procedure to prepare and purify DNA, DNA sequencing using Big Dye chemistry, ethanol precipitation to clean up reaction products and equipment such as the Genetix QPIX, Beckman Coulter Biomek FX, and Applied Biosystems 3730 xl DNA analyzer. For this reason our FMA methods can be readily adapted to analyze other similar sequencing platforms. Extending our present study to other library types commonly sequenced in the high-throughput community, such as shotgun or Serial Analysis of Gene Expression (SAGE) should offer further information regarding template specific failures. This failure mode analysis provides information on distinguishing between process and template-related attributes that may lead to downstream failed sequence. It can therefore be a useful tool used to audit overall sequencing procedures and identify key problematic steps in a process pipeline. Making proper adjustments to the pipeline based on the results will likely result in increased efficiency, enhanced data quality, and decreased cost.Samples are processed in duplicate, with quality assurance and observational checks to account for status of each read after every procedure leading to the final sequence read. These recorded observations facilitate downstream systematic analysis of failed sequence data. The regular core production pipeline was not altered for this study, as the purpose is to assess sequence failure modes under ordinary conditions in a high-throughput sequencing environment. An overview of the observational checkpoints within the FMA pipeline is outlined in Figure Populus trichocarpa cDNA libraries were transformed by electroporation into 40 μl of E. coli DH10B T1 resistant cells (Invitrogen). Transformed cells were recovered using 1 mL of SOC medium (prepared in house) and plated onto 22 cm × 22 cm agar plates (Genetix) containing 2xYT agar and 100 μg/μl Ampicillin. Agar plates were incubated overnight at 37°C for 14 hours. Bacterial colonies were picked from the agar plates and arrayed into 384-well microtiter plates (Genetix) containing 60 μl of 2xYT medium + 7.5% glycerol (made in house) using the Genetix QPIX automated colony picker (Genetix). A total of six 384-well microtiter plates, three plates for each of the two cDNA libraries, were picked. The plates were incubated overnight at 37°C for 16 hours then each microtiter plate was inspected for wells that contained no growth. The positions of all failed cultures were recorded. To further verify growth in each well after the incubation period, a disposable 384-well replicator was used to stamp bacterial culture from each 384-well microtiter plate onto a new 22 cm × 22 cm agar plate containing 2xYT agar and 100 μg/ul ampicillin. Agar plates were incubated overnight at 37°C for 14 hours then inspected the next day for colonies from every well. The positions of all failed growths were recorded.One microliter of ligation mix from each of two Two microliters of bacterial culture was transferred from the 384-well microtiter plate into a 240 μl 384-well deep well diamond plate (Axygen) containing 60 μl of 2xYT medium and 100 μg/ml ampicillin using a 384-well slotted inoculator (V&P Scientific). This was done in duplicate to create two sets of six 384-well deep well inoculated diamond plates. Inoculated plates were sealed with AirPore™ tape (Qiagen) and placed into a 37°C shaking incubator (New Brunswick Scientific C25 Incubator Shaker) at 350 rpm for 18 hours. After the incubation period, cultures were removed and each plate inspected for growth and contamination. All failed cultures were recorded. Cultures from both replicates were then placed onto separate multi-tube floor vortexers (VWR) at maximum speed for approximately 5 minutes until all cells were resuspended. Cultures were stored at 4°C until ready for DNA preparation. DNA was prepared using alkaline lysis [DNA preparations were evaluated by agarose gel electrophoresis. A 1.5 μl aliquot of purified DNA was combined with 1.5 μl of bromophenol blue loading buffer  and 2 μl was loaded onto a 1.2% agarose gel. Samples were loaded using a 12-channel loader (Hamilton) beside 3 ng of 1 kb plus DNA marker (Invitrogen). Gels were run at 120 volts for 90 minutes in TAE (Tris/Acetate/EDTA) buffer followed by staining for 35 minutes in SybrGreen Nucleic Acid stain (Cambrex). Gels were scanned using a Fluorimager 595 (Molecular Dyanmics) scanner. The image was visually examined for genomic DNA, as well as presence and quality of DNA. All empty lanes and observations were recorded and entered into the FMA database.DNA Sequencing reactions were assembled in 384-well clear optical reaction plates (Applied Biosystems) using a Biomek FX workstation (Beckman-Coulter). In each 5 μl reaction  the following were added: 3 μl of purified plasmid DNA (~45 ng/μl), 0.26 μl of sequencing primer (5 pmol/μl, Invitrogen), 0.43 μl of 5X reaction buffer (Applied Biosystems Big Dye Terminator 5X Sequencing Buffer), 0.77 μl of Ultrapure water (Gibco), and 0.54 μl of BigDye v.3.1 ready reaction mix (Applied Biosystems). Each well of the reaction plate was visually inspected for appropriate volumes after both reaction mix and DNA addition. Volumes were manually adjusted using a single channel pipet (Gilson) where required and all observations were recorded. Sequence data were obtained using universal M13 Forward (5'-TGTAAAACGACGGCCAGT-3') and M13 Reverse (5'-CAGGAAACAGCTATGAC-3') primers on each set of replicate plates. Thermal cycling was performed on PTC-225 thermal cyclers (MJ Research) with parameters of 35 cycles at 96°C for 10 seconds, 52°C for 5 seconds using M13 Forward primer or 43°C for 5 seconds using M13 Reverse, 60°C for 3 minutes, followed by incubation at 4°C. Reaction products were precipitated by adding 2 μl of 125 mM EDTA (pH8) and 18 μl of 95% ethanol per well followed by centrifugationat 2750 × g for 30 minutes in an Eppendorf 5810R centrifuge. The EDTA/ethanol was immediately decanted and reaction products washed with 70% ethanol. The 384-well cycle plates were allowed to dry inverted for 15 minutes. Samples were resuspended in 10 μl of Ultrapure water and analyzed using a 3730XL DNA analyzer (Applied Biosystems). The performance of each capillary on the four DNA analyzers used in this experiment were validated using one 384-well control plate for each instrument. Each 384-well plate contained our in-house control standard, a full-length human cDNA clone obtained from the I.M.A.G.E. Consortium . Blocked capillaries were recorded into the FMA database and traces originating from these capillaries flagged. Sequence data were evaluated using PHRED software [GSY was responsible for design and execution of this study, plus analysis of data and drafting of the manuscript. RAH conceived of the study and directed the analysis. JMS and SAB generated the data described in the study and participated in study design. DS developed several of the key protocols used in this study. MB provided quality assurance of the experimental procedures and participated in study design. MAM participated in the establishment of the production pipeline and read the manuscript and provided comments.


The practice of continuous, long-term monitoring of human joint motion is one that finds many applications, especially in the medical and rehabilitation fields. There is a lack of acceptable devices available to perform such measurements in the field in a reliable and non-intrusive way over a long period of time. The purpose of this study was therefore to develop such a wearable joint monitoring sensor capable of continuous, day-to-day monitoring.A novel technique of incorporating conductive fibers into flexible, skin-tight fabrics surrounding a joint is developed. Resistance changes across these conductive fibers are measured, and directly related to specific single or multi-axis joint angles through the use of a non-linear predictor after an initial, one-time calibration. Because these sensors are intended for multiple uses, an automated registration algorithm has been devised using a sensitivity template matched to an array of sensors spanning the joints of interest. In this way, a sensor array can be taken off and put back on an individual for multiple uses, with the sensors automatically calibrating themselves each time.The wearable sensors designed are comfortable, and acceptable for long-term wear in everyday settings. Results have shown the feasibility of this type of sensor, with accurate measurements of joint motion for both a single-axis knee joint and a double axis hip joint when compared to a standard goniometer used to measure joint angles. Self-registration of the sensors was found to be possible with only a few simple motions by the patient.After preliminary experiments involving a pants sensing garment for lower body monitoring, it has been seen that this methodology is effective for monitoring joint motion of the hip and knee. This design therefore produces a robust, comfortable, truly wearable joint monitoring device. Long-term measurement of human movement in the field is an important need today . For manMany specific medical applications benefit from the information provided by continuous human movement monitoring. To better develop and optimize total joint replacements, for instance, a detailed record of a patient's daily activities after such a replacement is required . The meaPresently, there is no satisfactory solution for long-term, human movement monitoring in the field. The use of video and optical motion analysis systems offer the most precise evaluation of human motion, but obviously restrict measurements to a finite volume . Body moElectrogoniometers are frequently used to measure dynamic, multi-axis joint angle changes in individuals, providing continuous joint movement information. These devices, however, are not desirable for long-term monitoring of daily living, since they are exoskeletal devices that cross the joint, potentially interfering with movement. Furthermore, any shift from their original placement leads to errors in angle estimations . Such coOther types of goniometric devices have been developed for measuring particular parts of the body. Electronic gloves -13, for For all types of body-mounted sensors, the issues of comfort and wearability are of major importance, if a patient has to wear the monitoring device for extended periods of time. Furthermore, such home-use wearable sensors need to be put on and off every day without close supervision of a medical professional. Proper registration of the sensor is therefore a crucial requirement for deploying wearable sensors to the home environment.The goal of this paper is therefore to develop a new method for continuous monitoring of human movement by measuring single or multi-axis joint angles with a wearable sensing garment that is non-intrusive and non-cumbersome and that can be properly registered for reliable monitoring. A new method is presented here for joint monitoring using conductive fibers incorporated into comfortable, flexible fabrics. All that is needed is a one-time calibration with a standard goniometer, and a conductive fiber sensor garment is then able to continuously detect joint movement and measure specific single or multi-axis joint angles. With an array of sensors incorporated into a sensing garment, registration of the sensor occurs automatically each time the garment is worn through only a few simple motions by the wearer. This type of wearable sensor would allow extended home monitoring of a patient, and is no harder to put on than a typical article of clothing.In the following, the principle and design details of this wearable device will be presented, along with effective algorithms for allowing a patient to perform long-term, unsupervised monitoring in the home environment. Experimental feasibility tests will also be presented on a prototype wearable sensor for both single-axis and multi-axis joints.The basic principle behind the wearable sensors presented in this paper is as follows: when a particular joint on the human body moves, skin around the joint stretches, along with any clothing surrounding the joint as well. A former study by the textile industry has shown that body movements about joints require specific amounts of skin extension. Lengthwise across the knee for example, the skin stretches anywhere from 35–45% during normal joint movement .When a particular joint moves, fabric around the joint will either expand or contract accordingly, assuming the fabric is form fitting to the skin, and has the necessary elastic properties. To assure comfort and freedom of body movement, stretchability of 25 to 30 percent is recommended for fabrics fitting closely to the body . By incoA in the figure. Along the conductive fiber, there is a wire contact point at B that is permanently stitched into the fabric. The other end of the conductive fiber, point C, is kept in tension by a coupled elastic cord, which is permanently attached to the remote side of the joint, point D. Therefore, any stretching in this coupled material will take place in the highly elastic cord, CD, and not in the conductive fiber AC. As the joint moves, the elastic cord will change length, causing the coupled conductive fiber to freely slide past the wire contact point at B that is stationary. The conductive fiber always keeps an electrical contact with this wire, but the length of conductive thread between points A and B will change as the joint rotates. The resistance, which is linearly related to length, is then measured continuously across these two points A and B.Figure θ1, θ2, and θ3. Our goal is to estimate these joint angles based upon the output of sensors 1, 2, and 3.Consider Figure Preliminary experiments have shown a clear relationship between joint angle and sensor output for individual sensors about various joints of the body. Figure It is desired to design a filter that receives sensor signals as inputs, and predicts the joint angle(s) of interest. In the proposed method, each joint angle being monitored has a corresponding single sensor that is situated about that particular joint for maximum sensitivity, as in Figure N axis sensors for measuring N joints, each consisting of a single thread sensor, as shown in Figure Consider  is the N × 1 vector of N joint angle predictions,  is its bias term, y = (y1 … yN)T is the N × 1 vector of corresponding sensor readings, and G and  are, respectively, the N × N matrix and the N × 1 vector experimentally determined to relate the inputs and the outputs.where Since there is a slight amount of curvature in the preliminary data of Figure whereG' is an N × N(N-1)/2 experimentally determined matrix. The three terms on the right hand side of the above equation can be incorporated into a homogeneous expression using augmented matrix and vector:and W and Y arewhere W = (θ0 G G')     (5)W, a least squares regression is performed using m sets of experimental data from a collection of sensors on an individual patient. Let P be a N × m matrix consisting of m sets of experimentally measured joint angles,To determine the parameter matrix B be a {1 + N(N + 1)/2} × m matrix containing the corresponding sensor outputs and their quadratic terms:and B = (Y(1) … Ym)     (8)W* that minimizes the squared prediction errors is given byThe optimal regression coefficient matrix W* = PBT (BBT)-1     (9)BBT non-singular.if the data are rich enough to make the matrix product N axis sensors. In practice, however, they can be reduced to a compact expression with lower orders. First the offset  can be eliminated from the coefficient matrix W, if the sensor outputs are zeroed at a particular posture, e.g. the one where the extremities are fully extended. Second, although the matrix G contains off-diagonal elements representing cross couplings among multiple joints, some joints have no cross coupling with other joints. For example, the measurement of the knee joint can be performed separately from that of the hip joints. If the j-th joint is decoupled from all others, it can be treated separately as:The above expressions are the most general forms for G, their second-order cross coupling terms, e.g. yj yk, can be negligibly small with proper design of individual sensors. In such a case, two coupled joints, say j and k, can be written as:where the offset is eliminated. Third, although multiple joints are coupled to each other having non-zero, first-order off-diagonal coefficients in matrix where the offset terms have been eliminated. Thus the number of parameters to identify through calibration experiments is reduced. In consequence, the dimension of the optimal coefficient matrix must be reduced accordingly. The same calibration procedure is performed for both single axis and multiple axis cases, and need be performed only once for a specific set of sensors on an individual.Although one sensor is sufficient to capture single-axis joint motion, any misalignment of such a sensor from use to use will lead to erroneous measurements. From a practical standpoint, it is obvious that a method is needed to adjust for any shifting of a sensor about the joint that will take place from one use to the next. It is both undesirable and impractical to recalibrate the whole sensor every time the patient takes off the sensing garment and places it back again. To take care of such registration problems, an array of multiple sensor threads is used. By incorporating multiple threads in a known pattern, a template-matching algorithm can be performed to determine a sensor's offset from calibration. In this way, measurement errors due to sensor misalignment are significantly reduced. The details of this method are described in the next section.The goal in designing these wearable sensors is to create a device that is ultimately self-registering for subsequent uses after the initial one-time calibration experiments. This means that no additional equipment is needed to register the sensors for each use. Also, it is important that any procedures that are needed for self-registration are simple, and able to be preformed by the patient without supervision. To achieve these goals, a multi-thread sensor array design is presented.M sensors covering a single-axis joint as shown in Figure d. This multi-thread sensor array is used to estimate a single-axis joint angle, θj. To develop a registration procedure let us first calibrate each sensor thread individually. Let  be the estimate of the j-th joint based on the i-th thread sensor given byFirst, consider an array of where is the 1 × 2 regression vector that is optimized for the i-th single-thread sensor of the j-th joint placed at a home position.and α, from the original position where calibration was performed. See Figure α. Assuming that the individual sensor threads are identical other than being separated by a distance d, we can conclude that the pattern of the sensitivity array is a shifted version of the calibrated one, as shown in the simplified plots of Figure Now consider the situation where the sensor array has been removed, and placed back on the joint for more measurements. The sensor array is now offset an unknown distance,  will no longer be the appropriate regression matrix to estimate θj from Yj(i). A new, unknown vector  will instead relate the sensor output to θj: is unknown, each individual sensor in the array should ideally give the same estimate for the actual joint angle at any time, so thatAlthough If the shifting of the sensor array were to happen in a discrete fashion,α = nd     (16)n is an integer value, it is seen thatwhere n is an unknown, it is desired to find an n that satisfies (15) and (17), rewritten asSince n is assumed to be |n| <M - 1. Namely, the sensor array, although shifted, can still cover the joint, having an overlap with the original sensor at the home position.where n that can be found to exactly solve (18). Unfortunately, for practical usage, n will not be a discrete integer. Furthermore, n cannot be explicitly found since process and measurement noise will cause the sensor outputs to deviate from their "ideal" values. With the knowledge of  for i = 1 ~ M, however, it is possible to find the optimal integer n that best solves (18).In the ideal, theoretical case, there will exist an integer n as follows :Let us first define the average joint angle estimate for M threads of sensor outputs for a given integer n is found by minimizing the average squared error between each sensor's estimate and the average estimate with respect to n (i.e. reducing the variance in the estimated angle as a function of n):The best estimate for n = -M+2, -M+3, ..., M-3, M-2. The value for  found from (20c) is then used in (17) to approximate each sensor's predictor regression matrix for this new offset position of the array. In the ideal discrete case, where α = nod, no is the discrete offset of the sensor array,  = no, and Rj = 0.Equations (20a) and (20b) are solved for a is not a discrete multiple of d, the minimum variance is not zero, Rj  ≠ 0, but it will decrease as M increases, and d decreases.For the non-ideal case, where θj. Furthermore, since  can always be approximated using this algorithm, a one-time calibration is all that is needed for these wearable sensors to be used by a patient.Creating a denser sensor array in this way leads to more accurate estimates of sensor sensitivities, which in turn leads to more accurate estimates of  is converged upon. Again, the denser the array of sensors used, the better the estimate obtained. Following this, the algorithm need not be performed as often, as long as the sensor array remains stationary for an individual use. To begin monitoring, it is assumed that  = 0.The registration algorithm takes place in real time as the sensor is in use. All that is needed for a patient to begin using these sensors is to first "zero" the sensor output with the joint fully extended in the 0° position, and then freely move the joint to obtain non-zero data. This non-zero data will then allow the self-registration to take place. While registration is not needed at all times, it should be performed during initial operation until an appropriate d. The j-th joint array is placed so that it is most sensitive to changes in θj, while the k-th joint array is situated so that it is most sensitive to changes in θk.In the double axis case, two sensor arrays are placed around a predominantly two-axis joint such as the hip. As in the single-axis case, each array contains M sensor threads equally spaced by a distance θ1 alone. This hip flexion/extension causes significant changes to sensor array 1, y1(i), i = 1 ~ M. Next the patient is instructed to make hip abduction/adduction (θ2) alone, which causes significant changes to sensor array 2, as shown in Figure θ1 or θ2, has been moved, since sensor array 1 is most sensitive to θ1, and sensor array 2 for θ2. Once the individual axis movements are detected, the same registration procedure as that of a single axis can be applied to determine the misalignment of each sensor array. Once the misalignment is determined, the corrected, optimal predictor can now be used for verifying whether the registration has been performed correctly based on individual axis movements.For registration, let the patient move only one axis at a time. As illustrated in Figure This registration method reduces the multi-axis problem to individual single axis procedures. However, the single axis procedures do not have to be repeated for all axes, if they are tightly related. For the two hip axes in Figure All experiments have been conducted under a protocol approved by the Massachusetts Institute of Technology Committee on the Use of Humans as Experimental Subjects .Figure In these particular pants, an array of eleven sensors spans across the knee joint, each separated by a distance of 5 mm, and each with an unstretched length of 55 cm. The sensors threads were silver plated nylon 66 yarn, which had an impedance of approximately 3.6 Ω/cm. Single sensors span both the posterior and side of the hip as well to capture two axes of hip motion. These single sensors are not seen in the view of Figure To get an idea of the capabilities of existing technology available for joint monitoring, tests were initially performed using a standard electrogoniometer. Figure The goniometer was taken off and placed back on the knee joint eight separate times. Each time the goniometer was put on, the leg was extended (Position 1) and the goniometer output was set to 0°. The leg was then slowly bent to Position 2 (50°) and the goniometer output was recorded. The average rms error between the goniometer output, and the known joint angle (50°) for these tests was 3.5° with a standard deviation of 2.6°. Even with the goniometer placed on the same joint by the same person, these results illustrate the fact that slight changes in how the goniometer is attached can lead to varying measurements. It will be important to keep errors such as these in mind when the results from the conductive fiber sensor are analyzed.Having just discussed the possible errors introduced by a standard electrogoniometer, it is important to also highlight the possible errors introduced by a conductive fiber thread sensor. Consider again Figure As can be seen, there is a significant amount of variation possible in sensor output for a given joint angle. In particular, for threads over the knee joint, the average rms error between curves such as those shown in Figure The pants sensing garment was first used to estimate single-axis knee angle measurements. For the following single-axis experiments, a rotary potentiometer firmly attached to the leg was used as a goniometer, and this was the standard for which to compare joint angles. In each experiment, the potentiometer was "zeroed" with the leg in the full extension position.A calibration was performed to find the optimal regression matrix for both the linear and nonlinear predictors, and a sequence of knee movements was then monitored with the sensors. Figure The performance of the pants sensors can be seen to be quite good, accurately capturing the joint movement patterns over time. The average rms error between the pants sensor estimate and the potentiometer using the linear predictor was 5.4°, while that for the quadratic predictor was significantly better, at just 3.2°.It is important that these sensors are able to measure all types of motion, including higher frequency motion. To determine the frequency capabilities of the prototype fiber sensors, tests were performed where the leg was swung back and forth at different frequencies. The resulting sensor estimations, and errors when compared to the potentiometer, are summarized in Figure From these results, it is seen that the sensors are able to track the joint motion for frequencies as high as 2 Hz, but significantly larger errors result as the frequency is increased. Since most gross human motion takes place below these frequencies in a typical day, these sensors are suitable for everyday measurements, but such limitations should be considered if more accurate measurements are desired.Since these sensors are to be worn multiple times by a user, the reliability of registration is important every time the sensors are worn. Therefore, it is important that using the template-matching algorithm with an array of sensors will give an accurate registration each time the sensors are taken off and put back on. To verify this, an initial repeatability test was performed on the prototype sensor pants. The pants sensors were taken off and put back on four separate times to simulate four future uses of the sensors after an initial calibration test. The knee joint was moved over a wide range of motion in each instance. The joint angles measured by the fiber sensors for each test are shown in 11. The errors between these measurements and the potentiometer measurements are summarized in Table Again, the sensors are able to capture the overall motion of the knee in each case, but appear to give less accurate results each time the pants are worn. For this reason, while a completely self-calibrating sensor is always desirable, it may be necessary to re-calibrate the sensors after many uses for more accurate measurements.y1 and y2, spanning the posterior and lateral side of the hip, respectively . In the second segment, leg movement was allowed only in the frontal plane, while the subject performed an abduction/adduction movement three times (θ2 varies, θ1 = 0).Figure In each case, the sensor spanning the axis in which the angle changes took place was the most sensitive to change, as expected. Each joint motion also produced small, but not insignificant, cross-coupling outputs in the "remote" sensors as well, showing that a single sensor output is dependent on multiple joint angles, and not one single angle.The pants sensor threads about the hip joint were then calibrated with the twin-axis goniometer. Table After initial calibration, random leg movements were then monitored with the sensors. Figure The pants sensors were again able to capture the joint movement patterns over time, in this case for two axes of motion. The average rms error between the pants sensors' estimate of hip flexion angle and the goniometer's was 2.5° using the linear predictor and 2.4° using the quadratic predictor. For hip abduction, these errors were 2.1° and 1.7° respectively. In this double axis case, the differences between the linear and quadratic predictors were not very significant over the typical ranges of hip joint angles measured.Previously, the assumption was made for the double axis hip joint that both sensor arrays would be offset from their calibration position by the same amount for each use. This allowed the double axis registration to be reduced to a single axis registration. To verify this assumption, a simple experiment was performed on the pant's hip sensors. The pants were taken off and put back on ten times. Each time, the distance around the waist between the sensor thread on the side of the hip, and the sensor thread on the rear of the hip was measured  movement of the fiber across the wire contact point may affect sensor output due to uncertainty in the area being contacted, and dynamic effects of the constant rubbing action; 2) although the elastic cord takes up a majority of the sensor tension, slight changes will also take place in the fiber tension as the joint is moved, and this will affect fiber resistance; and 3) different sections of even the same fibers will exhibit slightly different resistance characteristics due to the slightly inhomogeneous nature of such fibers. In spite of all these sources of uncertainty, it is still possible to accurately calibrate a set of sensors, and achieve acceptable joint measurements with minimal errors. These effects are minimized through careful selection of the particular fibers used as sensors, and in manufacturing the garment.While two specific predictor models have been presented for the calibration of a set of sensors, there are of course many more candidates that could be used as well. The linear and quadratic models used in this paper were the simplest choices, and the experimental results showed no advantage to adding more terms. Doing so only increased the computational requirements unnecessarily. This is why the models were presented as they were.A few more words should also be said about the registration algorithm. As presented, this algorithm only accounts for shifting of a set of sensors in one direction . It is felt that this is appropriate due to the construction of the sensing garment. With the sensors instrumented in a "vertical" fashion, the user is responsible for visually checking that they put the garment on with no twist. This is relatively easy to do with the fibers oriented vertically. Furthermore, as long as the sensors span well beyond the local effects of skin movement around a joint, small shifts in the vertical direction will theoretically have little to no effect on the sensor output. Requiring a patient to "zero" the sensor output with all joints in the 0° position each time the garment is worn further eliminates any errors due to sensor drift.Finally, the wearability of the pants sensing garment must be addressed. What makes this sensing garment "more wearable" than existing joint measurement devices is that it is simply a pair of pants that people already wear on a regular basis.The extra sensors and wires added to these pants are compact and lightweight, almost negligible to the wearer. These sensors are easy to use, requiring much less skill and carefulness by the user, in general, than a typical goniometer.A wearable joint movement sensor design has been presented that uses conductive fibers incorporated into a fabric that is form fitting to a joint. Resistance changes in the fibers caused by fiber movement as the joint is moved can be related to angular joint position. Using multiple fiber sensors, multi-axis joint angles can be determined, in addition to single-axis angles, after a one-time calibration procedure performed by a therapist/physician. Implementing a nonlinear predictor model, continuous joint angle measurements can be made during daily activities, with the sensor able to be taken off and put back on at any time with no need for manual recalibration. Sensor offsets due to misregistration can be accounted for through the use of a sensor array spanning the joints of interest. This allows the sensors to self-calibrate, with only a few simple motions of the patient.After preliminary experiments involving a pants sensing garment for lower body monitoring, it has been seen that this methodology is feasible for monitoring joint motion of the hip and knee. Multiple sensor arrays are used at multi-d.o.f. joints, where each sensor output is coupled to multiple joint angle changes. This design therefore produces a robust, comfortable, truly wearable joint monitoring device. This paper outlines the development of this sensor from initial idea to working prototype. Future effort is needed in developing a completely wearable, highly accurate sensor, though. This would include making the sensors wireless, and therefore "tether-free." More precise textile manufacturing techniques would also be needed to further reduce measurement errors.The author(s) declare that they have no competing interests.PTG developed the ideas discussed in this paper under the guidance of HHA. PTG carried out all experiments. Both authors read and approved the final manuscript.


The management of burn injuries is reported as painful, distressing and a cause of anxiety in children and their parents. Child's and parents' pain and anxiety, often contributes to extended time required for burns management procedures, in particular the process of changing dressings. The traditional method of pharmacologic analgesia is often insufficient to cover the burnt child's pain, and it can have deleterious side effects ,2. InterThis study investigates whether playing a virtual reality game, decreases procedural pain in children aged 5–18 years with acute burn injuries. The paper reports on the findings of a pilot study, a randomised trial, in which seven children acted as their own controls though a series of 11 trials. Outcomes were pain measured using the self-report Faces Scale and findings of interviews with parent/carer and nurses.The average pain scores  for pharmacological analgesia only was, 4.1 (SD 2.9), while VR coupled with pharmacological analgesia, the average pain score was 1.3 (SD 1.8)The study provides strong evidence supporting VR based games in providing analgesia with minimal side effects and little impact on the physical hospital environment, as well as its reusability and versatility, suggesting another option in the management of children's acute pain. Pain is a noxious stimulus which can be interpreted in many ways by different individuals but as yet the mechanisms by which the body manages it are not completely understood. Pain has been defined by the International Association for the Study of Pain as 'an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage' . Though Children with burn injuries undergo significant physical and emotional trauma, initially from their injury, and subsequently from the dressing changes and related treatment they undergo throughout the healing phase. The latter is referred to, in this article, as 'procedural pain'. Clinicians involved in the care of children with acute burns use the best available methods to reduce procedural pain. Adequate and appropriate pain management is essential to ensure that symptoms secondary to pain experiences do not become habitual . Moreoveanalgesics, muscle relaxants and hypnotics. These drugs help in reducing procedural pain experienced by children, however they frequently have unwanted side effects such as drowsiness, nausea, reduced postural control and lethargy [The burns service at the Women's and Children's Hospital  collaborated with University of South Australia, on this project. Pain management comprises administration of a number of medications, including lethargy ,8.Pain management of children in hospitals during dressing changes has been reported as inadequate ,2 and isVirtual Reality (VR) was initially conceived as a tool for pain modulation by Hoffman et al -18 who fWhy virtual reality? VR can be considered intermediary to reality and computer technology. Owing to its ability to allow the user to immerse and interact with the artificial environment that he/she can visualize, the game-playing experience is very engrossing -18,20-22This project was an interdisciplinary and inter-sectorial  collaboration between the Centre of Allied Health Evidence, and the  Department of Computer and Information Sciences, both at the University  of South Australia (UniSA), Australia, and Women's and Children's  Hospital(WCH), Adelaide, South Australia.Ethical approval for this project was obtained from both the institutions (WCH, Adelaide and UniSA).All children admitted to one specific ward (Newlands Ward), WCH, aged between 5 and 18 years, having burns to more than three percent of their body surface area, and requiring dressing changes, were eligible for inclusion in the study. Children with burns to their hands, face or head, past history of epilepsy and reduced intellectual capacity were not included, as they would have been unable to appropriately use the VR equipment.All eligible subjects were identified by ward staff, and were invited to participate in the project by the project team. Written child and parent consent was obtained at every contact.The test administrations of routine pharmacological analgesia or routine pharmacological analgesia coupled with virtual reality were randomly assigned to each half of the burns dressing change  following a coin toss determining the sequence. The child and parents were given a standard explanation about the VR administration and the VR game. If required, subjects were allowed a short preview to assist them to understand how to play the game.• The VR equipment constituted a laptop (Dell Inspiron 5100, Pentium 4 2.4 Ghz CPU with a Radeon Mobility 7500 Video Card) with the game software, developed by the Department of Computer and Information Sciences, UniSA (Based on the game 'Quake' by ID Software), a head-mount display (HMD) (IOGlasses Head Mount Display with a SVGA video resolution of 800 × 600 16 million colours), with a tracking system , to allow interaction with the virtual environment by moving the head and neck and decoder and a mouse used as a trigger. The game involved a visual simulation giving the children a feel of being on a track, using a pointer to aim and shoot monsters. Figure • The developers considered the applicability of the game through varying age groups, gender, intelligence and intellectual capacities, while designing the game. The game tried to achieve effective distraction via immersion without violence and a simplified game structure requiring minimal control by the player, to allow the smallest possible movement during the dressing change procedure.A within-subject design was used in which the children acted as their own controls.There was no interference with the dosage or type of analgesia which was administered to the children 30 to 45 minutes prior to dressing removal.The burns dressing changes would normally occur every Wednesday morning before the hospital ward, grand rounds. The dressing change involved administration of prescribed medication and application of olive oil on the dressing (if adhesive tape was covering the wound), approximately 30 to 45 minutes prior to the actual procedure. The first treatment half constituted the removal of the adhesive tape/bandages and the under-dressing (acticoat/silver oxide dressing) and the second half comprised of the wound being debrided and a fresh dressing applied, after being assessed by the consultant medical officer/s.Following the completion of each half of the dressing change (with or without administration of VR), the researcher obtained scores for average pain using the Face Scale Figure , and intThe subjects were asked to score their average pain experience at the end of each phase of the dressing change procedure . Pain was scored using a modified self-report Faces pain scale . The scaThe data was analysed by a blinded assessor, to reduce any biases and increase the rigour with which a de-identified and coded dataset was probed.There were nine eligible, consenting child subjects (6 boys and 3 girls) in the sample. The average age for both boys and girls was 10.0 years (SD 3.7 and 4.1 respectively), age ranging between 5 to 16 years. The average percent of body surface area burnt was 5.3% (SD 3.4%) and there was no significant gender or age difference in body area burnt.For boys, two had burns from contact with a silencer (muffler) on a four wheeled motor bike, two were burnt from a hot water bag bursting, one was burnt while playing with petrol and fire, and the remaining boy was burnt with hot oil from a BBQ. For the girls, all three were burnt by overturned fluids. All participants were experiencing burns for the first time, and when enrolled into the study, their burns were at either second or third dressing change. Every participants' pain, prior to enrolling in this study, had been managed either with no pain relief, or by pharmacological means.For every child participant, one parent or guardian with one exception (was not available to observe the dressing change) provided data on the effectiveness of the VR for every post-session interview. One key nurse involved in the burns management was also interviewed following each session.Overall, 13 trials were undertaken from nine children . The results of two subjects were withdrawn for further analysis as the respective participants were too drowsy from the effects of analgesia to participate appropriately in the VR section of the session. Thus the remaining seven child subjects were included for analysis, with a total of 11 useable trials . The seven participants in the included trials had an average age of 11.1 years (SD 3.5).p <0.05) in time taken in the two treatment halves . The average difference in administering the two treatment halves was approximately 2 minutes , over all included trials was 4.1 (SD 2.9), whilst for VR coupled with pharmacological analgesia, the average pain score was 1.3 (SD 1.8). Because of the small number of child subjects in the study, the data was considered per child, and per trial. Over all included trials, the mean pain score difference between administrations was 3.2 (SD 2.1), which was significant using paired t-tests (p < 0.01). This indicated the importance of the effect of using VR  in reducing pain experiences during burns dressing changes. The per trial pain responses to VR and analgesia, and analgesia alone, compared with the average trial response per administration is shown in Figure For each child subject who completed an eligible trial, the average per-child difference in pain scores between administrations of VR & Pharmacological Analgesia, or Pharmacological Analgesia alone, suggested that every child but one obtained an improvement in pain scoring of at least 2 points on the Faces Scale, attributable to VR, as demonstrated in Figure All nurses or parents agreed that VR helped distract the children  and was helpful in reducing pain and there were no negative comments  regarding the application of VR. (note - delete 'interestingly')The overwhelming response from the nursing staff was that VR administration was helpful to the child. Comments from the interviews are provided below as evidence of this."... probably VR helped to take concentration off ... probably helped take away a lot of the anticipation away from the treatment"."... from my past experience, I can tell that it (changing burns dressing) can be a real problem. It was not a problem today. He did not even flinch while the dressing was being taken off.""... communication was good – he understood what I asked him to do. I did not find it invasive or intrusive...""... it was great to do the changing (of dressing) without dumping him with medication.""... no (communication was not effected) ... he responded well to the requests and commands.""yes (pain was significantly less)... he had lot more pain with cleaning (of the wound) than taking the dressing off... He seemed to cope better with VR than without.""... cleaning the dressing in the bathroom made him more anxious, when he saw the wound – felt more pain.""Yes, he was more anxious when VR was not on.""... he felt worse when he was looking at it (the wound) compared to when he was not.""... was more relaxed and concentrated on the game. You could tell that he could feel the pain, but focussed on the game."The perception of the assisting nurses were the children were more cooperative and distracted from the administration of VR, which helped reduce the difficulty in changing the burns dressing compared to when routine analgesia was used by itself. There appeared to be no problem in physically using VR within the environmental constraints of the burns dressing area, and in no instance did VR impede communication with the child.All parents agreed with the positive effects of VR in pain management for their child. They all commented that the child's anxiety level was perceptibly less when using VR, and the child looked forward to playing the VR game. Comments below from the parent interviews support the positive VR effects:"... was a lot calmer and enjoyed the VR.""...absolutely, she did not remember about the last dressing that was taken off. She had to be prompted, to remind her of the game and change of dressing.""... much happier than usual. He reckoned he felt it but I think he did not. He did not show any of the same signs.""...she was not as anxious. Was afraid before hand, but she was all right after the game was switched on. It took her mind, off the pain.""...was smiling while playing the game.""... compared to the medication which left him groggy, disoriented, lost track of time and anxious, I think this (VR) allows the continuity of time and reduces anxiety.""Yes (pain was significantly less) ... probably judging it from yesterday – medication made him worse – uncooperative and pig-headed, compared to when he was playing the game.""Yesterday he was whinging thinking about the dressing change, this morning, when I told him that you were coming; he had a grin on his face..."However, the current game appeared to have a reasonable level of  complexity and engaged the participating children of different age  groups.The above comments were randomly selected from the questionnaire deployed at the completion of each trial (to interview the nurses, parents and children participating in the study).This is the first published randomised clinical trial to our knowledge reporting the use of VR for children with burns. It concurs with the findings of Hoffman et al ,16 who tGiven that the application of VR as a method for pain control in the clinical setting is very simple, the results of this study are encouraging with respect to future use. The present prototype VR game being used appears to be cumbersome due to the number of wires attaching to the laptop and the HMD, but this could be simplified considerably so that the equipment required is simply a console with a trigger and a head mount. Applying this equipment would be as simple as providing medication prior to the dressing change procedure. The equipment is reusable and requires minimal technical knowledge for use. Provided a number of different games were available to cater for different age groups, it could be widely applied, and will allow children to relocate themselves to 'another world' during dressing changes, decreasing their attention to painful stimuli. It was noted by nurses during several trials that communication was never a problem; they were able to instruct the child to change, or assist change in position without any difficulty in a compliant and relatively pain-free manner. On the other hand, children without VR were often distressed and crying in pain, decreasing their ability to listen and cooperate.A number of factors resulted in a small sample size. There were relatively fewer children with burns than anticipated, who fitted the inclusion – exclusion criteria, and who were admitted to the Women's and Children's Hospital, Adelaide, during the data collection period. Several potentially eligible children  had such severe side effects from the medication (particularly drowsiness) that they were not able to participate, and two refused to participate. The reduced sample size limits ability to generalise the results, and a study with a larger sample size may provide better understanding of the usefulness of VR as a treatment adjunct for pain relief.Another potential limitation is that some children were tested more than once – in the first instance immediately after their burn and then subsequently after surgery or during another dressing change later in the healing phase. In these cases, there may have been a learning effect which modified the pain scores, or simply an overall decrease in pain due to healing. It was also noted from the feedback that older children found the game too simple and therefore not as absorbing or distracting as the younger children found it.Finally, waterproofing of the VR equipment would allow data collectors to examine pain responses through an entire dressing change including when subjects had their wound debridement carried out in the bathroom.There appears to be considerable scope for further research into the potential for using VR in the clinical setting. Larger trials could be conducted, using games appropriate for the varying age groups. The next stage would be to test VR alone against pharmacological pain relief, to investigate whether VR is as effective in isolation, and could decrease use of analgesia, thus avoiding the side effects associated with medication. Another avenue of future research would be to investigate the exact mechanisms by which VR assists pain modulation. It is hypothesized that it works by distracting a child's attention from painful stimuli, which in turn reduces the perceived intensity of pain.The author(s) declare that they have no competing interestsDAD was involved with coordination and recruitment of trial  subjects, application of the trial, collection of data and writing up  the paper. KAG was involved with statistical analysis of the data and  supervising the scientific conduction of trials. ALS was a key medical  consultant for the trial subjects and coordinated efforts within the  hospital to recruit trial subjects. SEM was the first contact with the  prospective trial subjects. She identified and provided them with  initial information and got verbal consent from them. She also acted as  a coordinator for all the nursing staff members on the ward  participating in the trial. BAT provided IT support for building and  helped in maintaining the inventory (VR equipment) and the game  software.The pre-publication history for this paper can be accessed here:Pain scale scoringClick here for fileCaregiver interviewClick here for fileNurse interviewClick here for file